B1 SOX Coordinate Cell Specification with Patterning and Morphogenesis in the Early Zebrafish Embryo
[['The developing embryo must control gene expression to coordinate various embryonic processes such as cell fate specification , embryo patterning and morphogenesis .', 'During the embryonic stages from the blastula to neurula , the coupling of cell lineage specification and gastrulation cell movements is particularly evident .', 'There is also now an increased understanding of the regulatory mechanisms underlying each cell state and each morphogenetic process , but the precise mechanisms that coordinate these events have remained elusive .', 'The group B1 SOX transcription factors are good candidates as coordinators of these embryonic processes .', 'Indeed , they have been implicated in cell fate specification in the early embryo [1]–[7] and also patterning and morphogenetic processes [8]–[10] .', 'B1 Sox comprises sox1a/1b/2/3/19a/19b in zebrafish and Sox1/2/3 in amniotes [11] .', 'The sox19a/19b genes are evolutionary orthologs of mammalian Sox15 ( group G ) , although Sox15 has now been shown to have functionally diversified from the authentic B1 Sox paralogs [11] .', 'Overall , the regulatory functions of B1 sox genes appear to be conserved as a group across vertebrate species , although the paralogs are often differentially employed in a particular process [12] .', 'In zebrafish , sox3/19a/19b are expressed in the blastula [11] , whereas the corresponding early expression in mice is covered by Sox2 [1] .', 'Following this stage , the B1 sox genes are thought to be important for specification of the embryonic ectoderm into the neuroectoderm lineage .', 'During this process , their expression becomes confined to the neuroectoderm [11] .', 'As development proceeds to the neurula stage , expression of the B1 sox genes continues in neural precursors , where they function to maintain the neural progenitor states [13]–[15] .', 'The similarities in the characteristics of the B1 SOX proteins as transcriptional regulators [11] , [15] suggest redundant functions in tissues where they are coexpressed .', 'In support of this notion , single Sox1 or Sox3 knockout mice display only mild abnormalities in the central nervous system ( CNS ) , presumably because of extensive coexpression of Sox1/2/3 [16]–[18] , whereas Sox2-null mouse embryos die around implantation , reflecting its exclusive expression in the ICM [1] .', 'Consistently , a single sox2 or sox3 knockdown ( KD ) in zebrafish causes only mild developmental abnormalities [19] , [20] .', 'Xenopus studies utilizing dominant-negative forms of SOX2 indicate a specific role of Sox2 in neuroectoderm differentiation [2] .', 'To date , however , the overall functions of the B1 sox genes have not been systematically investigated from the blastula to early neurula stages .', 'An important characteristic of the B1 SOX proteins is that they form a complex with co-DNA-binding partner factors to target specific sequences and this enables them to participate in the regulation of various cell states [21] .', 'The SOX2-Oct3/4 ( Pou5f1 ) complex is a central player in regulatory networks in the ICM and ES cells [1] , [22] , [23] .', 'Potential target genes of SOX2 and Oct3/4 in ES cells have been identified through genome-wide chromatin immunoprecipitation ( ChIP ) and microarray expression analyses [22] , [24] .', 'The involvement of other B1 SOX-partner combinations in the regulation of specific cell states has also been reported , e . g . , B1 SOX-POUIII factors in neural precursors [15] and B1 SOX-Pax6 in lens cells [25] .', 'However , neither B1 SOX-dependent regulatory processes nor B1 SOX target genes in the developing early embryo have been extensively investigated .', 'In our present study , we performed single to quadruple knockdowns of sox2/3/19a/19b in zebrafish embryos and confirmed that these four genes are functionally redundant in early development .', 'More importantly , phenotypic analyses of the sox2/3/19a/19b quadruple KD embryos uncovered developmental process-specific functions of B1 sox .', 'In the blastula , B1 sox genes regulate the activation of the bmp2b/7 genes , which is critical for dorsoventral ( DV ) patterning .', 'During gastrulation , B1 sox also regulate the expression of pcdh18a/18b and wnt11 , a non-canonical Wnt ligand gene , which together play a role in convergence and extension ( C&E ) movements .', 'In neural development , the B1 sox genes are essential for the proper regulation of neural bHLH genes of both the her/Hes and proneural