Maternally provided LSD1/KDM1A enables the maternal-to-zygotic transition and prevents defects that manifest postnatally
At fertilization , the epigenome of the developing zygote undergoes widespread changes in DNA methylation and histone methylation ( Aoki et al . , 1997; Arico et al . , 2011; Bultman et al . , 2006; Burton and Torres-Padilla , 2010; Lindeman et al . , 2011; Smith et al . , 2012; Vastenhouw et al . , 2010; Wu et al . , 2011 ) . This reprogramming is driven by the deposition of maternal proteins and RNA into the zygote ( Tadros and Lipshitz , 2009 ) . After fertilization , zygotic genes become transcriptionally active , while the maternal transcriptional program is silenced ( Tadros and Lipshitz , 2009 ) . In mice , this maternal-to-zygotic transition ( MZT ) occurs between the one- and two-cell ( 1-2C ) stage ( Aoki et al . , 1997; Hamatani et al . , 2004; Xue et al . , 2013 ) . Somatic cell nuclear transfer ( SCNT ) experiments in Xenopus demonstrated that the oocyte has the capacity to reprogram a differentiated somatic nucleus into a cloned embryo ( Gurdon et al . , 1958 ) . This epigenetic reprogramming capacity of the oocyte is also conserved in mammals ( Campbell et al . , 1996 ) . It is thought that the epigenetic reprogramming capacity of the oocyte enables the MZT . Nevertheless , the enzymes involved in maternal epigenetic reprogramming , and the consequences of failure to reprogram , largely remain a mystery . Recently , work in C . elegans implicated LSD1/SPR-5/KDM1A ( previously referred to as LSD1 , hereafter referred to as KDM1A ) in maternal reprogramming at fertilization . Di-methylation of lysine 4 on histone H3 ( H3K4me2 ) is acquired in gamete genes during the specification and maintenance of the germline . The H3K4me2 demethylase KDM1A is maternally deposited into the zygote at fertilization and required to prevent H3K4me2 from being inherited transgenerationally . Without KDM1A , H3K4me2 accumulates at gamete genes across generations and results in the inappropriate expression of these genes . This correlates with increasing sterility in the population over time ( Katz et al . , 2009 ) . The requirement to reprogram H3K4 methylation is also suggested to be a critical step in SCNT . During SCNT in Xenopus , the cloned embryos often inappropriately express genes from the tissue where the somatic nucleus was derived ( Ng and Gurdon , 2005 ) . This transcriptional memory can be eliminated by overexpressing histone H3 with a mutated K4 residue ( Ng and Gurdon , 2008 ) . This work implies that H3K4 methylation may be the carrier of transgenerational transcriptional memory , and that complete reprogramming may require the removal of this modification . Genomic imprinting is the monoallelic expression of a small number of genes based on their parent of origin ( Bartolomei and Tilghman , 1997 ) . Imprinted genes are dependent upon DNA methylation at small imprinting control regions ( ICRs ) associated with these loci ( Bartolomei and Tilghman , 1997 ) . At ICRs , CpG methylation is established in the gametes and maintained throughout the development of the offspring ( Bartolomei , 2009 ) . In mammals , there are two amine-oxidase histone demethylases , LSD2/KDM1B ( hereafter referred to as KDM1B ) and KDM1A ( Ciccone et al . , 2009; Shi et al . , 2004 ) . KDM1B is expressed mainly in the oocyte and required for the establishment of maternal imprints at imprinted loci ( Ciccone et al . , 2009; Stewart et al . , 2015 ) . Without KDM1B , embryos derived from these oocytes exhibit a maternal effect embryonic lethality phenotype prior to mid-gestation ( Ciccone et al . , 2009 ) . This demonstrates that , similar to C . elegans , amine oxidase-type histone demethylases can function maternally in mammals . KDM1A is a component of several protein complexes . As part of the CoREST complex , it specifically demethylates H3K4me1/2 , but not H3K4me3 ( Shi et al . , 2004; You et al . , 2001 ) . Alternatively , when associated with the Androgen Receptor complex KDM1A has been shown to demethylate H3K9me2 in vitro ( Metzger et al . , 2005 ) . KDM1A is an essential gene in mammalian development , as homozygous mutants fail to develop properly after implantation and die prior to embryonic day 8 ( e8 ) ( Foster et al . , 2010; Macfarlan et al . , 2011; Wang et al . , 2007 ) . To determine whether KDM1A may also be involved in maternal reprogramming , we conditionally deleted Lsd1/Kdm1a ( previously referred to as Lsd1 , hereafter referred to as Kdm1a ) in mouse oocytes with three different maternal Cre transgenes . Deletion of Kdm1a maternally with either Zp3-Cre or Gdf9-Cre results in embryonic lethality primarily at the 1-2C stage and these embryos fail to undergo the MZT . This suggests that KDM1A plays a conserved role in maternal reprogramming . Surprisingly , deletion of Kdm1a maternally with Vasa-Cre results in an incomplete loss of KDM1A in oocytes . This uncovers a hypomorphic effect in which some surviving animals exhibit long-range developmental defects , including perinatal lethality and behavioral abnormalities . These defects are associated with disruption of the epigenetic landscape , including aberrant DNA methylation and expression at imprinted loci . These results demonstrate that defects in epigenetic reprogramming between generations can lead to abnormalities later in development .
Based on the previously demonstrated maternal role of KDM1A in C . elegans and KDM1B in mice , we first asked if KDM1A is expressed in mouse oocytes ( Ciccone et al . , 2009; Katz et al . , 2009 ) . RNA-seq datasets from ovulated oocytes and 2C-stage embryos suggested abundant KDM1A transcripts in these cells ( Macfarlan et al . , 2012 ) . Immunofluorescence ( IF ) and immunohistochemistry ( IHC ) with an antibody raised against KDM1A confirms that KDM1A is expressed in the oocyte nucleus and in the surrounding follicle cells throughout oocyte development ( Figure 1A , Figure 1—figure supplement 1A–L ) . Therefore , to determine if KDM1A functions in oocytes , we conditionally deleted Kdm1a by crossing Kdm1afl/fl mice ( Wang et al . , 2007 ) to three different maternal Cre transgenic lines , Ddx4/Vasa-Cre ( Gallardo et al . , 2007 ) , Gdf9-Cre ( Lan et al . , 2004 ) and Zp3-Cre ( de Vries et al . , 2000 ) ( oocytes and embryos from Kdm1afl/Δ::Ddx4/VasaCre , Kdm1afl/Δ::Gdf9-Cre , and Kdm1afl/Δ::Zp3-Cre mice will be referred to hereafter as Kdm1aVasa , Kdm1aGdf9 and Kdm1aZp3 respectively ) . Vasa-Cre is expressed in the germline beginning at e18 and induces full deletion by birth ( Figure 1B ) ( Gallardo et al . , 2007 ) . Gdf9-Cre is expressed in oocytes beginning at postnatal day 3 ( P3 ) , including in primordial follicles ( Lan et al . , 2004 ) , while Zp3-Cre is also expressed in oocytes , but beginning at P5 in primary follicles ( Figure 1B ) ( de Vries et al . , 2000 ) . KDM1A IHC and IF demonstrate that deletion of Kdm1a with either Gdf9-Cre or Zp3-Cre results in the complete loss of KDM1A from the oocyte nucleus ( Figure 1C–F , I ) . Crossing the Kdm1afl/fl allele to Vasa-Cre also results in complete maternal deletion , as can be determined by the 100% segregation of the maternally deleted allele to the offspring ( all of offspring from Kdm1aVasa mothers crossed to wild-type fathers result in heterozygous -/+ offspring ) ( Figure 1—figure supplement 2 ) . This demonstrates that , similar to deletion via Gdf9-Cre or Zp3-Cre , crossing the Kdm1afl/fl allele to Vasa-Cre also effectively deletes Kdm1a maternally . However , for reasons that are not clear , deletion of Kdm1a with Vasa-Cre results in a hypomorphic effect , where 33 . 3% of oocytes completely lack KDM1A protein , but 66 . 7% retain a low level of KDM1A ( Figure 1G–I ) . This incomplete effect is surprising because the Vasa-Cre transgene is reported to be expressed earlier in the germline than either Gdf9-Cre or Zp3-Cre ( Figure 1B ) ( Gallardo et al . , 2007; Lan et al . , 2004 ) . It is possible that the low level of KDM1A remaining in some oocytes is due to delayed deletion of Kdm1a , though the reason for this potential delay is unknown . 10 . 7554/eLife . 08848 . 003Figure 1 . Maternal expression and coditional deletion of Kdm1a in mouse oocytes . ( A ) Wild-type mouse oocyte nucleus ( white arrowhead ) and surrounding follicle cells ( white asterisks ) stained with anti-KDM1A ( green ) antibody and DAPI ( red ) . ( B ) Developmental timeline of maternal Cre expression ( Vasa-Cre , Gdf9-Cre and Zp3-Cre transgenes ) and corresponding oogenesis stages . ( C , D ) Immunohistochemistry ( IHC ) with anti-KDM1A ( brown ) antibody and hematoxylin ( blue ) showing KDM1A nuclear expression ( black arrowhead ) and absence of expression ( white arrowheads ) in Kdm1aGdf9 control ( C ) and mutant ( D ) oocytes . ( E , F ) Immunofluorescence ( IF ) with anti-KDM1A ( green ) antibody , phalloidin ( red ) and DAPI ( blue ) showing KDM1A nuclear expression ( black arrowhead ) and absence of expression ( white arrowheads ) in Kdm1aZp3 control ( E ) and mutant ( F ) oocytes . ( G , H ) IHC with anti-KDM1A ( brown ) antibody and hematoxylin ( blue ) showing KDM1A nuclear expression ( black arrowhead ) , absence of expression ( white arrowhead ) and reduced expression ( pink arrowhead ) in Kdm1aVasa control ( G ) and mutant ( H ) oocytes . ( I ) Percentage of oocytes with KDM1A ( green ) , reduced KDM1A ( red ) or no KDM1A blue ) staining in Kdm1aGdf9 and Kdm1aVasa heterozygous control versus mutant oocytes . Scale bars represent 50 μm . n=number of oocytes analyzed with percentages indicated for each category . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 00310 . 