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Ribosomes are essential nanomachines responsible for protein production. Although ribosomes are present in every living cell, ribosome biogenesis dysfunction diseases, called ribosomopathies, impact particular tissues specifically. Here, we evaluate the importance of the box C/D snoRNA-associated ribosomal RNA methyltransferase fibrillarin (Fbl) in the early embryonic development of Xenopus laevis. We report that in developing embryos, the neural plate, neural crest cells (NCCs), and NCC derivatives are rich in fbl transcripts. Fbl knockdown leads to striking morphological defects affecting the eyes and craniofacial skeleton, due to lack of NCC survival caused by massive p53-dependent apoptosis. Fbl is required for efficient pre-rRNA processing and 18S rRNA production, which explains the early developmental defects. Using RiboMethSeq, we systematically reinvestigated ribosomal RNA 2’-O methylation in X. laevis, confirming all 89 previously mapped sites and identifying 15 novel putative positions in 18S and 28S rRNA. Twenty-three positions, including 10 of the new ones, were validated orthogonally by low dNTP primer extension. Bioinformatic screening of the X. laevis transcriptome revealed candidate box C/D snoRNAs for all methylated positions. Mapping of 2’-O methylation at six developmental stages in individual embryos indicated a trend towards reduced methylation at specific positions during development. We conclude that fibrillarin knockdown in early Xenopus embryos causes reduced production of functional ribosomal subunits, thus impairing NCC formation and migration., Author summary Ribosomes are essential nanomachines responsible for protein production in all cells. Ribosomopathies are diseases caused by improper ribosome formation due to mutations in ribosomal proteins or ribosome assembly factors. Such diseases primarily affect the brain and blood, and it is unclear how malfunctioning of a process as general as ribosome formation can lead to tissue-specific diseases. Here we have examined how fibrillarin, an enzyme which modifies ribosomal RNA by adding methyl groups at specific sites, affects early embryonic development in the frog Xenopus laevis. We have revealed its importance in the maturation of cells forming an embryonic structure called the neural crest. Fibrillarin depletion leads to reduced eye size and abnormal head shape, reminiscent of other conditions such as Treacher Collins syndrome. Molecularly, the observed phenotypes are explainable by increased p53-dependent programmed cell death triggered by inhibition of certain pre-rRNA processing steps. Our systematic investigation of the ribosomal RNA 2’-O methylation repertoire across development has further revealed hypomodification at a late stage of development, which might play a role in late developmental transitions involving differential translation by compositionally different ribosomes.

Ribosomes are essential nanomachines responsible for protein production. Although ribosomes are present in every living cell, ribosome biogenesis dysfunction diseases, called ribosomopathies, impact particular tissues specifically. Here, we evaluate the importance of the box C/D snoRNA-associated ribosomal RNA methyltransferase fibrillarin (Fbl) in the early embryonic development of Xenopus laevis. We report that in developing embryos, the neural plate, neural crest cells (NCCs), and NCC derivatives are rich in fbl transcripts. Fbl knockdown leads to striking morphological defects affecting the eyes and craniofacial skeleton, due to lack of NCC survival caused by massive p53-dependent apoptosis. Fbl is required for efficient pre-rRNA processing and 18S rRNA production, which explains the early developmental defects. Using RiboMethSeq, we systematically reinvestigated ribosomal RNA 2’-O methylation in X. laevis, confirming all 89 previously mapped sites and identifying 15 novel putative positions in 18S and 28S rRNA. Twenty-three positions, including 10 of the new ones, were validated orthogonally by low dNTP primer extension. Bioinformatic screening of the X. laevis transcriptome revealed candidate box C/D snoRNAs for all methylated positions. Mapping of 2’-O methylation at six developmental stages in individual embryos indicated a trend towards reduced methylation at specific positions during development. We conclude that fibrillarin knockdown in early Xenopus embryos causes reduced production of functional ribosomal subunits, thus impairing NCC formation and migration.AUTHOR SUMMARYRibosomes are essential nanomachines responsible for protein production in all cells. Ribosomopathies are diseases caused by improper ribosome formation due to mutations in ribosomal proteins or ribosome assembly factors. Such diseases primarily affect the brain and blood, and it is unclear how malfunctioning of a process as general as ribosome formation can lead to tissue-specific diseases. Here we have examined how fibrillarin, an enzyme which modifies ribosomal RNA by adding methyl groups at specific sites, affects early embryonic development in the frog Xenopus laevis. We have revealed its importance in the maturation of cells forming an embryonic structure called the neural crest. Fibrillarin depletion leads to reduced eye size and abnormal head shape, reminiscent of other conditions such as Treacher Collins syndrome. Molecularly, the observed phenotypes are explainable by increased p53-dependent programmed cell death triggered by inhibition of certain pre-rRNA processing steps. Our systematic investigation of the ribosomal RNA 2’-O methylation repertoire across development has further revealed hypomodification at a late stage of development, which might play a role in late developmental transitions involving differential translation by compositionally different ribosomes.