classes , and also for the activation of region-specific transcription factor genes such as hesx1 , zic1 and rx3 .', 'Moreover , the activity of B1 sox is required for the neural expression of various signaling pathway genes: cyp26a1 in RA signaling , oep in Nodal signaling , shh , and also mdkb .', 'ChIP analysis of the her3 , hesx1 , neurog1 , pcdh18a and cyp26a1 genes suggests their direct regulation by B1 SOX .', 'These findings indicate that B1 SOX proteins play a central role in coordinating cell fate specification , embryo patterning and morphogenesis by controlling a wide variety of developmental regulators in the early embryo .', 'We have also found an interesting overlap between the early phenotypes of the B1 sox quadruple KD embryos and the maternal-zygotic ( MZ ) spg embryos that are devoid of pou5f1 activity [26]–[29] .', 'This highlights a broad role of the B1 SOX-Pou5f1 complex from the blastoderm to early neural stages of development .'], ['Among the B1 sox genes of zebrafish , sox2/3/19a/19b are expressed at high levels during early development with extensive regional overlaps [11] .', 'sox19b mRNA is maternally supplied .', 'sox3 and sox19a are activated around the 1000-cell stage , and sox2 around the 30% epiboly ( 30%E ) stage [11] .', 'The expression of sox3/19a/19b initially covers the entire blastoderm , but gradually disappears at the embryonic margin after 30%E ( Figure S1A ) .', 'At the shield stage , the expression of sox2/3/19a/19b covers the future ectoderm , but then becomes confined to the presumptive neuroectoderm [11] .', 'Expression of sox1a/1b is initiated only during late gastrulation stages ( Figure S1B ) .', 'These expression patterns suggest that sox2/3/19a/19b are involved in early processes of zebrafish development .', 'To investigate the function of B1 sox in early stage embryos , we knocked down sox2/3/19a/19b either individually or in combination using morpholino antisense oligonucleotides ( MO ) .', 'Two different MOs were simultaneously used to block translation of each B1 sox gene , which ensures efficient knockdown even when using reduced amounts of MOs [20] .', 'With this double MO strategy , an approximately 90% reduction in translation was achieved using 1 . 8 ng of a 1∶1 mixture of two MOs , as judged by their effects on luciferase reporters carrying MO-targeting 5′-UTR sequences ( sox2 [20]; sox3/19a/19b , Figure S2A ) .', 'By western blotting , we confirmed the efficient inhibition of the synthesis of endogenous B1 SOX proteins ( Figure S2B ) .', 'No gross abnormalities were observed in the embryo morphology when any one of sox2/3/19a/19b was knocked down ( single KD , Figure 1B ) , although the development of the CNS may be slightly perturbed and 75% of the sox2 morphants showed an up-turned tail phenotype ( Figure 1Ba ) .', 'When any three of sox2/3/19a/19b were simultaneously knocked down ( triple KD ) , a range of morphological abnormalities was observed depending on the combination of KD targets ( Figure 1C ) .', 'Triple KDs of sox2/19a/19b and sox2/3/19b caused only mild morphological defects ( Figure 1Cc and 1Cd ) , presumably because the remaining sox3 and sox19a genes , respectively , mostly cover the B1 sox expression domains .', 'sox3/19a/19b morphants often showed stronger yet variable defects in their posterior structures ( Figure 1Ca and 1Cb ) , presumably reflecting the weak sox2 expression in the posterior neuroectoderm .', 'sox2/3/19a morphants appeared normal during gastrulation , but later developed morphological abnormalities ( Figure 1Ce ) , likely because sox19b expression decreases in later stages .', 'In contrast to the triple KDs , the quadruple knockdown of sox2/3/19a/19b ( hereafter called QKD ) using a total of 7 . 2 ng MOs resulted in very severe developmental abnormalities , suggesting essential functions of B1 sox in early embryogenesis ( Figure 1Da–1Df ) .', 'This result also lends support to a model whereby in the triple KD embryos the remaining B1 sox gene compensates for the loss of the other three to a large extent .', 'Taken together , our initial observations indicate that the B1 sox genes are largely functionally redundant in early zebrafish embryos .', 'This is further corroborated by rescue experiments to be described in the next section .', 'By reducing the amount of MOs used for QKD , hypomorphic phenotypes were produced to different extents depending on the MO levels ( Figure 1Dg and 1Dh ) .', 'Embryos injected with a 50% concentration of MOs for QKD ( 3 . 