Dealing with an uncertain economy is never easy, especially for small businesses. Unlike their larger counterparts, small businesses rarely have the resources to monitor and take corrective action for every trend and issue. And even those owners who have weathered numerous business cycles may be faced with new circumstances that confound their otherwise successful instincts and knowledge.
While there is no crystal ball that accurately predicts the future, small business owners can take a number of steps that will help their enterprises endure the worst of times, and position them for success when conditions inevitably improve.
In the face of the ongoing credit crunch, consider an alternative to borrowing funds from a commercial bank. – Consider a funding line from Charter Capital, a preferred provider of accounts receivable factoring services. Charter Capital has vast experience in economic cycles, and can advise you on issues specific to your business and industry. It may be helpful to arrange for an invoice factoring or funding line. You may not need it for several months or at all. But if a lag in cash flow occurs, you will have a ready source of bridge money in place.
Make sure you have good relations with your vendors. – You may find it necessary to renegotiate terms, but overdue bills and inconsistent payment practices will not help your position. What's more, your vendors may be experiencing financial difficulties as well. Any flexibility will hinge on whether they perceive you as a reliable partner, or a risk. A factoring or funding line from Charter Capital will help establish you as a reliable partner.
Similarly, keep a close watch on your receivables. – Follow up with whoever owes your company money and make sure they are meeting their terms. Be firm when dealing problem accounts, but also be willing to negotiate where appropriate. It may take only a matter of months for a struggling customer to become a highly stable source of income. If you can accommodate such situations without adversely affecting your company's financial position, everybody wins. A factoring or funding line from Charter Capital will give you the freedom to extend even more generous payment terms to your valuable customers.
Step up your marketing efforts. – Many businesses mistakenly see marketing as a luxury when money is tight. The truth is that this is the time you need marketing most. Along with reassuring your current customers that you are still there to serve them, marketing can help you reach new markets that will sustain your business now, and facilitate its growth in the future. A factoring or funding line from Charter Capital will give you peace of mind to accelerate your marketing effort knowing that you have the cash flow to support your increase in sales.
Make sure expenditures can be justified, and that they contribute to the financial health of your business. – You may find it necessary to redirect money to areas that will enhance business performance. If you carry an inventory of products, check the accuracy of your records and procedures to prevent losses. Do not feel pressured to hold down sales for lack of inventory. A factoring or funding line from Charter Capital will give you freedom to invest in even more inventory than you might otherwise be able to afford.
Review your operations and expenses on a regular basis. – If you monitor your profitability on a monthly basis, it's a good idea to do it weekly or biweekly during slow economic times. Likewise, you should review your business plan more often. Monthly or quarterly reviews will make it easier for you to make adjustments and keep your business on track. A factoring or funding line from Charter Capital will give you a certain amount of flexibility in meeting your cash obligations. Obtain cash to negotiate prompt pay discounts with your vendors. Meeting your payroll and payroll tax obligations on a timely basis will most certainly save you additional expense.


John Essay The TV Program That I Like My favorite entertainment is to watch TV, but I won't see all kinds of program. I only choose.Favorite TV series essaysThat is definitely a tough question. When it comes to TV series I am a kid. My favorites are all cartoons. The one series that stands.Free Essays on Essay My Favourite Tv Programme. Get help with your writing. 1 through.It is a favourite past-time of many people, However, whenever I watch my favourite TV programme she doesn't complain. In fact, (Descriptive Essay) Money.Free Essays on My Favorite Tv Programme. Get help with your writing. 1 through.Nov 04, 2008 · My favorite TV programs Essay on picnic Essay on rainy season Travelling as a means of education Describe a village Difficulties of learning english.Jun 05, 2009 · Essay topic: My favorite TV programs; Essay topic: Talk about an embarrassing moment. Topic: With a big sum of money, will you purchase.We've Got Lots of Free Essays. research of the TV program to support his essay. Essay "My Favourite TV Programme" » Pullion pullion.com/news-media/933-essay-my.Essays - largest database of quality sample essays and research papers on My Favorite Tv Show Essay.Hello.Please rate my essay on A television program i like most You can judge my capacity to express my point of view. A television program.The television (TV) is part and parcel of many households. Therefore, watching television is a culture of today's modern society. It is a favourite past-.My favourite television program is an old television series which was Describe a TV programme that you watched and would Describe a famous TV program.It is a favourite past-time of many people, However, whenever I watch my favourite TV programme she doesn't complain. In fact, (Descriptive Essay) Money.
DESCRIPTIVE ESSAY: MY FAVOURITE TV PROGRAMME The television (TV) is part and parcel of many households. Therefore, watching television is a culture of today's.Do you like watching television? What are your favourite TV programmes? How much TV do you watch each week? Do you have a TV in your bedroom.My favourite tv programme essay in english. Contents1 Essay On Summer Season: My Favourite Season1.1 Read Also :Short Essay On Importance of Exams1.2 Incoming search.IELTS Cue Card Sample 171 - Talk about your favourite TV channel Details Last Updated: Saturday, 22 October 2016 04:51 Written by IELTS Mentor.English Compositions: Next TOEFL Vocabulary: English Conversation: My Favourite TV Cartoon Programme: Of all the TV cartoon programmes I watch.The television (TV) is part and parcel of many households. Therefore, watching television is a culture of today's modern society. It is a favourite past-.Kalle America today vs. brave new world flammable and protean Airt your essay on your favourite tv programme greeting or meteorologically.My Favourite Television Programme Essay For Kids. Write an English essay on My Favourite Television Programme in your words. Complexity: Easy: Grade/Class.Subject: Write an English essay on My Favourite Television Programme in your words. Complexity: Easy: Grade/Class: 1 st to 5th standard: Number of words.Everybody likes to watch TV and everybody has a favourite TV programme. I want to tell you about my favourite TV programme. I like to watch TV programme "Week.Check out our top Free Essays on My Favourite Tv Programs to help you write your own Essay.Free Essays on My Favorite Tv Serial Essay Of Tarak Mehta Ka Oolta Chasma. Get help with your writing. 1 through.My Favourite Cartoon 'Tom and Jerry' Essay For Class 3 ( 404 Words): This is one of my favourite episode of Tom and Jerry series.
Essay "My Favourite TV Programme" Essay Essay-Go Back. Related news. Composition "Television in Our Life" Composition "My Going to the Cinema" Composition "News.Essay My Favourite TV Programme Essay Essay-Go Back. Related news. Composition Television in Our Life Composition My Going to the Cinema Composition.DESCRIPTIVE ESSAY: MY FAVOURITE TV PROGRAMME The television (TV) is part and parcel of many households. Therefore, watching television is a culture of today's.Report Abuse Home Opinion Movies / Music / TV My Favorite Television Shows My Favorite Television Shows. By Anonymous. Image Credit: Bekah R., Snellville.My favorite TV programs As a senior high school student, I am very busy with my studies. Although I am very fond of watching television, I can only spend.Essay topic: My favorite TV programs; Essay topic: Talk about an embarrassing moment. Topic: With a big sum of money, will you purchase.Guardian and Observer writers pick their favourite TV programmes.Essay Writing Guide. Favourite TV - National It is a touching and heart-warming TV programme and I will not hesitate to recommend it to others.Check out our top Free Essays on Essay On My Favourite Tv Programme to help you write your own Essay.My favourite TV programme is Polònia. It's on TV3 on Thursdays evening, after the news. It's a comedy. Toni Soler is the director and the programme is about.My favorite tv show essay Prescott December 06, 2015 In trump we should just like trump's notice kovaleski is my gear's broken, is my favourite tv programs.Essay On Television. Forums Essay, we love your television essay and we would like you to write to us the smiths at the most popular programme is still.Essay Writing Guide. Favourite TV - National It is a touching and heart-warming TV programme and I will not hesitate to recommend it to others.
It is a favourite past-time of many people, However, whenever I watch my favourite TV programme she doesn't complain. In fact, (Descriptive Essay) Money. Essays - largest database of quality sample essays and research papers on My Favorite Tv Show Essay. My favourite television program is an old television series which was Describe a TV programme that you watched and would Describe a famous TV program.
English Compositions: Next TOEFL Vocabulary: English Conversation: My Favourite TV Cartoon Programme: Of all the TV cartoon programmes I watch. Subject: Write an English essay on My Favourite Television Programme in your words. Complexity: Easy: Grade/Class: 1 st to 5th standard: Number of words.
Report Abuse Home Opinion Movies / Music / TV My Favorite Television Shows My Favorite Television Shows. By Anonymous. Image Credit: Bekah R., Snellville. Essay Writing Guide. Favourite TV - National It is a touching and heart-warming TV programme and I will not hesitate to recommend it to others. Essay On Television. Forums Essay, we love your television essay and we would like you to write to us the smiths at the most popular programme is still.


In a large skillet, brown the ground beef, and then drain. Add tomato sauce, onion, and bell pepper.
In a large bowl, combine the noodles with the cream cheese, sour cream, garlic salt, and the sugar, taking some care to not destroy the noodles.
In a 9 X 13-inch baking dish, layer half the meat mixture, then half the noodle mixture. Repeat.
Cook in the oven for, let's say........30 to 45 minutes or so at 375 degrees F. Top with shredded cheese and bake another 7 minutes. After that, it's time to grub!


text:
Why should Malaysia's politicians be emulating Trump?
@fatbidin
Photos comparing the crowd that attended U.S. President Donald Trump's inauguration (L) on Jan 20, 2017, and Barack Obama's in 2009 at the National Mall in Washington DC. Source: Reuters/Lucas Jackson (L), Stelios Varias
ON SUNDAY, Malaysia's Deputy Prime Minister Ahmad Zahid Hamidi advised members of ruling party Umno (United Malay National Organisation) to learn how U.S. President Donald Trump manages public perception by mastering the use of social media.
The nation's Number Two pointed to how the business magnate, despite his controversies, managed to defy the odds to clinch the U.S. polls and become the 45th President of the United States. Zahid also said the same of India's Prime Minister, Narendra Modi, noting that both men have been successful in changing the public's perception of them.
Zahid went on to say that false news should never be repeated because they can become accepted as truth, and that Umno members should devise a strategy to counter the negative perception of them created by social media users.
Before Zahid's remarks, Malaysian Communication and Multimedia Minister, Salleh Said Keruak, also from Umno, has raised the issue of fake news several times before. He has accused the local news media of disseminating false information, although he hasn't quite backed up his claims with concrete evidence.
It is funny how Trump is regarded a good example to follow when he has quite a reputation for how he treats the media. He is often reported accusing the media of spinning the truth – like the time he refused to answer a CNN reporter's question after alleging that the entire organisation was churning out fake news.
https://www.youtube.com/watch?v=Vqpzk-qGxMU
Video can't be loaded: Donald Trump shuts down CNN reporter: "You're fake news" (https://www.youtube.com/watch?v=Vqpzk-qGxMU)
And remember what he did right after his inauguration last Friday? He immediately berated the press. He accused the media of lying in reports claiming the turnout for the ceremony was poor. He insisted that the crowd stretched across the National Mall and that about 1.5 million people were there.
[RT] [AT]foreigncorr1: Trump's speech in front of the CIA is frankly off the radar pic.twitter.com/XxdiCzeZfh
— Ai WeiWei (english) (@aiww_en) January 22, 2017
His press secretary, Sean Spicer, also held a press conference at the White House to say that the turnout was the biggest ever at a presidential inauguration and that the actual numbers weren't available. He accused the press of deliberately lying about the size of the crowd.
What's funny is that all the press did was to place actual photographs of former President Barack Obama's first inauguration in 2009 and those taken during Trump's inauguration side by side (see above).
The pictures, of course, show a significant difference, leaning to the advantage of Obama.
The media also reported that according to numbers obtained from Washington DC's train system, it clearly showed more used the trains during Obama's swearing in. In 2009, it is estimated that 1.8 million people turned up for the presidential inauguration.
