The transcription factor Pitx2 positions the embryonic axis and regulates twinning
In most invertebrates and anamniote vertebrates ( fishes and amphibians ) , embryonic polarity is first established by localisation of maternal determinants in the cytoplasm and/or cortex of the fertilised egg . This generates differences between the blastomeres that will form by cell division from the egg , and which will culminate in specifying the orientation of the embryonic axes ( Wilson , 1898 ) . Separation of the first two blastomeres can lead to twinning: the formation of genetically identical , complete individuals ( Driesch , 1892 ) . Separation of blastomeres after the four-cell stage , however , does not generate twins; in most cases it interferes with development of even a single embryo owing to the removal of important determinants that have by then segregated to different cells . This is known as the mosaic mode of development . Among the vertebrates , amniotes ( birds and many mammals , and possibly also reptiles ) have a remarkably extended capacity to give rise to twins . Some species of the armadillo genus Dasypus generate quadruplets or octuplets from a single fertilisation event , as a result of two or more sequential ‘splitting’ events of the embryo at a stage when it is already highly multicellular ( Newman and Patterson , 1910; Loughry et al . , 1998; Enders , 2002; Eakin and Behringer , 2004 ) . Conjoined ( ‘Siamese’ ) twins occur in mammals including humans ( Chai and Crary , 1971; Vanderzon et al . , 1998; Kaufman , 2004 ) and are also seen in reptiles ( Cunningham , 1937 ) and birds ( Ulshafer and Clavert , 1979 ) ; most of these are thought to arise from splitting of the embryo relatively late in development ( Kaufman , 2004 ) . Perhaps the most dramatic example is seen in the chick , where cutting an embryo into fragments at the blastoderm stage ( when the embryo contains as many as 20 , 000–50 , 000 cells ) can lead to each fragment generating a complete embryo; up to eight embryos have been generated from a single blastoderm by experimental splitting , right up to the time of appearance of the primitive streak ( Lutz , 1949; Spratt and Haas , 1960 ) . The ability of higher vertebrate embryos to retain a regulative model of development until such a late stage strongly suggests that localisation of maternally inherited determinants is not an essential component of the mechanisms specifying embryo polarity ( Stern and Downs , 2012 ) . Moreover , since a single blastoderm can generate multiple embryos , mechanisms must exist that suppress this ability in regions of the embryo that do not normally initiate axis formation ( Bertocchini and Stern , 2002; Bertocchini et al . , 2004 ) . In chick embryos , the earliest symmetry breaking event known is the localised expression of cVg1 , the chick orthologue of mammalian growth differentiation factor 1 ( GDF1 ) —a member of the transforming growth factor beta ( TGFβ ) superfamily of secreted proteins—encoding a Nodal/Activin-type molecule that signals through Smad2/3 ( Weeks and Melton , 1987; Thomsen and Melton , 1993; Kessler and Melton , 1995; Seleiro et al . , 1996; Shah et al . , 1997; Kessler , 2004; Birsoy et al . , 2006; Chen et al . , 2006; Andersson et al . , 2007 ) . Before primitive streak stages , cVg1 is expressed in the posterior marginal zone ( PMZ ) , an extraembryonic region adjacent to where the primitive streak will form; misexpression of cVg1 in other ( anterior or lateral ) parts of the marginal zone is sufficient to induce a complete axis from adjacent embryonic cells ( Seleiro et al . , 1996; Shah et al . , 1997; Skromne and Stern , 2001 , 2002 ) . The mechanisms that position cVg1 in the PMZ are unknown . Moreover , when a blastoderm is cut in half at right angles to the future primitive streak axis , cVg1 expression spontaneously initiates in the marginal zone adjacent to the cut edge , in either the right or left side at equal frequency , foreshadowing the appearance of the primitive streak a few hours later ( Bertocchini et al . , 2004 ) . This observation shows that the mechanisms that position cVg1 are active in the blastoderm stage embryo . Here we take advantage of these observations to design a molecular screen for new genes involved in the earliest stages of specifying embryo polarity; together with bioinformatic analysis and embryological experiments we identify the transcription factor Pitx2 as a direct and essential regulator of cVg1 expression both during normal development and in embryonic regulation ( induced twinning ) .
To search for putative upstream regulators of cVg1 , we took advantage of two of its properties: that it is expressed in the PMZ at early stages of development and that when a blastoderm is cut in half at right angles to the axis of the future primitive streak , cVg1 expression is initiated stochastically on either the left or the right corner ( adjacent to the cut edge ) of the isolated anterior half ( Bertocchini et al . , 2004 ) . We therefore performed two screens . First , we dissected the PMZ and an equivalent anterior explant ( anterior marginal zone , AMZ ) from 40 embryos ( in triplicate ) and analysed their transcriptomes using Affymetrix microarrays ( Figure 1A–B , Figure 1—figure supplement 1 ) . At this stage of development , it is not possible to predict the polarity of the embryo with complete certainty . To prevent contamination of the samples , we designed a verification strategy by which the predicted posterior and anterior explants were collected , the rest of the embryo immediately fixed and then processed for in situ hybridisation for cVg1 , developing the colour reaction for long enough to detect residual cVg1 expression around the posterior explant site . From each set of 40 embryos , approximately 36 had been dissected correctly; the explants from the remainder ( 3 × 4 ) were discarded ( Figure 1—figure supplement 1 ) . Each set of verified PMZs and AMZs ( 3 × 36 of each ) was then pooled and run on Affymetrix 30K chicken microarrays . 10 . 7554/eLife . 03743 . 003Figure 1 . Microarray screens for upstream regulators of cVg1 . ( A ) Diagram of the first screen: the posterior marginal zone ( PMZ ) and anterior marginal zone ( AMZ ) were dissected from embryos at stage XI–XII; the remaining embryo was then fixed and stained for cVg1 by in situ hybridisation ( ISH ) to confirm that the explants had been obtained from the correct regions . This was done from 40 embryos for each of three biological replicates , which were then run on microarrays . The diagram is accompanied by an example of an embryo after ISH . All 120 embryos are shown in Figure 1—figure supplement 1 . ( B ) Hierarchical clustering of differentially expressed genes for this experiment , and a plot of where cVg1-like probes ( enriched in PMZ ) are displayed in red and cVg1-unlike ( ‘downregulated’ ) probes shown in green across triplicate samples ( A1–A3 for AMZ , P1–P3 for PMZ ) . The scatter plot relates normalised log2 mean signal intensities and log2 fold changes of probes from both samples ( AMZ and PMZ ) . Probes identified as upregulated in the PMZ with a log2 fold change cut-off of 0 . 263 ( linear fold change 1 . 2 ) are displayed in red and those identified as downregulated in the PMZ with the same cut-off are displayed in blue . ( C ) Diagram of the second screen . An embryo at stage XI–XII was cut in half at a right angle to the future midline; the posterior half was fixed for ISH with cVg1 to confirm the orientation ( an example is shown ) , and the isolated anterior half cultured for 7 hr . At this point , a small explant was dissected from the marginal zone adjacent to the left and right side of the cut , and the remaining anterior half-embryo fixed for ISH with cVg1 ( an example is shown ) . This allowed identification of the ‘cVg1-like’ and ‘cVg1-unlike’ explants , which were then pooled appropriately . This was done for 70 embryos for each of three biological replicates; all 210 posterior and anterior fragments are shown in Figure 1—figure supplement 2 after ISH for cVg1 . ( D ) Hierarchical clustering of the probes expressed differentially in this assay , and corresponding scatter plot; details similar to ( B ) ≠V1 , ≠V2 , and ≠V3 correspond to each of the triplicate samples that do not express cVg1 and = V1 , =V2 , and = V3 correspond to explants that express cVg1 . ( E ) Venn diagrams showing the intersection of upregulated and downregulated probes common to both the PMZ and isolated anterior cut halves . A total of 122 upregulated probes and 78 downregulated probes were found to be common in both experiments using both p value and fold change as the criteria . The complete dataset has been submitted to ArrayExpress where it has been assigned the Accession number E-MTAB-3116 . DOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 00310 . 7554/eLife . 03743 . 004Figure 1—figure supplement 1 . The 120 embryos used for the first screen ( AMZ vs PMZ ) , after excision of the explants and ISH for cVg1 . The three sets of 40 embryos correspond to those used for each Affymetrix microarray . DOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 00410 . 7554/eLife . 03743 . 