7554/eLife . 08848 . 004Figure 1—figure supplement 1 . KDM1A expression in stged oocytes . ( A–L ) Immunohistochemistry ( IHC ) of primordial follicles ( A–C ) , primary follicles ( D–F ) , secondary follicles ( G-I ) and pre-antral and antral follicles ( J–L ) stained with anti-KDM1A ( brown ) antibody and hematoxylin ( blue ) . The oocyte nucleus is indicated with black arrowheads . Scale bars represent 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 00410 . 7554/eLife . 08848 . 005Figure 1—figure supplement 2 . Generation of Kdm1a mutant and control animals . Kdm1a animals were generated by crossing multiple generations of Kdm1afl/fl animals with either Gdf9- , Zp3- , or Vasa-Cre transgenic animals . Blue indicates Mus castaneus control animals . Purple indicates Kdm1a mutant females . Green indicates B6/Cast hybrid control progeny . Red indicates Kdm1a maternal effect progeny ( MEP ) . Orange indicates progeny resulting from intercrossing 2 MEP adult animals . All labelled progeny were used in crosses and assays presented in subsequent figures ( color-coding matches animals used and graphed in each figure ) . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 005 To determine if there is a functional requirement for maternal KDM1A in mice , we crossed Kdm1aVasa , Kdm1aGdf9 and Kdm1aZp3 females to wild-type males to generate heterozygous offspring ( Figure 1—figure supplement 2 ) . In mice , zygotic transcription begins in the 1C embryo just prior to the first cleavage to the 2C stage ( Aoki et al . , 1997; Hamatani et al . , 2004; Xue et al . , 2013 ) . The heterozygous offspring from the maternally deleted mothers have a normal Kdm1a gene on the paternal allele . Thus , crossing maternally deleted mothers to wild-type fathers enables us to isolate the maternal function of KDM1A ( Maternal- , Zygotic+ , hereafter referred to as M-Z+ ) . M-Z+ heterozygous embryos derived from Kdm1aGdf9 mutant mothers are hereafter referred to as Kdm1aGdf9 M-Z+ embryos , while M+Z+ heterozygous embryos derived from littermate control mothers that are Cre minus are hereafter referred to as Kdm1aGdf9 M+Z+ embryos . Kdm1aGdf9 M-Z+ embryos exhibit embryonic arrest at the 1-2C stage ( Figure 2—figure supplement 1A–I ) . Specifically , in control Kdm1aGdf9 M+Z+ embryos at embryonic day 1 . 5 ( e1 . 5 ) , we observe 7% fragmented/degraded embryos , 65% 1-cell embryos and 28% 2-cell embryos ( n=135 , Figure 2—figure supplement 1I ) . In contrast , in Kdm1aGdf9 M-Z+ embryos at e1 . 5 we observe 40% fragmented/degraded embryos , 59% unfertilized oocytes or 1-cell embryos , and only 1% 2C embryos ( n=134 , Figure 2—figure supplement 1I ) . The vast majority of the non-degraded Kdm1aGdf9 M-Z+ embryos are clearly fertilized and arrested at the 1C stage . However , we do occasionally observe unfertilized oocytes . In addition , we sometimes observe embryos that are highly abnormal morphologically and are difficult to clearly assign to a particular category . As a result , we quantified these Kdm1aGdf9 M-Z+ embryos together . Nevertheless , compared to Kdm1aGdf9 M+Z+ embryos , Kdm1aGdf9 M-Z+ embryos have a large increase in the number of fragmented/degraded embryos at the expense of normal 2C embryos ( Figure 2—figure supplement 1I ) . Also , the remaining 1C and 2C Kdm1aGdf9 M-Z+ embryos do not progress beyond the 1-2C stage , as we never observe any later stage embryos even at e2 . 5 ( Figure 2—figure supplement 1C , G , H ) . M-Z+ heterozygous embryos derived from Kdm1aZp3 mothers ( hereafter referred to as Kdm1aZp3 M-Z+embryos ) undergo a phenotype that is similar to Kdm1aGdf9 M-Z+ embryos . At e1 . 5 , 95% ( n=20 ) of control Kdm1aZp3 M+Z+ embryos have reached the 2C stage ( Figure 2E ) . In contrast , at e1 . 5 only 35% ( n=57 ) of Kdm1aZp3 M-Z+ embryos reach the 2C stage ( Figure 2E ) , though this is still much higher than the 1% of Kdm1aGdf9 M-Z+ embryos that reach the 2C stage ( Figure 2—figure supplement 1I ) . It is not clear why there is a difference in the number of Kdm1aZp3 versus Kdm1aGdf9 M-Z+ embryos that cleave to the 2C stage . However , this may be due to subtle differences in developmental timing , strain background , or Cre specific differences . In addition , we have observed two Kdm1aZp3 M-Z+ embryos that develop past the 2C stage to the 3C- and 4C- stage ( Figure 2—figure supplement 1J , K ) , though like the Kdm1aGdf9 M-Z+ embryos , no Kdm1aZp3 M-Z+ embryos survive to the blastocyst stage ( data not shown ) . Taken together , the results from Kdm1aGdf9 and Kdm1aZp3 mice suggest that the embryonic arrest , primarily at the 1-2C stages is the KDM1A maternal loss-of-function phenotype . 10 . 7554/eLife . 08848 . 006Figure 2 . Kdm1aZp3 embryos arrest at the 1–2 cell stage . ( A–D ) Brightfield images of ( A , C ) M+Z+ and ( B , D ) M-Z+ 1- and 2-cell embryos derived from Kdm1aZp3 control and mutant mothers at e1 . 5 . ( E ) Percentage of 1-cell ( green ) and 2-cell ( yellow ) embryos derived from Kdm1aZp3 control and mutant mothers at e1 . 5 . n = 40 for Kdm1aZp3 M+Z+ embryos from 3 litters . n = 57 for Kdm1aZp3 M-Z+ embryos from 6 litters . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 00610 . 7554/eLife . 08848 . 007Figure 2—figure supplement 1 . Lack of normal Kdm1aGdf9 and Kdm1aZp3 embryos at embryonic day 1 . 5 and 2 . 5 . ( A , B , D , E , F ) Brightfield images of embryonic day 1 . 5 ( e1 . 5 ) M+Z+ 1-cell ( A ) and 2-cell ( B ) embryos and M-Z+ 1-cell ( D ) , 2-cell ( E ) , and fragmented ( F ) embryos derived from Kdm1aGdf9 control and mutant mothers . ( C , G , H ) Brightfield images of e2 . 5 M+Z+ 8-cell ( C ) embryo and M-Z+ abnormal 1-cell ( G ) , and fragmented ( H ) embryos derived from Kdm1aGdf9 control and mutant mothers . ( I ) Percentage of fragmented ( purple ) , unfertilized oocyte or 1C ( green ) , and 2C ( yellow ) embryos from Kdm1aGdf9 control and mutant mothers . n = 123 for Kdm1aGdf9 M+Z+ control embryos from 8 litters . n = 104 for Kdm1aGdf9 M-Z+ embryos from 8 litters . ( J ) Brightfield image of 3-cell M-Z+ embryo derived from a Kdm1aZp3 mutant mother . ( K ) Brightfield image of 4-cell M-Z+ embryo derived from a Kdm1aZp3 mutant mother . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 007 To determine the maternal function of KDM1A , we considered two possibilities; ( 1 ) that KDM1A affects transcription in the oocyte , or ( 2 ) that maternal KDM1A affects transcription post-fertilization during the MZT . To determine if KDM1A affects transcription in the oocyte , we first compared the transcriptome of control Kdm1afl/fl ( Macfarlan et al . , 2012 ) versus Kdm1aZp3 mutant oocytes . We found relatively few transcriptional changes , with 195 genes over expressed and 281 genes under expressed in Kdm1aZp3 oocytes ( Figure 3A , Figure 3—source data 1A , Figure 3—figure supplement 5 ) . In addition , amongst the 875 repeat types extracted from the RepeatMasker database ( including LINEs , SINEs , etc . ) there were only 2 repeat families that were activated and 2 repressed in Kdm1aZp3 oocytes ( Figure 3A , Figure 3—source data 1A , Figure 3—figure supplement 5 ) . Manual inspection revealed that few of these genes/repeats had both large fold-changes and low p-values , with the exception of the Mt1 gene , which was very highly activated in Kdm1aZp3 oocytes . Furthermore , we found several mitochondrially encoded genes ( like mtCo3 and Atpase6 ) that had small but significantly reduced levels in Kdm1aZp3 oocytes . These mitochondrial genes are unlikely to be directly regulated by KDM1A , but their reduced levels could indicate a subtle metabolic defect in the mutants . Taken together , these results demonstrate that loss of KDM1A has little effect on transcription in oocytes . 10 . 7554/eLife . 08848 . 008Figure 3 . The MZT is impaired in Kdm1aZp3 mutants . ( A , B ) Differential expression of mRNAs in Kdm1afl/fl versus Kdm1aZp3 oocytes ( A ) or Kdm1afl/fl M+Z+ versus Kdm1aZp3 M-Z+ 2C embryos ( B ) as determined by RNA-seq . Genes/repeats highlighted in red are significant with the number of significant gene/repeats show . GO enrichment using the Up_tissue database was performed on Kdm1afl/fl M+Z+ 2C enriched and Kdm1aZp3 M-Z+ 2C enriched mRNAs , with a list of the most enriched categories displayed . ( C , D ) Differential expression of mRNAs in Kdm1afl/fl M+Z+ 2C embryos versus Kdm1afl/fl oocytes ( C ) or Kdm1aZp3 M-Z+ 2C embryos versus Kdm1afl/fl oocytes ( D ) . The numbers of zygotically activated ( 2C enriched ) genes/repeats and zygotically repressed ( oocyte enriched ) genes/repeats are highlighted in each comparison . ( E ) Hierarchical cluster dendrogram of transcriptomes in Kdm1afl/fl oocytes , Kdm1aZp3 oocytes , Kdm1afl/fl M+Z+ 2C embryos , and Kdm1aZp3 M-Z+ 2C embryos . ( F ) Heat map of gene expression of principal component 1 ( PC1 ) genes in Kdm1afl/fl oocytes , Kdm1afl/fl M+Z+ 2C embryos , and Kdm1aZp3 M-Z+ 2C embryos . The most GO Up_tissue enriched terms are displayed for the 2 categories of PC1 genes . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 00810 . 7554/eLife . 08848 . 009Figure 3—source data 1 . Gene Table comparison of Kdm1afl/fl oocytes and Kdm1aZp3 oocytes . Deseq output for expression of genes used for pairwise comparisons . Column labeled a represents average normalized expression of Kdm1afl/fl control oocytes and b represents average normalized expression of Kdm1aZp3 mutant oocytes . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 00910 . 7554/eLife . 08848 . 010Figure 3—source data 2 . Gene Table comparison of Kdm1aZp3 M+Z+ 2C and Kdm1aZp3 M-Z+ 2C embryos . Deseq output for expression of genes used for pairwise comparisons . Column labeled a represents average normalized expression of Kdm1aZp3 M+Z+ 2C embryos and b represents average normalized expression of Kdm1aZp3 M-Z+ 2C embryos . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 01010 . 7554/eLife . 08848 . 011Figure 3—source data 3 . Gene Table comparison of Kdm1aZp3 M+Z+ 2C and Kdm1afl/fl oocytes . Deseq output for expression of genes used for pairwise comparisons . Column labeled a represents average normalized expression of Kdm1aZp3 M+Z+ 2C embryos and b represents average normalized expression of Kdm1afl/fl control oocytes . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 01110 . 7554/eLife . 08848 . 012Figure 3—source data 4 . Gene Table comparison of Kdm1aZp3 M-Z+ 2C and Kdm1afl/fl oocytes . Deseq output for expression of genes used for pairwise comparisons . Column labeled a represents average normalized expression of Kdm1aZp3 M-Z+ 2C embryos and b represents average normalized expression of Kdm1afl/fl control oocytes . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 01210 . 7554/eLife . 08848 . 013Figure 3—figure supplement 1 . The MZT is impaired in Kdm1aZp3 mutants . ( A–H ) Differential expression of mRNAs in Kdm1afl/fl versus Kdm1aZp3 oocytes ( A , E ) , Kdm1afl/fl M+Z+ versus Kdm1aZp3 M-Z+ 2C embryos ( B , F ) , Kdm1afl/fl M+Z+ 2C embryos versus Kdm1afl/fl oocytes ( C , G ) , and Kdm1aZp3 M-Z+ 2C embryos versus Kdm1afl/fl oocytes ( D , H ) as determined by RNA-seq . Differential expression represented in mean difference plots ( A–D ) and normalized FPKM values on XY scatter plots ( E–H ) . Genes/repeats highlighted in red are significant . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 01310 . 7554/eLife . 08848 . 014Figure 3—figure supplement 2 . Principal component analysis of Kdm1aZp3 2C embryos . ( A ) Principal Component 1 is plotted on x-axis and Principal Component 2 is plotted on y-axis . Variance due to each component for Kdm1aZp3 M-Z+ 2C embryos ( red ) , Kdm1afl/fl M+Z+ 2C embryos ( green ) , and Kdm1afl/fl oocytes ( purple ) are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 01410 . 7554/eLife . 08848 . 015Figure 3—figure supplement 3 . Expression of epigenetic regulators in Kdm1aZp3 2C embryos . Sequenced RNA-seq reads showing relative expression from Kdm1afl/fl M+Z+ 2C embryos and Kdm1aZp3 M-Z+ 2C embryos aligned to the genome for Lsd1/Kdm1a ( A ) , Tet1 ( B ) , Trim28 ( C ) , Zfp57 ( D ) , Dppa3/stella ( E ) , Dnmt1 ( F ) and Uhrf1 ( G ) . Gene tracks visualized using Integrative Genomics Viewer . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 01510 . 7554/eLife . 08848 . 016Figure 3—figure supplement 4 . Relative expression of epigenetic regulators in Kdm1aZp3 2C embryos . Quantitative RT-PCR analysis of epigenetic regulators including Trim28 ( A ) , Zfp57 ( B ) Dppa3/stella ( C ) , and Dnmt1 ( D ) in Kdm1aZp3 M+Z+ 2C embryos compared to Kdm1aZp3 M-Z+ 2C embryos . Y-axis represents average fold change . All gene expression was normalized to Hprt expression . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 01610 . 7554/eLife . 08848 . 017Figure 3—figure supplement 5 . Expression of epigenetic regulators in Kdm1afl/fl and Kdm1aZp3 oocytes . Sequenced RNA-seq reads showing relative expression from Kdm1afl/fl oocytes and Kdm1aZp3 mutant oocytes aligned to the genome for Lsd1/Kdm1a ( A ) , Tet1 ( B ) , Trim28 ( C ) , Zfp57 ( D ) , Dppa3/stella ( E ) , Dnmt1 ( F ) and Uhrf1 ( G ) . Gene tracks visualized using Integrative Genomics Viewer . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 017 Since we did not see a significant affect on oocytes , we considered the possibility that loss of KDM1A affects transcription post-fertilization during the MZT . To determine if this is the case , we isolated fertilized Kdm1aZp3 M-Z+ embryos that cleaved to the 2C stage , and compared them to Kdm1afl/fl M+Z+ 2C embryos ( Macfarlan et al . , 2012 ) . We chose to focus on embryos from Kdm1aZp3 mothers because a substantially higher proportion of these embryos reach the 2C stage . We found a dramatic alteration of the transcriptome , with 1527 genes over expressed and 2794 genes under expressed in Kdm1aZp3 M-Z+ 2C embryos ( Figure 3B , Figure 3—source data 1B ) . In addition , there were 103 repeat families that were repressed in Kdm1aZp3 2C M-Z+ embryos , compared with only 5 that were activated ( Figure 3B , Figure 3—source data 1B ) . These findings are in contrast to what has been observed in Kdm1a mutant ES cells that display a general de-repression of both genes and repeats ( Macfarlan et al . , 2011; Macfarlan et al . , 2012 ) . GO analysis of over and under expressed genes in Kdm1aZp3 2C M-Z+ embryos demonstrated an enrichment of genes normally expressed in unfertilized oocytes , and a reduction in genes associated with embryonic development and tissue specific gene expression . This suggests that Kdm1aZp3 M-Z+ 2C embryos may fail to undergo the MZT ( Figure 3B , Figure 3—source data 1B ) . To test this hypothesis further , we compared the expression of mRNAs from control Kdm1afl/fl M+Z+ 2C embryos ( Macfarlan et al . , 2012 ) or Kdm1aZp3 M-Z+ 2C embryos with Kdm1afl/fl oocytes ( Macfarlan et al . , 2012 ) . If the Kdm1aZp3 M-Z+ 2C embryos fail to undergo the MZT , then we would expect them to be more similar to Kdm1afl/fl oocytes than Kdm1afl/fl M+Z+ 2C embryos . In Kdm1afl/fl M+Z+ 2C embryos relative to Kdm1afl/fl oocytes , there are massive changes in the mRNA profile , with >3 , 000 genes ( and 387 repeat families ) becoming zygotically activated and ~3000 maternal genes ( and 17 repeat families ) which are suppressed . This demonstrates that MZT has occurred ( Figure 3C , Figure 3—source data 1C ) . In contrast , Kdm1aZp3 M-Z+ 2C embryos fail to properly activate the zygotic genome , with only 666 genes becoming activated ( and 110 repeat families ) , and only ~1200 maternal genes repressed ( 13 repeat families ) ( Figure 3D , Figure 3—source data 1D ) . Hierarchical clustering of Kdm1afl/fl oocytes , Kdm1aZp3 oocytes , Kdm1afl/fl M+Z+ 2C embryos and Kdm1aZp3 M-Z+ 2C embryos confirms this failure to activate the zygotic genome . Specifically , though Kdm1afl/fl oocytes are similar to Kdm1aZp3 oocytes , Kdm1aZp3 M-Z+ 2C embryos are more similar to Kdm1afl/fl oocytes than Kdm1afl/fl M+Z+ 2C embryos ( Figure 3E ) . In addition , principal component analysis ( PCA ) demonstrated that ~40% of the variance between Kdm1afl/fl oocytes , Kdm1afl/fl M+Z+ 2C embryos , and Kdm1aZp3 M-Z+ 2C embryos can be explained by a single component ( PC1 , Figure 3—figure supplement 2 ) , and the heat maps generated from PC1 genes confirm that Kdm1aZp3 M-Z+ 2C embryos have expression profiles more similar to Kdm1afl/fl oocytes than Kdm1afl/fl M+Z+ 2C embryos ( Figure 3F ) . GO analysis demonstrated that PC1 genes fall into two categories , those associated with unfertilized oocytes and are highly expressed in oocytes and Kdm1aZp3 M-Z+ 2C embryos , and those associated with early embryonic development which are enriched only in Kdm1afl/fl M+Z+ 2C embryos ( Figure 3F ) . Taken together , these data confirm that the major defect in the transcriptome of Kdm1aZp3 M-Z+ 2C embryos is a failure to undergo the MZT . Deletion of Kdm1a with either Gdf9-Cre or Zp3-Cre results in 100% of oocytes completely lacking KDM1A ( Figure 1C–F ) . In contrast , 66 . 7% of Kdm1aVasa mutant oocytes retain some KDM1A protein , though the amount is much lower than heterozygous control oocytes ( Figure 1G–I ) . To determine if this lower amount of KDM1A retained in some Kdm1aVasa mutant oocytes gives rise to a hypomorphic phenotype , we mated Kdm1aVasa females to wild-type males to generate heterozygous M-Z+ offspring ( Figure 1—figure supplement 2 ) . As is the case with Kdm1aGdf9 and Kdm1aZp3 M-Z+ offspring , these heterozygous M-Z+ offspring ( hereafter referred to as Kdm1aVasa M-Z+ embryos ) have a normal paternal Kdm1a allele . Thus , any phenotypic effects in these Kdm1aVasa heterozygous M-Z+ offspring are due to reduced maternal KDM1A . Similar to Kdm1aGdf9 and Kdm1aZp3 M-Z+ embryos , the majority of Kdm1aVasa M-Z+ embryos arrest prior to the blastocyst stage ( Figure 4A–E ) . In control Kdm1aVasa M+Z+ embryos at e3 . 5 , 55% of embryos have reached the blastocyst stage , 35% are multicellular ( >2C ) and 10% are fragmented/degraded ( n=37 , Figure 4E ) . In contrast , at e3 . 5 85% of the Kdm1aVasa M-Z+ embryos have fragmented/degraded ( n=35 , Figure 4E ) . However , unlike Kdm1aGdf9 and Kdm1aZp3 M-Z+ embryos , which undergo complete embryonic arrest at the 1-2C stage , by e3 . 5 6% of Kdm1aVasa M-Z+ embryos have reached the multicellular stage ( >2C ) and 5% are blastocysts ( n=35 , Figure 4E ) . Remarkably , some of these Kdm1aVasa M-Z+ embryos survive to birth . The average litter size born from Kdm1aVasa mutant mothers is 2 . 3 ( n=20 ) , versus 6 . 2 ( n=32 ) born from littermate control mothers ( Figure 4F ) . However , the vast majority of time vaginal plugged Kdm1aVasa mutant mothers give rise to no viable progeny . Thus , this average litter size is undoubtedly a vast overestimate of the survival of Kdm1aVasa M-Z+ embryos overall . 10 . 7554/eLife . 08848 . 018Figure 4 . Hypomorphic phenotype in Kdm1aVasa progeny . ( A–D ) Brightfield images of M+Z+ . ( A ) and M-Z+ ( B–D ) embryos derived from Kdm1aVasa control and mutant mothers at embryonic day 3 . 5 ( e3 . 5 ) . Panels show blastocysts ( A , B ) , a multicellular embryo ( C ) and a fragmented embryo ( D ) . ( E ) Percentage of fragmented ( purple ) , 1-cell ( green ) , multi-cellular ( blue ) and blastocyst ( yellow ) embryos from Kdm1aVasa control and mutant mothers at e3 . 