PR homology domain-containing member 12 (PRDM12) belongs to a family of conserved transcription factors implicated in cell fate decisions. Here we show that PRDM12 is a key regulator of sensory neuronal specification in Xenopus. Modeling of human PRDM12 mutations that cause hereditary sensory and autonomic neuropathy (HSAN) revealed remarkable conservation of the mutated residues in evolution. Expression of wild-type human PRDM12 in Xenopus induced the expression of sensory neuronal markers, which was reduced using various human PRDM12 mutants. In Drosophila, we identified Hamlet as the functional PRDM12 homolog that controls nociceptive behavior in sensory neurons. Furthermore, expression analysis of human patient fibroblasts with PRDM12 mutations uncovered possible downstream target genes. Knockdown of several of these target genes including thyrotropin-releasing hormone degrading enzyme (TRHDE) in Drosophila sensory neurons resulted in altered cellular morphology and impaired nociception. These data show that PRDM12 and its functional fly homolog Hamlet are evolutionary conserved master regulators of sensory neuronal specification and play a critical role in pain perception. Our data also uncover novel pathways in multiple species that regulate evolutionary conserved nociception.

The maternal-zygotic transition (MZT) is an embryonic event that overlaps with and plays key roles in primary germ layer specification in vertebrates. During MZT, maternally supplied mRNAs are degraded while zygotic transcripts are synthesized to either reinforce the already specified cell fate or to trigger new cell identity. Here, we show that forced expression of the RNA-binding protein, XSeb4R, in animal pole blastomeres of Xenopus embryos, inappropriately stabilizes transcripts there, including maternal Sox3. This leads to the impaired ability of the ectodermal progenitors to respond to factors regulating brain patterning and their eventual loss by apoptosis. XSeb4R protein binds specifically to the 3′UTR of Sox3 mRNA. XSeb4R gain-of-function in ectodermal explants reveals increased stability of the maternal Sox3 transcripts, associated with a robust Sox3 protein production. Conversely, whereas XSeb4R depletion abolishes VegT expression, the amount of the maternal Sox3 mRNA is rather increased but without augmentation in the amount of Sox3 protein. Moreover, XSeb4R protein knockdown leads to the modification of the ectoderm–mesoderm boundary, marked by expanded/shifted expression of the mesodermal marker genes such as Xbra and Apod, followed by an expression inhibition of Epi. K., an ectodermal marker. Overall, our data suggest XSeb4R as a novel player in gene expression regulation, acting at the posttranscriptional level during ectoderm specification in Xenopus.

The Zic transcription factors play critical roles during embryonic development. Mutations in the ZIC2 gene are associated with human holoprosencephaly, but the etiology is still unclear. Here, we report a novel function for ZIC2 as a regulator of β-catenin·TCF4-mediated transcription. We show that ZIC2 can bind directly to the DNA-binding high mobility group box of TCF4 via its zinc finger domain and inhibit the transcriptional activity of the β-catenin·TCF4 complex. However, the binding of TCF4 to DNA was not affected by ZIC2. Zic2 RNA injection completely inhibited β-catenin-induced axis duplication in Xenopus embryos and strongly blocked the ability of β-catenin to induce expression of known Wnt targets in animal caps. Moreover, Zic2 knockdown in transgenic Xenopus Wnt reporter embryos led to ectopic Wnt signaling activity mainly at the midbrain-hindbrain boundary. Together, our results demonstrate a previously unknown role for ZIC2 as a transcriptional regulator of the β-catenin·TCF4 complex.

A complex set of extracellular signals is required for neural crest (NC) specification. However, how these signals function to coordinate cell-cycle progression and differentiation remains poorly understood. Here, we report in Xenopus a role for the transcription factor signal transducers and activators of transcription-3 (Stat3) in this process downstream of FGF signalling. Depletion of Stat3 inhibits NC gene expression and cell proliferation, whereas overexpression expands the NC domain and promotes cell proliferation. Stat3 is phosphorylated and activated in ectodermal cells by FGFs through binding with FGFR4. Stat3 activation is also modulated by Hairy2 and Id3 proteins that, respectively, facilitate and disrupt Stat3-FGFR4 complex formation. Furthermore, distinct levels of Stat3 activity control Hairy2 and Id3 transcription, leading to Stat3 self-regulation. Finally, high Stat3 activity maintains cells in an undifferentiated state, whereas low activity promotes cell proliferation and NC differentiation. Together, our data suggest that Stat3, downstream of FGFs and under the positive and negative feedback regulation of Hairy2 and Id3, plays an essential role in the coordination of cell-cycle progression and differentiation during NC specification.