005Figure 1—figure supplement 2 . The 210 embryos used for the second screen ( lateral marginal zone from isolated anterior half embryos , cultured for 7 hr , then sorted into cVg1-like or cVg1-unlike expression ) . The procedure shown in Figure 1C was used . Embryos ( stage XI–XII ) were bisected at right angles to the midline , the posterior half ( shown at the bottom of each panel ) fixed immediately and processed by ISH for cVg1 to confirm the orientation of the cut . The anterior half was cultured for 7 hr , a piece of marginal zone adjacent to the cut edge excised from each side , and the remainder of the anterior half fixed and processed for cVg1 expression ( upper image in each panel ) . The pieces corresponding to the side expressing cVg1 were pooled together ( ‘cVg1-like’ ) , and those on the opposite side pooled with each other ( ‘cVg1-unlike’ ) . Seventy embryos were used for each microarray , and the experiment performed in triplicate . The sets of embryo fragments shown here correspond to each of the three microarrays . DOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 00510 . 7554/eLife . 03743 . 006Figure 1—figure supplement 3 . Analysis of microarray datasets for posterior marginal zone ( PMZ ) and isolated anterior cut halves . Volcano plots showing the correlation between p values and fold change of probes for each microarray experiment . The left graphs displays gene expression data from whole embryos ( PMZ vs AMZ ) , and the second graph displays data from the anterior cut halves ( cVg1-like and -unlike ) . Probes that pass a p value threshold of 0 . 05 together with a fold change cut-off of 1 . 2 ( 0 . 263 log2 ( FC ) ) are coloured red . Those that do not pass the p value threshold of 0 . 05 but pass the fold change cut-off of 1 . 2 ( 0 . 263 log2 ( FC ) ) are blue . Those that pass the p value threshold of 0 . 05 but not the fold change cut-off of 1 . 2 ( 0 . 263 log2 ( FC ) ) are green . The heat map on the right side of the figure uses hierarchical clustering to compare the 122 upregulated ( red ) and 78 downregulated ( green ) probes from the PMZ ( P vs A ) and the isolated anterior cut halves expressing cVg1 ( =V vs ≠V ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 006 Next , we used a similar strategy for isolated anterior halves of embryos . It was previously reported that cVg1 starts to be expressed in either the left or right corner of the cut anterior half around 6 hr after bisection ( Bertocchini et al . , 2004 ) . We chose to collect the left and right corners of the marginal zone adjacent to the cut edge 7 hr following bisection to ensure that surrounding cVg1 expression could be detected after excision of the fragment . To confirm that all embryos had been cut at exactly right angles to the future axis , the posterior half of each embryo was fixed immediately after cutting and subjected to in situ hybridisation for cVg1 . The anterior half was cultured for 7 hr , the left and right corners of the margin were dissected , and the remainder was fixed and processed for cVg1 expression ( with extended reaction time to detect weak expression ) . We estimated that 70 explants would be needed for microarray analysis of each sample; this was done in triplicate ( Figure 1C–D; Figure 1—figure supplement 2 ) . Samples were designated ‘cVg1-like’ or ‘cVg1-unlike’ based on this and pooled accordingly . This strategy randomised any left–right asymmetric genes unrelated to axial polarity and regulation , and enriched those for the cells in which cVg1 was just starting to be expressed de novo in one sample , and their contralateral equivalents ( not expressing cVg1 ) in the other . RNA from the pooled explants ( approximately 3 × 63 of each type ) was analysed using Affymetrix chicken microarrays . The intersection between the two datasets from the above screens was used to identify genes co-regulated with cVg1 , as well as those that are enriched in equivalent regions not expressing cVg1 , both in normal embryos and during regulation ( Figure 1B , D , E and Figure 1—figure supplement 3 ) . Using a threshold of just 1 . 2-fold change and p < 0 . 05 , this strategy identified 122 sequences ( corresponding to 85 genes ) with putative ‘cVg1-like’ expression ( a cVg1-synexpression group ) and 78 sequences ( 52 genes ) expressed more highly in the ‘Vg1-unlike’ explants ( cVg1 negative ) . A list of the top common genes ranked by fold change is shown in Table 1 . Comparison of the top ‘Vg1-like’ candidates from whole embryos ( PMZ vs AMZ ) with their counterparts from half-embryos shows highly significant correlation ( Spearman's rank Rho = 0 . 73; p = 0 . 00036 ) . Confirming that the screen was performed appropriately , cVg1 ( incorrectly annotated as GDF3 instead of GDF1 in the current version of the chicken genome , Galgal4 ) itself appears among the top genes: it is upregulated 4 . 3 fold , with a p value of 0 . 00006 ( rank 11 ) in whole embryos , and 1 . 71 , p = 0 . 0062 in the isolated anterior half ( rank 21 ) . Among all genes , Pitx2 immediately stands out as the best candidate , being very strongly co-regulated with cVg1 and the top transcription factor on the list . In the PMZ of whole embryos Pitx2 is upregulated almost 10-fold compared to the AMZ explants ( three different probes , ranking two , three and eight on the list; p = 0 . 0001 , 0 . 00019 and 0 . 003 respectively ) , whereas in the cut halves it is upregulated by about 2 . 4 fold ( three probes ranking four , six and eight on the list; p between 0 . 002–0 . 008 ) . 10 . 7554/eLife . 03743 . 007Table 1 . Genes identified from the screens , sorted according to their ranking in PMZ>AMZ in whole embryosDOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 007Posterior vs anteriorAnterior half , Vg1-likeGene symbolProbe IDFold changep ValueRankFold changep ValueRankADMPGga . 354 . 1 . S1_at13 . 0950 . 008013 . 7710 . 06862PITX2Gga . 3398 . 2 . S1_a_at9 . 5860 . 000122 . 1620 . 00698PITX2Gga . 3398 . 1 . S1_a_at8 . 5280 . 000132 . 4290 . 00874THPOGgaAffx . 21801 . 1 . S1_at7 . 3980 . 000942 . 2720 . 02677ST6-GAL2Gga . 14379 . 1 . S1_at6 . 8960 . 074452 . 5060 . 00033TGga . 3772 . 1 . S1_a_at6 . 3880 . 086765 . 5440 . 02511PKDCCGga . 12157 . 1 . S1_at5 . 6180 . 000671 . 5720 . 204833PITX2Gga . 3398 . 1 . S1_at5 . 2630 . 003082 . 3200 . 00266MIXL1Gga . 426 . 1 . S1_s_at5 . 1970 . 016092 . 3780 . 01855n/aGga . 2705 . 1 . S1_at5 . 0190 . 00002101 . 4150 . 023763GDF3Gga . 4324 . 2 . S1_a_at4 . 3090 . 00006111 . 7100 . 006221PMEPA1Gga . 6268 . 1 . S1_at4 . 1410 . 0805122 . 0320 . 010312PMEPA1GgaAffx . 12721 . 1 . S1_at3 . 7770 . 0007131 . 7450 . 000319TBX6Gga . 466 . 1 . S1_at3 . 6940 . 0004142 . 0300 . 021013FGF8Gga . 661 . 1 . S1_at3 . 2770 . 0052151 . 6900 . 085922OvoinhibitorGga . 6976 . 1 . S1_at3 . 2130 . 0015161 . 4670 . 063350ELK3Gga . 4498 . 1 . S1_s_at3 . 0940 . 0096171 . 5030 . 001043LITAFGga . 3383 . 1 . S2_at3 . 0660 . 0031181 . 6700 . 011025LITAFGga . 3383 . 1 . S1_at2 . 8950 . 0019191 . 8220 . 002717n/aGga . 13092 . 1 . S1_at2 . 8230 . 0025201 . 4060 . 028466List of the top 20 common upregulated probes expressed in both the PMZ of whole embryo and isolated anterior cut halves . Entries in red are probes that pass a fold change cut-off of 1 . 2 as well as a p value cut-off of 0 . 05; those in blue pass the fold change cut off of 1 . 2 but not the p value cut-off of 0 . 05; and those in black pass the p value cut-off but not the fold change . Common genes are ranked according to the fold change of genes expressed in the PMZ ( Spearman's rank Rho = 0 . 72 , p = 0 . 00048 ) . To confirm the microarray results , we examined 53 of the differentially expressed genes by whole-mount in situ hybridisation at pre-primitive streak stages X-XIII ( Eyal-Giladi and Kochav , 1976 ) ( 23 of these are shown in Figure 2 ) . Apart from Pitx2 three other genes show a similar expression to cVg1 at stage XII: Elk3 ( an Ets-domain protein also known as SRF accessory protein-2 ) , PKDCC ( protein kinase domain containing cytoplasmic protein ) and LITAF ( lipopolysaccharide-induced tumor necrosis factor-alpha ) . Others are expressed in cells adjacent to the PMZ ( and are therefore likely to represent early axial cells ) , such as ADMP , Brachyury ( T ) , Mixl1 , Tbx6 , FGF8 , and CHRD , or are expressed much later ( stage XIV ) , such as DENND5B . A final group is virtually undetectable , such as Thrombopoietin , Ovoinhibitor and PMEPA . All of these rank lower than Pitx2 ( see above and Table 1 , Table2 , Table3 , Table 4 ) : Elk3 ranks 17th in whole embryos and 43rd in cut halves , PKDCC ranks 7th in whole embryos and 33rd in the anterior half , and LITAF ranks 18–19th in whole embryos and 17th and 25th in anterior halves . Pitx2 is therefore the strongest candidate as a putative regulator of cVg1 . The ‘cVg1-unlike’ genes ( Figure 2P–W ) give less obvious information . Comparison of the top genes identified from differential expression in whole embryos ( stronger in AMZ than in PMZ ) with their counterparts in the corners of anterior halves reveals weak correlation ( Spearman's rank Rho between −0 . 02 and 0 . 17; p = 0 . 44–0 . 93 ) . These genes include those encoding extracellular matrix proteins as well as glucose- , glutamate- , glycine- , GABA- and LDL transporters and receptors , the transcriptional repressor ID3 , and BASP1 , which has been reported to act as a transcriptional co-suppressor for WT1 ( Carpenter et al . , 2004 ) , among others . In situ hybridisation for these genes does not show enrichment in the AMZ or any other obvious pattern consistent with a putative role as an inhibitor of cVg1 expression in the PMZ at the appropriate stages of development ( Figure 2P–W ) . Pitx2 therefore remains as the most likely candidate . 10 . 7554/eLife . 03743 . 