So it appears that in this case, the facts are really on the press' side and Trump and his aide have likely been spouting unsubstantiated claims. At the very least, they could have provided something to prove their remarks true and the media wrong.
So this is how Trump handles negative perception – repeating untruths regardless how many times they are disproved.
How can this be a good example for Malaysian politicians to follow? Should our leaders take the Trump route – persevering by repeating what appears to be half-truths and hoping their constituents would eventually buy into it?
At least in the U.S., media outlets aren't so easily cowed into submission. Judging from reports filed in the aftermath of Trump's tantrum, they have instead banded together, each one demonstrating in their own way how they would not be intimidated by the world's most powerful man. To do this, outlets have been republishing facts and evidence to prove the president and his aide wrong.
First press conference is a blatant lie to the public, another word for a lie is #alternativefact #trump #inauguration pic.twitter.com/0Lae19FzUO
— US Kare (@USKare27) January 23, 2017
They have also come together to express disdain for Trump's treatment of the press, saying it was not something the country's president should be doing. From the many opinion pieces, articles defending previous reports on the inauguration turnout and comments issued so far, their message is clear – they won't back down until Trump backs off.
But for the rest of the world, this episode could still prove dangerous.
When the leader of the free world makes moves that appear to infringe on key democratic principles upon which the U.S. is founded, it sets a precedent for others to follow. It tells others that attacking the press is the new norm, and that it is okay to do so.
SEE ALSO: Newspapers warn Asia-Pacific countries to brace for Trump
In Malaysia, politicians across the divide have been berating the press for a long time. The difference is that our local outlets usually obey, fearing they'd lose their licenses and be out of jobs. So while the press may be united, they don't go out to name and shame the politicians who try to dictate the news.
In fact, it looks like the local media is so conditioned by this that they accept it as the norm. And now with the President of the United States and his administration doing it too, it just reaffirms to the politician that this is acceptable.
Now that, to me, is rather unsettling and frankly, quite scary.
** This is the personal opinion of the writer and does not reflect the views of Asian Correspondent
{"total":9,"error":"","stumbleupon":0,"facebook_total":7,"google":1,"pinterest":0,"linkedin":0,"buffer":1,"twitter":0,"vk":0}
Topics covered: donald trump fake news Trump US presidency

redpajama_set_name: RedPajamaCommonCrawl


In the globalised world we live in today, migration flows are heavier than they have ever been. In Ireland, economic booms and busts have always had a significant impact on emigration and immigration, and this latest cycle since the mid-2000s has been no different.
The economy has improved dramatically in the past four years since emigration out of Ireland peaked in 2012, with unemployment now at just 6.4 per cent. But Irish people continue to leave Ireland in very significant numbers, according to new data from the Central Statistics Office, which shows 30,800 Irish people emigrated in the year to April 2017, outnumbering those returning home by 3,400.
But how reliable are those figures?
Keeping track of the numbers coming and going from Ireland is a complex task for statisticians. Unlike many other countries, Ireland does not have a population register requiring people to log their place of residence.
The migration statistics published by the CSO each year are usually estimates, based on data gathered from a sample of 26,000 households in the Quarterly National Household Survey which are grossed up to give a national estimate. When the census is carried out every five years, more specific data from every household in the Republic is used to correct the estimates retrospectively.
Last year, before the census data had been crunched, the CSO estimated that 60,500 Irish people had moved home between April 2012 and April 2016, when in fact, according to census-corrected figures published on Thursday, 99,500 did. It is a very significant difference of more than 64 per cent.
The rise in the figures will certainly be welcomed by groups working to encourage Irish emigrants to return home. But how could the CSO estimates be so wrong? And what implications could this have had on policy?
According to CSO demographic statistician James Hegarty, recording the number of Irish nationals returning to the country from abroad poses a particular challenge.
Between censuses, they have to rely on a combination of diverse data sets, including visa data from popular destinations such as Canada and Australia, national insurance numbers in the UK, figures from the Garda National Immigration Bureau, and Irish PPSN data. The latter is the most accurate indicator, but only for non-Irish nationals.
Between the 2011 and 2016 censuses, the CSO population estimates were out by 65,000 in total, which, given the population of Ireland in 2016 was 4.76 million, is an error of 1.4 per cent overall in the five-year period.
"In that context I think we are doing okay, but always striving to improve," Hegarty says.
Following the 2016 census results, the CSO is now looking at alternative data sources in an effort to improve accuracy, especially in the way it measures returning Irish citizens. One way to do this is to include the full suite of migration questions as part of other CSO social studies, such as a major upcoming survey on the health of the nation which will be carried out on a much larger sample than the quarterly survey.
The overall figures may not have been too far off, but the biggest anomalies are in the figures for returning Irish emigrants, who may have specific support needs. Many more have returned than we previously thought, meaning much higher numbers have been affected by issues such as their no-claims bonuses not being recognised for car insurance, difficulty accessing mortgages and other loans, and other issues which are very specific to them.
The Government commissioned an economic report on these barriers in August, but by the time the findings are published, and policy changed, it could be too late to make a difference to the majority of "generation emigration" who may have already returned, or indeed, come and gone again.


text:
Vicky Phelan
Vicky Phelan's powerful speech about smear test scandal highlights importance of cervical checks
The Limerick mother now has terminal cancer
Emma Costello
Vicky Phelan pictured speaking to the media on leaving the Four Courts (Image: Collins Courts)
Vicky Phelan has made a powerful speech to the Public Accounts Committee (PAC) this evening.
The Limerick woman shared her story with RSVP Live earlier this year revealing she has cervical cancer.
She has been praised across the country for highlighting the failures in the cervical cancer screening programme.
Speaking previously with RSVP Live, the mother revealed she was diagnosed with cancer three years after her smear test results of 2011 were incorrectly reported as clear.
She had hopes of an experimental treatment to save her life but she has been told by doctors she has six to 12 months left as her cancer is now terminal.
Free smear tests in private clinics available for Irish women to alleviate cervical cancer fears
Vicky told the committee she "never missed a smear" and had undergone the tests since she old enough – even before the CervicalCheck screening programme was rolled out in 2008.
She told TDs; "I want to see change happen, we never want to see this happen again."
She has told the Dáil's PAC that she wants to see the people involved held accountable and the HSE overhauled "from the ground up" as well as a commitment that the failures that have been exposed never happen again.
She added that "over my dead body, I'm going to keep at this. Simple as that."
Vicky explained that she went to see her doctor in 2014 after experiencing bleeding and had another test. She was diagnosed with cancer that year.
The cancer spread beyond her cervix and she underwent a five week course of radiation and chemotherapy.
She described that time as "five weeks of hell."
Her treatment ended on her 40th birthday but she has constant back pain since it finished.
Then in September 2017 she found out her cancer returned after a check up.
(Image: Vicky Phelan)
It had spread and was affecting vital organs and Ms Phelan was told it was terminal.
She also discovered that her 2011 smear test had been incorrect and began her legal action in January.
She said there are issues of open disclosure in her case as there was 15 months of correspondence about who would tell her about the incorrect smear test.
Vicky told TDs: "The misdiagnosis in my case it has cost me my life. I've got terminal cancer.
"I don't believe I'm going to die but I have to fight for my life every day."
She said the incorrect 2011 smear test was either as a result of incompetence or it wasn't looked at at all as it was "full of cancer."
Vicky Phelan's daughter Amelia meets her hero Ed Sheeran in Cork
She continued that 18 women who have been affected by the smear test scandal have died.
She said: "At least I'm still here to tell the tale and that's why I'm fighting with everything in my being."
Speaking to Neil Prendeville on Cork's Red FM earlier this year, Vicky urged listeners that, despite the smear test scandal at present, they should still go for smear tests and they should go privately.
She said: "Please continue to go for smears. You can [also] pay for a private smear. I would encourage women to be honest to go more often.
"I think three years is too long. Pay for a private smear if you have any concerns.
"The thing to look out for in my case is bleeding between periods, dull lower back pain that doesn't go away and if you bleed after sex.
"If you bleed after sex 100% go to the doctor. What I don't want is women not going for smears. Cervical screening does save lives."
Can mumps make you infertile?
5 signs your job is damaging your health
Most weight you can gain in a day
"Coffee makes your hangover worse"


text:
Q: Group Policy settings remain after GPO was removed I have a Windows 7 Professional I had in an UO called "Testing" using Windows Server 2008 R2. In the OU a GPO setting to disable the registry access was set:
User Configurations/Administrative templates/System/Prevent access to the registry - Enabled
I have since removed this GPO from this OU and no other GPO is altering that settings. That GPO is no longer linked in the OU and it is removed. Problem is when I try to access the registry on the machine I am still blocked. Looking into the RSOP it shows the computer is still playing the setting. How can it still be set if no GPO is telling it to remain set? And how can I remove this setting from being set and bring it back to "Not Configured"?

A: Is your default domain policy enabled on this OU?  If yes, add:
User Configurations/Administrative templates/System/Prevent access to the registry - Disabled
Then 'gpupdate /force' and test.

A: try to run the following as admin
RD /S /Q "%WinDir%\System32\GroupPolicyUsers"
RD /S /Q "%WinDir%\System32\GroupPolicy"
gpupdate /force

redpajama_set_name: RedPajamaStackExchange


text:
Issue in progress
Latest complete issue
Archive by article type
Editors and Board
Editor biographies
Travelling Fellowships
Journal Meetings
Presubmission enquiries
Cover suggestions
Promoting your paper
Biology Open transfer
Media policies
DEVELOPMENT AND DISEASE
Mice lacking sister chromatid cohesion protein PDS5B exhibit developmental abnormalities reminiscent of Cornelia de Lange syndrome
Bin Zhang, Sanjay Jain, Haengseok Song, Ming Fu, Robert O. Heuckeroth, Jonathan M. Erlich, Patrick Y. Jay, Jeffrey Milbrandt
Development 2007 134: 3191-3201; doi: 10.1242/dev.005884
Bin Zhang
Haengseok Song
Ming Fu
Robert O. Heuckeroth
Jonathan M. Erlich
Patrick Y. Jay
Jeffrey Milbrandt
Supp info
PDS5B is a sister chromatid cohesion protein that is crucial for faithful segregation of duplicated chromosomes in lower organisms. Mutations in cohesion proteins are associated with the developmental disorder Cornelia de Lange syndrome (CdLS) in humans. To delineate the physiological roles of PDS5B in mammals, we generated mice lacking PDS5B (APRIN). Pds5B-deficient mice died shortly after birth. They exhibited multiple congenital anomalies, including heart defects, cleft palate, fusion of the ribs, short limbs, distal colon aganglionosis, abnormal migration and axonal projections of sympathetic neurons, and germ cell depletion, many of which are similar to abnormalities found in humans with CdLS. Unexpectedly, we found no cohesion defects in Pds5B-/- cells and detected high PDS5B expression in post-mitotic neurons in the brain. These results, along with the developmental anomalies of Pds5B-/- mice, the presence of a DNA-binding domain in PDS5B in vertebrates and its nucleolar localization, suggest that PDS5B and the cohesin complex have important functions beyond their role in chromosomal dynamics.
