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Somites sequentially form with a regular interval by the segmentation from the anterior region of the presomitic mesoderm (PSM). The expression of several genes involved in the somite segmentation is switched off at the transition from the anterior PSM to somites. Zebrafish Ripply1, which down-regulates a T-box transcription factor Tbx6, is required for the suppression of segmentation gene expression. However, the functional roles of the Ripply-mediated suppression of segmentation gene expression at the anterior PSM remain elusive. In this study, we generated ripply1 mutants and examined genetic interaction between ripply1/2 and tbx6. Zebrafish ripply1-/- embryos failed to form the somite boundaries as was observed in knockdown embryos. We found that somite segmentation defects in ripply1 mutants were suppressed by heterozygous mutation of tbx6 or partial translational inhibition of tbx6 by antisense morpholino. We further showed that somite boundaries that were recovered in tbx6+/-; ripply1-/- embryos were dependent on the function of ripply2, indicating that relative gene dosage between ripply1/2 and tbx6 plays a critical role in the somite formation. Interestingly, the expression of segmentation genes such mesp as was still not fully suppressed at the anterior PSM of tbx6+/-; ripply1-/- embryos although the somite formation and rostral-caudal polarity of somites were properly established. Furthermore, impaired myogenesis was observed in the segmented somites in tbx6+/-; ripply1-/- embryos. These results revealed that partial suppression of the segmentation gene expression by Ripply is sufficient to establish the rostral-caudal polarity of somites, and that stronger suppression of the segmentation gene expression by Ripply is required for proper myogenesis in zebrafish embryos.

High expression of the B1 Sox genes, Sox2 and Sox3, is associated with the development of definitive neural primordia, the neural plates, in early stage embryos. However, in the caudal lateral epiblast (CLE) where axial stem cells reside, Sox2 and Sox3 are expressed at low levels, together with Brachyury. Because axial stem cells are the bipotential precursors of the neural plate and paraxial mesoderm, we investigated the possibility that low-level B1 Sox expression in CLE may regulate the fate of axial stem cells. We combined the genetic conditions of Sox3-null and Sox2 N1 enhancer homozygous deletion (Sox2(ΔN1/ΔN1)) to decrease B1 Sox expression in CLE. At 5-7 somite stages of mouse embryogenesis, these genetic manipulations caused approximately 30% higher production of paraxial mesodermal precursors, resulting in the development of larger somites. Analysis of mitotic cell populations suggested that decrease of B1 Sox expression in CLE does not activate cell proliferation but promotes cell migration into the mesodermal compartment. Thus, the low-level B1 Sox expression in CLE regulates axial stem cells to adjust the production of paraxial mesoderm precursors to an appropriate level.

International audience; The high mobility group (HMG) proteins constitute a superfamily of nuclear proteins that regulate the expression of a wide range of genes through architectural remodeling of the chromatin structure, and the formation of multiple protein complexes on promoter/enhancer regions, but their function in germ layer specification during early development is not clear. Here we show that hmgb genes regulate mesoderm formation and dorsoventral patterning both in zebrafish and Xenopus early embryos. Overexpression of hmgb3 blocks the expression of the pan-mesoderm gene no tail/Xbra and other ventrolateral mesoderm genes, and results in embryos with shortened anteroposterior axis, while overexpression of hmgb3EnR, which contains the engrailed repressor domain, most potently repressed no tail expression and mesoderm formation. However, hmgb3VP16, which contains the transcriptional activation domain of VP16, had an opposite effect, indicating that hmgb3 may function as a repressor during mesoderm induction and patterning. In addition, we show that hmgb3 inhibits target gene expression downstream of mesoderm-inducing factors. Furthermore, using reporter gene assays in Xenopus whole embryos, we show that hmgb3 differentially regulates the activation of various mesendoderm reporter genes. In particular, it up-regulates the goosecoid, but inhibits the Xbra reporter gene activation. Therefore, our results suggest that hmgb genes may function to fine-tune the specification and/or dorsoventral patterning of mesoderm during zebrafish and Xenopus development.