008Figure 2 . Expression of ‘cVg1-like’ and ‘cVg1-unlike’ genes , verified by in situ hybridisation . Embryos at stage X–XIII ( the earliest stage at which differential expression was detected is shown ) were processed using in situ hybridisation for genes co-regulated with cVg1 ( ‘cVg1-like’ , Table 1A–B ( A–O ) and genes expressed at lower level in the cVg1-positive region than in its counterpart ( ‘cVg1-unlike’ , Table 1C–D ) ( P–W ) , from the two microarray screens ( see Table 1C–D ) . The expression of 23 genes ( 15 ‘cVg1-like’ and 8 ‘cVg1-unlike’ ) is shown here . DOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 00810 . 7554/eLife . 03743 . 009Table 2 . Genes identified from the screens , sorted according to their ranking in cVg1-like > -unlike in isolated anterior halvesDOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 009Anterior half , Vg1-likePosterior vs anteriorGene symbolProbe IDFold changep ValueRankFold changep ValueRankTGga . 3772 . 1 . S1_a_at5 . 5440 . 025116 . 3880 . 08676ADMPGga . 354 . 1 . S1_at3 . 7710 . 0686213 . 0950 . 00801ST6GAL2Gga . 14379 . 1 . S1_at2 . 5060 . 000336 . 8960 . 07445PITX2Gga . 3398 . 1 . S1_a_at2 . 4290 . 008748 . 5280 . 00013MIXL1Gga . 426 . 1 . S1_s_at2 . 3780 . 018555 . 1970 . 01609PITX2Gga . 3398 . 1 . S1_at2 . 3200 . 002665 . 2630 . 00308THPOGgaAffx . 21801 . 1 . S1_at2 . 2720 . 026777 . 3980 . 00094PITX2Gga . 3398 . 2 . S1_a_at2 . 1620 . 006989 . 5860 . 00012DENND5BGga . 16679 . 1 . S1_at2 . 1510 . 002692 . 3410 . 195227WNT8AGga . 886 . 1 . S1_at2 . 0780 . 0042101 . 9310 . 025953CHRDGga . 490 . 1 . S1_at2 . 0640 . 0739112 . 2670 . 095930PMEPA1Gga . 6268 . 1 . S1_at2 . 0320 . 0103124 . 1410 . 080512TBX6Gga . 466 . 1 . S1_at2 . 0300 . 0218133 . 6940 . 000414HOXB1Gga . 18352 . 1 . S1_at2 . 0150 . 0298141 . 7490 . 002973AREGBGgaAffx . 6867 . 1 . S1_at1 . 8910 . 0526151 . 6400 . 119585SOHO-1Gga . 770 . 1 . S1_at1 . 8840 . 0256161 . 4170 . 1282106LITAFGga . 3383 . 1 . S1_at1 . 8220 . 0027172 . 8950 . 001919MLLT6Gga . 11449 . 1 . S1_at1 . 7620 . 0194182 . 2180 . 009132PMEPA1GgaAffx . 12721 . 1 . S1_at1 . 7450 . 0003193 . 7770 . 000713FAM19A1Gga . 11944 . 2 . S1_a_at1 . 7370 . 0948202 . 1020 . 095641List of the top 20 common upregulated probes expressed in both the PMZ of whole embryo and isolated anterior cut halves . Entries in red are probes that pass a fold change cut-off of 1 . 2 as well as a p value cut-off of 0 . 05; those in blue pass the fold change cut off of 1 . 2 but not the p value cut-off of 0 . 05; and those in black pass the p value cut-off but not the fold change . Common genes are ranked according to the fold change of genes in the anterior cut halves ( Spearman's rank Rho = 0 . 73 , p = 0 . 00036 ) . 10 . 7554/eLife . 03743 . 010Table 3 . Genes identified from the screens , sorted according to their ranking in AMZ > PMZ in whole embryosDOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 010Posterior vs anteriorAnterior half , Vg1-likeGene symbolProbe IDFold changep ValueRankFold changep ValueRankLPLGga . 4248 . 1 . S1_at−3 . 5780 . 03501−1 . 2170 . 019169UPK1BGga . 17532 . 1 . S1_at−2 . 5730 . 06622−1 . 5190 . 004615UPK1BGga . 17532 . 1 . S1_s_at−2 . 5500 . 03833−2 . 1330 . 00032SPOCK3GgaAffx . 6009 . 1 . S1_at−2 . 5310 . 02844−1 . 4610 . 017424SLC1A3GgaAffx . 25896 . 1 . S1_at−2 . 3330 . 21685−1 . 3380 . 027247SLC5A1Gga . 8594 . 1 . S1_at−2 . 2840 . 28146−2 . 2530 . 08571BASP1Gga . 3179 . 1 . S1_at−2 . 2760 . 09167−1 . 1700 . 007975ID3Gga . 4048 . 1 . S1_at−2 . 2430 . 00048−1 . 4220 . 046334ATP13A4GgaAffx . 12270 . 1 . S1_at−2 . 1720 . 04869−1 . 2020 . 014871DIO3Gga . 552 . 1 . S1_at−2 . 1490 . 005110−1 . 6560 . 037612UPK1BGga . 12930 . 1 . S1_at−2 . 1340 . 064011−2 . 1230 . 00143COL4A2Gga . 3104 . 1 . S1_at−2 . 1150 . 102412−1 . 2860 . 005958SLC1A3GgaAffx . 25896 . 1 . S1_s_at−2 . 0890 . 319213−1 . 2410 . 036360LRP2GgaAffx . 23355 . 1 . S1_at−2 . 0520 . 063714−1 . 3060 . 014455EFNB2Gga . 13001 . 1 . S1_s_at−2 . 0492 . 652715−1 . 3600 . 022042---Gga . 15960 . 1 . S1_at−2 . 0050 . 000716−1 . 4370 . 020329---Gga . 18649 . 1 . A1_at−1 . 9910 . 009217−1 . 3580 . 006843GABRA1Gga . 17167 . 1 . S1_at−1 . 9550 . 106018−1 . 4870 . 097820GLRA3GgaAffx . 6806 . 1 . S1_at−1 . 9510 . 000119−1 . 6720 . 020910DIO2Gga . 1819 . 1 . S1_at−1 . 9080 . 121720−1 . 4570 . 002525List of the top 20 downregulated probes common to both the PMZ of whole embryo and isolated anterior cut halves . Entries in red are probes that pass a fold change cut-off of −1 . 2 as well as a p value cut-off of 0 . 05; those in blue pass the fold change cut off of −1 . 2 but not the p value cut-off of 0 . 05; and those in black pass the p value cut-off but not the fold change cut-off . Common genes are ranked according to the fold change of genes in the PMZ ( Spearman's rank Rho = −0 . 02 , p = 0 . 93 ) . 10 . 7554/eLife . 03743 . 011Table 4 . Genes identified from the screens , sorted according to their ranking in Vg1-unlike > Vg1-like in isolated anterior halvesDOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 011Anterior half , Vg1-likePosterior vs anteriorGene symbolProbe IDFold changep ValueRankFold changep ValueRankSLC5A1Gga . 8594 . 1 . S1_at−2 . 2530 . 08571−2 . 2840 . 28146UPK1BGga . 17532 . 1 . S1_s_at−2 . 1330 . 00032−2 . 5540 . 03833UPK1BGga . 12930 . 1 . S1_at−2 . 1230 . 00143−2 . 1340 . 064211EDNRBGga . 3306 . 1 . S1_s_at−1 . 9350 . 14914−1 . 7730 . 234832FSTL4Gga . 13574 . 1 . S1_at−1 . 8780 . 01525−1 . 4960 . 001451GABARB2Gga . 17131 . 1 . S1_at−1 . 8140 . 04916−1 . 5860 . 064745KCNAB1Gga . 4971 . 1 . S1_at−1 . 7630 . 01857−1 . 3020 . 023872MYLKGga . 6776 . 1 . S1_at−1 . 7580 . 01268−1 . 8220 . 177730CBLN4GgaAffx . 4848 . 1 . S1_s_at−1 . 7020 . 04249−1 . 6410 . 056440GLRA3GgaAffx . 6806 . 1 . S1_at−1 . 6720 . 020910−1 . 9510 . 000119SLC14A2Gga . 7955 . 1 . S1_at−1 . 6630 . 009411−1 . 8970 . 056122DIO3Gga . 552 . 1 . S1_at−1 . 6560 . 037612−2 . 1490 . 005110KCNMA1Gga . 19342 . 1 . S1_at−1 . 5990 . 206813−1 . 3840 . 009763GRB10GgaAffx . 8324 . 2 . S1_at−1 . 5340 . 056914−1 . 2130 . 023676UPK1BGga . 17532 . 1 . S1_at−1 . 5190 . 004615−2 . 5730 . 06622CALD1GgaAffx . 21386 . 1 . S1_s_at−1 . 5180 . 005416−1 . 7270 . 110633MAFAGga . 974 . 1 . S1_at−1 . 5180 . 259717−1 . 8550 . 014429OLFML3Gga . 1150 . 2 . S1_a_at−1 . 5090 . 003518−1 . 3820 . 014962---Gga . 18986 . 1 . S1_at−1 . 4870 . 010919−1 . 5220 . 031248GABRA1Gga . 17167 . 1 . S1_at−1 . 4870 . 097820−1 . 9550 . 106718List of the top 20 downregulated probes common to both the PMZ of whole embryo and isolated anterior cut halves . Entries in red are probes that pass a fold change cut-off of −1 . 2 as well as a p value cut-off of 0 . 05; those in blue pass the fold change cut off of −1 . 2 but not the p value cut-off of 0 . 05; and those in black pass the p value cut-off but not the fold change cut-off . Common genes are ranked according to the fold change of genes in the anterior cut halves ( Spearman's rank Rho = 0 . 17 , p = 0 . 44 ) . To determine the temporal relationship between Pitx2 and cVg1 in whole embryos and during embryonic regulation , we compared their expression in time-course . In normal embryos cVg1 is first detected at around stage XI ( Bertocchini and Stern , 2012 ) . We detected Pitx2 in the PMZ by the time of laying , stage X ( Figure 3A ) , where it remains until early streak stages ( Figure 3B–F ) . In isolated anterior halves , we increased the sensitivity of the assay by developing the NBT/BCIP colour reaction for several days to ensure that even weak expression could be detected . With this strategy we detected cVg1 expression 4–5 hr after cutting ( Figure 3L–P ) , 1–2 hr earlier than in previous reports ( Bertocchini et al . , 2004 ) , while Pitx2 appeared even earlier , just 3 hr after embryo bisection ( Figure 3G–K ) . Taken together , these results implicate Pitx2 as a good candidate for an upstream regulator of cVg1 expression: it is a transcription factor , it is expressed in the same domain as cVg1 in whole embryos and in bisected embryo marginal zone , and it is expressed before cVg1 . 10 . 7554/eLife . 03743 . 012Figure 3 . Time-course of expression of Pitx2 in whole embryos and in isolated anterior halves , and comparison with cVg1 . ( A–F ) Time-course of Pitx2 expression in whole embryos . Expression is restricted to the posterior marginal zone ( PMZ ) already at the time of laying ( stage X ) ; this intensifies over the subsequent stages . At primitive streak stages ( HH2–3 ) , expression is seen in the primitive streak itself as well as in the PMZ ( F ) . ( G–P ) Comparison of the time-course of Pitx2 ( G–K ) and cVg1 ( L–P ) upregulation in isolated anterior halves . cVg1 is first detectable 4–5 hr after cutting ( M ) , whereas Pitx2 can be detected an hour earlier ( G ) . Both Pitx2 and cVg1 expression appears randomly on the left or the right corner of the marginal zone adjacent to the cut edge . The series chosen for this figure shows upregulation on the right side of all embryo fragments . DOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 012 To determine whether Pitx2 is important for embryonic regulation and for controlling cVg1 expression , we used targeted electroporation of morpholino oligonucleotides ( MOs ) . When a translation-blocking Pitx2-MO was targeted to the right edge of an isolated anterior half embryo , the frequency of axis formation shifted to the opposite side ( Figure 4A–D; Figure 4—source data 1A , B ) : embryos electroporated with control-MO at the right edge had random cVg1 expression on the right , left or neither edges in equal proportion . In contrast , Pitx2-MO on the right generated a majority ( 9/10 , p = 0 . 