PDS5
APRIN
Sister chromatid cohesion
Congenital defects
Primordial germ cells
Sister chromatid cohesion, the linkage of sister chromatids from S phase until onset of anaphase, is important for accurate chromosome segregation during cell division and is mediated by a highly conserved multi-protein complex, called cohesin. The cohesin complex consists of core components, SMC1, SMC3, SCC1 and SCC3, and cohesin activity is regulated by its regulatory factors, PDS5, SCC2, SCC4 and ECO1 (Hartman et al., 2000; Nasmyth and Haering, 2005). In addition to chromosomal dynamics, cohesin and its regulatory factors also play important roles in development. For example, Mau-2, a recently identified metazoan homolog of yeast SCC4, not only participates in sister chromatid cohesion, but also guides cell movement and axonal outgrowth during development (Benard et al., 2004; Seitan et al., 2006; Watrin et al., 2006). In Drosophila, mutants of cohesion factors (e.g. SMC1 and NIPPED-B) regulate transcription of cut and other genes encoding developmental regulators through attenuating cohesin-mediated blocking of long-range enhancer and promoter communication (Dorsett et al., 2005; Rollins et al., 2004). This additional developmental function has recently been extended to humans with the discovery that mutations in NIPBL, SMC1A and SMC3 are responsible for ∼50% of cases of Cornelia de Lange syndrome (CdLS). CdLS patients present with a variety of developmental anomalies, including dysmorphic facial features, mental retardation, growth delay, limb reduction, genital anomalies and cardiac defects (Deardorff et al., 2007; Krantz et al., 2004; Musio et al., 2006; Tonkin et al., 2004).
PDS5 is a regulatory component of cohesin and interacts genetically or physically with the cohesion factors SCC2, ECO1, SMC1, SMC3, SCC1 and SCC3 (Losada et al., 2005; Tanaka et al., 2001) to ensure proper sister chromatid cohesion during mitosis and meiosis (Hartman et al., 2000; Panizza et al., 2000; van Heemst et al., 1999). The N-terminal region of PDS5 contains several HEAT repeats, a motif that is involved in protein-protein interactions and is present in other cohesin subunits and regulatory factors (i.e. SCC2) (Neuwald and Hirano, 2000; Panizza et al., 2000). In vertebrates, there are two homologs of PDS5, PDS5A and PDS5B, that both contribute to cohesion dynamics (Losada et al., 2005). Interestingly, in addition to its regulation of chromatid cohesion, PDS5 also appears to directly regulate transcription of a number of developmental genes. For example, similar to SCC2/NIPPED-B and SMC1, PDS5 regulates transcription of the Drosophila cut gene during wing development (Dorsett et al., 2005). In humans because PDS5B (also known as APRIN in human and mouse) is located in a region where loss of heterozygosity is commonly detected in tumors (13q12.3), it may act as a tumor suppressor (Geck et al., 2000). Furthermore, PDS5B regulates androgen-induced differentiation of prostate epithelial cells (Geck et al., 2000). Finally, lethality in yeast Pds5 mutants can be rescued by overexpression of topoisomerase II, however, TOPII does not compensate for the cohesion deficit in these mutants, indicating that PDS5 has crucial functions in yeast beyond those related to chromosome cohesion (Aguilar et al., 2005).
To delineate physiological roles of PDS5B in mammals we generated Pds5B-deficient mice using homologous recombination. Loss of Pds5B results in a number of developmental defects, including dysmorphic facies, cleft palate, skeletal patterning and bone development defects, cardiac malformation, distal enteric nervous system (ENS) aganglionosis, abnormal autonomic nervous system formation, and depletion of primordial germ cells. Together, these results indicate that PDS5B and perhaps the cohesin complex itself is a critical regulator of multiple aspects of organogenesis. The association of CdLS with mutations in several different cohesion proteins and the constellation of developmental defects in Pds5B-/- mice indicate the widespread role of the cohesion factors in normal development, and provide a possible mechanism for their role in the pathogenesis of CdLS and related developmental disorders. As the first mouse model resembling CdLS, the Pds5B-deficient mice will be useful in delineating how PDS5B and cohesin regulate developmental processes.
Generation of Pds5B knockout mice
Pds5B-deficent mice were generated by homologous recombination and genotypes were determined by Southern and PCR analyses.
The Pds5B recombination construct encompasses genomic regions surrounding the first coding exon. A 7.9 kb 5′ arm upstream of the first coding exon of Pds5B containing 20 nt of 5′ UTR and a 6.3 kb 3′ arm downstream of the first coding exon of Pds5B were generated by PCR (See below for primer sequences) and cloned into pRS315 by yeast gap repair (Le et al., 2005). The final targeting construct was generated using yeast gap repair by co-transforming yeast with linearized 7.9 kb 5′ arm in pRS315, 6.3 kb 3′ arm in pRS315, the β-gal-PGK-Neo (β-gal-Neo) cassette, and two adapters. Two adapters were generated by annealing adapter forward and reverse primers and filled by Herculase DNA polymerase (Stratagene). For cloning the 5′ arm and 3′ arm by gap repair, pRS315 was digested with SacI and XhoI. For cloning the final target construct, 5′ arm pRS315 and 3′ arm pRS315 were digested with XhoI and MluI, respectively. The final Pds5B recombination construct was confirmed by sequencing analysis to ensure veracity of the Pds5B arm regions.
The Pds5B targeting vector was linearized with KpnI and transfected by electroporation into R1 embryonic stem cells. Homologous recombinants were screened by Southern blot hybridization using a probe downstream of the 3′ arm. Two properly targeted ES cell clones were injected into blastocysts to generate chimeric mice. The chimeras were mated and successful germline transmission of the targeted allele was detected by Southern blotting. Heterozygous mice grew normally and were mated toβ -actin Cre mice to remove the PGK-Neo cassette. The phenotypes of two independent lines (before or after excision of the PGK-Neo cassette) were similar. All animal procedures were performed according to our institutional approved protocols. Experiments were performed on mice from a mixed 129Sv/B6 background. The genotypes of Pds5B mutant mice were determined using Southern blotting or PCR using primers P1, P2, P3 (95°C 30 seconds, 58°C 60 seconds, 72°C 90 seconds; 35 cycles). Wild-type and mutant alleles were amplified with P1-P2 (390 nt amplicon) and P1-P3 (450 nt amplicon) primer pairs, respectively. (See below for primer sequences.)
Primers, containing Pds5B genomic and associated pRS315 sequences, used for PCR amplification of the 5′ arm and 3′ arm of the targeting construct were as follows:
5′ arm forward primer: TTAATGCGCCGCTACAGGGCGCGTCACGCGTCTGTGCTGGAGCTCACATGACCTGTCCT; 5′ arm reverse primer: ACAGCTATGACCATGATTACGCCAACTCGAGGATGGAAATGTCTGTACCCCTAAAGCAAGAAAA; 3′ arm forward primer: TTAATGCGCCGCTACAGGGCGCGTCACGCGTCAGTGTATGCTGCAGACAGCTGGGCAGTT; 3′ arm reverse primer: ACAGCTATGACCATGATTACGCCAACTCGAGCAGCCTGGTTTGCAGAGAGTTCTAGCACA.
Primers used to generate adapters for the final targeting construct using yeast gap repair:
Adapter 1 forward primer: GATCCCCCAAGCTTACT TAG ATCTCGAGCTAGCACGCGTGATGGAAATG; adapter 1 reverse primer: TTTTCTTGCTTTAGGGGTACAGACATTTCCATCACGCGTGCTAGC. Adapter 2 forward primer: ATACATTATACGAAGTTATAC CGGTTAATTAAGGGCTCGAGCTAGCGGGCCC; Adapter 2 reverse primer: CACACAAACTGCCCAGCTGTCTGCAGCATACACTGGGGCCCGCTAGCTCGAGCCCTTAA.
Primers used to genotype Pds5B mutant mice: P1, CCAGACCTGAAGAATTGGTGGAGGA;P2,ACCCTCCTGGAGTCAAGGAAA; P3, ACCCTCCTGGAGTCAAGGAAA.
Generation of polyclonal antibody against mouse PDS5B
Rabbit anti-PDS5B polyclonal antibodies were raised using C-terminal peptide immunogen NH2-CATKENDSSEEMDV-COOH (Pacific Immunology, CA, USA). The same peptide was crosslinked to a solid support by using SulfoLink coupling Gel (Pierce Biotechnology, IL, USA) and used for affinity purification of antibodies from serum.
Immunohistochemistry and western blot analysis
Tissues used in immunohistochemistry were fixed in paraformaldehyde or Bouin's fixative. For antigen retrieval, paraffin-embedded sections were deparaffinized in xylene, hydrated and boiled in 1 mM EDTA solution for 30 minutes prior to immunostaining (Jain et al., 2004; Naughton et al., 2006). Primary antibodies used in this study include sheep tyrosine hydroxylase (TOH; Chemicon), rabbit neuron-specific class IIIβ -tubulin (TuJ1; Covance), rat GCNA1 (George C. Enders, PhD, University of Kansas Medical Center, Kansas City, KS, USA), mouse BrdU (Roche), which were visualized using donkey anti-sheep HRP (Jackson ImmunoResearch), donkey anti-rabbit Alexa Fluor 488 (1:100; Molecular Probes), goat anti-rat Cy3 (Jackson ImmunoResearch), or goat anti-mouse Alexa Fluor 488 (Molecular Probes) secondary antibodies. Bisbenzimide (2 μg/ml; Sigma) was used for nuclear staining.
Western blot analysis was performed using standard techniques using proteins derived from embryonic day (E) 14.5 forelimbs. The limbs were lysed in 2× SDS protein lysis buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 1× protease inhibitor cocktail; Roche). The western blots were probed with rabbit anti-PDS5B antibody (1:500 dilution) and PDS5B was visualized using chemiluminescence substrate (Pierce). For a loading control, mouse anti-β-tubulin antibody (DSHB, University of Iowa, IA, USA) was also used at 1:20,000 dilution.
Histological analysis of bone and palate
Alizarin Red S and Alcian Blue staining of newborn mice was performed as previously described (Wellik and Capecchi, 2003). Briefly, newborn mice were skinned and dehydrated in 95% ethanol. They were then stained in Alcian Blue solution for 3 days, rehydrated and incubated in 1% KOH for 2 days, and stained with Alizarin Red S for 3 days. Embryonic and newborn heads were fixed in 4% paraformaldehyde at 4°C for 16 hours. The fixed heads were properly oriented in paraffin, coronal sections were prepared, sections were stained with Hematoxylin and Eosin (H&E) and examined microscopically for evidence of cleft palate.
Analysis of cardiac malformations
The thoraxes of newborn mice was fixed in 4% formaldehyde before the heart was dissected free and embedded in paraffin. Entire neonatal hearts were serially sectioned at 6 μm thickness, stained with H&E, and inspected for defects. Two people (J.M.E. and P.Y.J.) examined the set of sections from every heart under a 5× microscope objective. The two examiners independently made the same diagnosis for every heart in this study.
Peripheral nervous system analysis
For analysis of the enteric nervous system, the E12.5 or E18.5 whole gut was dissected from the mouse and fixed with 4% paraformaldehyde at 25°C for 30 minutes. After fixation, explants were washed three times in TBST (1 mM Tris, 150 mM NaCl, 0.2% Triton X-100) and blocked with 4% donkey serum in TBST (1 hour, 25°C) before incubation with primary antibody (4°C, overnight) in TBST. Primary antibody (rabbit anti-Tuj1, 1:100; Covance) staining was visualized using donkey anti-rabbit Alexa Fluor 488 (1:100; Molecular Probes) secondary antibody after washing three times with TBST (Fu et al., 2006). For cross-sectional analysis, post-natal day (P)0 gut was fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin sections were stained with anti-Tuj1 (rabbit; Covance; 1:500) and visualized using goat anti-rabbit Cy3 (1:500; Jackson ImmunoResearch). P0 gut staining with acetylcholinesterase and analysis of the percentage of distal colon colonized by ENS precursors was performed as previously described (Jain et al., 2004).
The sympathetic nervous system of embryos and newborns was analyzed using whole-mount TOH immunohistochemistry as described previously (Enomoto et al., 2001).
Analysis of germ cells
Embryonic gonads were dissected, fixed in Bouin's solution overnight at 4°C, embedded in paraffin, and 6 μm sections were prepared. Primordial germ cells were examined by H&E staining, alkaline phosphatase histochemistry, or GCNA1 immunohistochemistry. For germ cell identification, anti-GCNA1 antibody (1:200, rat polyclonal) was used, whereas Sertoli cells were identified using anti-GATA4 antibody (1:200, goat Sertoli cells polyclonal; Santa Cruz Biotechnology) (Naughton et al., 2006).