6 ng in total ) still showed more severe abnormalities than any of the triple KD embryos .', 'However , injection of only 25% MOs for QKD ( 1 . 8 ng in total ) resulted in phenotypes similar to the triple KD embryos in terms of severity , further demonstrating the dose-dependent knockdown effects .', 'The specificity of the MOs we used was confirmed by injection of 5-base mismatch MOs , which caused no developmental abnormalities ( Figure 1Di ) .', 'In addition , the observed QKD phenotypes were not altered by coinjection of p53-MO ( Figure 1Dj ) , which relieves MO-induced non-specific neural cell death [30] .', 'Efficient elimination of B1 SOX activity under this QKD condition was also confirmed by the loss of nestin enhancer activity ( Figure S3 ) , which is regulated by B1 SOX and POU [15] .', 'The earliest detectable morphological abnormality of the QKD embryos was a delay in epiboly , notably after the shield stage ( Figure 1D ) .', 'At 10 hour post-fertilization ( hpf ) , when normal embryos reach the tailbud stage , the QKD embryos were still in late epiboly .', 'The thickening of the anterior head region was less prominent in the QKD embryos ( Figure 1Dc and 1Dd ) , suggesting impairment of CNS development .', 'Impaired CNS development was also indicated by the loss of hesx1 expression in the anterior-most neuroectoderm ( Figure 2Bd; see also Figure 3Ca ) and by the anterolateral displacement of the pax2a expression domains that mark the midbrain-hindbrain boundary ( MHB ) ( Figure 2Bd ) .', 'An early phase of neurogenesis was also affected in the QKD embryos as indicated by the loss of proneural neurog1 expression ( Figure 2Bf ) .', 'The QKD embryos further displayed a shortened anterior-posterior ( AP ) axis with a broadened neural plate ( marked by hoxb1b ) and broadened mesodermal structures including notochord ( marked by ntl ) ( Figure 1D and Figure 2B ) .', 'Consistently , the gap between the prechordal plate ( marked by hgg1 ) and notochord was reduced in the QKD embryos ( Figure 2Be ) .', 'These abnormalities commonly occur in zebrafish embryos when C&E movements are impaired during gastrulation [31] , [32] .', 'The effects of the B1 sox KDs suggested that the B1 SOX proteins act equivalently in transcriptional regulation in the zebrafish embryo .', 'Indeed , SOX1/2/3/19 all activate the nestin and δ-crystallin enhancers in cooperation with Brn2 and Pax6 , respectively , in cultured cells [11] .', 'We therefore tested whether injection of a single B1 sox mRNA could rescue the QKD phenotype .', 'Moderate amounts ( 20–30 pg ) of B1 sox mRNAs lacking the MO-target 5′ UTR sequences were individually injected with the MOs for QKD .', 'Coinjection with any one of the sox2/3/19a/19b mRNAs dramatically rescued the QKD phenotype , as judged by the recovery of a normal morphology ( Figure 2Ca–2Cc ) .', 'By measuring the AP axis length of the embryos at 15–16 hpf , we confirmed the recovery of axial elongation in the B1 sox mRNA-injected QKD embryos ( Figure 2D ) .', 'In these embryos also , the expression of hesx1 , dlx3b ( neural plate border ) and neurog1 was recovered and the expression patterns of pax2a and hoxb1b were restored ( Figure 2Cd–2Cf ) .', 'In addition , the C&E movements indicated by the expression patterns of hgg1 , ntl and dlx3b [31] were normalized in the B1 sox mRNA-injected embryos ( Figure 2Ce ) .', 'This phenotypic rescue was efficient only to early somitogenesis stages , likely because of the gradual decrease in the exogenously supplied SOX expression [19] .', 'Simultaneous injection of sox2/3/19a/19b mRNAs ( 5 pg each ) had essentially the same rescue effects ( data not shown ) .', 'These observations indicate that the function of the B1 SOX proteins is interchangeable during early zebrafish development .', 'To further explore the functions of B1 sox in early zebrafish embryogenesis , we characterized our QKD embryos by focusing on their defects in neural development and gastrulation movements .', 'In these QKD embryos , expression domains of otx2 ( anterior neuroectoderm ) and zic2b ( entire neuroectoderm ) were expanded ventrally at 75%E , whereas expression of both foxi1 and dlx3b ( non-neuroectoderm ) was reduced ( Figure 3Aa–3Ad ) .', 'Consistently , the neuroectodermal expression of B1 sox was also expanded in the QKD embryo ( Figure 3Ae–3Ah ) .', 'These gene expression changes are reminiscent of the dorsalized phenotype seen in BMP-pathway mutants [33] , [34] , suggesting impairment of this pathway in the QKD embryos .', 'At the tailbud stage in the QKD embryos , expression domains of pax2a , gbx1 ( hindbrain ) and hoxb1b ( posterior neuroectoderm ) were anterolaterally shifted , which encompass the otx2 expression domain ( Figure 2Bd and Figure 3B ) .', 'A similar change was seen for eng2a expression in the MHB at the 3-somite stage ( Figure 3Bf ) .', 'These observations indicate that some characteristics of the early neural plate can develop even under a severe reduction of B1 SOX activity .', 'However , our initial analyses revealed that expression of many neural genes is abolished in the QKD embryos , including zic1 ( forebrain ) , rx3 ( eye field ) , nkx1 . 