05 ) of embryos with no expression and the remaining embryo with expression on the opposite side 7 hr post-electroporation ( Figure 4B , Figure 4—source data1B ) . After 16 hr ( Figure 4—source data1B ) embryos electroporated with control-MO at the right edge formed a primitive streak expressing Brachyury randomly; with Pitx2-MO on the right ( Figures 4B ) , 8/10 embryos formed a streak on the left side and 2 had two streaks ( one from each corner ) , but no embryo formed a single streak arising from the MO-transfected side ( hypergeometric exact test with 2 × 3 contingency table , p = 0 . 026 ) . Equivalent results were observed with MO electroporations on the left ( Figure 4C–D; Figure 4—source data1A , B ) : control-MO embryos transfected on the left and examined for cVg1 expression had random expression on the right , whereas for Pitx2-MO cVg1 expression was biased towards the left . After 16 hr , control embryos transfected on the left formed a Bra-expressing streak on the right , again randomly , while Pitx2-MO embryos formed a streak on the right in 2/8 cases , none on the left , 1/8 on both sides and 5/8 with no streak ( p = 0 . 003 ) ( Figure 4D , Figure 4—source data1B ) . 10 . 7554/eLife . 03743 . 013Figure 4 . Pitx2 is required for cVg1 expression and for axis formation . ( A–D ) A morpholino ( MO ) targeting Pitx2 applied to one side of the marginal zone of an isolated anterior half shifts axis formation to the opposite marginal zone . ( A–B ) The experiment done on the right side ( A = control-MO , B=Pitx2-MO ) , ( C–D ) the equivalent done on the left marginal zone . The top row shows diagrams of the experiment , the middle row shows embryo fragments 7 hr after electroporation/cutting , hybridised for cVg1 ( purple ) and stained with anti-fluorescein ( in the MO , brown ) , and the lower row shows fragments processed for Brachyury ( T ) in purple and fluorescein in brown . ( E–F ) In whole embryos , Pitx2-MO has a transient effect . At 5 hr , cVg1 expression is lost , at 12 hr a primitive streak ( T-expressing ) is sometimes seen especially at the edge of the electroporated domain , but by 16 hr embryos appear essentially normal . ( E ) Shows embryos electroporated with control-MO , F are embryos transfected with Pitx2-MO . ( G–H ) . Embryos electroporated with MOs targeting both Pitx2 and Pitx1 do not recover: at 5 hr , no cVg1 is seen; at 12–16 hr there is a high proportion of embryos with either no streak or two T-expressing streaks arising from outside the MO-electroporated domain ( as shown in G ) . This effect can be rescued by co-electroporation of Pitx2 with the mixture of Pitx1-MO/Pitx2-MO ( H ) . ( I–J ) Likewise , in isolated anterior half-embryos , the effects of electroporation of the Pitx1-MO/Pitx2-MO combination ( I ) can be rescued by co-electroporation with Pitx2 alone ( J ) : cVg1 expression is now seen on the electroporated side . Arrowheads point to sites of expression . DOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 01310 . 7554/eLife . 03743 . 014Figure 4—source data 1 . Numbers of embryos displaying different types of results in the Pitx loss-of-function experiments illustrated in the main Figure . Each type of experiment is summarised in a separate sub-table , where each row refers to a particular column ( A–J ) and row ( first , second , and third ) of panels in Figure 4 . The table columns show each type of result obtained: ‘Vg1 R’ refers to embryos showing cVg1 expression on the right side , ‘streak L’ when there is a Brachyury-expressing primitive streak on the left , ‘streak L/R’ when there are two streaks ( one arising from each side ) , etc . DOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 01410 . 7554/eLife . 03743 . 015Figure 4—figure supplement 1 . Pitx1 is expressed posteriorly in normal embryos , and upregulated in Pitx2 morphants . The upper row of images show embryos at stages X , XI and XII processed for in situ hybridisation for Pitx1 . The left lower embryo shows upregulation of Pitx1 ( blue ) , 5 hr after electroporation of Pitx2-MO ( brown from anti-fluorescein staining ) . The right lower image shows lack of effect of similar electroporation of a control-MO . DOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 015 In intact embryos electroporated at stage X , Pitx2-MO affected cVg1 expression and streak formation ( Figure 4E–F; Figure 4—source data 1C–D ) . Control-MO targeted to the PMZ did not alter cVg1 expression after 5 hr or streak formation after 12–16 hr ( Figure 4E; Figure 4—source data1C , D ) . With Pitx2-MO , cVg1 expression was affected after 5 hr ( 0/6 embryos expressing; p < 0 . 001 ) . Embryos started to recover , however , at later time points: at 12 hr , 3/12 had a normal streak , 3/12 had a displaced streak , 3/12 had two streaks , and the remaining 3/12 had no streaks ( p = 0 . 1—not significantly different ) . By 16 hr , the majority of the embryos were normal ( 7/8; the remaining embryo had a displaced streak ) ( Figure 4F; Figure 4—source data 1D ) . These results suggest that while knockdown of Pitx2 affects cVg1 expression , embryos tend to recover by 12–16 hr . We reasoned that functional redundancy with another Pitx gene , or compensatory upregulation of such a gene in response to Pitx2 knockdown , could account for this recovery . To test this , we examined Pitx1 expression in normal embryos at stages X–XII and in embryos electroporated with Pitx2-MO . Pitx1 is barely detectable in the PMZ at stage X–XII ( Figure 4—source data 1 ) . After Pitx2-MO electroporation , expression increased considerably ( 6/6 embryos; Figure 4—source data 1 ) . We therefore repeated the targeting experiments in whole embryos using a mixture of two MOs targeting the translation start site of Pitx2 and an internal splice junction of Pitx1 , respectively . Pitx1+2-MOs caused loss of cVg1 at 5 hr in 6/6 cases ( p = 0 . 005 ) . By 12 hr 1/9 embryos had a normal streak , 3/9 had a displaced streak , 3/9 had two streaks and 2/9 had none ( p = 0 . 043 ) . By 16 hr no recovery was observed: 4/4 embryos had double streaks , neither arising from the targeted site ( p = 0 . 029; Figure 4G; Figure 4—source data 1E , F ) . This effect could be rescued by supplying Pitx2 alone: co-electroporation of Pitx1+2-MO together with a Pitx2 expression construct lacking the MO target sequence led to normal axis formation: in 5/6 cases cVg1 expression was restored after 5 hr incubation and by 16 hr 12/16 embryos displayed a normal streak ( Figure 4H; Figure 4—source data 1G–H ) . Likewise in isolated anterior halves , the effects of Pitx1+2-MO ( Figure 4I; Figure 4—source data 1I , J ) could be rescued by Pitx2 . After electroporation on the right , cVg1 expression was seen on the right in 4/10 , on the left in 3/10 , and in neither in 3/10 cases ( Figure 4J; Figure 4—source data 1K ) . After 16 hr a primitive streak developed on the right in 3/8 cases , on the left in 2/8 and no streak in 3/8 cases ( Figure 4—source data l ) . In conclusion , Pitx2 is required for expression of cVg1 in the PMZ as well as for formation of the normal primitive streak . In isolated anterior halves , Pitx2 is required both for cVg1 expression and for the later formation of a primitive streak . Knockdown of Pitx2 is followed by upregulation of the related transcription factor Pitx1 , which can partly compensate for the loss of Pitx2 . The cVg1 gene ( erroneously annotated as GDF3 in the chick genome; its orthologue is human GDF1 , as confirmed by synteny; see Figure 5 ) is located on chicken chromosome 28 . As a parallel approach to the above to identify putative upstream regulators , we applied a recently described pipeline ( Khan et al . , 2013 ) , starting with prediction of conserved , constitutive CTCF-binding sites ( CTCF is an 11 zinc-finger transcriptional repressor protein that co-localizes with cohesin and acts to delimit chromatin loops ) ( Holwerda and de Laat , 2013; Merkenschlager and Odom , 2013 ) around this locus which could act as insulators . This was followed by algorithms to identify conserved motifs in non-coding regions that are order independent , modified from the Enhancer Discovery using only Genomic Information ( EDGI ) tool described for Drosophila ( Sosinsky et al . , 2007 ) . 10 . 7554/eLife . 03743 . 016Figure 5 . CTCF insulator analysis , enhancer identification and synteny of the cVg1 locus . The chicken cVg1 locus with computationally predicted conserved CTCF-binding sites in chick , human , and mouse is shown ( genes represented in blue ) . This putative insulator region lies ∼200 kB upstream and ∼100 kB downstream of cVg1/GDF3 and harbours other genes such as CERS1/Lass1 , COPE , DDX49 and HOMER3 upstream and UPF1 downstream of cVg1 . Six putative enhancer regions ( E1–E6 ) , predicted using DREiVe , are displayed in pink . In chick , E1 ( galGal4 genomic coordinates: chr28:3502783-3504834 ) and E2 ( chr28:3504993-3508041 ) lie in the first intron of the bicistronic CERS1/Lass1 gene . E3 ( chr28:3510154-3511032 ) and E4 ( chr28:3 , 511 , 413-3 , 511 , 725 ) lie in intron 4 of CERS1/Lass1 and E5 ( chr28:3471200-3471520 ) and E6 ( chr28:3 , 471 , 946-3 , 472 , 230 ) respectively lie in introns 1 and 2 of HOMER3 . E1 and E3 each contain conserved Pitx1 ( black ) and Pitx2 ( green ) binding sites . E2 and E4 do not contain any Pitx sites and E5 and E6 contain Pitx2-binding sites but no Pitx1-binding sites . The orthologous regions of human ( chromosome 19 ) and mouse ( chromosome 8 ) genomes are also shown . Note that the corresponding human region has been inverted , and that although all six elements are found within it , these appear in different order and are associated with different introns and intergenic regions than in chick and mouse . DOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 01610 . 