For BrdU and GCNA1 double immunostaining, timed pregnant females were injected with 200 mg/kg body weight BrdU 2 hours prior to sacrifice (Jain et al., 2004). Paraffin-embedded sections of embryonic testis were incubated with BrdU primary antibody (1: 200; Roche) and visualized using goat anti-mouse immunoglobulin labeled with Alexa Fluor 488 (1:500; Molecular Probes) secondary antibody, followed by staining with GCNA1 (1:200) and goat anti-rat immunoglobulin labeled with Cy3 (1:500, Jackson ImmunoResearch). TUNEL was performed as previously described (Jain et al., 2004).
Testes transplantation
Testes from E16.5 mutant and wild-type mice were transplanted subcutaneously onto the back and/or flank of castrated 4- to 8-week-old male nude mice (Taconic No. NCRNUM, Germantown, NY, USA) as previously described (Honaramooz et al., 2002). The grafted donor testes were harvested and processed for histological evaluation at the indicated time points.
Metaphase spread analysis
Chromosome analysis was performed in the Chromosome Analysis Laboratory at University of Southern California directed by Chih-Lin Hsieh, PhD. Mouse embryonic fibroblasts from wild-type and Pds5B mutant mice (passage 3) were cultured in DMEM with 10% fetal bovine serum. Chromosomes were harvested the day after plating after growth for 1 hour in colcemid (1μ g/ml) using standard hypotonic (0.075 M KCl) treatment and fixed in methanol:acetic acid (3:1). Slides were prepared and chromosomes were analyzed using the Geimsa-trypsin-Wrights (GTW) banding method. Two cells were fully analyzed from each line, and all were found to have normal karyotypes. Twenty-two to 25 cells were examined for precocious sister chromatid separation (PSCS). To avoid selection bias, the first 22-25 metaphases encountered were included in the analysis regardless of the length of the chromosomes or differences in the quality of banding.
Mouse cDNA fragments corresponding to Pds5B (probe 1: nt 2903-3438 and probe 2: nt 4561-5098) were generated by RT-PCR and cloned into the BamHI and HindIII sites of pBluescript II KS+ vector. Sense or antisense 35S-labeled cRNA probes were generated from these Pds5B fragments and in situ hybridization was performed as previously described (Song et al., 2002). Frozen sections (12 μm) were mounted on poly-L-lysine-coated slides, fixed in cold 4% paraformaldehyde in PBS, acetylated and hybridized at 45°C for 4 hours in hybridization buffer containing the 35S-labeled antisense cRNA probes. After hybridization, the sections were treated with RNase A (20 μg/ml) at 37°C for 20 minutes. RNase A-resistant hybrids were detected by autoradiography. Sections hybridized with the sense probes served as negative controls.
Quantitative RT-PCR
RNA was prepared from mouse adult and embryonic tissues using Trizol (Invitrogen) and quantified by the Ribogreen fluorometric assay (Molecular Probes). Reverse transcription was performed using M-MLV reverse transcriptase (Invitrogen) and oligo(dT) and random hexamers as primers. qRT-PCR was performed using a Model 7700 instrument (Applied Biosystems) using Sybr Green I fluorescence (Molecular Probes) as described previously (Svaren et al., 2000). Target genes were analyzed using standard curves to determine relative levels of gene expression. Individual RNA samples were normalized according to the levels of GAPDH or 18S mRNA. (See below for primer sequences.)
Primers used for qRT-PCR analysis: mouse Pds5B, ACCCTCCTGGAGTCAAGGAAA and CAGAGTCCTGGTCCAT GT CCAT; mouse GAPDH, TGCCCCCATGTTTGTGATG and TGTGG TCATGAGCCCTTCC; mouse 18S ribosomal RNA, CGGCTA CCA CATCCAAGGAA and GCTGGAATTACCGCGGCT.
Plasmids and cell culture
Flag-tagged human PDS5A in pCR3.1 expression vector and the human PDS5B (KIAA0979) cDNA were gifts from Kasid Usha (Georgetown University, DC, USA) and Kazusa DNA Research Institute (Chiba, Japan), respectively. To generate the carboxyl-terminal EGFP-PDS5B fusion protein, KIAA0979 was digested with BamHI and EcoRI, blunted with Klenow, and cloned into the blunted XhoI site in pEGFP-C1 (Clontech). The EGFP-FLAG-PDS5A fusion protein was generated by digesting the Flag-PDS5A construct with PmeI and inserting this fragment into the blunted BglII site of pEGFP-C1. HeLa cells were cultured in DMEM with 10% fetal bovine serum. Transient expression of EGFP-tagged PDS5A and PDS5B was achieved using Lipofectamine-mediated transfection of plasmids.
Generation of Pds5B-deficient mice
As an initial investigation of PDS5B function we surveyed Pds5B mRNA expression levels in adult and embryonic mouse tissues. In the adult we found the highest levels of Pds5B expression in brain and testis (Fig. 1A). At the cellular level, in situ hybridization experiments revealed high Pds5B expression in neurons of adult hippocampus and dentate gyrus (Fig. 1C). In the testis, high PDS5B expression was detected at the periphery of the seminiferous tubules in a pattern consistent with immature germ cells (Fig. 1B). In the embryo, Pds5B expression was comparable in all tissues examined (data not shown), and in situ hybridization revealed that Pds5B was ubiquitously expressed at the cellular level (Fig. 1D).
The expression of PDS5B, a regulatory factor of cohesin, in post-mitotic as well as dividing cells, suggested that it participates in multiple processes in mammals. Therefore, to determine its physiological roles in mice, we generated Pds5B null mice by homologous recombination (Fig. 1E-G). To confirm the absence of Pds5B expression in the homozygous Pds5B mice, we quantified Pds5B mRNA in E14.5 Pds5B-/- brain and examined PDS5B levels in Pds5B-/- limbs by western blotting using PDS5B antibodies. We did not detect Pds5B mRNA or protein (Fig. 1H,I; see Fig. S1 in the supplementary material) in embryos homozygous for the mutant Pds5B allele, indicating that it is a true null allele. Pds5B-/- embryos were present at the expected Mendelian ratio until age E16.5. However, only about 75% of the expected number of Pds5B-/- fetuses were born alive and none of the mice homozygous for the mutant Pds5B allele survived beyond P1. The newborn animals had signs of respiratory distress, including labored breathing, use of accessory muscles of respiration as indicated by head bobbing and intercostal and abdominal retractions, cyanosis and pallor. They all died shortly after birth, most probably from cardiac and/or respiratory abnormalities (see below).
Pds5B deficiency results in growth retardation, abnormal skeletal patterning and cleft palate
Wild-type and Pds5B-/- mutant embryos were of similar size until E16.5, but then the mutants began to show signs of growth retardation. Newborn Pds5B-/- mice were smaller than their wild-type littermates, and had short stature, short limbs, a small head and facial dysmorphisms (short snout, short low chin and thin upper lip; Fig. 2A-C,K). These abnormalities, which are similar to those reported for CdLS patients, along with the fact that mutations in other cohesion proteins are associated with CdLS, suggested to us that the Pds5B-deficient mice could be a valuable model of this developmental syndrome. To substantiate this idea, we searched for other abnormalities in Pds5B-/- mice that are commonly found in patients with CdLS.
Expression ofPds5Band generation ofPds5B-deficient mice. (A) Quantitative RT-PCR analysis of Pds5B mRNA levels in adult mouse tissues normalized to 18S RNA. (Pr, prostate; Te, testis, St, stomach; Lu, lung; Sp, spleen; He, heart; Br, brain; Li, liver; Ki, kidney; Th, thymus). Error bars represent ± s.e.m. (B,C,D) Radioactive in situ hybridization (ISH) using 35S-CTP-labeled probes was performed using WT adult testis (B), adult brain (C), and E13.5 embryo (D). Silver grains indicate Pds5B mRNA. The green background is the Eosin counterstain visualized by darkfield microscopy. Note the high signal at the periphery of the seminiferous tubules (ST) consistent with Pds5B expression in immature germ cells (arrows in B), in hippocampus (arrow in C) and dentate gyrus (arrowhead in C), and wide spread expression in the E13.5 mouse embryo (D). In D, the sagittal sections are probed with antisense (AS, left) and sense control (S, right) probes. (E) Schematic representation of homologous recombination between Pds5B gene (top) and the targeting construct (middle) to give the mutant Pds5B allele (bottom). The coding region of the second exon of Pds5B is replaced by a β-gal/PGK-Neo (βGal Neo) cassette. The floxed PGK-Neo cassette was later excised by mating to β-actin Cre mice. K, KpnI; N, NheI; S, SacI. Black bars with numbers indicate exons and P1-P3 indicate positions of primers used for genotyping. (F) Southern blot analysis depicting successful targeting of the Pds5B locus using genomic DNAs from tails of wild-type (+/+), heterozygous (+/-), homozygous (-/-) mice. The DNA samples were digested with NheI and hybridized with the radiolabeled DNA probe, indicated in E. (G) PCR genotyping analysis of the Pds5B mutation was performed with primers P1, P2, and P3 indicated in E. (H) Relative amounts of Pds5B mRNA from wild-type (+/+), heterozygous (+/-) and homozygous (-/-) embryonic E14.5 brains using qRT-PCR normalized to GAPDH mRNA level. Error bars represent the s.e.m. *P<0.001, Student's unpaired t-test. (I) Western blot on E14.5 forelimb tissue lysates was probed with antibodies to PDS5B and β-tubulin. Note undetectable PDS5B protein in Pds5B-/- lysate.
To test for skeletal abnormalities similar to those in CdLS patients, we stained wild-type and mutant embryos with Alizarin Red S and Alcian Blue to highlight the skeleton, and found that several bones including the mandible, ulna, radius, humerus and scapula were shorter in the Pds5B-/- mice (Fig. 2D-F). Shortened limbs are associated with mild cases of CdLS (Ireland et al., 1993); however, we did not observe the limb truncations or fused digits (syndactyly) that are observed in more severely affected CdLS patients. All Pds5B-/- mice exhibited sternal malformations [wild type (WT): 0/24; Pds5B-/-: 37/37] characterized by either two unfused ossification centers or lack of ossification of the sternebrae (Fig. 2G). In addition, the sternums of Pds5B-/- mice were shorter than those of wild-type mice (Fig. 2G). We also found other abnormalities in skeletal patterning, with incomplete penetrance and variable expressivity, including delayed ossification of digital bones and vertebrae (data not shown), fusion of extensively ossified rib anlage at C7 and the costal cartilage of the T1 rib (WT: 0/10; Pds5B-/-: 10/18; Fig. 2H), loss or hypoplasia of the 13th ribs (WT: 0/10; Pds5B-/-: 20/20), and unfused ossification centers in the vertebrae (WT: 0/10; Pds5B-/-: 5/18; Fig. 2I) and hyoid bone (data not shown).
Another developmental abnormality observed in some children with CdLS is cleft palate. An examination of this region in Pds5B-/- mice revealed a complete cleft of the secondary palate (Fig. 2J,K) and failure of the posterior palatal bones to extend fully and meet at the midline (Fig. 2L,M; WT: 0/24; Pds5B-/-: 25/33). To further characterize the dysmorphogenesis of the palatal shelves in Pds5B mutant embryos, we examined the palatal shelves of wild-type and Pds5B-/- embryos at E12.5, E14.5 and E18.5. Whereas palatal shelves (PS) in Pds5B-/- embryos were seemingly normal at E12.5 (Fig. 2N,Q), by E14.5 they were clearly abnormal (Fig. 2O,R). In E18.5 wild-type embryos, the medial-edge epithelia of the two PS have normally fused and then degenerated (Fig. 2P), however, the PS of E18.5 Pds5B-/- embryos were shorter and failed to fuse (Fig. 2S). Using BrdU and TUNEL analysis on E13.5 embryonic palates, we did not observe significant changes in cell proliferation or apoptosis in palatal shelves of Pds5B-/- embryos compared to WT or Pds5B+/- embryos (see Fig. S2 in the supplementary material). The cleft palate observed in Pds5B-/- mice could be the result of abnormal reorientation of palatal shelves or failure of epithelial-mesenchymal transition of medial edge epithelia at a later stage. Clearly, cleft palate is a distinctive feature of Pds5B-/- mice and could cause aspiration and subsequent asphyxiation, thus contributing to the neonatal lethality of these mice.