2la ( posterior neuroectoderm ) and krox20 ( rhombomere [r] 3/5 ) as well as hesx1 and neurog1 ( Figure 2B and Figure 3C ) .', 'Injection of the MOs for QKD into embryos of the nr2f2 enhancer-trap line [35] confirmed the impairment of brain development at later stages ( Figure S4A ) .', 'These findings together suggest that B1 sox activity is critical for neural development , although it is dispensable for the expression of some early neural genes .', 'The normal expression levels of ntl and myod1 ( somite ) in the QKD embryos suggest that mesodermal differentiation per se can proceed ( Figure 3D ) .', 'However , the broadened expression domains of these genes indicate defects in convergence movements , which is consistent with the widened expression of neural genes such as gbx1 and hoxb1b .', 'Movement of the anterior prechordal plate ( marked by hgg1 ) was also impaired in the QKD embryos ( Figure 2Be ) , which is characteristic of defective C&E movements .', 'However , hatching gland precursor cells were commonly found to aberrantly move in a dorsal direction and penetrated the ectoderm during mid-somite stages ( Figure 1De’ ) .', 'In the severe morphants , these hatching gland cells remained as a single ball-like structure in the head ( Figure S4B ) .', 'This phenotype is unique to the QKD embryo and may reflect a decreased adhesion of ectodermal cells , as also suggested by cell dissociation from the dorsal trunk region ( Figure 1De’ and 1Df’ ) .', 'Interestingly , the QKD embryos show an increase in transcript levels of sox2/3/19a/19b and also sox1b at early developmental stages ( Figure 3A and Figure S1B ) .', 'This implies a negative autoregulation of transcription among the B1 sox members , although a stabilization of these mRNAs by the MOs could not be ruled out .', 'It is also noteworthy that even with these elevated levels of B1 sox transcripts in the QKD embryos , the MO-mediated knockdowns were effective in inhibiting their translation as revealed by western blotting ( Figure S2B ) .', 'To further characterize the phenotype of the QKD embryos , we examined the changes in gene expression in greater detail by the combined use of in situ hybridization , RT-PCR ( summarized in Table S1 ) and microarray analysis ( Figure S5 , Table S2 and ) .', 'Overall , these analyses indicated that a wide range of developmental processes were affected in the QKD embryos and the major phenotypes can be categorized into: ( 1 ) the early dorsoventral patterning defects; ( 2 ) defects in gastrulation movements; ( 3 ) dysregulation of early neural and neuronal regulatory genes; ( 4 ) neural patterning defects associated with the abrogated expression of signaling pathway genes; and ( 5 ) early defects resembling those observed for MZspg embryos .', 'Further details of these phenotypes are described below .', 'Early DV patterning of the embryo relies on a gradient of Bmp signaling , in which bmp2b/7 play a major role in zebrafish [36] , [37] .', 'The phenotypic similarities between the QKD embryos and bmp pathway mutants described above prompted us to examine the genes in this pathway .', 'Expression of bmp2b/7 was found to be reduced in the QKD embryos from the beginning ( Figure 4Aa–4Af ) .', 'bmp4 expression levels were more or less normal initially , but were downregulated at late epiboly stages in the QKD embryos ( Figure 4Ag; data not shown ) .', 'Consistently , expression of the gata2 , szl and eve1 genes , which are immediately downstream of Bmp signaling , was also reduced in the QKD embryos ( Figure 4Ah–4Aj ) .', 'The dorsal identity of the zebrafish embryo requires activation of maternal β-catenin , which then activates expression of gsc and the Bmp antagonist genes chd and nog1 at the dorsal side .', 'In the QKD embryos , these genes were initiated normally , although the expression of chd was slightly ventrally expanded at 30%E ( Figure 4Ak ) , which is likely secondary to the reduced expression of bmp2b/7 .', 'At later stages in the QKD embryos , however , chd