7554/eLife . 03743 . 017Figure 5—figure supplement 1 . Expression of genes adjacent to cVg1/GDF3 on chick chromosome 28 . Expression of Lass1/CERS1 , HOMER3 , COPE and UPF1 is shown . All are expressed posteriorly although the patterns are not quite identical to each other . DOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 017 The insulator-predicting software identifies strongly conserved CTCF-binding sites about 200 kB upstream and about 100 kB downstream of cVg1/GDF3 ( Figure 5 ) . The cVg1/GDF3 gene itself is bicistronic , the upstream exons encoding Lass1/CERS1 and the last two exons containing the cVg1 sequence ( Wang et al . , 2007 ) . Several other genes lie in this region , including COPE , DDX49 and HOMER3 upstream and UPF1 downstream . If the predicted CTCF-binding sites are indeed insulators , we would expect these genes to be co-regulated with cVg1 . To test this , we examined their expression . Strikingly , all genes examined ( CERS1/LASS1 , COPE , HOMER3 , and UPF1 ) are expressed in a similar domain of the PMZ as cVg1 ( Figure 5—figure supplement 1 ) . We then applied the DREiVe tool ( Discovery of Regulatory Elements in Vertebrates , the vertebrate version of EDGI ) ( Sosinsky et al . , 2007; Khan et al . , 2013 ) , to identify de novo conserved sequence motifs around this region . This identifies six domains ( designated E1–E6 ) , ranging in size from 600–3000 bases , located within the introns of CERS1/LASS1 and of the neighbouring Homer3 in chick ( Figure 5 ) . Analysis of these regions using Position Frequency Matrices from JASPAR and TRANSFAC databases together with the algorithms Matrix-Scan ( from RSAT ) and Clover predicts four of these regions ( E1 , E3 , E5 , and E6 ) to contain one or more putative binding sites for Pitx2 and/or the related factor Pitx1 ( Figure 5 ) . The power of DREiVe as a tool for discovering regulatory elements is highlighted by the observation that it is able to identify homologous non-coding regions in the human genome , where the syntenic region ( on chromosome 19 ) is not only inverted but also the orthologous elements are found in a different order , within introns of different neighbouring genes ( Figure 5 ) . In mouse ( chromosome 8 ) the arrangement is similar to chick ( Figure 5 ) . To determine whether any of the predicted regions bind Pitx2 in the PMZ of normal embryos , we conducted chromatin-immunoprecipitation experiments ( ChIP ) , assessing precipitation by real-time quantitative polymerase chain reaction ( qPCR ) analysis using primers targeting the predicted regions . We compared chromatin from the AMZ and PMZ ( Figure 6 ) . A monoclonal antibody against Pitx2 precipitated chromatin from the PMZ more effectively than from the AMZ for all predicted enhancers that contained consensus Pitx1/2-binding sites ( especially E3 , E5 , and E6 ) but not those that do not ( E2 and E4 ) . These findings suggest that Pitx2 is differentially bound to putative enhancer sites E3 , E5 , and E6 in the PMZ of normal embryos . We also tested each of the six putative enhancer regions for acetylation of Lys-27 of Histone-3 ( H3K27ac ) , which is associated with active enhancers ( Creyghton et al . , 2010 ) . Enhancers E5 and E6 showed the greatest differential activity in the PMZ relative to the AMZ . Together , these results suggest that E3 , E5 , and E6 are the most likely enhancers driving expression in the PMZ . 10 . 7554/eLife . 03743 . 018Figure 6 . Chromatin immunoprecipitation to test for active histone marks and Pitx2 binding to predicted enhancers . Relative immunoprecipitation around each of the putative six enhancers by an antibody to Pitx2 ( diagonal hatching ) or an antibody to acetyl-lysine-27 of Histone-H3 ( grey shading ) , expressed as a ratio of the amount precipitated from posterior and anterior marginal zone ( PMZ and AMZ ) chromatin . Primers were used to target each of the putative enhancers and precipitated chromatin measured by the quantitative polymerase chain reaction ( qPCR ) . Each data bar represents the average of at least three true biological replicates and the error bars indicate standard error of the mean . Amplification from input DNA from each of the same samples is also shown ( solid black shading ) . Note that enhancers that contain Pitx2-binding sites ( E1 , E3 , E5 , and E6 ) are precipitated much more strongly from the PMZ than the AMZ . DOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 018 To test whether these putative enhancers do indeed direct transcription in the correct endogenous domain , we generated reporter constructs based on a vector designed by the group of Kondoh ( Uchikawa et al . , 2003 ) . Each construct contained one candidate enhancer ( E1–E6 ) , a minimal promoter ( TK ) , and a reporter fluorescent protein ( EGFP or RFP ) and was electroporated either in a very broad domain including the PMZ and lateral marginal zones of normal embryos , or encompassing the entire cut edge ( including both corners ) of bisected embryos and the anterior half subsequently cultured . A ubiquitous reporter ( pCAβ with either EGFP or RFP ) was co-electroporated with each construct to reveal the extent of the electroporated domain ( Figures 7 and 8 ) . Embryos were then photographed live to reveal the electroporated and expressing domains , then fixed and processed for in situ hybridisation for cVg1 to determine whether the side with reporter activity corresponds to the cVg1 expressing region . In whole embryos , E3 and E5 are most efficient in driving expression of the reporter in the PMZ ( Figure 7 ) . The same two enhancers also drive expression in the cVg1-expressing side in isolated anterior halves ( Figure 8 ) . 10 . 7554/eLife . 03743 . 019Figure 7 . Enhancers E3 and E5 drive expression in the posterior marginal zone of whole embryos . Embryos were electroporated with a construct containing a candidate enhancer ( E1–E6 ) , a minimal promoter ( TK ) and a fluorescent reporter ( GFP or RFP ) , together with a ubiquitous marker ( pCAβ-EGFP or DS-RedExpress ) to reveal the electroporated area . After 5–9 hr culture the embryos were observed by fluorescence ( first 4 columns ) and then fixed and processed to reveal cVg1 expression by in situ hybridisation ( last column ) . The position of Koller's sickle is marked with a curved white line . Enhancers E3 and E5 faithfully recapitulate cVg1 expression in the posterior marginal zone ( PMZ ) , whereas E1 drives expression inside the embryo ( but not in the PMZ ) and the remaining enhancers show little or no detectable activity . In all cases , the electroporated area appears red and the activity of the specific enhancer construct in green . DOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 01910 . 7554/eLife . 03743 . 020Figure 7—figure supplement 1 . Effects of mutations in Pitx-binding sites on activity of cVg1 enhancers in whole embryos . Each of the enhancers containing Pitx1- or Pitx2-binding sites was mutated either singly or , for those enhancers containing more than one discrete site , both together . The mutation is shown diagrammatically on the left column . In columns 2–5 , the area electroporated ( DS-RedExpress reporter ) appears in red and expression driven by the specific enhancer construct in green . The last column shows cVg1 expression revealed by in situ hybridisation in the same embryos . Enhancer E1 is unaffected by mutation of either Pitx1- or Pitx2-binding sites , and continues to be active inside the embryo but not in the PMZ . E6 is also unchanged by mutation of the Pitx2-binding site ( no expression is seen from the intact reporter , see main Figure 7 ) . The activity of enhancers and E3 , E5 is destroyed by mutation of any of the Pitx1/2-binding sites . DOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 02010 . 7554/eLife . 03743 . 021Figure 8 . Enhancers E3 and E5 drive expression in the cVg1-expressing corner of the marginal zone at the cut edge of isolated anterior half-embryos . Embryos were electroporated with the same vectors as described in Figure 7 , then bisected . The anterior half was then cultured for 5–7 hr and viewed under fluorescence ( first 4 columns ) , then fixed and processed for cVg1 expression ( last column ) . Enhancers E3 and E5 drive expression of the reporter at the cVg1-expressing edge of isolated anterior half-embryos . Note that unlike what is found in whole embryos , Enhancer E1 does not appear to drive expression in the area pellucida of the isolated anterior half . DOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 02110 . 7554/eLife . 03743 . 