Pds5Bdeficiency results in growth retardation, abnormal skeletal patterning and cleft palate. (A) The weight of Pds5B-/- P0 mice is lower than that of wild-type littermates (WT, n=43 and Pds5B-/- mice, n=35). Error bars represent s.e.m. *P<0.001, Student's unpaired t-test. (B,C) Morphology of wild-type and mutant mice. (B) Note short stature, facial dysmorphism, short limbs (arrow), short snout (arrowhead), and small head in Pds5B-/- compared to wild-type P0 mice. (C) Short mandible (arrow) in Pds5B-/- neonate (right) compared to wild-type (left). (D-I) Alizarin Red S and Alcian Blue staining of newborn mouse skeleton. Red indicates bone and blue indicates cartilage. (D) Mandibles; (E) ulna, radius and humerus of forelimbs; (F) scapula of forelimbs. Bone lengths (square bracket) are indicated by numbers (lengths in mm ± s.d.). Note the length of mandible, ulna, radius and humerus is significantly shorter in Pds5B-/- compared to wild-type. Sample sizes for each genotype range from 18 to 34. P<0.001, Student's unpaired t-test. (G) Sternum. Arrows, unfused ossification centers; arrowheads, delayed or missing ossification centers. (H) Ribs. The arrow points to the C7-T1 fusion. (I) Thoracic (T) and lumbar (L) vertebrae. The arrow points to the unfused L3 vertebra and arrowheads point to the hypoplastic 13th ribs in Pds5B-/- neonate. (J,K) Complete cleft palate (arrow) and thin upper-lip (arrowhead) of a Pds5B-/- neonate (K) compared with wild-type control (J). (L,M) Alizarin Red S and Alcian Blue staining of neonate skulls. The edges of palatal bones are marked by dashed lines. Posterior parts of the palatal bones in Pds5B-/- mutant failed to meet at the midline (arrows, M), compared with wild-type littermate (L). (N-S) Palatogenesis defects illustrated using H&E stained coronal sections of wild-type and Pds5B-/- E12.5, E14.5, and E18.5 embryos. Wild-type (N-P); Pds5B-/- (Q-S). ps, palatal shelf; n, nasal bone; to, tongue. Arrow in S points to the unfused palatal shelves. Scale bars:1 mm.
PDS5B is important for cardiac development
Because children with CdLS often have congenital heart defects and Pds5B-/- mice suffer respiratory distress and perinatal lethality, we searched for cardiac malformations. The incidence of congenital heart defects of any type was significantly higher in Pds5B-/- compared to wild-type mice (15/24 versus 0/14, P<0.001; Fig. 3A-G). The defects included those that are commonly found in CdLS patients (Jackson et al., 1993). We identified atrial septal defects of the secundum type (ASD, 6/24), ventricular septal defects (VSD, 12/24; ten perimembranous, one muscular, one inlet), and a common atrioventricular canal defect (1/24; Fig. 3C-F) in these mice. Of note, one Pds5B-/- mouse had a large perimembranous VSD with anterior malalignment of the outlet septum, causing the aorta to shift over the VSD toward the right ventricle (Fig. 3C). This mouse did not have right ventricular outflow tract obstruction and thus had the forme fruste of tetralogy of Fallot, a common malformation in CdLS. The VSDs and common AV canal defects would be expected to cause pulmonary overcirculation and edema from left-to-right shunting of blood at the ventricular level, thus resulting in respiratory distress and potentially contributing to early postnatal lethality of Pds5B-/- mice.
PDS5B-deficiency causes abnormal peripheral nervous system development
The high expression of Pds5B in post-mitotic neurons, the role of other cohesion components such as Mau-2 in axonal guidance and neuronal migration, and the profound mental retardation associated with CdLS, encouraged us to examine the nervous system in Pds5B-/- mice. Consistent with clinical reports showing that CdLS patients have relatively normal brain anatomy, we did not observe any gross or microscopic structural abnormalities in the brain of Pds5B-deficient mice. This suggests that the cohesin complex does not play a major role in CNS development; instead, it may influence neuronal function or connectivity. Unfortunately, the neonatal lethality of Pds5B-/- mice currently prevents us from further examining this hypothesis.
An examination of the peripheral nervous system revealed that the sympathetic chain ganglia were well developed with neuronal projections appearing normal in Pds5B-/- mice (see Fig. S3 in the supplementary material). We also examined the superior cervical ganglia (SCG), which provides sympathetic innervation to the ocular muscles. Abnormalities in the SCG are associated with ptosis, a common condition in CdLS. We found a number of abnormalities in the SCG and its projections to target organs in the Pds5B-/- mice (Fig. 4A). For example, in 40% (5/13) of Pds5B-/- mice, the SCG was located far caudal of its normal position (Fig. 4B,C). Furthermore, all Pds5B-/- mice had thin carotid nerves (Fig. 4D,E) and 30% (4/13) of these mice had unilateral or bilateral absence of the carotid nerve (Fig. 4F,G). These SCG defects are similar to those observed in mice deficient for the neurotrophic factor artemin, which commonly manifest unilateral or bilateral ptosis (Honma et al., 2002). These results suggest that SCG innervation defects are the likely cause of the ptosis observed in many CdLS patients (Jackson et al., 1993).
Congenital heart defects inPds5B-/- mice mimic those observed in CdLS patients. (A,B) Sections of wild-type mouse hearts demonstrating (A) the intact ventricular septum and relationships of the aortic, tricuspid and mitral valves, and (B) an intact atrial septum. (C-F) Sections of mutant mouse hearts showing: (C) a large perimembranous VSD with the aorta riding over both ventricles; (D) a secundum ASD; (E) a muscular VSD; (F) a common atrioventricular canal defect. Note the single atrioventricular valve and the common defect of the atrial and ventricular septum. Arrows indicate the respective defects. AoV, TV, MV, aortic, tricuspid and mitral valve; RA, LA, right, left atrium; RV, LV, right, left ventricle. Scale bar: 1 mm. (G) Summary of congenital heart defects in Pds5B-/- mice. P<0.001 is calculated by a Z-test for significant differences in total incidence between WT and Pds5B homozygotes.
We next examined the enteric nervous system (ENS), a complex network of neurons and glia within the bowel that is derived from migrating neural crest cells (NCCs). The NCCs migrate in a proximal to distal direction along the length of the bowel and normally reach the mid-colon by E12.5 (Fig. 4H). By contrast, in Pds5B-/- mice, ENS precursors failed to migrate past the ileocecal (IC) junction by this age (Fig. 4I). Furthermore, even in regions of the distal bowel colonized by NCCs, the neuronal density was lower in Pds5B-/- mice compared to wild-type animals (data not shown).
To determine if the enteric neuron defects occur because of delayed migration of NCCs into the colon, we examined the ENS at E18.5 and P0 using the Tuj1 antibody, which recognizes a neuronal form of tubulin, or acetylcholinesterase (AchE) staining (Fig. 4J-M; see Fig. S3 in the supplementary material). As expected, enteric neurons colonize the entire length of the wild-type neonatal bowel (Fig. 4J,L), whereas 70% (8/12) of Pds5B-/- mice exhibited variable degrees of abnormal innervation and ganglion formation in the distal colon (Fig. 4K,M). By contrast, small bowel innervation appeared grossly normal in Pds5B-/- mice (data not shown). Thus, the majority of Pds5B-/- mice had significant defects in the distal enteric nervous system that resemble Hirschsprung disease in humans. This contrasts with CdLS where distal colonic aganglionosis has not been reported, but where other gastrointestinal problems including persistent emesis and feeding problems are common, occurring in 62% and 77%, respectively, of infants with CdLS (Jackson et al., 1993). These observations suggest that defects in the ENS such as hypoganglionosis may underlie the gastrointestinal symptoms of CdLS patients.
PDS5B regulates primordial germ cell proliferation
The high expression of Pds5B in adult and embryonic testis, the role of PDS5 in mitosis and meiosis in lower organisms, and the high frequency of genital anomalies seen in people with CdLS, encouraged us to examine germ cell development in Pds5B-/- mice. We found a severely reduced number of germ cells in testes and ovaries of newborn Pds5B-/- mice (Fig. 5A-D; see Fig. S4 in the supplementary material) that appeared to be more severe in males. We therefore focused on male germ cell development to further investigate mechanisms underlying germ cell depletion. At E16.5, Pds5B-/- male mice had an 80% reduction in germ cells in the testis (Fig. 5E,F,I). To determine if the remaining germ cells could undergo spermatogenesis, we performed testis transplantation to propagate E16.5 testis of wild-type and Pds5B-/- mice as explants in nude mice (Naughton et al., 2006). Using GCNA1 immunohistochemistry, we found a complete depletion of spermatogonial stem cells at 2 weeks after transplantation (Fig. 5G,H). By 6 weeks after transplantation, the explanted Pds5B-/- testes contained testicular cords that only contained Sertoli cells (see Fig. S4 in the supplementary material). The Pds5B-/- Sertoli cells were morphologically normal as determined by GATA4 immunohistochemistry (data not shown). These studies indicate that the germ cells remaining at E16.5 in Pds5B-/- mice were incapable of sustaining spermatogenesis.
The embryonic loss of germ cells at E16.5 in Pds5B-deficient mice suggested that PDS5B is important for primordial germ cell (PGC) development. PGCs are derived from a founder population of ∼45 cells in the extraembryonic mesoderm posterior to the primitive streak around E7.25 (Ginsburg et al., 1990). These cells migrate to the genital ridge by E11.5 (Gomperts et al., 1994), and continue proliferating until E13.5 in males (McLaren, 2000). To identify the mechanism underlying the reduced number of germ cells in male Pds5B-/- mice, we evaluated the migration, proliferation and apoptosis of these germ cell precursors. At E12.5, a time point when PGCs enter the genital ridge, there were already significantly fewer numbers of germ cells in the Pds5B-/- mice. We hypothesized that overt migration defects in Pds5B-/- PGCs would result in failure of these precursors to reach the genital ridge by E11.5. However, alkaline phosphatase staining of E10.5 gonads to identify PGCs revealed no significant differences in PGC location between wild-type and Pds5B-/- embryos (data not shown), indicating that PGC migration at this early stage must be relatively normal. In addition, TUNEL analysis revealed no changes in the number of apoptotic germ cells in E12.5 or E16.5 mutant embryos (data not shown). However, when we examined germ cell proliferation in E12.5 embryos using BrdU incorporation, we found a 30% reduction in the number of BrdU-positive germ cells in testes of Pds5B-/- versus wild-type mice (Fig. 5J; see Fig. S4 in the supplementary material). Although it is possible that germ cell depletion could be partially due to apoptosis or delayed migration at stages in development that we did not examine, it is clear that the reduced proliferation is a major factor in the reduced number of germ cells.