expression was rather decreased ( Figure 4Am ) , contrasting to the bmp pathway mutants [38] .', 'To determine the relationship between the dorsalized phenotype of the QKD embryos and Bmp signaling , we injected a mixture of bmp2b/7 mRNAs ( 20 or 40 pg each ) together with the MOs for QKD .', 'This bmp2b/7 injection rescued the expression of the Bmp downstream genes gata2 , szl and eve1 ( Figure 4B ) , indicating that signaling components acting downstream of Bmp2b/7 are not affected in the QKD embryos .', 'Consistently , the mRNA levels of Bmp receptor genes ( acvrl1 , bmpr1aa , bmpr1ab and bmpr1ba ) and smad5 were found to be normal in the QKD embryos by microarray ( GEO accession number GSE18830 ) .', 'These observations , together with the normal initiation of the dorsal pathway in the QKD embryos , indicate that the DV patterning defects of the QKD embryos primarily result from the reduction of bmp2b/7 expression .', 'Components of non-canonical Wnt signaling and cell adhesion molecules are major regulators of gastrulation movements , including epiboly and C&E movements [32] , [39] .', 'Since these movements are severely impaired in the QKD embryos , we investigated expression profiles of genes related to these processes .', 'wnt11 and wnt5b are major Wnt ligand genes involved in C&E movements [31] , [32] .', 'In the QKD embryos , upregulation of wnt11 that normally occurs during early gastrulation was not observed and expression of wnt5b was slightly reduced at late epiboly stages ( Figure 4C ) .', 'We also observed decreased expression of wnt11r and wnt4a , which are important for convergence movements at later stages [40] .', 'These data suggest that the reduction of non-canonical Wnt ligands contributes to the impairment of C&E movements .', 'Both classical cadherins and protocadherins are involved in gastrulation movements [39] .', 'We found that expression of pcdh18a/18b was significantly reduced in the QKD embryos ( Figure 4C ) .', 'These genes are expressed in the epiblast at the shield stage and later in the neuroectoderm in an overlapping manner in normal embryos [41] , [42] .', 'To examine how reduced activity of Pcdh18a/18b affects embryogenesis , we knocked down these two genes .', 'Although only mild gastrulation defects were observed when these genes were knocked down separately , simultaneous KD caused a delay in epiboly and also C&E defects ( Figure S6; for pcdh18a single KD , see also [41] ) .', 'Delayed epiboly has also been reported for the hypomorphic cdh1 mutants [43] , [44] , but its expression was not altered in our QKD embryos ( Figure 4C ) .', 'These observations indicate that multiple mechanisms involved in gastrulation movements are simultaneously affected in the QKD embryos .', 'Several neuronal genes were found to be abnormally upregulated in the QKD embryos .', 'stmn2a is strongly expressed in CNS neurons from mid-somitogenesis stages in wild-type embryos [45] and also weakly expressed throughout the embryo at epiboly stages ( Figure 5A and Figure S7A ) .', 'The latter early stage expression was found to be aberrantly upregulated in the QKD embryos ( Figure 5A and Figure S7A ) .', 'The neuronal tuba1 gene was also upregulated from 75%E ( Figure 5A ) .', 'These observations suggest that a portion of the neuronal differentiation programs is precociously initiated in the QKD embryos .', 'Neural bHLH transcription factors are key players in the neuronal differentiation programs .', 'Hes/her genes encode repressor-type bHLH proteins , are expressed in undifferentiated neural progenitor cells and maintain their cell state [46] .', 'Among the zebrafish her genes , her3 , an ortholog of mammalian Hes3 , is initiated in the dorsal region of the epiblast at about 30%E , and its expression continues in bilateral inter-proneuronal domains [47] .', 'This her3 expression is totally lost in the QKD embryos ( Figure 5A and 5Ba–5Bc ) .', 'Proneural genes encoding activator-type bHLH proteins and participating in neurogenesis are also affected in the QKD embryos .', 'In normal zebrafish embryos , neurog1 expression initially marks primary neurons at the end of gastrulation and then covers the proneuronal domains in a fashion complementary to her3 expression .', 'In the QKD embryos , neurog1 expression is also lost ( Figure 2Bf and Figure 5A ) .', 'However , not all proneural genes behave in this manner as for example ascl1a is transiently upregulated at about 75%E in a broad area of the neuroectoderm in the QKD embryos ( Figure 5A and 5B ) .', 'Proneural genes are known to be repressed by