BBP proteins constitute a subclass of CUL3 interacting BTB proteins whose in vivo function remains unknown. Here, we show that the Xenopus BBP gene BTBD6 and the single Drosophila homologue of mammalian BBP genes lute are strongly expressed in the developing nervous system. In Xenopus, BTBD6 expression responds positively to proneural and negatively to neurogenic gene overexpression. Knockdown of BTBD6 in Xenopus or loss of Drosophila lute result in embryos with strong defects in late neuronal markers and strongly reduced and disorganized axons while early neural development is unaffected. XBTBD6 knockdown in Xenopus also affects muscle development. Together, these data indicate that BTBD6/lute is required for proper embryogenesis and plays an essential evolutionary conserved role during neuronal development.

International audience; Loss of function studies have shown that the Xenopus helix-loop-helix transcription factor Hairy2 is essential for neural crest formation and maintains cells in a mitotic undifferentiated state. However, its position in the genetic cascade regulating neural crest formation and its relationship with other neural crest regulators remain largely unknown. Here we find that Hairy2 is regulated by BMP, FGF and Wnt and that it is only required downstream of BMP and FGF for neural crest formation. We show that Hairy2 overexpression represses neural crest and upregulates neural border genes at early stages while it expands a subset of them in later embryos. We show that Hairy2 downregulates Id3, another essential HLH neural crest regulator, through attenuation of BMP signaling. Knockdown and rescue experiments indicate that Id3 protein, which physically interacts with Hairy2, negatively regulates Hairy2 activity. However, Id3 is required to allow Hairy2 to promote neural crest formation. Together, our results provide evidence that Hairy2 acts downstream of FGF and BMP signals at the neural border to maintain cells in an undifferentiated state, and that Hairy2-Id3 interactions play an essential role in neural crest progenitor specification.

VegT represents a localized maternal determinant essentially required for endoderm formation in Xenopus. Here, we report on the identification of the RNA-binding protein XSeb4R as a positive regulator of VegT. XSeb4R interacts directly with the 3′-untranslated region of VegT mRNA, stabilizes it, and stimulates translation. Ablation of XSeb4R activity results in impairment of endoderm and mesoderm formation, while ectopic expression of XSeb4R in ectodermal cells induces endodermal and mesodermal gene expression. These observations unravel a novel mode of VegT regulation at the post-transcriptional level that is essential for germ layer formation in Xenopus.

The DNA-binding transcription factor Smad-interacting protein-1 (Sip1) (also named Zfhx1b/ZEB2) plays essential roles in vertebrate embryogenesis. In Xenopus, XSip1 is essential at the gastrula stage for neural tissue formation, but the precise molecular mechanisms that underlie this process have not been fully identified yet. Here we show that XSip1 functions as a transcriptional repressor during neural induction. We observed that constitutive activation of BMP signaling prevents neural induction by XSip1 but not the inhibition of several epidermal genes. We provide evidence that XSip1 binds directly to the BMP4 proximal promoter and modulates its activity. Finally, by deletion and mutational analysis, we show that XSip1 possesses multiple repression domains and that CtBPs contribute to its repression activity. Consistent with this, interference with XCtBP function reduced XSip1 neuralizing activity. These results suggest that Sip1 acts in neural tissue formation through direct repression of BMP4 but that BMP-independent mechanisms are involved as well. Our data also provide the first demonstration of the importance of CtBP binding in Sip1 transcriptional activity in vivo.

International audience; V1 interneurons are inhibitory neurons that play an essential role in vertebrate locomotion. The molecular mechanisms underlying their genesis remain, however, largely undefined. Here, we show that the transcription factor Prdm12 is selectively expressed in p1 progenitors of the hindbrain and spinal cord in the frog embryo, and that a similar restricted expression profile is observed in the nerve cord of other vertebrates as well as of the cephalochordate amphioxus. Using frog, chick and mice, we analyzed the regulation of Prdm12 and found that its expression in the caudal neural tube is dependent on retinoic acid and Pax6, and that it is restricted to p1 progenitors, due to the repressive action of Dbx1 and Nkx6-1/2 expressed in the adjacent p0 and p2 domains. Functional studies in the frog, including genome-wide identification of its targets by RNA-seq and ChIP-Seq, reveal that vertebrate Prdm12 proteins act as a general determinant of V1 cell fate, at least in part, by directly repressing Dbx1 and Nkx6 genes. This probably occurs by recruiting the methyltransferase G9a, an activity that is not displayed by the amphioxus Prdm12 protein. Together, these findings indicate that Prdm12 promotes V1 interneurons through cross-repressive interactions with Dbx1 and Nkx6 genes, and suggest that this function might have only been acquired after the split of the vertebrate and cephalochordate lineages.