Pds5B-/- mice display multiple defects in the peripheral nervous system. (A) Summary of carotid nerve and superior cervical ganglion (SCG) abnormalities in Pds5B-deficient mice. SCG AML, SCG delayed migration or abnormal localization; TCN, thin carotid nerve; CNM, carotid nerve missing. *P<0.05 and **P<0.001 determined by a Z-test for significant differences in incidence between WT and Pds5B homozygotes. (B-G) TOH whole-mount staining of sympathetic neurons of neonate mice. (B,C) Abnormal caudal location of SCG in Pds5B-/- mice compared to wild-type mice (SCG, arrows). Dashed circle indicates the expected normal location of SCG (C). Note the severe reduction or absence of carotid nerves in Pds5B-/- (E,G) compared to WT mice (D,F). Carotid nerves are indicated by arrows in D and E, and by two dashed lines in F and G. ey, eye; co, cochlea. Scale bars: 400 μm (B-E); 200 μm (F,G). (H,I) E12.5 enteric neurons in the distal bowel of whole gut demonstrated by TuJ1 immunohistochemistry (H, wild type; I, Pds5B-/-). *, ileocecal junction. Note that the enteric neuron migration wave front did not pass the ileocecal junction in Pds5B-/- mice (I). Scale bars: 10 μm (H,I). (J,K) TuJ1 staining of P0 paraffin sections of distal colon from wild-type (J) and Pds5B-/- mice (K), showing the lack of clustered enteric neuron cell bodies in Pds5B-/- mice. Scale bars: 20 μm. (Insets) High magnification of TuJ1 staining of P0 distal colons. Scale bars: 10 μm. Arrowheads point to the clustered enteric neuron cell bodies. (L,M) Tuj1 whole-mount staining of E18.5 guts from wild-type mice (L) and Pds5B-/- (M). Wild-type mice show normal formation of enteric plexus with clustered neuronal cell bodies (arrowheads) in the mid-colon, whereas Pds5B-/- mice lack enteric neuron cell bodies and show only a few thin nerve fibers (arrows). Scale bar: 100 μm. (N) Schematic representation of ENS defects in Pds5B-/- mice [E12.5: Pds5B-/- (n=7) and WT (n=9); P0: Pds5B-/- (n=12) and WT (n=13)]. The scale at the top corresponds to the percentage of the respective intestinal segment (small or large intestine) successfully colonized by neurons.
Pds5B-deficient mice manifest germ cell defects. GCNA1 staining of germ cells of WT (A) and Pds5B-/- (B) neonatal ovaries, and WT (C) and Pds5B-/- (D) neonatal testes show reduced germ cells in Pds5B-/- mice. Arrows point to empty testicular cords in D. (E,F) GCNA1 immunohistochemical analysis of E16.5 germ cells in WT (E) and Pds5B-deficient (F) testes shows a reduction in germ cells during embryogenesis. (G,H) GCNA1 staining of two-week testes transplants of E16.5 wild-type (G) and Pds5B-/- mice (H). In contrast to the presence of germ cells in the periphery of WT seminiferous tubules (G), note complete loss of germ cells in Pds5B-/- testis explant (H). *Non-specific auto-fluorescence signal; red, GCNA1; blue, bisbenzimide. (I) Quantitative analysis of germ cells in E16.5 testes of WT and Pds5B-deficient mice (n=4 for each genotype). Error bars represent SEM. *P<0.001, Student's unpaired t-test. (J) Proliferation index measured by BrdU incorporation in GCNA1-positive germ cells at E12.5 (n=3 for each genotype and 100 GCNA1-positive cells were counted for each animal). Error bars represent s.e.m. *P<0.05, Student's paired t-test. Scale bars: 50 μm.
PDS5B loss does not alter sister chromatid cohesion
PDS5 is crucial for sister chromatid cohesion (Dorsett et al., 2005; Hartman et al., 2000; Losada et al., 2005; Panizza et al., 2000; van Heemst et al., 1999; Wang et al., 2003; Wang et al., 2002). To test for cohesion defects in Pds5B-deficient mammalian cells, we performed GTW metaphase chromosome banding of mouse embryonic fibroblast (MEF) chromosomes. Two MEF lines were obtained from both Pds5B-deficent and wild-type mice. Quantitative RT-PCR and western blot analyses were performed and we found that these cells were indeed devoid of both Pds5B mRNA and protein (see Fig. S1 in the supplementary material). We analyzed a total of 22-25 metaphase cells per line (two WT and two Pds5B-/- MEF lines; detailed results are available on request). No obvious cohesion defects such as precocious sister chromatid separation (PSCS) or gross chromosomal abnormalities were observed in the Pds5B-/- MEFs (Fig. 6A,B). The lack of sister chromatid cohesion abnormalities in Pds5B-deficient cells suggests that PDS5B has novel functions other than sister chromatid cohesion that are important for mammalian embryonic development.
Pds5B-/- MEFs lack sister chromatid cohesion defects. (A,B) Metaphases from mouse embryonic fibroblasts (MEFs) derived from WT and Pds5B-/- mice show no differences in sister chromatid cohesion. (C) Multi-species alignment of AT hook domains of PDS5B. Hs, Homo sapiens; Pa, Pan troglodytes; Ca, Canis familiaris; Mm, Mus musculus; Rn, Rattus norvegicus; Ga, Gallus gallus; Xe, Xenopus tropicalis; Da, Danio rerio; Te, Tetraodon nigroviridis. (D) Subcellular localization of human homologs of PDS5 using EGFP-tagged PDS5A and PDS5B fusion proteins. HeLa cells were transfected with EGFP-PDS5A and EGFP-PDS5B using Lipofectamine. At 24 hours later, the EGFP signals were examined. Left panels are phase-contrast images of HeLa cells; right panels are images of GFP signals. Note that EGFP-PDS5B is present in the nucleus and highly enriched in the nucleolus (arrows), whereas EGFP-PDS5A is localized to the nucleus but excluded from the nucleolus (arrows). Scale bar: 5 μm.
The wide ranging deficits in the Pds5B-/- mice suggested that PDS5B has acquired additional functional domains during evolution. To examine this possibility, we performed motif searching of mouse PDS5B using Blocks and Pfam motif libraries with the default cut-off scores. We identified the previously reported HEAT repeats within the N terminus (Losada et al., 2005; Neuwald and Hirano, 2000; Panizza et al., 2000) and two AT hook domains in the C terminus that closely match the high mobility group protein (HMGY) signature (Block IPB000116A). Further analysis revealed that these AT hook domains are present in PDS5B from fish to human (Fig. 6C), but are not present in the yeast, worm and fly orthologs. The AT hook domain was initially identified in the HMG-I/Y family as a domain that binds to AT tracts in the minor groove of DNA (Maher and Nathans, 1996). The identification of AT hook domains in PDS5B is the first recognizable DNA binding motif reported in a sister chromatid cohesion factor, indicating that PDS5B and the cohesin complex may regulate gene transcription through direct cohesin complex-DNA interactions mediated by PDS5B.
There are two PDS5 homologs in vertebrates (Losada et al., 2005). The N-terminal regions of these proteins both contain HEAT repeats and are closely related, but PDS5A lacks the AT hook domains present in the C-terminal region of PDS5B. Despite their similarity, the severe abnormalities caused by PDS5B deficiency clearly show that PDS5A cannot compensate for at least some of the physiologic roles of PDS5B. We investigated whether their distinct cellular functions could stem, at least in part, from differential subcellular localization of these two proteins. We generated EGFP-tagged PDS5A and PDS5B fusion proteins and determined their subcellular localization in interphase cells using fluorescence microscopy. Whereas both PDS5A and PDS5B were found in the nucleus, PDS5B appeared to be more concentrated in the nucleolus than in the rest of the nucleus. By contrast, PDS5A was less abundant in the nucleolus than in the rest of the nucleus (Fig. 6D).
Mutations in four different cohesion proteins (NIPBL, SMC1A, SMC3 and ESCO2) have been found in patients with CdLS and Roberts syndrome/SC phocomelia (RBS/SC), and are thought to be responsible for the broad spectrum of prenatal and postnatal developmental anomalies (Deardorff et al., 2007; Krantz et al., 2004; Musio et al., 2006; Schule et al., 2005; Tonkin et al., 2004; Vega et al., 2005). These observations clearly indicate that sister chromatid cohesion factors are important for early development of multicellular organisms, either through abnormalities in chromosome dynamics or via alternative functions.
In this work, we generated mice deficient in Pds5B, a sister chromatid cohesion factor. These mice manifest a spectrum of developmental defects similar to those found in human CdLS, including abnormal skeletal patterning, cardiac anomalies and cleft palate. Other phenotypes that are associated with CdLS such as mental retardation, hearing loss and gastroesophageal reflex are difficult to evaluate in this animal model particularly since Pds5B-/- mice are not viable past P0. However, the striking defects in the enteric and autonomic nervous systems of these mice may be related to the frequent occurrence of ptosis and gastrointestinal problems in CdLS patients. Because of significant phenotypic overlap in Pds5B-/- mice and people with CdLS (Table 1), and the fact that mutations in other cohesion factors have been identified in CdLS patients, we believe that Pds5B-/- mice represent the first mouse model resembling human Cornelia de Lange syndrome. In fact, the broad spectrum of symptoms with incomplete penetrance and variable expressivity in these mice is also a feature typically observed in CdLS. These studies and the previously known association of mutations in other cohesion factors and CdLS suggest that screening CdLS patients for PDS5B mutations is warranted. PDS5B mutations, unless they produce dominant negative mutants, may be rare because they would be recessive and most CdLS cases are dominantly inherited (Krantz et al., 2004; Tonkin et al., 2004), and because they may result in lethality unless they are hypomorphic.
Comparison of clinical and phenotypic features between Cornelia de Lange syndrome and in Pds5B−/− mice
Although the molecular function of PDS5 is still under investigation, several organisms require diverse function of PDS5 for chromatid cohesion (Losada et al., 2005). For example, it may act as a regulator of the SMC ATPase through the HEAT motifs shared by SCC2 (Kimura and Hirano, 2000) and thereby modulate the dynamics of the cohesin ring. Alternatively, PDS5 may act as a scaffolding protein and promote protein-protein interactions between adjacent cohesin complexes (Losada et al., 2005). An examination of the chromosomes from Pds5B-deficient mouse cells did not show any abnormalities in sister chromatid cohesion (i.e. PSCS). This result was in contrast to those obtained in budding yeast, worm and Drosophila, where PDS5 plays an important role in chromosome segregation (Dorsett et al., 2005; Hartman et al., 2000; Wang et al., 2003). It is possible that our assay in which a total of 50 cells were examined may not have detected subtle defects. Indeed, only mild cohesion defects were found in HeLa cells in which PDS5B was knocked down by RNAi (Losada et al., 2005). Furthermore, our results are consistent with reports of the lack of sister chromatid cohesion defects in cells from CdLS patients (Tonkin et al., 2004; Vrouwe et al., 2007) or only mild cohesion defects in 40% of CdLS patients (Kaur et al., 2005). Overall, these results are consistent with our hypothesis that the abnormalities present in patients with CdLS and related diseases probably result from abnormal cohesin-mediated functions other than sister chromatid cohesion (Dorsett, 2007; Krantz et al., 2004; Tonkin et al., 2004). In this regard, it has been hypothesized that the cohesin complex directly binds to regulatory cis-elements and transcriptionally regulates developmental gene expression (Krantz et al., 2004; Rollins et al., 1999; Tonkin et al., 2004). Support for this idea comes from a number of studies in lower organisms. For example, severe cohesion defects have been observed in Drosophila Pds5 homozygous mutants, but not in heterozygotes, where only defects in cut gene expression and wing (limb) development are observed (Dorsett et al., 2005). In this regard, direct interactions of the cohesin complex with multiple sites between the wing margin enhancer and promoter lead to transcriptional regulation of the developmental gene cut (Dorsett et al., 2005). Yet another example of alternative Pds5B function is found in yeast Pds5 mutants, in which topoisomerase II can rescue the lethality, but not the cohesion defect of these mutants (Aguilar et al., 2005). Furthermore, the cohesion protein, MAU-2, is highly expressed in the mammalian adult nervous system and is crucial for neuronal migration and axonal guidance (Seitan et al., 2006; Watrin et al., 2006). PDS5B is also expressed at high levels in neurons, yet its function in adult neurons is unlikely to be related to sister chromatid cohesion as these cells are post-mitotic and thus do not undergo cell division. How cohesin complexes could directly regulate transcription has remained elusive because no DNA binding motifs have been identified in proteins that comprise the cohesin complex or its regulatory factors. Our analysis of PDS5B has identified two AT hook domains, the motif that constitutes the DNA binding domain of HMG proteins (Maher and Nathans, 1996). We propose that PDS5B may constitute an important link in cohesin regulation of transcription through its ability to bind AT rich regulatory sequences and bring the cohesin complex in proximity to promoter elements to regulate transcription.