Hes/Her [46] , but exogenous injection of her3 mRNA into QKD embryos did not repress aberrant ascl1a expression ( data not shown ) , indicating that the loss of her3 was not causal to this upregulation .', 'The neuronal repressor REST has been implicated in suppression of Ascl1 as well as Stmn2 [48] .', 'However , rest expression was unchanged in the QKD embryos ( Figure 5A ) , although Rest is suggested to be downstream of SOX2 in ES cells [24] , indicating that rest is not involved in the aberrant regulation of ascl1a and stmn2a .', 'Taken together , our results indicate that the proper operation of the neuronal differentiation programs , including regulatory networks involving the neural bHLH genes , is highly dependent on the activity of B1 sox .', 'Regional identities of the neural plate are specified through regulatory networks involving various signaling pathways and transcriptional regulators .', 'Genes that are critical for these networks are severely affected in the QKD embryos .', 'As described earlier , in the QKD embryos , expression of the transcription factor genes hesx1 , zic1 , and rx3 , which are required for forebrain and eye development [49]–[51] , is lost throughout early embryogenesis ( Figure 3C and Figure 5A ) .', 'The MHB itself was established , as judged from the expression of pax2a and eng2a , but the anterolaterally-shifted expression patterns of these genes suggest an improper formation of the axes of the anterior neural plate ( Figure 2Bd and Figure 3Bf ) .', 'The expression domain of otx2 was expanded and encircled by those of pax2a , eng2a and gbx1 ( Figure 2Bd and Figure 3B ) , which is likely due to the dorsalized phenotype caused by the decreased expression of bmp2b/7 , as the bmp2b/7 mutant embryos show similar patterns of gene expression [33] , [34] .', 'In addition , the QKD embryos lacked the anterior neuroectoderm expression of cyp26a1 ( Figure 5C ) , which encodes an RA degrading enzyme and thereby plays a role in hindbrain patterning [52] .', 'Hence , the reduction of cyp26a1 expression partly accounts for the hindbrain defects in the QKD embryos , e . g . , expansion of hoxb1a in r4 ( Figure 5Cc ) as observed in the cyp26a1 mutant [53] .', 'However , B1 sox also seem to be more directly involved in gene regulation in hindbrain development as evidenced by severe downregulation of mafba ( r5/6 ) ( Figure 5Cd ) and the loss of krox20 expression in r3/5 ( Figure 3Ce ) .', 'Nodal and Sonic hedgehog signaling are crucial for the development of ventral brain structures [54]–[56] .', 'In normal embryos , oep , an ortholog of mouse Cripto , is strongly expressed in the anterior neural plate and is essential as a coreceptor for receiving Nodal signals [54] .', 'Interestingly , oep expression in the ectoderm at the shield stage and the neuroectoderm at later stages is selectively lost in the QKD embryos ( Figure 5Db–5Dd ) , whereas its early zygotic and mesodermal expression was maintained ( Figure 5Da–5Dd ) .', 'Moreover , expression of shha and shhb in the ventral floor of the brain is also lost in the QKD embryos , leaving only shhb expression in the prechordal plate ( Figure 5Df–5Di ) .', 'These findings suggest that the loss of oep and shha/b expression leads to defective ventral brain development in the QKD embryos .', 'It is known that shha expression in the neuroectoderm is regulated by Nodal signals from the mesoderm [57] .', 'However , a more direct link between B1 SOX action and shh regulation is suggested , as the exogenous injection of oep mRNA into the QKD embryos did not restore the shha expression ( data not shown ) , although Nodal-encoding ndr2 is normally expressed ( Figure 5De ) .', 'Defects in the anterior neural plate development in the QKD embryos also include the loss of mdkb expression ( Figure 5E ) .', 'Consistent with the proposed role of mdkb in the specification of neural crest cells [58] , foxd3 expression in the neural crest was reduced in the QKD embryos ( Figure 5Ec ) .', 'As the B1 SOX proteins primarily function as transcriptional activators [11] , [13] , [15] , [23] , [25] , the expression of direct target genes is expected to be decreased in response to the QKD .', 'However , the upregulation of several neuronal genes such as stmn2a raised the possibility that B1 SOX might also act as repressors .', 'To test this , we utilized dominant activator and repressor forms of SOX3 , SOX3-VP16 and SOX3-EnR ( Figure 6A ) and compared the effects of these variants under QKD conditions with those of SOX3 .', 'As anticipated , genes that were downregulated