Notch signaling has been shown to play a role in cell fate decisions in the Xenopus pronephros anlagen. Here, we show that the XenopusHairy-related transcription factor (HRT) gene XHRT1, and the Hairy/Enhancer of split (HES) genes Xhairy1, Xhairy2b, esr9and esr10, have distinct restricted dynamic expression patterns during pronephros development, and that their expression is regulated by Notch. XHRT1, which is the earliest and strongest gene expressed in the pronephric region, is initially transcribed predominantly in the forming glomus, where it is downregulated by antisense morpholino oligonucleotide inhibition of xWT1. Later, it is activated in the most dorsoanterior part of the pronephros anlagen that gives rise to the proximal tubules. In agreement with this dynamic expression profile, we found that early activation of Notch favors glomus, whereas only later activation promotes proximal tubule formation. We show that, among the bHLH-O factors tested, only XHRT1 efficiently inhibits distal tubule and duct formation, and that only its translational inhibition causes a reduction of the expression of proximal tubule and glomus markers. Using domain swap experiments, we found that the XHRT1 C-terminal region is crucial for its activity. Together, our results provide evidence that XHRT1 plays an important role in glomerular development and early proximodistal patterning that is distinct from those of the other pronephric bHLH repressors.

We have isolated a Xenopus homologue of the mammalian hairy and Enhancer of split related gene HRT1. XHRT1 expression in late gastrula and early neurula embryos is restricted to two stripes of cells in the medial neural plate and in dorsal endodermal cells. At later stages, XHRT1 is expressed in the floor plate, in hypochord cells and in the somitogenic and anterior presomitic mesoderm. By tailbud stage, XHRT1 is also highly expressed in the dorsal hindbrain, telencephalon and eye vesicles, olfactory placodes, pronephros, branchial arches and tail fin. We also show that XHRT1 expression in medial neural cells is induced by Notch signaling and that there are differences in the way XHRT1 and other H/E(spl) genes are regulated.

The Notch signaling pathway plays an important role in many cell-fate decisions during development. Here we investigate the regulation and function of the conserved gene XNAP, which is a member of the Delta-Notch synexpression group in Xenopus. XNAP encodes a small protein with two C-terminal tandem ankyrin repeats which is expressed in the neurectoderm and in the presomitic mesoderm in a pattern that resembles that of other component of the Notch pathway. When a myc-tag form of XNAP is overexpressed in Xenopus or Hela cells, XNAP protein is detected both in the nucleus and the cytoplasm. In embryos and in animal cap assays, XNAP expression is activated, perhaps directly, by the Notch pathway and this activation appears to be Su(H) dependent. Overexpression of XNAP in embryos decreases Notch signaling, which leads to an increase in the number of primary neurons that form within the domains of the neural plate where neurogenesis normally occurs. In culture Hela cells, XNAP overexpression interferes with ICD activation of a Notch regulated reporter gene. Together, these data indicate that XNAP is a novel target of the Notch pathway that may, in a feedback loop, modulate its activity.

In contrast with the wealth of data involving bHLH and homeodomain transcription factors in retinal cell type determination, the molecular bases underlying neurotransmitter subtype specification is far less understood. Using both gain and loss of function analyses in Xenopus, we investigated the putative implication of the bHLH factor Ascl1 in this process. We found that in addition to its previously characterized proneural function, Ascl1 also contributes to the specification of the GABAergic phenotype. We showed that it is necessary for retinal GABAergic cell genesis and sufficient in overexpression experiments to bias a subset of retinal precursor cells towards a GABAergic fate. We also analysed the relationships between Ascl1 and a set of other bHLH factors using an in vivo ectopic neurogenic assay. We demonstrated that Ascl1 has unique features as a GABAergic inducer and is epistatic over factors endowed with glutamatergic potentialities such as Neurog2, NeuroD1 or Atoh7. This functional specificity is conferred by the basic DNA binding domain of Ascl1 and involves a specific genetic network, distinct from that underlying its previously demonstrated effects on catecholaminergic differentiation. Our data show that GABAergic inducing activity of Ascl1 requires the direct transcriptional regulation of Ptf1a, providing therefore a new piece of the network governing neurotransmitter subtype specification during retinogenesis., SCOPUS: ar.j, info:eu-repo/semantics/published

We have identified a Xenopus bHLH gene, Xath2, which is the homologue of the murine MATH-2/NEX-1 gene, using a functional expression screening approach. Overexpression of this gene in neurula embryos induces the expression of the N-tubulin neuronal marker but does not stimulate the expression of the X-ngnr-1 and NeuroD proneural genes. Expression of Xath2 begins in stage 32 embryos and is restricted to the dorsal telencephalon. Within the neuroepithelium of the dorsal telencephalon, Xath2 expression is detected in postmitotic cells located more laterally than those expressing several other related bHLH neuronal regulators.