An alternative explanation for the absence of sister chromatid cohesion defects in Pds5B-/- mice could be redundancy with its homolog PDS5A (Losada et al., 2005). Physiological evidence for this will require generation of Pds5A-/- animals, but our preliminary studies indicate significant overlap in PDS5A and PDS5B mRNA expression (B.Z. and J.M. unpublished observations). Indeed, we found overlapping expression in a number of organ systems where Pds5B-/- mice manifest developmental defects, suggesting differential sensitivity to PDS5B loss in various organs or perhaps reflecting differences in subcellular localization (and function) of these two closely related proteins.
Both PDS5A and PDS5B have previously been reported to be localized in the nucleus by immunohistochemistry (Kumar et al., 2004; Maffini et al., 2002). In these studies, we found that PDS5B is localized to the nucleus with higher levels in the nucleolus. Interestingly, PDS5B lacking the C-terminal 142 residues containing the second AT hook motif was localized to the nucleus but was not concentrated in the nucleolus (B.Z. and J.M., unpublished observation), suggesting that this domain may contribute to its nucleolar localization. Nucleolar localization of PDS5B indicates that PDS5B and/or cohesin complexes containing PDS5B could regulate rRNA metabolism and ribosome biogenesis. Interestingly, other cohesion proteins (e.g. NIPBL and SMC1A) have also been detected in nucleoli (Andersen et al., 2005), and yeast PDS5 can interact with NIP7 and NOP7, both of which are involved in ribosomal RNA (rRNA) biosynthesis (Davierwala et al., 2005; Krogan et al., 2006). Further evidence of cohesion protein involvement in ribosomal biogenesis comes from studies in yeast, where an additional copy of NOG2, which encodes a GTPase required for 60S ribosomal subunit maturation, is able to suppress the defects caused by mutation of the cohesin protein Scc3 (Bialkowska and Kurlandzka, 2002). In addition, the cohesin complex is recruited by Sir2 to silenced chromatin in the rDNA loci to maintain rDNA repeat copy number (Kobayashi et al., 2004). Finally, normal development can be disrupted by defects in rRNA metabolism, a process largely confined to the nucleolus. For example, Tcof1, which encodes a nucleolar protein, is mutated in Treacher Collins syndrome, an autosomal dominant disorder of craniofacial development (Dixon et al., 2006). Haploinsufficiency of Tcof1 in mice causes a reduction of mature ribosomes that results in deficient neural crest cell migration and proliferation that leads to craniofacial abnormalities, including cleft palate and mandibular hypoplasia (Dixon et al., 2006). Deficits in the nucleolar functions of cohesion proteins could also contribute to the abnormalities observed in patients with CdLS or related diseases.
In summary, we have discovered that the cohesion protein PDS5B is a critical regulator of multiple aspects of organogenesis, probably via roles unrelated to its ancient function in sister chromatid cohesion. This Pds5B-deficient mouse provides the first mammalian model to study the molecular mechanisms of developmental functions of the cohesin complex and the pathogenesis of CdLS and related disorders with mutations in cohesion factors.
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/134/17/3191/DC1
We are deeply indebted to Robert H. Baloh and Jason A. Gustin for careful review of the manuscript and/or helpful discussions of the data. We thank Kelli Simburger, Shelly Audrain, Amber Nielson, Jack Shields, Jennifer Armon, Amy Strickland, Amanda Knoten, Tatiana Gorodinsky and Nina Panchenko for technical assistance. We are grateful to George C. Enders for GCNA1 antibody, Kasid Usha for PDS5A plasmid, Kazusa DNA Research Institute for KIAA0979 cDNA, and Mark Johnston for pRS315 plasmid and help with yeast gap repair. We thank Chih-Lin Hsieh for providing the chromosomal analysis. This work was supported by NIH grants CA111966, AG13730 and NS039358 (to J.M.), HD047396 (to S.J.), DK57038 and DK6459201 (to R.O.H.), O'BRIEN center for Kidney Disease Research (P30DK079333), and Washington University Cancer Biology Pathway Fellowship (to B.Z.). P.J. is a Scholar of the Child Health Research Center of Excellence in Developmental Biology at Washington University School of Medicine (HD001487).
Accepted June 22, 2007.
Aguilar, C., Davidson, C., Dix, M., Stead, K., Zheng, K., Hartman, T. and Guacci, V. (2005). Topoisomerase II suppresses the temperature sensitivity of Saccharomyces cerevisiae pds5 mutants, but not the defect in sister chromatid cohesion. Cell Cycle 4,1294 -1304.
Andersen, J. S., Lam, Y. W., Leung, A. K., Ong, S. E., Lyon, C. E., Lamond, A. I. and Mann, M. (2005). Nucleolar proteome dynamics. Nature 433,77 -83.
Benard, C. Y., Kebir, H., Takagi, S. and Hekimi, S. (2004). mau-2 acts cell-autonomously to guide axonal migrations in Caenorhabditis elegans. Development 131,5947 -5958.
Bialkowska, A. and Kurlandzka, A. (2002). Additional copies of the NOG2 and IST2 genes suppress the deficiency of cohesin Irr1p/Scc3p in Saccharomyces cerevisiae. Acta Biochim. Pol. 49,421 -425.
Davierwala, A. P., Haynes, J., Li, Z., Brost, R. L., Robinson, M. D., Yu, L.,Mnaimneh, S., Ding, H., Zhu, H., Chen, Y. et al. (2005). The synthetic genetic interaction spectrum of essential genes. Nat. Genet. 37,1147 -1152.
Deardorff, M. A., Kaur, M., Yaeger, D., Rampuria, A., Korolev, S., Pie, J., Gil-Rodriguez, C., Arnedo, M., Loeys, B., Kline, A. D. et al. (2007). Mutations in cohesin complex members SMC3 and SMC1A cause a mild variant of cornelia de Lange syndrome with predominant mental retardation. Am. J. Hum. Genet. 80,485 -494.
Dixon, J., Jones, N. C., Sandell, L. L., Jayasinghe, S. M., Crane, J., Rey, J. P., Dixon, M. J. and Trainor, P. A. (2006). Tcof1/Treacle is required for neural crest cell formation and proliferation deficiencies that cause craniofacial abnormalities. Proc. Natl. Acad. Sci. USA 103,13403 -13408.
Dorsett, D. (2007). Roles of the sister chromatid cohesion apparatus in gene expression, development, and human syndromes. Chromosoma 116, 1-13.
Dorsett, D., Eissenberg, J. C., Misulovin, Z., Martens, A., Redding, B. andMcKim, K. (2005). Effects of sister chromatid cohesion proteins on cut gene expression during wing development in Drosophila. Development 132,4743 -4753.
Enomoto, H., Crawford, P. A., Gorodinsky, A., Heuckeroth, R. O., Johnson, E. M., Jr and Milbrandt, J. (2001). RET signaling is essential for migration, axonal growth and axon guidance of developing sympathetic neurons. Development 128,3963 -3974.
Fu, M., Vohra, B. P., Wind, D. and Heuckeroth, R. O. (2006). BMP signaling regulates murine enteric nervous system precursor migration, neurite fasciculation, and patterning via altered Ncam1 polysialic acid addition. Dev. Biol. 299,137 -150.
Geck, P., Maffini, M. V., Szelei, J., Sonnenschein, C. and Soto, A. M. (2000). Androgen-induced proliferative quiescence in prostate cancer cells: the role of AS3 as its mediator. Proc. Natl. Acad. Sci. USA 97,10185 -10190.
Ginsburg, M., Snow, M. H. and McLaren, A. (1990). Primordial germ cells in the mouse embryo during gastrulation. Development 110,521 -528.
Gomperts, M., Garcia-Castro, M., Wylie, C. and Heasman, J. (1994). Interactions between primordial germ cells play a role in their migration in mouse embryos. Development 120,135 -141.
Hartman, T., Stead, K., Koshland, D. and Guacci, V. (2000). Pds5p is an essential chromosomal protein required for both sister chromatid cohesion and condensation in Saccharomyces cerevisiae. J. Cell Biol. 151,613 -626.
Honaramooz, A., Snedaker, A., Boiani, M., Scholer, H., Dobrinski, I. andSchlatt, S. (2002). Sperm from neonatal mammalian testes grafted in mice. Nature 418,778 -781.
Honma, Y., Araki, T., Gianino, S., Bruce, A., Heuckeroth, R., Johnson, E. and Milbrandt, J. (2002). Artemin is a vascular-derived neurotropic factor for developing sympathetic neurons. Neuron 35,267 -282.
Ireland, M., Donnai, D. and Burn, J. (1993). Brachmann-de Lange syndrome. Delineation of the clinical phenotype. Am. J. Med. Genet. 47,959 -964.
Jackson, L., Kline, A. D., Barr, M. A. and Koch, S. (1993). de Lange syndrome: a clinical review of 310 individuals. Am. J. Med. Genet. 47,940 -946.
Jain, S., Naughton, C. K., Yang, M., Strickland, A., Vij, K., Encinas, M., Golden, J., Gupta, A., Heuckeroth, R., Johnson, E. M., Jr et al. (2004). Mice expressing a dominant-negative Ret mutation phenocopy human Hirschsprung disease and delineate a direct role of Ret in spermatogenesis. Development 131,5503 -5513.
Kaur, M., DeScipio, C., McCallum, J., Yaeger, D., Devoto, M., Jackson, L. G.,Spinner, N. B. and Krantz, I. D. (2005). Precocious sister chromatid separation (PSCS) in Cornelia de Lange syndrome. Am. J. Med. Genet. A 138, 27-31.
Kimura, K. and Hirano, T. (2000). Dual roles of the 11S regulatory subcomplex in condensin functions. Proc. Natl. Acad. Sci. USA 97,11972 -11977.
Kobayashi, T., Horiuchi, T., Tongaonkar, P., Vu, L. and Nomura, M. (2004). SIR2 regulates recombination between different rDNA repeats, but not recombination within individual rRNA genes in yeast. Cell 117,441 -453.
Krantz, I. D., McCallum, J., DeScipio, C., Kaur, M., Gillis, L. A., Yaeger, D.,Jukofsky, L., Wasserman, N., Bottani, A., Morris, C. A. et al. (2004). Cornelia de Lange syndrome is caused by mutations in NIPBL, the human homolog of Drosophila melanogaster Nipped-B. Nat. Genet. 36,631 -635.
Krogan, N. J., Cagney, G., Yu, H., Zhong, G., Guo, X., Ignatchenko, A., Li, J.,Pu, S., Datta, N., Tikuisis, A. P. et al. (2006). Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 440,637 -643.
Kumar, D., Sakabe, I., Patel, S., Zhang, Y., Ahmad, I., Gehan, E. A.,Whiteside, T. L. and Kasid, U. (2004). SCC-112, a novel cell cycle-regulated molecule, exhibits reduced expression in human renal carcinomas. Gene 328,187 -196.
Le, N., Nagarajan, R., Wang, J. Y., Araki, T., Schmidt, R. E. and Milbrandt, J. (2005). Analysis of congenital hypomyelinating Egr2Lo/Lo nerves identifies Sox2 as an inhibitor of Schwann cell differentiation and myelination. Proc. Natl. Acad. Sci. USA 102,2596 -2601.
Losada, A., Yokochi, T. and Hirano, T. (2005). Functional contribution of Pds5 to cohesin-mediated cohesion in human cells and Xenopus egg extracts. J. Cell Sci. 118,2133 -2141.