022Figure 8—figure supplement 1 . Effects of mutations in Pitx-binding sites on activity of cVg1 enhancers in isolated anterior halves . Each of the enhancers containing Pitx1- or Pitx2-binding sites was mutated either singly or , for those enhancers containing more than one discrete site , both together . The mutation is shown diagrammatically on the left column . Details as in Figure 7—figure supplement 1 . The specific activity of E3 and E5 is destroyed by mutation of any of the Pitx-binding sites . DOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 022 To test whether the Pitx-binding sites are required within these enhancers to direct expression to the appropriate domain , we generated reporter constructs for enhancers E1 , E3 , E5 , and E6 containing mutations in each Pitx1- or Pitx2-binding site as well as constructs where all Pitx-binding sites were mutated . In whole embryos , mutations in either of the Pitx-binding sites ( Figure 7—figure supplement 1A–B ) or in both binding sites ( Figure 7—figure supplement 1C ) of E1 still showed GFP expression without selectivity , in the middle of the embryo . For E3 and E5 , however , mutations in either a single or both binding sites of each reporter completely abolished expression in the PMZ ( Figure 7—figure supplement 1D–G ) . Un-mutated reporter E6 , which did not show any activity ( see above ) , was not altered by a mutation of its Pitx2-binding site ( Figure 7—figure supplement 1H ) . Similar results were found in isolated anterior half-embryos: mutations of any of the Pitx-binding sites in E3 or E5 completely eliminated expression in the cVg1-expressing corner of the isolated half ( Figure 8—figure supplement 1 ) . Taken together , these results suggest that cVg1 expression is regulated directly by Pitx2/Pitx1 binding to an enhancer ( E5 ) within an intron of the HOMER3 gene , adjacent to cVg1/GDF3 , and to an intronic enhancer ( E3 ) within the Lass1/CERS1 locus . The ChIP experiments suggest that Pitx2 binding to an additional intronic enhancer in HOMER3 ( E6 ) may also be functional in the PMZ , although this was not seen when a reporter construct containing this element was electroporated into the PMZ . The same enhancers ( E3 and E5 ) are involved in controlling cVg1 expression in the normal PMZ as in the portion of the lateral marginal zone where cVg1 is upregulated a few hours after isolation of a portion of embryo . These results implicate the transcription factor Pitx2 as the earliest gene described to date that regulates the position of the embryonic axis as well as embryonic regulation/regeneration and twinning .
The PMZ of the chick embryo is the equivalent of the Nieuwkoop centre of amphibians: it can induce a complete axis including the organiser from neighbouring cells , without making a cellular contribution to the axis ( Azar and Eyal-Giladi , 1979; Khaner and Eyal-Giladi , 1989; Bachvarova et al . , 1998 ) . The Nodal/Activin-related TGFβ superfamily member cVg1 ( homologous to mammalian GDF1 ) is expressed in the PMZ . When ectopically applied to another region of the marginal zone , cVg1 is sufficient to initiate formation of a complete embryonic axis from adjacent embryonic ( area pellucida ) cells ( Seleiro et al . , 1996; Shah et al . , 1997 ) . To act , cVg1 requires canonical Wnt , which seems to be provided mainly by cWnt8C , expressed all around the marginal zone ( Skromne and Stern , 2001 ) . A target of cVg1 and Wnt is Nodal , transcribed in area pellucida cells next to the cVg1+Wnt expression domain ( Skromne and Stern , 2002 ) . The anterior end of the embryo also has an early identity , defined by the expression of GATA binding protein 2 ( GATA2 ) ( Sheng and Stern , 1999; Bertocchini and Stern , 2012 ) . However , unlike cVg1 , GATA2 is not a sufficient determinant of polarity and at best only acts as a bias ( Bertocchini and Stern , 2012 ) . While amphibian and teleost embryos lose their ability to generate complete , independent embryos if fragmented after the first few cell divisions , amniotes have huge regulative capacity . Dividing a chick blastoderm even just before primitive streak formation ( when the embryo may have as many as 50 , 000 cells ) into up to eight fragments can lead to formation of as many complete embryos ( Lutz , 1949; Spratt and Haas , 1960 ) . In an isolated anterior half , a visible primitive streak ( and expression of Brachyury and Snail2 ) can be detected about 12 hr after cutting . This appears randomly from either the left or right area pellucida adjacent to the cut edge of the marginal zone ( Spratt and Haas , 1960 ) , preceded at least 6 hr earlier by expression of cVg1 in the right or left marginal zone ( Bertocchini et al . , 2004 ) . Blocking cVg1 on one side of the marginal zone of an isolated anterior half will cause the axis to arise from the opposite side . A similar manipulation in the PMZ will cause a streak to arise from outside the MO-electroporated domain ( Bertocchini et al . , 2004; Bertocchini and Stern , 2012 ) . Thus , cVg1 expression in the marginal zone is both necessary and sufficient to initiate formation of a primitive streak . There was no information , however , about what positions cVg1 expression in the PMZ of normal embryos or in isolated fragments . This led us to undertake a screen for upstream regulators . We took two complementary approaches: a molecular screen , designed to identify genes co-expressed with cVg1 both in the normal PMZ and in the cVg1-expressing edge of the marginal zone in isolated anterior halves at the time when cVg1 first becomes detectable , and a bioinformatics-based approach , predicting and analysing putative enhancers of cVg1 situated on chromosome 28 . A particular difficulty of the molecular screen was that it is impossible to know , in a single embryo , which of the two edges of the isolated anterior half will express cVg1 . This required analysis of each individual fragment just after excising left and right edge explants , then pooling them appropriately according to whether they derived from the cVg1-expressing side or the opposite edge . This has the additional advantage of removing any intrinsic left–right differences that may exist in the embryo at this stage . Selecting genes that are co-expressed with cVg1 when this is first upregulated at one edge of an isolated fragment , and also co-expressed with cVg1 in the normal PMZ , turned out to be a powerful strategy to reduce the number of relevant genes , thereby avoiding the ‘cherry-picking’ approaches often associated with molecular screens . This combination of molecular screens and bioinformatic analysis converged towards a single strong candidate: the transcription factor Pitx2 . The regulatory role of Pitx2 on cVg1 expression was then confirmed by loss-of-function experiments showing that Pitx2 is required for cVg1 activation and axis formation both in normal development and during embryonic regulation , and that it binds directly to four non-coding regions around the cVg1 locus ( E1 , E3 , E5 , and E6 ) , two of which ( E3 and E5 ) are sufficient to direct expression specifically to the PMZ and cVg1-expressing edge of an isolated anterior fragment . Moreover , mutation of any of the Pitx-binding sites in enhancers E3 and E5 abolishes the activity of these enhancers in the PMZ . These findings strongly implicate Pitx2 as a direct and essential regulator of cVg1 expression . Pitx transcription factors are characterised by possessing a Lysine residue at position 50 of the homeodomain , an unusual property shared with the vertebrate genes Goosecoid and OTX-1/2 and with the founder gene of the family , Drosophila Bicoid . In Drosophila , Bicoid is a critical specifier of ‘anterior’ identity and essential for setting up head–tail polarity of the early embryo . Although it is tempting to speculate that a Bicoid/Pitx system may have an ancient function in the specification of head–tail polarity , the Bicoid gene does not appear to have direct orthologues in species other than schizophoran flies ( closely related to Drosophila ) , so this may be either a coincidence or convergent evolution . The four key components that form part of this gene regulatory network initiating axis formation , Pitx2 , cVg1/GDF1 , Tbx6 , and Nodal , are also involved a little later in development ( from the late primitive streak stage ) in specifying left–right asymmetry in different vertebrate classes ( Levin et al . , 1995; Hyatt et al . , 1996; Hyatt and Yost , 1998; Logan et al . , 1998; Meno et al . , 1998; Piedra et al . , 1998; Ryan et al . , 1998; St Amand et al . , 1998; Yoshioka et al . , 1998; Zhu et al . , 1999; Rankin et al . , 2000; Wall et al . , 2000; Levin , 2005; Raya and Izpisua Belmonte , 2006; Tanaka et al . , 2007; Hadjantonakis et al . , 2008 ) . Indeed , these are the main conserved components of the left–right pathway among different vertebrates . These observations raise the possibility that the left–right pathway may have evolved by co-opting a more ancient mechanism for initiating formation of the gastrular axis . This is supported by the finding that a Nodal/Pitx2 loop is involved in specifying both left–right asymmetry and mesendoderm formation ( oral-aboral polarity ) in the sea urchin , a non-vertebrate deuterostome ( Duboc et al . , 2004; Hibino et al . , 2006; Warner et al . , 2012 ) . Pitx2 , Vg1 , Nodal , and Tbx6 are also involved in early mesendoderm development in anamniotes , although Vg1 is maternal ( Thomsen and Melton , 1993; Kessler and Melton , 1995; Faucourt et al . , 2001 ) . In the mouse , double mutants for Pitx1 and Pitx2 lead to early lethality ( at pre- or peri-implantation stages ) of the embryo ( Marcil et al . , 2003 ) . Only a single embryo was ever recovered that had survived to E10–E12 ( Drouin , personal communication ) . Expression of these transcription factors has to date only been studied in detail at later stages of mouse development ( L'Honore et al . , 2007; Lanctot et al . , 1997 ) and it will therefore be interesting to see if they are indeed expressed as in the chick at pre-primitive streak stages . Certainly , the strong conservation of the active enhancers ( E3 and E5 , including the Pitx-binding sites contained therein ) near mouse and human GDF1 suggests that this is likely to be a conserved feature of birds and mammals , and despite the fact that the peculiar geometry of rodent embryos at these early stages has led to some differences