We describe the structural organization of a gene, termed XFDL 141/156, that is transiently activated during early Xenopus development. XFDL 141/156 is first transcribed at the midblastula transition (MBT) and during early gastrulation events. A roughly 200 nucleotide fragment immediately 5' to the transcription start site is sufficient for transient, early zygotic activation of gene expression. The primary transcript is subject to alternative splicing. Corresponding cDNAs encode two structurally related but completely distinct C2H2-type zinc finger proteins of unknown biological function.

X-MyT1 is a C2HC-type zinc finger protein that we find to be involved in the primary selection of neuronal precursor cells in Xenopus. Expression of this gene is positively regulated by the bHLH protein X-NGNR-1 and negatively regulated by the Notch/Delta signal transduction pathway. X-MyT1 is able to promote ectopic neuronal differentiation and to confer insensitivity to lateral inhibition, but only in cooperation with bHLH transcription factors. Inhibition of X-MyT1 function inhibits normal neurogenesis as well as ectopic neurogenesis caused by overexpression of X-NGNR-1. On the basis of these findings, we suggest that X-MyT1 is a novel, essential element in the cascade of events that allows cells to escape lateral inhibition and to enter the pathway that leads to terminal neuronal differentiation.

The Homez gene encodes a protein with three atypical homeodomains and two leucine zipper motifs of unknown function. Here we show that during neurula stages, Xenopus Homez is broadly expressed throughout the neural plate, the strongest expression being detected in the domains where primary neurons arise. At later stages, Homez is maintained throughout the central nervous system in differentiating progenitors. In accordance with this expression, Homez is positively regulated by neural inducers and by Ngnr1 and negatively by Notch signaling. Interference with Homez function in embryos by injection of an antisense morpholino oligonucleotide results in the specific disruption of the expression of late neuronal markers, without affecting the expression of earlier neuronal and early neurectodermal markers. Consistent with this finding, Homez inhibition also interferes with the expression of late neuronal markers in Ngnr1 overexpressing animal cap explants and in Notch inhibited embryos. In gain of function experiments, Homez inhibits the expression of late neuronal markers but has no effect on earlier ones. These data suggest a role for Homez in neuronal development downstream of proneural/neurogenic genes.

The evolutionarily conserved IA1 (Insm1) gene is strongly expressed in the developing nervous system. Here, we show that IA1 is expressed during Xenopus laevis embryogenesis in neural plate primary neurons as well as in a population of uncharacterized anteroventral cells that form in front of the cement gland and that we identified as noradrenergic neurons. We also show that the formation of those anteroventral cells is dependent on BMPs and inhibited by Notch and that it is regulated by the transcription factors Xash1, Phox2, and Hand2. Finally, we provide functional evidence suggesting that IA1 may also play a role in their formation. Together, our results reveal that IA1 constitutes a novel player downstream of Xash1 in the formation of a previously unidentified population of Xenopus noradrenergic primary neurons.

International audience; The ecotropic viral integration site 1 (Evi1) and related MEL1 (MDS1/Evi1-like gene 1) genes are zinc finger oncogenic transcription factors involved in myeloid leukaemia. Here, we show that in Xenopus, Evi1 and MEL1 have partially overlapping restricted embryonic expression profiles. Within the pronephros, Evi1 and MEL1 are sequentially expressed within the distal tubule and duct compartments, Evi1 transcription being detected prior to any sign of pronephric morphogenesis. In the pronephros of zebrafish embryos, Evi1 expression is restricted to the posterior portion of the duct, the anterior portion having characteristics of proximal tubules. In the Xenopus pronephros, Evi1 expression is upregulated by retinoid signaling and repressed by overexpression of xWT1 and by Notch signaling. Overexpression of Evi1 from late neurula stage specifically inhibits the expression of proximal tubule and glomus pronephric markers. We show that the first zinc finger and CtBP interaction domains are required for this activity. Overexpression of a hormone-inducible Evi1-VP16 antimorphic fusion with activation at neurula stage disrupts distal tubule and duct formation and expands the expression of glomus markers. Although overexpression of this construct also causes in many embryos a reduction of proximal tubule markers, embryos with expanded and ectopic staining have been also observed. Together, these data indicate that Evi1 plays a role in the proximo-distal patterning of the pronephros and suggest that it may do so by functioning as a CtBP dependent repressor.