5 . n = 58 for Kdm1aVasa M+Z+ embryos from 7 litters . n = 79 for Kdm1aVasa M-Z+ embryos from 10 litters . ( F ) Litter sizes of Kdm1aVasa control and mutant mothers . Average litter size for each indicated by red line . Each circle indicates one litter and n=number of litters analyzed . p-values calculated using an unpaired t-test with **** = p<0 . 0001 indicating statistical significance . ( G ) Percentage of newborn pups from Kdm1aVasa heterozygous control and mutant mothers that died perinatally . n = number of litters analyzed . p-values calculated using an unpaired t-test with **** = p<0 . 0001 indicating statistical significance . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 018 The transition from in utero development to postnatal development , during the first 48 hr immediately following birth , is a highly stressful time for mice . If a mouse has a subtle defect during embryogenesis , the newborn pup may die perinatally . This is reflected in the large number of mouse models that exhibit perinatal lethality ( Turgeon and Meloche , 2009 ) . Therefore , to determine if lower maternal KDM1A might give rise to more subtle embryonic defects , we carefully monitored the first 48 hr following birth in Kdm1aVasa M-Z+ progeny . Consistent with this being a major developmental transition , even in genotypically normal mice , we observe that 5% of offspring ( n=216 ) born from littermate Kdm1aVasa control mothers die perinatally during the first 48 hr after birth ( Figure 4G ) . However , in Kdm1aVasa M-Z+ progeny , the percentage of perinatal lethality is significantly increased ( 26% , n=47 ) ( Figure 4G ) . It is unclear why these animals are dying perinatally . However , this increase in perinatal lethality is consistent with these animals having more subtle developmental defects . Of the small number of M-Z+ animals that are born from Kdm1aVasa mutant mothers , nearly 1/3 die perinatally in the first 48 hr after birth . However , the remaining 2/3 survive to adulthood and appear to be morphologically normal . Notably , because of the embryonic arrest and the perinatal lethality , this remaining 2/3 is a very small number of animals ( n=10 from 8 crosses ) . Since progeny from Kdm1aVasa mutant mothers have defects that can lead to perinatal lethality , we considered the possibility that the surviving animals from these Kdm1aVasa mutant mothers ( hereafter referred to as Kdm1aVasa M-Z+ adults ) could have phenotypic defects as well . To analyze these surviving animals , we crossed Kdm1aVasa mutant mothers ( C57BL/6 background ) to M . castaneus ( CAST ) fathers . This enabled us to use polymorphisms to examine subsequent molecular defects in a parent of origin specific fashion . Importantly , F1 hybrid ( B6/CAST ) offspring from Kdm1aVasa mutant mothers have the same hypomorphic phenotype that we observe in a B6 background , where a small number of animals survive until birth ( average litter size=1 . 6 , n=6 litters from 8 crosses ) . Mating the surviving Kdm1aVasa M-Z+ adults produced progeny , suggesting no gross defects in the specification of the germline or mating behavior . However , when we observed these Kdm1aVasa M-Z+ adults closely , we noticed that they exhibit abnormal behaviors ( Figure 5—figure supplement 1A , Video 1 ) . These abnormal behaviors include excessive scratching and digging , along with food harassing , where animals grind up all of the remaining food in the food hopper and incorporate it into the bedding ( Figure 5A–C ) . To quantify the food harassing behavior , we monitored both the weight of the food in the food hopper and the height of the bedding in the cage . Over a 3-day period , each individual Kdm1aVasa M-Z+ adult almost completely depleted the food in the food hopper due to food grinding and harassing ( Figure 5D and Figure 5—figure supplement 1B–D ) . This resulted in a correspondingly large increase in the height of the bedding over a 6-day period ( Figure 5E ) . These effects are not observed in Kdm1aVasa mutant mothers or the M . castaneus animals to which they were mated ( Figure 5D , E and Figure 5—figure supplement 1B , E ) , indicating that the behavior may be due to a maternal effect . To determine if this behavior is due to a maternal effect , we intercrossed the affected Kdm1aVasa M-Z+ adults . If the food-grinding behavior is due to a maternal effect , then mating two affected Kdm1aVasa M-Z+ adults should not produce progeny with abnormal behavior . This is because the Kdm1aVasa M-Z+ adults used as mothers in these crosses have a normal Kdm1a allele ( -/+ ) so the resulting intercrossed progeny ( hereafter referred to as F2 intercrossed M+Z+ adults ) are all M+ . Intercrossing affected M-Z+ F1 hybrids produced no F2 intercrossed M+Z+ adults with the food-grinding behavior , suggesting that the food-grinding behavior is dependent upon a maternal effect ( Figure 5D , E and Figure 5—figure supplement 1D , G ) . Nevertheless , it remains possible that the F1 hybrid background also contributes to the abnormal behavior . To test this possibility , we crossed control littermates of Kdm1aVasa mothers to CAST fathers to generate control B6/CAST hybrids ( hereafter referred to as B6/CAST M+Z+ controls ) . These control hybrids exhibit modest food-grinding behavior , suggesting that the hybrid strain background likely also contributes to the maternal food-grinding defect ( Figure 5D , E and Figure 5—figure supplement 1C , F ) . However , Kdm1aVasa M-Z+ adults are significantly more affected than B6/CAST M+Z+ hybrid controls ( Figure 5D , E and Figure 5—figure supplement 1C , F ) , suggesting that the abnormal food-grinding behavior is predominantly due to a maternal effect . Importantly , we also do not observe any Kdm1a haploinsufficiency defects . For example , the F2 intercrossed M+Z+ adults that are heterozygous for Kdm1a do not exhibit this abnormal behavior ( Figure 5D , E; Figure 5—figure supplement 1D , G ) . This suggests that the abnormal behavior is not due to Kdm1a haploinsufficiency . Finally , behavioral defects were observed in Kdm1aVasa M-Z+ survivors obtained from litters ranging from one to four animals . This suggests that the maternal behavioral abnormalities are not simply due to reduced litter size . 10 . 7554/eLife . 08848 . 019Video 1 . Stereotypical and abnormal behaviors in Kdm1aVasa M-Z+ adults . 0:00-0:27 M . castaneus ( CAST ) control playing behavior . 0:27-0:40 M . castaneus ( CAST ) control eating . 0:40-1:03 ( right ) Kdm1aVasa M-Z+ adult food harassing behavior . 1:03-1:28 ( right ) Kdm1aVasa M-Z+ adult abnormal scratching behavior . 1:28-1:45 ( right ) Kdm1aVasa M-Z+ adult food harassing behavior and ( left ) abnormal digging behavior . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 01910 . 7554/eLife . 08848 . 020Figure 5 . Abnormal behaviors in Kdm1aVasa M-Z+ adults . ( A–C ) Mouse cages at day 0 ( A ) and day 8 ( B ) from M . castaneus ( CAST ) controls compared to day 6 ( C ) from a Kdm1aVasa M-Z+ adult . ( D ) Quantification of change in weight of food in the hopper from CAST controls , B6/CAST hybrid M+Z+ controls , and F2 intercrossed M+Z+ adults versus Kdm1aVasa M-Z+ adults . Data are shown as mean for each day with error bars indicating ± S . E . M . ( E ) Quantification of change in bedding height from CAST controls , B6/CAST hybrid M+Z+ controls , and F2 intercrossed M+Z+ adults versus Kdm1aVasa M-Z+ adults . Data are shown as mean for each day with error bars indicating ± S . E . M . ( F–H ) Mouse cages before ( F ) and after ( G , H ) the marble burying assay was performed on a CAST control ( G ) compared to a Kdm1aVasa M-Z+ adult ( H ) . ( I ) Quantification of the number of marbles buried during the marble burying assay performed on CAST controls , B6/CAST hybrid M+Z+ controls , and F2 intercrossed M+Z+ adults versus Kdm1aVasa M-Z+ adults . Data are shown as quartiles with error bars indicating ± S . E . M . ( J , K ) Open field test performance in CAST controls versus Kdm1aVasa M-Z+ adults scored by number of center crosses ( J ) and time spent in center of cage ( K ) . Data are shown as quartiles with error bars indicating ± S . E . M . p-values calculated using an unpaired t-test with n . s . indicating p>0 . 05 , * = p<0 . 05 , ** = p<0 . 005 , *** = p<0 . 0005 . All asterisks indicate statistical significance . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 02010 . 7554/eLife . 08848 . 021Figure 5—figure supplement 1 . Abnormal behaviors in individual Kdm1aVasa M-Z+ adults . ( A ) Behavioral ethogram of M . castaneus ( CAST ) controls versus Kdm1aVasa M-Z+ adults . ( B ) Quantification of change in weight of food in the hopper of parents of Kdm1aVasa M-Z+ adults and CAST controls versus Kdm1aVasa M-Z+ adults . ( C ) Quantification of change in weight of food in the hopper of B6/CAST M+Z+ controls versus Kdm1aVasa M-Z+ adults . ( D ) Quantification of change in weight of food in the hopper of F2 intercrossed M+Z+ adults versus Kdm1aVasa M-Z+ adults . ( E ) Quantification of change in bedding height of parents of Kdm1aVasa M-Z+ adults and CAST controls versus Kdm1aVasa M-Z+ adults . ( F ) Quantification of change in bedding height of B6/CAST M+Z+ controls versus Kdm1aVasa M-Z+ adults . ( G ) Quantification of change in bedding height of F2 intercrossed M+Z+ adults versus Kdm1aVasa M-Z+ adults . The measurements for each individual animal ( B–D ) and ( E–G ) correspond to the averages shown in Figure 5 ( D , E ) . Yellow arrowheads represent animals heterozygous for Kdm1a . Data shown as mean for each day . p-values calculated using an unpaired t-test with * = p<0 . 05 , *** = p<0 . 0005 , **** = p<0 . 0001 . All asterisks indicate statistical significance . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 021 Observations of Kdm1aVasa M-Z+ adults indicated that these mice also exhibit excessive digging ( Figure 5—figure supplement 1A , Video 1 ) . To quantify this behavior , we assayed Kdm1aVasa M-Z+ adults and controls in a marble-burying assay . In this assay , individual mice are placed in a cage in which 20 marbles are arrayed on top of the bedding . Mice exhibiting excessive digging behavior will bury higher numbers of marbles . For example , Slitrk5 obsessive-compulsive mice bury approximately 50% of the marbles in 30 min , compared to approximately 25% in controls ( Shmelkov et al . , 2010 ) . Kdm1aVasa M-Z+ adults exhibit striking behavior in the marble-burying assay . Compared to M . castaneus controls , which bury 25% in 25 min ( Figure 5G , I , Video 2 ) , Kdm1aVasa M-Z+ adults bury 90% in the same time period ( Figure 5H , I , Video 2 ) . Similar to what was observed in the food-grinding assays , we do not observe this behavior in F2 intercrossed M+Z+ adults ( average 7 marbles , Figure 5I ) , but we do see slightly elevated marble-burying in B6/CAST hybrid controls ( average 10 , Figure 5I ) . Nevertheless , as in the food-grinding assay , Kdm1aVasa M-Z+ adults are significantly more affected than B6/CAST hybrid M+Z+ controls ( Figure 5D , E and Figure 5—figure supplement 1C , F ) . Taken together , these data indicate that the excessive digging behavior is also predominantly due to a maternal effect . Finally , we also performed the open-field test on Kdm1aVasa M-Z+ adults . In this assay , animals are placed in a much larger space than they are accustomed to . Animals that are more anxious or fearful cross the middle of cage fewer times and spend less time overall in the center ( Video 3 ) . Consistent with the behavioral defects that we observe in food-grinding and marble-burying , Kdm1aVasa M-Z+ adults perform significantly fewer movements across the middle of the cage ( Figure 5J ) and spend less time in the center overall than CAST controls ( Figure 5K ) . 10 . 7554/eLife . 08848 . 022Video 2 . Marble burying in Kdm1aVasa adults . 0:00-0:23 Clip 1 of M . castaneus ( CAST ) control in marble burying assay . 0:23-0:44 Clip 2 of M . castaneus ( CAST ) control in marble burying assay . 0:44-1:22 Clip 1 of Kdm1aVasa M-Z+ adult in marble burying assay . 1:22-1:36 Clip 2 of Kdm1aVasa M-Z+ adult in marble burying assay . 1:36-2:13 Clip 3 of Kdm1aVasa M-Z+ adult in marble burying assay . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 02210 . 7554/eLife . 08848 . 023Video 3 . Open field test on Kdm1aVasa M-Z+ adults . 0:00-0:30 M . castaneus control in open field test . 0:30-0:56 Kdm1aVasa M-Z+ adult in open field test . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 023 The perinatal lethality and behavioral defects observed in Kdm1aVasa M-Z+ progeny are remarkable because these animals have a normal Kdm1a allele . Thus the defects that we observe in Kdm1aVasa M-Z+ progeny must be due to a heritable effect originating from the low level of maternal KDM1A . As a result , we sought to determine the nature of this heritable defect . The Kdm1a homolog Kdm1b is expressed maternally in mice , and loss of Kdm1b results in a heritable embryonic lethality defect associated with a failure to maternally acquire DNA methylation at imprinted genes ( Ciccone et al . , 2009; Stewart et al . , 2015 ) . Therefore , we considered the possibility that the heritable defects could be due to DNA methylation defects at these loci , or in maternally methylated imprinted loci unaffected by the loss of KDM1B . In addition , in our RNA-seq data , we observe the misregulation of multiple genes that could potentially affect DNA methylation at imprinted loci . For example , four genes that are known to maternally affect DNA methylation at imprinted loci , Tet1 , Trim28 , Zfp57 and Dppa3/Stella ( Dawlaty et al . , 2013; Li et al . , 2008; Messerschmidt et al . , 2012; Nakamura et al . , 2007; Yamaguchi et al . , 2013 ) , are all misregulated in Kdm1aZp3 M-Z+mutants ( Tet1: -3 . 8 fold , Trim28: -3 . 3 fold , Zfp57: +3 . 2 fold and Dppa3/Stella: + . 58 fold , Figure 3—figure supplement 3; Figure 3—source data 1B , quantitative RT-PCR validation of Trim28 , Zfp57 and Dppa3/Stella in Figure 3—figure supplement 4 ) . Additionally , the DNA methyltransferase DNMT1 is overexpressed in Kdm1aZp3 M-Z+ mutants ( Dnmt1: +2 . 1 fold , Figure 3—figure supplement 3; Figure 3—source data 1B ) . DNMT1 is thought to primarily act as the maintenance DNA methyltransferase ( Li et al . , 1992; Pradhan et al . , 1999 ) , but when overexpressed , DNMT1 has been shown to have de novo DNA methyltransferase activity ( Vertino et al . , 1996 ) . Taken together , these observations raise the possibility that DNA methylation and monoallelic expression at imprinted genes could be altered in Kdm1aVasa M-Z+ progeny , either directly or indirectly . Interestingly , despite the clear loss of KDM1A in Kdm1aZp3 mutant oocytes , the regulatory proteins that are affected by the loss of KDM1A are either not expressed in oocytes ( Tet1 ) or not affected until after fertilization ( Trim28 , Zfp57 , Dppa3/Stella and Dnmt1 , Figure 3—figure supplements 4 , 5 ) . This is consistent with our previous observations that KDM1A primarily affects the MZT . To investigate DNA methylation and the expression of imprinted genes in Kdm1aVasa M-Z+ offspring , we performed bisulfite analysis and qRT-PCR on perinatal lethal Kdm1aVasa M-Z+ pups . We analyzed imprinted that are both maternally ( Zac1 , Impact , Igf2r , Mest , Snrpn ) and paternally methylated ( H19 ) . For these analyses , we took advantage of B6/CAST polymorphisms to determine whether individual alleles in F1 hybrid offspring came from the mother or the father . These F1 hybrid pups , derived from hypomorphic B6 Kdm1aVasa mothers mated to wild-type CAST fathers , are denoted as maternal effect progeny 1 and 2 ( MEP1 and MEP2 ) , and were compared to two stage matched B6/CAST hybrid controls . Two imprinted loci , Zac1 and Impact , are severely affected in Kdm1aVasa offspring . At Zac1 , DNA methylation is inappropriately acquired on the paternal allele in both MEP , though to a slightly lesser extent in MEP2 ( Figure 6A ) . This is associated with significantly less expression of Zac1 in both MEP ( Figure 6B ) . Moreover , Zac1 is normally expressed exclusively from the paternal allele ( Figure 6C ) . However , in MEP1 Zac1 is now expressed exclusively from the maternal allele , while in MEP2 , the slightly lower level of inappropriate paternal methylation is associated with biallelic expression ( Figure 6C ) . At Impact , the overall DNA methylation pattern is unchanged ( Figure 6D ) , but there is a dramatic change in expression . Impact is normally expressed exclusively from the paternal allele , but in both MEP1 and 2 , Impact is now expressed predominantly from the maternal allele ( Figure 6F ) . In addition , in both MEP1 and MEP2 , there is a significant decrease in the overall expression level of Impact ( Figure 6E ) . 10 . 7554/eLife . 08848 . 024Figure 6 . Imprinting defects in Kdm1aVasa progeny . ( A , D , G ) Allele-specific bisulfite analysis of Zac1 ( A ) , Impact ( D ) , and H19 ( G ) . Each line represents the clone of an allele . Each circle represents a CpG dinucleotide where closed circles indicate methylation and open circles indicate no methylation . Maternal and paternal alleles are indicated . ( B , E , H ) Relative expression analysis of Zac1 ( B ) , Impact ( E ) , and H19 ( H ) . Expression normalized to β-actin . Error bars indicate S . E . M . p-values calculated using an unpaired t-test with n . s . indicating p>0 . 05 , * = p<0 . 05 , ** = p<0 . 005 , **** = p<0 . 0001 . All asterisks indicate statistical significance . ( C , F ) Allele-specific expression of Zac1 ( C ) and Impact ( F ) . The polymorphic base is highlighted in yellow . For Zac1 , the maternal allele SNP is T ( red ) in highlighted position and paternal allele SNP is C ( blue ) in electrophoretogram . For Impact , the maternal allele SNP is A ( green ) in highlighted position and paternal allele SNP is G ( black ) in electrophoretogram . All analyses were performed on 2 staged matched B6/CAST hybrid control pups and 2 maternal effect progeny ( MEP ) exhibiting perinatal lethality . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 02410 . 7554/eLife . 08848 . 025Figure 6—figure supplement 1 . Imprinting analysis of Kdm1aVasa progeny . ( A , C , E ) Allele-specific bisulfite analysis of , Igf2r ( A ) , Mest ( C ) , and Snrpn ( E ) . Each line represents the clone of an allele . Each circle represents a CpG dinucleotide where closed circles indicate methylation and open circles indicate no methylation . Maternal and paternal alleles are indicated . ( B , D , F ) Relative expression analysis of Igf2r ( B ) , Mest ( D ) , and Snrpn ( F ) . Expression normalized to β-actin . Error bars indicate ± S . E . M . p-values calculated using an unpaired t-test with n . s . indicating p>0 . 05 , * = p<0 . 05 , ** = p<0 . 005 , **** = p<0 . 0001 . All asterisks indicate statistical significance . All analyses were performed on a stage matched B6/CAST hybrid control pup and 2 maternal effect progeny ( MEP ) exhibiting perinatal lethality . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 025 In addition to the altered imprinting at Zac1 and Impact , we observe a general disruption of several additional imprinted genes . At two of these additional imprinted loci ( H19 and Igf2r ) , DNA methylation is altered ( Figure 6G and Figure 6—figure supplement 1A ) . For example , at H19 DNA methylation is inappropriately lost on the paternal allele in MEP1 ( Figure 6G ) . There is also a slight acquisition of inappropriate methylation on the maternal allele of both MEP ( Figure 6G ) . At two additional imprinted genes ( Mest and Snrpn ) , DNA methylation appears normal ( Figure 6—figure supplement 1C , E ) . Furthermore , at three of the additional imprinted loci , we observe an overall decrease in gene expression . These include Igf2r , Mest , and Snrpn ( Figure 6—figure supplement 1B , D , F ) , while at H19 , expression levels are unaffected ( Figure 6H ) . However , the allele-specific expression of all of these additional imprinted loci remains unaffected ( data not shown ) . Taken together , the changes in DNA methylation and expression indicate a general disruption in the normal regulation of genomic imprinting .