in the QKD embryos , namely bmp2b/7 , pcdh18a/18b , her3 , hesx1 and zic1 , were efficiently recovered by the exogenous supply of either SOX3 or SOX3-VP16 but not by SOX3-EnR ( Figure 6B ) .', 'These genes are thus likely activation targets of B1 SOX .', 'In addition , the increased expression of stmn2a and ascl1a in the QKD embryos was suppressed in the same way .', 'This suggests an indirect regulation of these genes by B1 SOX through the activation of repressors .', 'However , SOX3-VP16 was less effective than SOX3 in the rescue of some genes such as pcdh18a/18b and ascl1a , suggesting that the activation process may require additional molecular interactions with the intact SOX3 protein .', 'It is noteworthy that the morphological rescue of the QKD embryos by SOX3-VP16 was much less complete when compared to that observed for SOX3 , and that the SOX3-VP16-injected embryos showed a rather ventralized phenotype ( Figure 6C ) .', 'In line with this observation , chd and nog1 were unexpectedly reduced in SOX3-VP16-injected embryos , but increased in SOX3-EnR-injected embryos , suggesting that the repressive action of B1 SOX may be required for the proper regulation of dorsally expressed BMP antagonist genes .', 'These findings together indicate that the B1 SOX proteins primarily act as activators in early embryos , whereas a context-dependent repressive action of these factors is also suggested .', 'To further explore whether the B1 SOX proteins directly regulate the potential downstream genes described above , we searched for possible B1 SOX binding sites ( containing the consensus sequence CATTGTT [21] , [59] or closely related sequences ) in the regulatory regions of these genes .', 'We identified potential SOX-binding sites in the regulatory sequences of her3 [47] , hesx1 , cyp26a1 [60] and neurog1 [61] and also in the conserved non-coding sequences upstream of pcdh18a ( Figure 7Aa ) .', 'To investigate the direct interaction of the B1 SOX proteins with these genomic sequences in vivo , ChIP experiments were performed using zebrafish embryos at the 70–80%E and tailbud to 2-somite stages .', 'ChIP analysis using anti-SOX2 antibody that weakly cross-reacts with SOX3/19A/19B revealed specific binding of B1 SOX to these regulatory sequences in the zebrafish embryo ( Figure 7Ab ) .', 'Similar results were obtained with anti-SOX3 antibody ( data not shown ) .', 'It is thus likely that these genes are direct downstream targets of B1 SOX .', 'To further investigate whether the activities of these regulatory sequences are dependent on B1 SOX , we created luciferase reporter vectors containing the promoter sequences for hesx1 and cyp26a1 [60] ( Figure 7Ba ) .', 'The 0 . 8-kb promoter sequence of the zebrafish hesx1 gene used here corresponds to the chicken Hesx1 promoter that has been shown to have anterior CNS-specific regulatory activity in chicken and also zebrafish embryos [62] .', 'The conserved non-coding sequence upstream of pcdh18a ( 412 bp ) was also cloned into the TK-luciferase reporter vector ( Figure 7Ba ) .', 'When these reporter vectors were injected with or without the MOs for QKD into zebrafish embryos , the promoter activities of hesx1 and cyp26a1 were significantly downregulated upon B1 sox QKD ( Figure 7Bb ) .', 'The 412-bp pcdh18a sequence showed an enhancer activity in normal embryos , whereas this activity was also reduced in the QKD embryos ( Figure 7Bb ) .', 'These data confirm that B1 SOX proteins regulate the hesx1 , cyp26a1 and pcdh18a genes through these regulatory elements .', 'Interestingly , POU binding sites were found abutting the SOX sites of her3 and hesx1 .', 'These genes were found to be commonly downregulated in the QKD embryos ( Figure 3C and Figure 5A and 5B ) and also MZspg mutants ( Table S7; see also [29] ) .', 'In the QKD embryos , pou5f1 is expressed at normal levels ( Figure S7B ) , indicating that Pou5f1 alone is insufficient to induce her3 or hesx1 .', 'These data together suggest that B1 SOX and Pou5f1 proteins synergistically cooperate to activate her3 and hesx1 .'], ['We found similarities in the gene expression profiles between the B1 sox QKD embryos and the MZspg embryos in a wider range of developmental stages from the blastoderm to early neural stages ( Figure 8 , Table S7 and [29] ) .', 'This strongly suggests that their cooperation is required not only for the blastoderm stage , which may be similar to the ES cell state , but also for the early neural stage .', 'In contrast to mouse knockouts of Sox2 or Oct3/4 , where impairment of the ICM/epiblast lineage