The zinc finger/homeo-domain transcription factor (zfh x 1) family in vertebrates consists of two members, deltaEF1 and SIP1. They have been proposed to display antagonistic activities in the interpretation of Smad-dependent TGFbeta signaling during mesoderm formation. We cloned Xenopus deltaEF1 cDNA, analyzed the expression profile of the gene, and compared the inducing and interacting properties of the protein to that of XSIP1. Whereas XSIP1 RNA is selectively expressed in the early developing nervous system, we show that XdeltaEF1 gene transcription is only activated during neurulation and that its expression is restricted to the paraxial mesoderm. From early tail bud stage, XdeltaEF1 and XSIP1 are coexpressed in migratory cranial neural crest, in the retina, and in the neural tube. Overproduction of XdeltaEF1 in RNA-injected embryos, like that of XSIP1, reduced the expression of BMP-dependent genes but only XSIP1 has the ability to induce neural markers. We find that XdeltaEF1 and XSIP1 can both form complexes, although with different efficiency, with Smad3, with the coactivators p300 and pCAF, and with the corepressor CtBP1. Together, these results indicate that deltaEF1 and SIP1 do not function as antagonists during Xenopus early embryogenesis but do display different repression efficiencies and interaction properties.

The embryonic pronephric kidneys of Xenopus and zebrafish serve as models to study vertebrate nephrogenesis. Recently, multiple subdomains within the Xenopus pronephros have been defined based on the expression of several transport proteins. In contrast, very few studies on the expression of renal transporters have been conducted in zebrafish. We have recently shown that the anterior and posterior segments of the zebrafish pronephric duct may correspond to the proximal tubule and distal tubule/duct compartments of the Xenopus and higher vertebrate pronephros, respectively. Here, we report the embryonic expression pattern of the Na(+)/PO(4) cotransporter SLC20A1 (PiT1/Glvr-1) gene encoding a type III sodium-dependent phosphate cotransporter in Xenopus and zebrafish. In Xenopus, SLC20A1 mRNA is expressed in the somitic mesoderm and lower level of expression is detected in the neural tube, eye, and neural crest cells. From stage 25, SLC20A1 is also detectable in the developing pronephros where expression is restricted to the late portion of the distal pronephric tubules. In zebrafish, SLC20A1 is transcribed from mid-somitogenesis in the anterior part of the pronephros where its expression corresponds to the rostral portion of the expression of other proximal tubule-specific markers. Outside the pronephros, lower level of SLC20A1 expression is also observed in the posterior cardinal and caudal veins. Based on the SLC20A1 expression domain and that of other transporters, four segments have been defined within the zebrafish pronephros. Together, our data reveal that the zebrafish and Xenopus pronephros have non-identical proximo-distal organizations.

Hairy-related transcription factors (HRTs) constitute a recently identified subfamily of basic-helix-loop-helix transcription factors containing an Orange domain (bHLH-O factors). As compared to the related HES proteins, HRTs exhibit distinct DNA-binding activities in vitro and the molecular mechanisms underlying their transcriptional activity remain poorly understood. We have identified here the sequence "ggCACGTGcc" as predominant binding site for Xenopus HRT1 (XHRT1). In transiently transfected 3T3 cells, XHRT1 represses the expression of a luciferase reporter gene under the control of multimerized XHRT1 binding sites. Deletion analysis indicated that repression by XHRT1 requires the presence of the DNA-binding bHLH motif and the Orange domain. However, the presence of the sequence motif YRPWGTEIGAF located at the very C-terminus of XHRT1 is dispensable. Accordingly, the groucho co-repressor, which is known to mediate transcriptional repression by HES factors through binding their C-terminal WRPW sequence, does not recognize the related YRPW motif present in the C-terminal part of XHRT1 significantly in vitro. As the C-terminus of HRTs is well conserved, our observation indicates that this part of HRTs, unlike the corresponding part of HES proteins, does not recruit the groucho co-repressor efficiently.

XHRT1 is a member of the HRT/Hey protein subfamily that are known as Notch effectors. XHRT1 is expressed in the developing floor plate and encodes a basic helix-loop-helix (bHLH) transcription repressor. Here, we show that XHRT1 misexpression in the neural plate inhibits differentiation of neural precursor cells and thus may be important for floor plate cells to prevent them from adopting a neuronal fate. Deletion analysis indicated that inhibition of differentiation by XHRT1 requires the DNA-binding bHLH motif and either the Orange domain or the C-terminal region. XHRT1 could efficiently homodimerize and heterodimerize with hairy proteins. Among those hairy genes, Xhairy2b shows extensive overlap of expression with XHRT1 in floor plate precursors and may be a biologically relevant XHRT1 partner. Dimerization is mediated through both the bHLH and downstream sequences, the Orange domain being particularly important for the efficiency of the interaction. Using chimeric constructs between XHRT1 and the ESR9 bHLH-O protein that does not interact with Xhairy1 and Xhairy2b, we found that both the bHLH domain and downstream sequences of XHRT1 were required for heterodimerization with Xhairy2b, while only the XHRT1 sequences downstream of the Orange domain are required for the interaction with Xhairy1. Together, these results suggest that XHRT1 plays a role in floor plate cell development and highlight the importance of the Orange and downstream sequences in dimerization and in the selection of the bHLH partners.