A major question in the field of epigenetics is whether histone methylation is regulated between generations , and whether potential defects in this process can lead to abnormalities in the resulting progeny . To address this question , we asked if the maternal reprogramming function of KDM1A , first demonstrated in C . elegans ( Katz et al . , 2009 ) , is conserved in mammals . As in C . elegans , KDM1A is heavily deposited maternally in the mouse oocyte , suggesting that KDM1A could play a conserved role in maternal reprogramming ( Figure 1 ) . To address this hypothesis directly , we conditionally deleted Kdm1a maternally in mouse oocytes using three separate Cre drivers . Deletion of Kdm1a in developing oocytes via Gdf9-Cre or Zp3-Cre completely eliminates maternal KDM1A and results in embryonic arrest prior to the blastocyst stage , with the vast majority of embryos never developing past the 2C stage ( Figures 1 , 2 ) . This result is also observed when a second conditional Kdm1a allele ( Zhu et al . , 2014 ) is deleted with Zp3-Cre in the accompanying paper by Ancelin et al . . Thus , maternal KDM1A is essential for the progression of mammalian embryogenesis . The observed 1-2C embryonic arrest does not rule out an additional role for KDM1A in oocyte maturation , as we observe that a small fraction of KDM1A deficient oocytes are unfertilized , fertilized with multiple sperm , or display other gross morphological defects , including loss of the zona pellucida . These occasional defects are also observed in the accompanying paper by Ancelin et al . Consistent with this , Kim et al . have recently reported that deletion of a different conditional Kdm1a allele with Zp3-Cre results in defects in meiotic progression ( Kim et al . , 2015 ) . However , in contrast to our results , Kim et al . find that most oocytes are affected and are not fertilized ( Kim et al . , 2015 ) . It is not entirely clear why the phenotypes are slightly different , but it could reflect subtle differences in the timing of deletion due to the use of different floxed alleles , or differences in strain background . Nevertheless , in Kdm1aGdf9 and Kdm1aZp3 mutants , we find that the majority of oocytes are fertilized normally , and many embryos undergo cleavage to reach the 2C stage ( Figure 2 ) . Also , in hypomorphic Kdm1aVasa animals we observe defects on the paternal allele of imprinted loci ( Figure 6 ) . These results suggest a requirement for KDM1A post-fertilization in the zygote . In mammals , zygotic transcription initiates between the one and two cell stage ( Aoki et al . , 1997; Hamatani et al . , 2004; Xue et al . , 2013 ) . As a result , the heterozygous offspring derived from our crosses likely begin to express KDM1A from paternal allele at the 2C stage . Taken together , these results pinpoint a requirement for maternal KDM1A reprogramming between fertilization and cleavage . Consistent with this conclusion , the accompanying paper by Ancelin et al . demonstrates that chemically inhibiting KDM1A function post-fertilization also results in a 2C arrest . This further suggests a zygotic requirement for KDM1A immediately post-fertilization . When Kdm1a is deleted with Gdf9-Cre , only one embryo reached the 2C stage . In contrast , when Kdm1a is deleted with Zp3-Cre , many embryos cleave to the 2C stage and we even observe rare embryos that reach the 3C and 4C stage ( Figure 2 ) . This slight difference in phenotype is likely due to the fact that the Gdf9-Cre acts earlier than the Zp3-Cre . Nevertheless , the difference in phenotype suggests that KDM1A may act maternally at multiple discrete steps . This conclusion is reinforced by the recent demonstration that loss of KDM1A also impairs meiotic progression ( Kim et al . , 2015 ) . Importantly , the observation that many maternally deleted embryos undergo cleavage , suggests that KDM1A is not just required for cell-cycle progression or cell viability ( Figure 2 ) . This conclusion is consistent with the observation that KDM1A null embryos survive until e8 ( Wang et al . , 2007 ) . Furthermore , the difference in phenotype between maternally deleted embryos and embryonically deleted embryos , which survive to e8 , clearly delineates the maternal requirement for KDM1A from the embryonic requirement . To determine the potential cause of the KDM1A maternal embryonic arrest , we profiled the expression of Kdm1afl/fl oocytes and Kdm1aZp3 M-Z+ embryos that underwent cleavage to the 2C stage . Compared to control 2C embryos that have robustly activated the zygotic transcriptional program , Kdm1aZp3 M-Z+ 2C embryos retain the expression of nearly 2 out of 3 oocyte genes , and fail to activate approximately 4 out of 5 zygotically activated embryonic genes ( Figure 3 ) . This indicates that maternally deleted Kdm1a embryos fail to undergo the MZT . Consistent with this being the primary defect in Kdm1aZp3 mutants , we observe relatively few transcriptional changes in Kdm1afl/fl oocytes ( Figure 3 ) . The requirement for KDM1A in facilitating the MZT is also demonstrated in the accompanying paper by Ancelin et al . , where it is shown that maternal KDM1A is necessary for the multiple waves of zygotic gene activation . Thus , we propose that KDM1A plays a major role in facilitating the progression of embryogenesis by enabling the MZT . Surprisingly , in Kdm1aVasa oocytes , we found an incomplete loss of KDM1A protein , despite the fact that 100% of the progeny inherited the deleted Kdm1a allele ( Figure 1 ) . We have previously observed that strain background differences and/or specific interactions between Cre drivers and certain floxed alleles can lead to different patterns of deletion , often not matching published patterns . Thus , it is possible that Kdm1aVasa oocytes have a small amount of KDM1A remaining because the Vasa-Cre transgene occasionally works much later than previously reported when crossed with the Kdm1a floxed allele . Similar to Kdm1aZp3 and Kdm1aGdf9 M-Z+ embryos , many of the hypomorphic Kdm1aVasa M-Z+ embryos undergo early embryonic arrest ( Figure 4 ) . However , the partial loss of KDM1A maternal reprogramming also uncovers a hypomorphic phenotype where a few embryos survive past the 2C stage until birth ( Figures 4–6 ) . Of these animals that are born , a significantly increased fraction die perinatally ( Figure 4 ) . Furthermore , on the rare occasions that more than one animal was born from Kdm1aVasa mutant mothers , the full litter either all died perinatally or all survived . Remarkably , the perinatal lethal pups are derived from crosses between Kdm1aVasa mothers and wild-type fathers ( M-Z+ ) . This suggests that the increased perinatal lethality is due to hypomorphic maternal KDM1A in the mother’s oocyte . It remains possible that the small Kdm1aVasa M-Z+ litter sizes also contribute to the perinatal lethality , due to maternal neglect . However , many Kdm1aVasa M-Z+ animals that are born in small litters survive to adulthood ( Table 1 ) . This suggests that the perinatal lethality is not simply due to small litter size . 10 . 7554/eLife . 08848 . 026Table 1 . Litter sizes for Kdm1aVasa animals Kdm1aVasa litters for mutant and control mothers indicating number of pups that died and entire litters that died . Kdm1aVasa adult , B6/Cast control , and F2 intercrossed control litter sizes that were assayed for behavioral defects . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 026Kdm1aVasa M+Z+Kdm1aVasa M-Z+B6/Cast M+Z+ controlsKdm1aVasa M-Z+ adultsF2 intercrossed M+Z+ adultsLitter size 18 ( 1 ) 1*727Litter size 27431Litter size 3724Litter size 474Litter size 571*Litter size 654Litter size 791*Litter size 88 ( 1 ) 5Litter size 9112*Litter size 1082*Litter size 115 ( 2 ) 2*Litter size 12104Litter size 1333Litter size 1491*Litter size 1553Litter size 1661*Litter size 1743Litter size 1831*Litter size 19111*Litter size 2082Litter size 212*Litter size 227Litter size 2310Litter size 248Litter size 258Litter size 263 ( 1 ) Litter size 279 ( 1 ) Litter size 2811Litter size 297Litter size 304Litter size 315 ( 1 ) Litter size 321 ( ) indicates pups that died . * indicates entire litter died . It is possible that subtle defects in maternal KDM1A reprogramming could also lead to behavioral abnormalities in the surviving adults . To investigate this possibility , we performed a number of behavioral assays on surviving Kdm1aVasa M-Z+ adult animals . These include monitoring food disappearance and bedding height to measure excessive food grinding , the marble burying assay to measure excessive digging behavior , and the open-field test , to measure anxiety . In all of these assays , we observe a maternal effect on behavior due to hypomorphic maternal KDM1A from Kdm1aVasa mothers ( Figure 5 ) . This suggests that hypomorphic KDM1A in the mother’s oocyte can lead to altered