in the processes leading to axis development ( Stern and Downs , 2012 ) . Monozygotic twins do not seem to occur commonly in the mouse and it is possible that these very small , cup-shaped embryos do not survive to term if more than one primitive streak appears within a single blastocyst . Together , these findings suggest that the retention of a regulative mode of development at late stages by amniote embryos ( that allows the formation of the types of monozygotic twins that arise relatively late in development , including Siamese twins ) evolved through novel uses of an ancient pathway , involved in both mesendoderm development and left–right asymmetry , but in slightly different ways . Our study also brings forward the time at which the earliest responses to cutting an embryo can be detected . First , we can now detect cVg1 expression at one edge of a cut anterior fragment 4–5 hr after cutting , about 2 hr earlier than the 6 hr reported previously ( Bertocchini et al . , 2004 ) ; Pitx2 appears even earlier , 3 hr after cutting . But this also begs the question of what lies upstream of Pitx2 . At some early point in the cascade , the regulators will no longer be controlled at the transcriptional level and it will be considerably more difficult to identify them . The present finding that Pitx2 is upregulated locally just 3 hr after cutting an embryo suggests that its regulators may not be differentially expressed mRNAs , but other asymmetries . Our studies allow us to predict the Pitx2/cVg1 ( GDF1 ) /Nodal pathway as a possible candidate to explain the obligate quadruplets of armadillos ( Newman and Patterson , 1910; Loughry et al . , 1998; Enders , 2002; Eakin and Behringer , 2004 ) and/or the high incidence of monozygotic and conjoined twins in certain human populations ( Cox , 1963; Hamamy et al . , 2004; Forsberg et al . , 2010 ) . Answering these questions represent interesting future challenges .
Fertile Brown Bovan Gold hens' eggs ( Henry Stewart , UK ) were incubated for 1–16 hr to obtain stages X–XIII ( Eyal-Giladi and Kochav , 1976 ) and stage 4 ( Hamburger and Hamilton , 1951 ) ( HH ) . Embryo manipulation was performed in Tyrode's solution . Anterior halves were obtained by cutting embryos with a hair loop . Unlike previous studies ( Bertocchini et al . , 2004 ) , here we did not use a strip of anterior area opaca to seal the cut edge . Embryos and fragments were set up in modified New culture ( New , 1955; Stern and Ireland , 1981 ) and incubated at 38°C as required . Whole mount in situ hybridisation was performed as described previously ( Stern , 1998; Streit and Stern , 2001 ) . The probes used were: chick cVg1 ( Shah et al . , 1997 ) , Brachyury ( Kispert et al . , 1995a; Kispert et al . , 1995b; Knezevic et al . , 1997 ) , and Pitx2 ( Logan et al . , 1998; Zhu et al . , 1999 ) . Two microarray screens were performed with tissues collected from stages X–XII ( Eyal-Giladi and Kochav , 1976 ) . A first screen was performed with triplicates of 40 pieces of PMZ and AMZ , dissected and individually frozen from whole embryos ( Figure 1A , Figure 1—figure supplement 1 ) . A second screen was done with triplicates of 70 pieces of the left and right corners of the marginal zone , dissected and frozen individually from anterior embryo halves that had been cultured for 7 hr ( Figure 1C , Figure 1—figure supplement 2 ) . After dissection , whole embryos and anterior halves were fixed in 4% PFA for in situ hybridisation with cVg1 . In both screens , in situ hybridisation was carried out for an extended period to detect low cVg1 expression adjacent to the excised pieces and confirm the orientation of the embryo ( see Results ) ; the orientation was ambiguous or incorrect in about 10% of the embryos , and explants obtained from them were therefore not included . The remaining validated PMZ and AMZ samples , cVg1-like and non-cVg1-like samples were pooled in TRIzol reagent ( Ambion , Invitrogen , UK ) . RNA was prepared and run for a complete Affymetrix analysis by ARK-Genomics . 500 ng of total RNA was required for the standard 3' IVT-Express protocol . Each label was quality checked through all stages of amplification and preparation for hybridisation on Affymetrix 30K chicken microarrays . Microarray raw data were analysed using Bioconductor in R ( Gentleman et al . , 2004 ) . Raw datasets were normalised using the Robust Multi-array Average ( RMA ) method ( Irizarry et al . , 2003 ) . Differentially expressed genes were then identified using the Limma package in R ( Smyth , 2005 ) with a fold change threshold of 1 . 2 and p < 0 . 05 . This strategy identified 122 sequences ( corresponding to 85 genes ) with putative cVg1-like expression ( ‘cVg1-like’ ) and 78 sequences ( 52 genes ) expressed in the cVg1-negative explants ( ‘cVg1-unlike’ ) . The complete dataset was deposited with ArrayExpress where it can be accessed under Accession number E-MTAB-3116 . A selection of genes from the top up- and downregulated genes was verified by in situ hybridisation: ADMP , PITX2 , THPO , PKDCC , PMEPA , TBX6 , FGF8 , Ovoinhibitor , ELK3 , LITAF , PITX1 , CAMK1G , DENND5B , MLLT6 , Homer3 , SEMA5B , PIK3R1 , GALNTL1 , SH2D4A , GLI3 , PDLIM5 , VANGL1 , LEF1 , FGF18 , LOC768709 , CXCL14 , PLCB1 , ZBTB1 , MED15 , SPOCK3 , BASP1 , LRP2 , GABRB2 , PDGFA , Autotaxin , SALL1 , MAFA , ESRRG , FOXD2 , FSTL4 , MALT1 , OLFM3 , GNAZ and GRB10; when not already available , probes were generated from the chick EST collection ( Boardman et al . , 2002 ) . Computational prediction of CTCF insulator elements was performed as previously described ( Khan et al . , 2013 ) . A PERL script ( http://www . ncbi . nlm . nih . gov/pmc/articles/PMC3664090/#SD1 ) was used to scan chromosome 28 ( location of GDF3/cVg1 ) from the galGal4 build of the chicken genome for occurrences of CTCF-binding sites with stringent parameters ( False Discovery Rate , FDR 0% ) . Equivalent regions of human chromosome 19 ( hg19 genome build , location of homologous GDF1 ) and mouse chromosome 8 ( mm10 genome build , location of homologous GDF1 ) were also scanned for CTCF-binding sites with the same FDR parameter . The nearest conserved CTCF-binding sites harbouring the same set of genes both upstream and downstream of GDF3/GDF1 in all three species were then identified and these domains were defined as regions bounded by putative insulators . Enhancer prediction was carried out using the software package Discovery of Regulatory Elements in Vertebrates ( DREiVe ) ( Khan et al . , 2013 ) , the vertebrate version of EDGI ( Sosinsky et al . , 2007 ) . Genomic coordinates for the predicted insulator region in human were used as the reference to predict order-independent conserved patterns of DNA sequences shared between human and any seven of the following species: horse ( Equcab2 ) , cow ( Bostau4 ) , rabbit ( Orycun2 ) , guinea pig ( Cavpor3 ) , mouse ( mm9 ) , opossum ( Mondom5 ) , platypus ( Taegut1 ) , chicken ( Galgal3 ) , and lizard ( Anocar1 ) . Parameters used included motif density of 6 matching nucleotides within a window length of 8 bp , where the minimum number of matching nucleotides in the motifs was set at 12 bp . The maximal cluster length ( maximum length of predicted enhancers ) was set at 3000 bp with a sequence conservation score cut-off of 2 . This set of parameters successfully predicted a series of conserved blocks , designated E1–E4 ( see Figure 5 ) , in human , chicken , mouse , and other species . Transcription factor binding site analysis of these predicted enhancers was carried out using the matrix-scan algorithm from the RSAT workbench ( http://rsat . ulb . ac . be/rsat/ ) ( Thomas-Chollier et al . , 2011 ) . Position frequency matrices from both Jaspar ( http://jaspar . cgb . ki . se/ ) and Transfac ( http://www . gene-regulation . com/pub/databases . html ) libraries were used in matrix-scan where the background model estimation method was based on a Markov order of 0 . Organism-specific ‘upstream no-orf’ background sequences were used ( galGal4 ) with a pseudo-frequency of 0 . 01 and an upper p-value threshold of 1e−4 . As a complementary approach , a modified methodology of Clover ( http://cagt . bu . edu/page/Clover_about ) was used to detect enhancers that shared order-independent transcription factor binding sites rather than DNA patterns . This approach uncovered two additional putative enhancers , E5 and E6 . Fluorescein-labelled MOs against Pitx2 , Pitx1 and a standard control-MO ( Gene Tools , Philomath , Oregon , USA ) were delivered to young embryos by electroporation as described ( Voiculescu et al . , 2007; Voiculescu et al . , 2008 ) . Pitx2-MO was designed to target the translational start site: ( 5′-CAAGTTTACGGCAGTTGGACTCCAT-3′ ) . A splice blocking Pitx1-MO was designed to target the start of exon-2: ( 5′-CTCTCTTTTTCTACGGTGGGATGTT-3′ ) . The coding sequence of chicken Pitx2 ( Logan et al . , 1998 ) was cloned into pCAβ-IRES-GFP to generate a Pitx2 expression construct . The expression of Pitx2 protein was confirmed by western blot with a Pitx2-antibody ( Abcam , UK , ab55599 ) . Candidate enhancers 1–6 ( E1–E6 ) were amplified from chick genomic DNA using the following primer pairs: ( E1 ) -forward ( 5′-CAGCCCCAGGCAGACAGGGCTGCAGGGAAGAAGGGG-3′ ) , reverse ( 5′-ACGGGACCCCCAGCCCTGCAGGATGCTGCCCGGGGT-3′ ) ; ( E2 ) -forward ( 5′-GGAGGTACCATAATTCATGCTTTCTGGGCTCGGGAC-3′ ) , reverse ( 5′-GTACTCGAGCAAAGGTATCCCAGACCCTGCTGTC-3′ ) ; ( E3 ) -forward ( 5′- GGAGGTACCACTCATTTGGTTTTAGCATTAATAAAC-3′ ) , reverse ( 5′- GTACTCGAGCTGCCAGGGCAGAGGGAGCAGGGTG-3′ ) ; ( E4 ) -forward ( 5′-CAGGGGATGAAGGGGGTGTTGGGGATCAAGCTCTTC-3′ ) , reverse ( 5′-CAGTCTGCTACAATCCCTTCCCATGGATTCCTGGGG -3′ ) ; ( E5 ) -forward ( 5′-GTGAGGTACCCTGTTCAGTC-3′ ) , reverse ( 5′- AACTCGAGGTACAAGCTCTGC -3′ ) ; ( E6 ) -forward ( 5′-GGTAGAGACCTGGTACCAGTAG-3′ ) , reverse ( 5′-GAAGGGAGCTCGAGTGTCAC -3′ ) . The amplified fragments were subcloned into pGEMT-easy ( Promega , Madison , Wisconsin , USA ) , excised with inserted KpnI and XhoI sites and cloned into ptkEGFP ( Uchikawa et al . , 2003 ) or ptkRFP ( kind gifts of H Kondoh ) . All constructs were checked by sequencing . 