Hairy-related transcription factor (HRT/Hey) genes encode a novel subfamily of basic helix-loop-helix (bHLH) transcription factors related to the Drosophila hairy and Enhancer-of-split (E(spl)) and the mammalian HES proteins that function as downstream mediators of Notch signaling. Using the yeast two-hybrid approach, a previously uncharacterized protein was identified in Xenopus that interacts with XHRT1 (originally referred to as bc8), one member of the HRT/Hey subclass. This protein is evolutionarily conserved in chordates. It binds to sequences adjacent to the bHLH domain of XHRT1 known as the Orange domain and has been named bc8 Orange interacting protein (BOIP). BOIP shows a rather uniform subcellular localization and is recruited to the nucleus upon binding to XHRT1. In Xenopus, XBOIP mRNA is detected by RNase protection analysis throughout embryogenesis. In the adult, the strongest expression is detected in testis. In the mouse, high levels of BOIP mRNA are also found in adult testis. No expression is detected in the embryo and in any of the other adult organs tested. In situ hybridization revealed that BOIP transcripts were detected almost exclusively in round spermatids and that this expression overlaps with that of Hey1 (HRT1), which is expressed throughout spermatogenesis. In view of the importance of the Orange domain for HRT/Hey function, the newly identified BOIP proteins may serve as regulators specifically of HRT1/Hey1 activity.

Xath3 encodes a Xenopus neuronal-specific basic helix–loop–helix transcription factor related to the Drosophila proneural factor atonal. We show here that Xath3 acts downstream of X-ngnr-1 during neuronal differentiation in the neural plate and retina and that its expression and activity are modulated by Notch signaling. X-ngnr-1 activates Xath3 and NeuroD by different mechanisms, and the latter two genes crossactivate each other. In the ectoderm, X-ngnr-1 and Xath3 have similar activities, inducing ectopic sensory neurons. Among the sensory-specific markers tested, only those that label cranial neurons were found to be ectopically activated. By contrast, in the retina, X-ngnr-1 and Xath3 overexpression promote the development of overlapping but distinct subtypes of retinal neurons. Together, these data suggest that X-ngnr-1 and Xath3 regulate successive stages of early neuronal differentiation and that, in addition to their general proneural properties, they may contribute, in a context-dependent manner, to some aspect of neuronal identity.

Our initial description of the Xenopus gene Eomesodermin (Eomes) indicated that it is expressed largely if not entirely in mesodermal cells of gastrula stage embryos. A more detailed examination, described here, shows that it continues to be expressed in the most anterior (future head) mesoderm during gastrula and neurula stages. However, during tail-bud stages, Eomes expression is re-born in the most anterior part of the brain, becoming strongly transcribed in the olfactory region of the telencephalon. This later Eomes expression marks a very localized region of the forebrain distinct from that of Otx-2, anterior to that of En-1 and overlapping that of Sox-3.

Genetic circuits responsible for the development of photoreceptive organs appear to be evolutionarily conserved. Here, the Xenopus homologue Xtll of the Drosophila gene tailless (tll), which we find to be expressed during early eye development, is characterized with respect to its relationship to vertebrate regulators of eye morphogenesis, such as Pax6 and Rx. Expression of all three genes is first detected in the area corresponding to the eye anlagen within the open neural plate in partially overlapping, but not identical, patterns. During the evagination of the optic vesicle, Xtll expression is most prominent in the optic stalk, as well as in the distal tip of the forming vesicle. In tadpole-stage embryos, Xtll gene transcription is most prominent in the ciliary margin of the optic cup. Inhibition of Xtll function in Xenopus embryos interferes specifically with the evagination of the eye vesicle and, in consequence, Xpax6 gene expression is severely reduced in such manipulated embryos. These findings suggest that Xtll serves an important regulatory function in the earliest phases of vertebrate eye development.

We have identified in Xenopus and in the mouse two highly related genes, Xiro3 and Irx3 respectively, that encode a Drosophila Iroquois-related homeobox transcription factor. Xiro3 in Xenopus and Irx3 in the mouse are expressed early in the prospective neural plate in a subset of neural precursor cells. In Xenopus, injection of Xiro3 mRNA expands the neural tube and induces ectopic neural tissue in the epidermis, based on the ectopic expression of early neural markers such as Xsox3. In contrast, the differentiation of the early forming primary neurons, as revealed by the expression of the neuronal marker N-tubulin, is prevented by Xiro3 expression. Activation of Xiro3 expression itself requires the combination of a neural inducing (noggin) and a posteriorizing signal (basic fibroblast growth factor). These results suggest that Xiro3 activation constitutes one of the earliest steps in the development of the neural plate and that it functions in the specification of a neural precursor state.