behavior in the resulting adult progeny . To investigate the potential molecular nature of the heritable defects , we analyzed DNA methylation and expression at imprinted loci . Both DNA methylation and expression are altered at imprinted genes in perinatal lethal Kdm1aVasa pups . In particular , we observe a striking disruption in the allele-specific expression of both Zac1 and Impact , along with decreased expression and altered DNA methylation at other imprinted loci ( Figure 6 ) . This suggests that subtle defects in maternal KDM1A can alter the epigenetic landscape in a heritable fashion . Also , though KDM1A and KDM1B may have some functional overlap , the distinct imprinting defects observed in each mutant demonstrate that they have functions that are not redundant ( Ciccone et al . , 2009; Stewart et al . , 2015 ) . Interestingly , both Zac1 and Impact have been associated with nervous system defects , and loss of Zac1 paternally results in perinatal lethality ( Chung et al . , 2011; Kosaki et al . , 2001; Pereira et al . , 2005; Roffe et al . , 2013; Schmidt-Edelkraut et al . , 2014; Varrault et al . , 2006 ) . Thus , it is tempting to speculate that defects in these genes could potentially contribute to the abnormalities that we observe in Kdm1aVasa M-Z+ progeny . Nevertheless , it remains unclear if this is the case . Loss of maternal KDM1A could lead to the observed phenomena ( imprinting defects , perinatal lethality and behavioral abnormalities ) either directly or indirectly . Evidence for an indirect mechanism can be inferred from the misregulation of several genes ( Dnmt1 , Tet1 , Trim28 , Zfp57 , Uhrf1 and Dppa/Stella ) that are known to regulate epigenetic information . For example , Tet1/Tet2 double mutants exhibit a partial perinatal lethality phenotype that is reminiscent of what we observe in Kdm1aVasa progeny ( Dawlaty et al . , 2013 ) . Also , mice that are haploinsufficient for Trim28 display abnormal exploratory behaviors ( Whitelaw et al . , 2010 ) . Alternatively , KDM1A may affect these phenomena by directly regulating histone methylation . For example , the removal of H3K4 methylation may be required to enable the activity of the DNMT3a/3b de novo methyltransferase complex ( Ooi et al . , 2007 ) . Such a model has been proposed to account for the imprinting defects that are observed when KDM1B is maternally deleted ( Ciccone et al . , 2009; Stewart et al . , 2015 ) . Consistent with this model , the accompanying paper by Ancelin et al . demonstrates that loss of KDM1A maternally leads to global changes in both H3K4 and H3K9 methylation following fertilization . However , Stewart et al . recently demonstrated that loss of maternal KDM1A in mice does not affect H3K4me2 , or the acquisition of DNA methylation at imprinted genes ( Stewart et al . , 2015 ) . Moreover , the variable disruption of imprinting that we observe in hypomorphic animals is more consistent with a general disruption of epigenetic information . Thus , we favor a model in which the general disruption of epigenetic reprogramming , along with secondary epigenetic defects due to the misregulation of epigenetic reprogramming enzymes during the MZT , combine to give rise to the phenomena that manifest postnatally in progeny . Currently , we are engineering a hypomorphic maternal Kdm1a allele that can be used to generate larger numbers of hypomorphic animals to further elucidate these mechanisms . In summary , our data highlights three important consequences of the loss of maternal KDM1A: ( 1 ) early embryonic arrest and the failure of embryos to undergo the MZT , ( 2 ) perinatal lethality and altered behavior in rare surviving progeny from hypomorphic mothers , and ( 3 ) a general disruption of genomic imprinting in the hypomorphic progeny ( Figure 7 ) . Although these individual phenotypes may be linked , it is possible that they are separate outcomes that depend on distinct activities of maternal KDM1A during the MZT . Nevertheless , our results establish a novel mammalian paradigm in which altered epigenetic reprogramming by a histone demethylase between generations leads to defects that manifest weeks later , highlighting the potential long-range consequences of epigenetic perturbations in the early mammalian embryo . 10 . 7554/eLife . 08848 . 027Figure 7 . Model . Loss of maternal LSD1 results in defects later in development in wild-type oocytes , after fertilization ( denoted by blue sperm encircling oocyte ) the fertilized egg undergoes the maternal to zygotic transition ( MZT; green to blue/purple ) at the 1–2 cell stage . These M+Z+ embryos proceed normally through development ( indicated by blastocyst , perinatal stage pup , and adult mouse ) . In contrast , when Lsd1 is deleted with either Gdf9- or Zp3-Cre , the resulting Lsd1Gdf9 and Lsd1Zp3 progeny become arrest at the 1–2 cell stage and never undergo the MZT ( green ) . When Lsd1 is deleted with Vasa-Cre , we observe 3 hypomorphic outcomes in resulting Lsd1Vasa progeny: ( 1 ) developmental arrest at the 1–2 cell stage , ( 2 ) perinatal lethality and ( 3 ) abnormal behavior in surviving adult animals . These outcomes are due to reduced LSD1 in the mothers oocyte , suggesting that lowered maternal LSD1 can result in defects much later in development . These long-range outcomes are associated with imprinting defects ( depicted as wild-type versus mutant changes in DNA methylation within the yellow region ) . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 027
The following mouse strains were used: Kdm1a/Lsd1fl/fl MGI: 3711205 ( Wang et al . , 2007 ) , Gdf9-Cre MGI: 3056522 ( Lan et al . , 2004 ) , Ddx4/Vasa-Cre MGI: 3757577 ( Gallardo et al . , 2007 ) , and Zp3-Cre MGI: 2176187 ( de Vries et al . , 2000 ) animals . To generate Kdm1a oocyte conditional knockout mice , Kdm1a/Lsd1fl/fl females were crossed to transgenic Cre males to generate Kdm1a/Lsd1Δ/+ F1 animals with Cre . F1 males were crossed to transgenic Cre females to generate Kdm1a/Lsd1Δ/+ F2 animals with Cre and F2 males were mated to Kdm1a/Lsd1fl/fl females to generate Kdm1a/Lsd1Δ/+ F2 animals with Cre . These maternally deleted Kdm1a/Lsd1Δ/Δ females were then mated to wild-type B6 or M . castaneus males to produce maternal effect progeny . Prior to the initial crosses , Kdm1a/Lsd1fl/fl were mated to C57BL/6 mice for several generations so that the genetic background is mostly B6 . For Gdf9-Cre and Zp3-Cre , Kdm1a/Lsd1Δ/+ F1 animals with Cre were mated directly with Kdm1a/Lsd1fl/fl females . A table is included with the litter sizes of all perinatal lethal animals and all animals assayed in the behavioral assays ( See Table 1 ) . All mouse work was performed under the approved guidelines of the Emory University , NIH and Salk Institute IACUC . Polymorphisms were identified through the UCSC Genome Browser and subsequently verified through sequencing of parental strains and hybrid progeny ( See Table 2 ) . 10 . 7554/eLife . 08848 . 028Table 2 . Allele-specific primers and polymorphisms , each primer and polymorphism used for allele-specific analysis bisulfite analysis and allele-specific expression analysis . DOI: http://dx . doi . org/10 . 7554/eLife . 08848 . 028Gene nameUse ( Bisulfite or qPCR ) Verified SNP ( rs# ) PrimersZac1Allele-Specific Bisulfite AnalysisA-B6/ G-Cast ( rs29364824 ) For- G‍ GGTAGGTAAGTAGTGATAARev- C‍ CTAAAACACCAAAATAACAqPCRT-B6/C-Cast ( rs33583472 ) For- C‍‍A‍TTTGTAGGCATGCCCGTCRev- G‍ TGGTAGCTGCATCTGGGGCTGGAImpactAllele-Specific Bisulfite AnalysisA-B6/G-Cast ( rs31057356 ) For- T‍TGTATAGTTTTGTTTTTATAAGTGRev- A‍ACCTACTCATATAACAATACAACqPCRA-B6/ G-Cast ( rs31052361 ) For- G‍AAGAAAACTGAAGAGGTTGRev- G‍CATAGATGTTGTGGGTGGCH19Allele-Specific Bisulfite AnalysisG-B6/ A-Cast ( verified from Bartolomei Lab ) For- A‍TTTATAAATGGTAATGTTGTGGRev- C‍CTCATAAAACCCATAACTATAAAATCqPCRFor- C‍CACTACACTACCTGCCTCAGAATCTGCRev- G‍GTGGGTACTGGGGCAGCATTGIgf2rAllele-Specific Bisulfite AnalysisG-B6/ A-Cast ( rs107811421 ) For- T‍AGAGGATTTTAGTATAATTTTAARev- T‍AACACTTTTAAATTCATCTCTqPCRFor- C‍TGGAGGTGATGAGTGTAGCTCTGGCRev- G‍AGTGACGAGCCAACACAGACAGGTCMestAllele-Specific Bisulfite AnalysisG-B6/ T-Cast ( rs245841095 ) ForMat- G‍GGTGTTTTATGTTTTTTAGGGT; ForPat- GGGTGTTTTATGTTTTTTAGGGGRev- C‍CCAAATTCTAATAAAAAAAACCTTCCCATqPCRFor- G‍CTGGGGAAGTAGCTCAGTRev- TTTCTTCTTAGCAAGGGCCASnrpnAllele-Specific Bisulfite AnalysisA-B6/T-Cast ( rs50790468 ) ForMat- GTA‍ATTATATTTATTATTTTAGATTGATAGTGAT; ForPat- GTAATT‍ ATATTTATTATTTTAGATTGATAGTGAGRev- A‍TAAAATACACTTTCACTACTAAAATCCqPCRFor- TGC‍ TCGTGTTGCTGCTACTGRev- GC‍AGTAAGAGGGGTCAAAAGCβ-actinqPCRFor- G‍ TGACGAGGCCCAGAGCAAGAGRev- C‍ GTACATGGCTGGGGTGTTGAAGG To establish phenotypes in KDM1A maternal mutants , timed matings were set up between control/mutant females and wild-type males . Superovulation was found to have little affect on phenotypes , so we used superovulation to collect enough embryos for RNA-seq . For super-ovulation , PMSG was injected into sexually mature females on day 1 . After 48 hr , females were injected with HcG and subsequently housed with stud males . Confirmation of natural or superovulatory matings on subsequent days was made via observation of a copulation plug . 1–2 cell embryos were flushed from ovarian tract at embryonic day 1 . 5 ( e1 . 5 ) , morulae on e2 . 5 and blastocysts on e3 . 5 . Flushed embryos were categorized and imaged . Isolated mouse ovaries were fixed for 1 hr in 4% PFA on ice then washed with PBS multiple times over a 2 hr period . Ovaries were allowed to sit in 30% sucrose solution at 4°C overnight and were then embedded in OCT compound . 