Maffini, M. V., Geck, P., Powell, C. E., Sonnenschein, C. and Soto, A. M. (2002). Mechanism of androgen action on cell proliferation: AS3 protein as a mediator of proliferative arrest in the rat prostate. Endocrinology 143,2708 -2714.
Maher, J. F. and Nathans, D. (1996). Multivalent DNA-binding properties of the HMG-1 proteins. Proc. Natl. Acad. Sci. USA 93,6716 -6720.
McLaren, A. (2000). Germ and somatic cell lineages in the developing gonad. Mol. Cell. Endocrinol. 163,3 -9.
Musio, A., Selicorni, A., Focarelli, M. L., Gervasini, C., Milani, D., Russo, S., Vezzoni, P. and Larizza, L. (2006). X-linked Cornelia de Lange syndrome owing to SMC1L1 mutations. Nat. Genet. 38,528 -530.
Nasmyth, K. and Haering, C. H. (2005). The structure and function of SMC and kleisin complexes. Annu. Rev. Biochem. 74,595 -648.
Naughton, C. K., Jain, S., Strickland, A. M., Gupta, A. and Milbrandt, J. (2006). Glial cell-line derived neurotrophic factor-mediated RET signaling regulates spermatogonial stem cell fate. Biol. Reprod. 74,314 -321.
Neuwald, A. F. and Hirano, T. (2000). HEAT repeats associated with condensins, cohesins, and other complexes involved in chromosome-related functions. Genome Res. 10,1445 -1452.
Panizza, S., Tanaka, T., Hochwagen, A., Eisenhaber, F. and Nasmyth, K. (2000). Pds5 cooperates with cohesin in maintaining sister chromatid cohesion. Curr. Biol. 10,1557 -1564.
Rollins, R. A., Morcillo, P. and Dorsett, D. (1999). Nipped-B, a Drosophila homologue of chromosomal adherins, participates in activation by remote enhancers in the cut and Ultrabithorax genes. Genetics 152,577 -593.
Rollins, R. A., Korom, M., Aulner, N., Martens, A. and Dorsett, D. (2004). Drosophila nipped-B protein supports sister chromatid cohesion and opposes the stromalin/Scc3 cohesion factor to facilitate long-range activation of the cut gene. Mol. Cell. Biol. 24,3100 -3111.
Schule, B., Oviedo, A., Johnston, K., Pai, S. and Francke, U. (2005). Inactivating mutations in ESCO2 cause SC phocomelia and Roberts syndrome: no phenotype-genotype correlation. Am. J. Hum. Genet. 77,1117 -1128.
Seitan, V. C., Banks, P., Laval, S., Majid, N. A., Dorsett, D., Rana, A., Smith, J., Bateman, A., Krpic, S., Hostert, A. et al. (2006). Metazoan Scc4 homologs link sister chromatid cohesion to cell and axon migration guidance. PLoS Biol. 4, e242.
Song, H., Lim, H., Paria, B. C., Matsumoto, H., Swift, L. L., Morrow, J.,Bonventre, J. V. and Dey, S. K. (2002). Cytosolic phospholipase A2alpha is crucial [correction of A2alpha deficiency is crucial] for `on-time' embryo implantation that directs subsequent development. Development 129,2879 -2889.
Svaren, J., Ehrig, T., Abdulkadir, S. A., Ehrengruber, M. U., Watson, M. A. and Milbrandt, J. (2000). EGR1 target genes in prostate carcinoma cells identified by microarray analysis. J. Biol. Chem. 275,38524 -38531.
Tanaka, K., Hao, Z., Kai, M. and Okayama, H. (2001). Establishment and maintenance of sister chromatid cohesion in fission yeast by a unique mechanism. EMBO J. 20,5779 -5790.
Tonkin, E. T., Wang, T. J., Lisgo, S., Bamshad, M. J. and Strachan, T. (2004). NIPBL, encoding a homolog of fungal Scc2-type sister chromatid cohesion proteins and fly Nipped-B, is mutated in Cornelia de Lange syndrome. Nat. Genet. 36,636 -641.
van Heemst, D., James, F., Poggeler, S., Berteaux-Lecellier, V. and Zickler, D. (1999). Spo76p is a conserved chromosome morphogenesis protein that links the mitotic and meiotic programs. Cell 98,261 -271.
Vega, H., Waisfisz, Q., Gordillo, M., Sakai, N., Yanagihara, I., Yamada, M., van Gosliga, D., Kayserili, H., Xu, C., Ozono, K. et al. (2005). Roberts syndrome is caused by mutations in ESCO2, a human homolog of yeast ECO1 that is essential for the establishment of sister chromatid cohesion. Nat. Genet. 37,468 -470.
Vrouwe, M. G., Elghalbzouri-Maghrani, E., Meijers, M., Schouten, P., Godthelp, B. C., Bhuiyan, Z. A., Redeker, E. J., Mannens, M. M., Mullenders, L. H., Pastink, A. et al. (2007). Increased DNA damage sensitivity of Cornelia de Lange syndrome cells: evidence for impaired recombinational repair. Hum. Mol. Genet. 16,1478 -1487.
Wang, F., Yoder, J., Antoshechkin, I. and Han, M. (2003). Caenorhabditis elegans EVL-14/PDS-5 and SCC-3 are essential for sister chromatid cohesion in meiosis and mitosis. Mol. Cell. Biol. 23,7698 -7707.
Wang, S. W., Read, R. L. and Norbury, C. J. (2002). Fission yeast Pds5 is required for accurate chromosome segregation and for survival after DNA damage or metaphase arrest. J. Cell Sci. 115,587 -598.
Watrin, E., Schleiffer, A., Tanaka, K., Eisenhaber, F., Nasmyth, K. and Peters, J. M. (2006). Human Scc4 is required for cohesin binding to chromatin, sister-chromatid cohesion, and mitotic progression. Curr. Biol. 16,863 -874.
Wellik, D. M. and Capecchi, M. R. (2003). Hox10 and Hox11 genes are required to globally pattern the mammalian skeleton. Science 301,363 -367.
Thank you for your interest in spreading the word on Development.
You are going to email the following Mice lacking sister chromatid cohesion protein PDS5B exhibit developmental abnormalities reminiscent of Cornelia de Lange syndrome
Message Subject (Your Name) has sent you a message from Development
Message Body (Your Name) thought you would like to see the Development web site.
Indirect modulation of Shh signaling by Dlx5 affects the oral-nasal patterning of palate and rescues cleft palate in Msx1-null mice
Regulation of lymphatic-blood vessel separation by endothelial Rac1
Coordination of sonic hedgehog and Wnt signaling determines ventral and dorsal telencephalic neuron types from human embryonic stem cells
Show more DEVELOPMENT AND DISEASE
Other journals from The Company of Biologists
Special Issue: development at the single cell level
Our Special Issue on single cell approaches to developmental biology contains a wealth of content, from research papers using various model systems to the latest cutting edge techniques and a selection of reviews and commentaries surveying the field and its future.
The people behind the papers
Meet New York-based Eduardo Leyva-Díaz and Oliver Hobert, authors of a recent Development paper on transcriptional autoregulation in C. elegans.
The Development team are going to be out and about through June and July. Do come and meet them! Going to the Gordon conference on Developmental Biology in South Hadley, USA, June 16 – 21? Meet our Executive Editor Katherine Brown. At the ISSCR 2019: The global stem cell event in Los Angeles, 26-29 June you can meet Senior Editor Seema Grewal. Reviews Editor Alex Eve and Online Editor Aidan Maartens will be at the SDB 78th Annual Meeting in Boston, USA, July 26-30.
The Company of Biologists Workshops - Chromatin-Based Regulation of Development
Watch a short video from this workshop, where we discussed what mechanisms related to chromatin biology inform developmental processes, explored plans to integrate unsolved developmental questions with exciting new avenues in chromatin biology, and which outstanding questions in gene regulation might be best revealed in the context of dynamic cell fate transitions. Find out about upcoming workshops here.
preLights – Developmentally regulated Shh expression is robust to TAD perturbations
Are TAD borders that important to prevent enhancer rewiring? preLighter Jesus Victorino highlights a preprint by Wendy Bickmore and Laura Lettice's team which shows that tissue-specific enhancers maintain robust control of Shh gene expression after challenging the architectural chromatin context.
Catch up with other recent preLights selected for the developmental biology community.
Articles of interest in our sister journals
Rho kinase-dependent apical constriction counteracts M-phase apical expansion to enable mouse neural tube closure
Max B. Butler, Nina E. Short, Eirini Maniou, Paula Alexandre, Nicholas D. E. Greene, Andrew J. Copp, Gabriel L. Galea
Suppressor of Fused regulates the proliferation of postnatal neural stem and precursor cells via a Gli3-dependent mechanism
Hector G. Gomez, Hirofumi Noguchi, Jesse Garcia Castillo, David Aguilar, Samuel J. Pleasure, Odessa R. Yabut
© 2019 The Company of Biologists Ltd Registered Charity 277992


Overall: Completely redesigned for next-gen console players, Tom Clancy`s Rainbow Six 3 for the xbox gives players the opportunity to command an elite, four-man international anti-terrorist squad, and features the trademark realism of the Clancy games, immediate, immersive close-quarters action, state of the art in-game cinematics, Tom Clancy`s Splinter Cell®-quality graphics and a nail-biting plot ripped from today`s headlines. A voice-activated command system and comprehensive offline and online multiplayer capabilities round out the most exciting experience xbox gamers will have this year.
Graphics: now the visual is also amazing outstanding graphics very realistic, the graphics will blast you out of this world.
Audio: The sounds are ok appart from the gun noises could have been made a little more realistic appart from that ok.
Graphics: The visual is good thats about it.
Audio: The sound is also not to good there is practacly only 2 sounds that go on in the game the gun fire and the bosses voice wich he says the same thing over and over again.
Audio: The sounds are ok sothing special the snow sound effects are very good and another good thing is you can upload your very own sound tracks.
Gameplay: The gameplay is ok nothing special its just the fact you are stuck in a plane all the game there is no vast exploretion.
Graphics: Its ok again nothing special, the looks of the how could i say ground are poor and the looks of the sky is poor and the looks of the surroundings are poor.
and also very good multiplayer.
Now the weapons are just so powerfull they will blast you out of your seat and blow your hands off with the recoil of the controll pads vibration.
There are a vast range of weapons from handgun to RPG (Rocket prepelled grenade).
So i say get out there and buy it!
Gameplay: The gameplay very easy to pick up its 1 of those games you just pick up the controller and learn it within a few minutes.


Like the Mountain Ramp Festival, the Sister Meal Festival (also called the "Sisters" or "Sister Rice Festival") is a way for young Miao men and women to match up for marriage. Although exact dates and customs vary between towns and village in Guizhou Province, the festival commonly occurs in the Spring on the fifteenth day of the third lunar month. Activities such as bullfighting, singing, dancing, and drinking rice wine make the festival increasingly popular with tourists in Southwest China.
In Taijiang County, Guizhou Province, the Sister Meal Festival happens every spring. This is a day for all young boys, girls, and relatives to stay together. In this old festival, married ladies come back to their parents' home to sing and to dance. Boys and girls celebrate together and find their beloved one in the party.
Their costumes are different from those who attend the Mountain Ramp Festival. Red, purple, and blue are the main colors on their clothing. Various patterns and embroidery styles appear on their costumes, e.g. dragons, phoenixes, birds, and flowers. The silver objects worn by the Miao ladies are for decoration and also for exorcising evil spirits. Middle-age ladies have a piece of white thread on their head to represent the river along their village.
Miao people have amazing costumes and beautiful voices for singing. They are very passionate and generous. Whenever there are guests, the host serves each of them with homemade rice wine, bowl by bowl. If you finish all the wine without any hesitation, you will definitely get drunk in a Miao people's home.