International audience; The mechanisms by which a subset of mesodermal cells are committed to a nephrogenic fate are largely unknown. In this study, we have investigated the role of retinoic acid (RA) signalling in this process using Xenopus laevis as a model system and Raldh2 knockout mice. Pronephros formation in Xenopus embryo is severely impaired when RA signalling is inhibited either through expression of a dominant-negative RA receptor, or by expressing the RA-catabolizing enzyme XCyp26 or through treatment with chemical inhibitors. Conversely, ectopic RA signalling expands the size of the pronephros. Using a transplantation assay that inhibits RA signalling specifically in pronephric precursors, we demonstrate that this signalling is required within this cell population. Timed antagonist treatments show that RA signalling is required during gastrulation for expression of Xlim-1 and XPax-8 in pronephric precursors. Moreover, experiments conducted with a protein synthesis inhibitor indicate that RA may directly regulate Xlim-1. Raldh2 knockout mouse embryos fail to initiate the expression of early kidney-specific genes, suggesting that implication of RA signalling in the early steps of kidney formation is evolutionary conserved in vertebrates.

The Dmrt (doublesex and mab-3 related transcription factor) genes encode a large family of evolutionarily conserved transcription factors whose function in sex specific differentiation has been well studied in all animal lineages. In vertebrates, their function is not restricted to the developing gonads. For example, Xenopus Dmrt4 is essential for neurogenesis in the olfactory system. Here we have isolated and characterized Xenopus Dmrt5 and found that it is coexpressed with Dmrt4 in the developing olfactory placodes. As Dmrt4, Dmrt5 is positively regulated in the ectoderm by neural inducers and negatively by proneural factors. Both Dmrt5 and Dmrt4 genes are also activated by the combined action of the transcription factor Otx2, broadly transcribed in the head ectoderm and of Notch signaling, activated in the anterior neural ridge. As for Dmrt4, knockdown of Dmrt5 impairs neurogenesis in the embryonic olfactory system and in neuralized animal caps. Conversely, its overexpression promotes neuronal differentiation in animal caps, a property that requires the conserved C-terminal DMA and DMB domains. We also found that the sea anenome Dmrt4/5 related gene NvDmrtb also induces neurogenesis in Xenopus animal caps and that conversely, its knockdown in Nematostella reduces elav-1 positive neurons. Together, our data identify Dmrt5 as a novel important regulator of neurogenesis whose function overlaps with that of Dmrt4 during Xenopus olfactory system development. They also suggest that Dmrt may have had a role in neurogenesis in the last common ancestor of cnidarians and bilaterians.

We have isolated a Xenopus homologue of the zinc finger/homeodomain- containing transcriptional repressor Smad-interacting protein-1 (SIP1) from mouse. XSIP1 is activated at the early gastrula stage and transcription occurs throughout embryogenesis. At the beginning of gastrulation, XSIP1 is strongly expressed in prospective neurectoderm. At the neurula stage, XSIP1 is highly expressed within the neural plate but weakly in the dorsal midline. At later stages of development transcripts are detected primarily within the neural tube and neural crest. In the adult, XSIP1 expression is detected at variable levels in several organs.

Mutations in ZFHX1B cause Mowat-Wilson syndrome (MWS) but the precise mechanisms underlying the aberrant functions of mutant ZFHX1B proteins (also named Smad-interacting protein-1, SIP1) in patients are unknown. Using mass spectrometry analysis, we identified subunits of the NuRD corepressor complex in affinity-purified Zfhx1b complexes. We find that Zfhx1b associates with NuRD through its N-terminal domain, which contains a previously postulated NuRD interacting motif. Interestingly, this motif is substituted by an unrelated sequence in a recently described MWS patient. We show here that such aberrant ZFHX1B protein is unable to recruit NuRD subunits and displays reduced transcriptional repression activity on the XBMP4 gene promoter, a target of Zfhx1b. We further demonstrate that the NuRD component Mi-2 beta is involved in repression of the Zfhx1b target gene E-cadherin as well as in Zfhx1b-induced neural induction in animal caps from Xenopus embryos. Thus, NuRD and Zfhx1b functionally interact, and defective NuRD recruitment by mutant human ZFHX1B can be a MWS-causing mechanism. This is the first study providing mechanistic insight into the aberrant function of a single domain of the multi-domain protein ZFHX1B/SIP1 in human disease.