3 µg of each enhancer construct were co-electroporated with pCAβ-RFP , DS-RedExpress ( Invitrogen , UK ) or pCAβ-IRES-GFP as appropriate . Mutations were introduced into each of the Pitx1- or Pitx2-binding sites of the four enhancers ( E1 , E3 , E5 , and E6 ) that contained such sites , with base changes highlighted in red ( Table 5 ) . The PCR primers used for site-directed mutagenesis are shown in Table 6 . For Enh5 , site 1 refers to the 5′ Pitx2 , while site 2 is the 3′ Pitx2-binding site ( see Figure 5 ) . Site-directed mutagenesis was performed as described ( Liu and Naismith , 2008 ) . Briefly , 100 ng of template Enhancer DNA was PCR amplified with 2 µM of each mutant primer pair , 200 µM dNTPs , 2 µl Phusion high-fidelity polymerase , and 5 µl 10X buffer in a total volume of 50 µl . The PCR programme was as follows: 94°C 3 min , 94°C 1 min , 52°C 1 min , 68°C 8 , 12 , or 24 min ( 500 bp/min ) , final extension 68°C 1 hr , then 4°C . 1/25th of each reaction was run on an Agarose gel to verify amplification , after which the remainder of the reaction was digested with DpnI to remove the parent template for 1 hr at 37°C . Another 1/25th of the DpnI digest was transformed into DH5α cells and clones selected and amplified in culture for DNA extraction and sequence verification of the introduced mutations . 10 . 7554/eLife . 03743 . 023Table 5 . Construction of mutations introduced to destroy Pitx-binding sitesDOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 023Enh PITX siteMutated basesE1 PITX2GCTGGTCATGACTTCTTE1 PITX1TGGTTTCGAGTTGAAAGE3 PITX2GGGTGTCATGAATGGCCE3 PITX1AGGGTTCGAGTAATGGCE5 PITX2 1TAAAATCCTGAAACTGCE5 PITX2 2AGAAATCATGAATAAATE6 PITX2TGTCTTCATGACTCCTCThe mutations ( highlighted in red ) introduced into the sequences of Pitx-binding sites in enhancers E1 , E3 , E5 and E6 . 10 . 7554/eLife . 03743 . 024Table 6 . Primers used for construction of mutations in Pitx-binding sitesDOI: http://dx . doi . org/10 . 7554/eLife . 03743 . 024Primer sequencePrimer nameaggggtttggttTCgaGttgaaagcgtgtacttctcacccattgaaactgccaggtctgtEnh1PitX1MutFctttcaaCtcGAaaccaaacccctcccatcacacacacaaactctactgcttcttcaaaccaEnh1PitX1MutRagggggctggTCatGacttcttgcaggtgccccaaaggcagggccaEnh1PitX2MutFaagaagtCatGAccagccccctacccgaccctgctgcacagggaatEnh1PitX2MutRggccattaCtcGAaccctgctccctctgccctggcagEnh3PitX2MutFctgccagggcagagggagcagggtTCgaGtaatggccEnh3PitX2MutRagtgcagtttCagGAttttactttatagtttttttattcctacttgccatgaaagtggagaacatEnh5PitX2MutFtaaaaTCctGaaactgcacttaacatagcgattacaaaacactcttggttagagtagcaacacaaEnh5PitX2MutRtgggctttcctatttattCatGAtttcttacagtagacaagaagattcacatttgtttgtgtttcatagctagagEnh5PitX22MutFtgtaagaaaTCatGaataaataggaaagcccaaatgttacaagcttcattgggcctccattgctggaagaaaagaEnh5PitX22MutRataacaaaggggaggagtCatGAagacattgattctaatttgttctacatttcagtttattagcaaagtgacaEnh6PitX2MutFaatcaatgtctTCatGactcctcccctttgttatttcattttatgtgctttctaattcatcttcaattaagaEnh6PitX2MutRThis table shows the primer combinations used to generate the mutations indicated in Table 5 . Micro ChIP experiments were performed with pools of 25 pieces of PMZ and their anterior counterparts ( AMZ ) from whole pre-streak stage X–XI embryos . Chromatin was cross-linked , extracted and sonicated to obtain 200–1000 bp fragments . Immunoprecipitation was done with rabbit anti-H3K27ac ( Abcam , UK ) , mouse anti-PITX2 ( Abcam , ab55599 ) , antibodies and rabbit IgG ( Millipore , Merck Millipore UK ) and mouse IgG ( Millipore , Merck Millipore UK ) bound to magnetic Dynabeads ( Life Technologies , Carlsbad , California , USA ) . The purified DNA was used as template for qPCR analyses with a Bio-Rad iCycler and Sybr Fast Q-PCR mix ( Kapa Biosystems , Wilmington , Massachusetts , USA ) . The following primer pairs were used: enhancer1—forward primer1 E1F1 ( 5′-gctctatcccgattccctgtgca-3′ ) reverse primer1 E1R1 ( 5′- ggtagggttcacttcattagggatg -3′ , forward primer2 E1F5 ( 5′-cactctgtgtccggagaatgctac-3′ ) reverse primer2 E1R5 ( 5′- caatgggtgagaagtacacgctttc-3′ ; enhancer2—forward primer1 E2F1 ( 5′- atgaagagcgcaagagtggcaaag-3′ ) reverse primer1 E2R1 ( 5′-tgggaaattccgacctggaagcag-3′ , forward primer2 E2F3 ( 5′- agctgagctgtttaacggtggatc-3′ ) reverse primer2 E2R3 ( 5′- agctgaccgctgctagttctct-3′ ) ; enhancer 3—forward primer E3F2 ( 5′- cctagctattacactctgctcttcc-3′ ) reverse primer E3R2 ( 5′- CAGTAGAGACGGAACCAGAACct-3′ ) ; enhancer 4—forward primer1 E4F1 ( 5′- gcttcttggtgctggaactgagaat-3′ ) reverse primer1 E4R1 ( 5′- atggagagacacagtcagccacga-3′ , forward primer2 E4F2 ( 5′- agctgcccaatgctctgaaagaag -3′ ) reverse primer2 E4R2 ( 5′- ggaatggaaaggtgaggattcatgg -3′ ) ; enhancer 5—forward primer E5F3 ( 5′- CGTGAGGCAGTCTGTTCAGT-3′ ) reverse primer E5R3 ( 5′- GCTATGAAACACAAACAAATGTGAA-3′ ) ; enhancer 6—forward primer E6F3 ( 5′- CACTGGGGTCCTGATGTAGTG-3′ ) , reverse primer E6R3 ( 5′- AGAAGGGAGCACAAATGTCA-3′ ) ; Vg1 -0 . 5 ATG—forward primer Vg1F1 ( 5′-agtgggtgctgattgctgtctgtg-3′ ) reverse primer Vg1R1 ( 5′-ctcccatcccttctgcatctccata-3′ ) . All qPCR reactions were run in triplicate and contained an input control for normalisation of chromatin concentration , and the appropriate IgG as control . The amplification products were quantified using the ΔΔCt method ( Livak and Schmittgen , 2001 ) .
Embryonic polarity of invertebrates , amphibians and fish is specified largely by maternal determinants , which fixes cell fates early in development . In contrast , amniote embryos remain plastic and can form multiple individuals until gastrulation . How is their polarity determined ? In the chick embryo , the earliest known factor is cVg1 ( homologous to mammalian growth differentiation factor 1 , GDF1 ) , a transforming growth factor beta ( TGFβ ) signal expressed posteriorly before gastrulation . A molecular screen to find upstream regulators of cVg1 in normal embryos and in embryos manipulated to form twins now uncovers the transcription factor Pitx2 as a candidate . We show that Pitx2 is essential for axis formation , and that it acts as a direct regulator of cVg1 expression by binding to enhancers within neighbouring genes . Pitx2 , Vg1/GDF1 and Nodal are also key actors in left–right asymmetry , suggesting that the same ancient polarity determination mechanism has been co-opted to different functions during evolution .
In warm-blooded animals , including chickens and humans , a single embryo can give rise to several separate individuals ( identical twins ) . Some species of armadillos routinely give birth to quadruplets in this way—and in experiments , up to eight identical chick embryos can be produced by cutting one embryo into smaller pieces ( a type of ‘experimental twinning’ ) . This ability of a developing embryo to subdivide into separate individuals ends when the embryo starts to form its first midline structure , called the ‘primitive streak’ . This is the first line of symmetry and defines where the head–tail axis will later develop . The steps that establish the axes of the embryo in birds and mammals , and the factors that prevent further splitting of the embryo to form twins after this point , are only just beginning to be understood . In chick embryos , the production of a protein called cVg1 is the first known step and precedes the development of a line of symmetry . A similar protein is produced in mammalian embryos and both proteins are members of an important family of signalling proteins . Now , Torlopp , Khan et al . have used a combination of techniques to search for other proteins that that control the production of the cVg1 protein . Genes that are active in the region of the embryo that will express cVg1 later in development were identified , both in normal embryos and during the process of experimental twinning . This search revealed Pitx2 as a protein that acts to switch on the expression of the gene that encodes cVg1 . When the Pitx2 protein is removed , the embryonic axis forms from the opposite side . Next , Torlopp , Khan et al . searched the chicken genome to identify stretches of DNA around the cVg1 gene where proteins that regulate gene expression might bind . Six potential sites were found , including four to which Pitx2 can bind . Further experiments confirmed that two of these regulatory sequences encourage the expression of the cVg1 gene at its correct position in the embryo . Pitx2 and related proteins were known to be involved with the development of left–right symmetry later in development; the findings of Torlopp , Khan et al . reveal , unexpectedly , that these proteins are also involved in first establishing the position at which the midline of the embryo will arise . It remains unclear what prevents most embryos from forming twins . But Torlopp , Khan et al . 's findings could help to explain some strange observations , made long ago , about left–right asymmetry in identical twins . For example , they could help explain why one of the twins in an identical twin pair is more likely to be left-handed than an individual in the general population , and why the direction of whorls of hair on the back of the head is often mirrored between identical twins .