10 micron cryosections were obtained for analysis . Immunostaining was performed using rabbit polyclonal anti-KDM1A ( 1:200 , Abcam , Cambridge UK , ab17721 ) and Alexa fluor conjugated secondary antibodies . Isolated mouse ovaries were fixed overnight in 4% PFA . Ovaries were then dehydrated in the following series of steps: 70% ethanol for 20 min 3 times; 85% ethanol for 45 min 2 times; 95% ethanol for 1 hr; 100% for 1 hr; xylenes overnight; xylenes:paraffin mix for 2 hr twice under vacuum; paraffin for 4 hr under vacuum; and paraffin under vacuum overnight . Oocytes were embedded in paraffin and 10 micron sections were taken for analysis . Immunostaining was performed using rabbit polyclonal anti-KDM1A ( 1:500 , Abcam , ab17721 ) . Oocytes were scored for presence of KDM1A signal with qualitative comparison to wild-type oocyte signal . DNA was isolated from sagittal sections of each perinatal pup . Bisulfite conversion was performed according to the Zymo ( Irvine , CA ) EZ DNA Methylation Kit protocol . Primer sets and polymorphisms used are listed in Table 1 . The BiQ Analyzer program was used in the analysis of bisulfite converted sequences ( Bock et al . , 2005 ) . RNA was isolated from sagittal sections of each perinatal pup using Trizol . SuperScript® III first-strand synthesis system was used to generate cDNA . The following cycling conditions were used: 95°C for 3 min , 95°C for 15 sec , 60°C for 30 sec , 72°C for 30 sec , 50 cycles . RNA-seq on KDM1A mutant and wild-type 2C embryos was performed as described ( Macfarlan et al . , 2012 ) . Briefly embryos were lysed in Prelude Direct Lysis buffer ( Nugen , San Carlos , CA ) and amplified cDNAs were prepared using the Ovation RNA-seq system V2 ( Nugen , 7102-32 ) . Paired end libraries were prepared according to the Tru-seq library construction protocol starting with Covaris fragmentation step . Libraries were then sequenced on an Illumina Hi-seq 2000 . For expression analysis , only first mate pair was used and reads were trimmed from 3' end to 50 bp . Trimmed reads were filtered to a minimum average base quality of 15 . We combined the knownGene , ensGene and refGene annotations for mm10 ( downloaded from UCSC Genome Browser ) with the full RepeatMasker annotation , also from UCSC Genome Browser , to build a single gene annotation . Redundant transcripts were filtered out using the gffread utility packaged with Cufflinks . The annotation was modified to include a common gene_id value for same-name repeat elements resulting in a total of 875 repeats . Sequences for all annotated features were extracted from the mouse genome and RNA-Seq reads were aligned to them using BBMap with 95% identity , up to a single INDEL and up to 2000 equally best alignments per read . Alignments to features were quantified by counting hits to gene loci and down-weighting read alignments that mapped to multiple gene loci by 1/N2 where N is equal to the number of gene loci . Reads aligned to repeat elements were counted in the same way but instead of using gene loci the repeat name was used for binning hits . If a read mapped to multiple repeat features all of the same name then it was counted as 1 hit to that repeat name . Raw read counts were loaded into R and median normalized for differential expression analysis . For each pair-wise test genes with raw counts less than 10 in all conditions were not tested . Differential expression was performed by testing the null hypothesis that a gene's fold change between conditions is zero . The observed dispersion-mean relationship was fit using a method similar to that of DESeq and the predicted dispersions were used as the minimum dispersion per gene . Fold-changes between conditions were tested by monte-carlo simulation using the observed and estimated means and dispersions to generate random negative binomial distributed count values ( the rnbinom method in R ) . In each monte-carlo iteration N samples were simulated per condition ( N = to the number of samples in each condition ) , averaged into conditions and the log fold-change was calculated . The resulting simulated log fold-change distributions are normally distributed . Z- scores were computed for each observed log fold-change compared to its corresponding monte-carlo simulated fold-change distribution and translated to two-tail p-values against the normal probability distribution . Raw p-values were adjusted using the Benjamini & Hochberg correction . Genes and repeats were tested together and marked significant if the adjusted p-value was less than 0 . 05 . RNA-seq data is deposited at GEO ( GSE66547 ) . Oocyte expression data used for comparisons was previously deposited at GEO ( GSE33923 . ) For the heat map plot , each gene found to be significantly associated with PC1 is standardized relative to its mean and scaled relative to its variance ( ie each gene is scaled separately to improve visualization . ) Each row is a gene which is standardized where mean is zero with standard deviation is 1 . This makes each row relative to its mean . The scale is relative to its variance . Rows are sorted by a clustering of the genes . Color scale is relative to mean expression level per gene . Each animal was placed in a mouse housing unit ( 32 . 8 × 18 . 6 × 13 . 6 cm ) with 5/8 inches of bedding . Over an 6 day period , the height of the bedding was measured in inches . In addition , 400 grams of standard mouse diet pellets was placed in each food hopper . The amount of food remaining in the food hopper was weighed each day over a 3 day period . A clean , transparent plastic cage ( 32 . 8 × 18 . 6 × 13 . 6 cm ) was filled 4 . 5 cm deep with corncob bedding material . 20 glass marbles ( 20 mm diameter ) were evenly spaced in a 5 × 4 grid on the surface of the bedding . During the testing phase , the mice were placed in the cage for 25 min and allowed to explore . At the end of the testing phase , mice were removed from the cage and the number of marbles that were buried 2/3 their height in the bedding were counted . Mice were placed in a clean , transparent plastic cage ( 55 . 5 × 32 . 5 × 19 . 5 cm ) with grid lines marked on bottom of cage . The center area was clearly marked for analysis ( 27 × 16 . 2 cm ) . After 10 min , video was scored for number of times center area was crossed and amount of time spent in center area .
Somatic cell nuclear transfer has established that the oocyte contains maternal factors with epigenetic reprogramming capacity . Yet the identity and function of these maternal factors during the gamete to embryo transition remains poorly understood . In C . elegans , LSD1/KDM1A enables this transition by removing H3K4me2 and preventing the transgenerational inheritance of transcription patterns . Here we show that loss of maternal LSD1/KDM1A in mice results in embryonic arrest at the 1-2 cell stage , with arrested embryos failing to undergo the maternal-to-zygotic transition . This suggests that LSD1/KDM1A maternal reprogramming is conserved . Moreover , partial loss of maternal LSD1/KDM1A results in striking phenotypes weeks after fertilization; including perinatal lethality and abnormal behavior in surviving adults . These maternal effect hypomorphic phenotypes are associated with alterations in DNA methylation and expression at imprinted genes . These results establish a novel mammalian paradigm where defects in early epigenetic reprogramming can lead to defects that manifest later in development .
During fertilization , an egg cell and a sperm cell combine to make a cell called a zygote that then divides many times to form an embryo . Many of the characteristics of the embryo are determined by the genes it inherits from its parents . However , not all of these genes should be “expressed” to produce their products all of the time . One way of controlling gene expression is to add a chemical group called a methyl tag to the DNA near the gene , or to one of the histone proteins that DNA wraps around . Soon after fertilization , a process called reprogramming occurs that begins with the removal of most of the methyl tags a zygote inherited from the egg and sperm cells . The zygote’s DNA is then newly methylated to activate a new pattern of gene expression . In mammals , some genes escape this reprogramming; these “imprinted” genes retain the methylation patterns inherited from the parents . Reprogramming is assisted by “maternal factors” that are inherited from the egg cell . Once reprogramming is completed , the maternal factors are destroyed as part of a process called the maternal-to-zygotic transition . A maternal factor called KDM1A can remove specific methyl tags from certain histone proteins , but how this affects the zygote is not well understood . Now , Wasson et al . ( and independently Ancelin et al . ) have investigated the role that KDM1A plays in mouse development . Wasson et al . genetically engineered mouse egg cells to contain little or no KDM1A . Zygotes created from egg cells that completely lack KDM1A die before or shortly after they have divided for the first time and fail to undergo the maternal-to-zygotic transition . Other egg cells that contain low levels of KDM1A can give rise to baby mice . However , many of these mice die soon after birth , and those that grow to adulthood behave in abnormal ways; for example , they display excessive chewing and digging . These disorders are linked to the disruption of DNA methylation at imprinted genes . The next challenge will be to further investigate the mechanisms by which defects in maternally deposited KDM1A exert their long-range effects on imprinted genes and altered behaviour . This is particularly important because of the recent discovery of three patients with birth defects that are linked to genetic variants in KDM1A .