development causes early embryonic lethality [1] , [63] , zebrafish embryos of the B1 sox QKD and MZspg mutants are viable although with severe developmental defects .', 'This enabled us to analyze the functions of B1 sox in later developmental stages as well as the blastoderm stage .', 'A group of key genes downstream of B1 SOX and Pou5f1 in the blastoderm was found to be bmp2b/7 , as the expression of bmp2b/7 is also severely reduced in MZspg embryos [28] .', 'In addition , an overlap of the expression domains of B1 sox , pou5f1 and bmp2b/7 in the blastoderm strongly suggests a direct regulation of bmp2b/7 by B1 SOX and Pou5f1 .', 'The dorsalized phenotype of the B1 sox QKD and MZspg embryos can to a large ext
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['The B1 SOX transcription factors SOX1/2/3/19 have been implicated in various processes of early embryogenesis .', 'However , their regulatory functions in stages from the blastula to early neurula remain largely unknown , primarily because loss-of-function studies have not been informative to date .', 'In our present study , we systematically knocked down the B1 sox genes in zebrafish .', 'Only the quadruple knockdown of the four B1 sox genes sox2/3/19a/19b resulted in very severe developmental abnormalities , confirming that the B1 sox genes are functionally redundant .', 'We characterized the sox2/3/19a/19b quadruple knockdown embryos in detail by examining the changes in gene expression through in situ hybridization , RT–PCR , and microarray analyses .', 'Importantly , these phenotypic analyses revealed that the B1 SOX proteins regulate the following distinct processes: ( 1 ) early dorsoventral patterning by controlling bmp2b/7; ( 2 ) gastrulation movements via the regulation of pcdh18a/18b and wnt11 , a non-canonical Wnt ligand gene; ( 3 ) neural differentiation by regulating the Hes-class bHLH gene her3 and the proneural-class bHLH genes neurog1 ( positively ) and ascl1a ( negatively ) , and regional transcription factor genes , e . g . , hesx1 , zic1 , and rx3; and ( 4 ) neural patterning by regulating signaling pathway genes , cyp26a1 in RA signaling , oep in Nodal signaling , shh , and mdkb .', 'Chromatin immunoprecipitation analysis of the her3 , hesx1 , neurog1 , pcdh18a , and cyp26a1 genes further suggests a direct regulation of these genes by B1 SOX .', 'We also found an interesting overlap between the early phenotypes of the B1 sox quadruple knockdown embryos and the maternal-zygotic spg embryos that are devoid of pou5f1 activity .', 'These findings indicate that the B1 SOX proteins control a wide range of developmental regulators in the early embryo through partnering in part with Pou5f1 and possibly with other factors , and suggest that the B1 sox functions are central to coordinating cell fate specification with patterning and morphogenetic processes occurring in the early embryo .']
['In the developing embryo , various processes such as cell fate specification , embryo patterning , and morphogenesis take place concurrently .', 'The embryo must control gene expression in order to coordinate these processes and thereby enable the proper organization of its structures .', 'The B1 sox transcription factor genes , exemplified by the “stem cell gene” sox2 , are thought to play a key role in these embryonic processes from the blastoderm stage to the neural stage .', 'However , the precise regulatory functions of these genes are largely unknown due to the lack of loss-of-function studies .', 'In our current study , we took advantage of the zebrafish system and successfully depleted B1 sox activity from the early embryo using antisense knockdown technology .', 'This approach enabled us to further uncover the regulatory functions of B1 sox in early embryos .', 'We found that the activity of the B1 sox genes is required for the expression of a wide range of developmental regulators including transcription factors , signaling pathway components , and cell adhesion molecules .', 'These findings suggest that the B1 sox functions are central to coordinating diverse embryonic processes , particularly those that occur during the development of the primordium of the central nervous system .']
['developmental biology/stem cells', 'developmental biology/morphogenesis and cell biology', 'genetics and genomics/gene expression', 'developmental biology/pattern formation', 'developmental biology/cell differentiation', 'neuroscience/neurodevelopment', 'developmental biology/neurodevelopment', 'developmental biology/developmental molecular mechanisms']