RNA-binding proteins play an important role to post-transcriptionally regulate gene expression. During early development they exhibit temporally and spatially regulated expression pattern. The expression of Xenopus laevis Seb4 gene, also known as RBM24 in other vertebrates, is restricted to the lateral and ventral mesoderm during gastrulation and then localized to the somitic mesoderm, in a similar pattern as XMyoD gene. Using a hormone-inducible form of MyoD to identify potential direct MyoD target genes, we find that Seb4 expression is directly regulated by MyoD at the gastrula stage. We further show that a 0.65 kb X. tropicalis RBM24 regulatory region contains multiple E boxes (CANNTG), which are potential binding sites for MyoD and other bHLH proteins. By injecting a RBM24 reporter construct into the animal pole of X. laevis embryos, we find that this reporter gene is indeed specifically activated by MyoD and repressed by a dominant negative MyoD mutant. Knockdown of Seb4 produces similar effects as those obtained by the dominant negative MyoD mutant, indicating that it is required for the expression of myogenic genes and myogenesis in the embryo. In cultured ectodermal explants, although overexpression of Seb4 has no obvious effect on myogenesis, knockdown of Seb4 inhibits the expression of myogenic genes and myogenesis induced by MyoD. These results reveal that Seb4 is a target of MyoD during myogenesis and is required for myogenic gene expression.

To study the developmental origin of the pancreas we used DiI crystals to mark regions of the early chick endoderm: this allowed correlations to be established between specific endoderm sites and the positions of their descendants. Endodermal precursor cells for the stomach, pancreas and intestine were found to segregate immediately after completion of gastrulation. Transplantation experiments showed that region-specific endodermal fates are determined sequentially in the order stomach, intestine, and then pancreas. Non-pancreatic endoderm transplanted to the stomach region generated ectopic pancreas expressing both insulin and glucagon. These results imply that a pancreas-inducing signal is emitted from somitic mesoderm underlying the pre-pancreatic region, and this extends rostrally beyond the stomach endoderm region at the early somite stage. Transplantation experiments revealed that the endoderm responding to these pancreatic-inducing signals lies within the pre-pancreatic region and extends caudally beyond the region of the intestinal endoderm. The results indicate that pancreatic fate is determined in the area of overlap between these two regions.

The roles of the FGF family growth factors and their receptors (FGFRs) in zebrafish embryos were examined using variously modified versions of the four FGFR genes (fgfr1-4). Constitutively active forms of all of the examined FGFRs (ca-FGFRs) caused dorsalization, brain caudalization, and secondary axis formation, indicating that the main FGF signal transduction downstream of the receptor is highly similar among FGFRs. All of the membrane-bound type of dominant-negative FGFRs (mdn-FGFRs) derived from the four fgfr genes, which interfere with endogenous FGFRs, produced posterior truncation, as previously reported in both Xenopus and zebrafish. mdn-FGFR3c had the strongest effects on embryos, progressively disrupting the posterior structure as the dose increased. At the highest dose, only the forebrain was formed. At lower doses, mdn-FGFR3c mainly suppressed the paraxial mesoderm. The co-injection of mRNA for different mdn-FGFRs and FGFs resulted in diverse suppression spectra of the respective FGFRs against FGFs. Only mdn-FGFR3c severely suppressed all of the FGFs examined. We also examined the effects of the secretory type of dominant-negative FGFRs (sdn-FGFRs), which are released from cells and trap FGF ligands. Only sdn-FGFR3c resulted in the characteristic effect of selectively disrupting the isthmic development, as well as the tailbud. The co-injection of the mRNA for sdn-FGFRs and FGFs suggested that sdn-FGFR3c inhibits FGFs of the FGF8 subfamily, which is consistent with its specific effects on development. We discuss the implications of our findings obtained in the present study.

Protein O-fucosyltransferase 1 (Pofut1), which catalyzes the addition of O-linked fucose to the EGF domains of the Notch receptor, is indispensable for Notch signaling activation. However, the mechanism of action of Pofut1 in mice is still unclear. Mouse embryos lacking Pofut1 shows defects in valve formation and trabeculation in the cardiovascular system, which are almost identical abnormalities to those of the RBP-Jk mutants. In our current study, we have examined the epistatic relationship between the functions of Pofut1 and activated-Notch1 (NICD1) by taking advantage of the fact that forced expression of NICD1 results in myocardial defects. These defects were still evident in NICD1-expressing embryos irrespective of the presence or absence of Pofut1, which indicates that Pofut1 is required for Notch signaling upstream of NICD1. We further found that Pofut1-null cells do not possess normally localized Notch1 receptors, which may results in their lack of interaction with the Dll1 ligand in the presomitic mesoderm where Notch signaling plays a pivotal role. We propose that altered trafficking pathways may account for the abnormal accumulation of the Notch1 receptor in the endoplasmic reticulum in Pofut1-null mouse embryos.

Bacterial artificial chromosome (BAC) modification technology is a powerful method for the identification of enhancer sequences and genetic modifications. Using this method, we have analyzed the Mesp1 and/or Mesp2 enhancers and identified P1-PSME, a PSM-specific enhancer of Mesp1, which contains a T-box binding site similar to the previously identified P2-PSME. Hence, Mesp1 and Mesp2 use different enhancers for their PSM-specific expression. In addition, we find that these two genes also use distinct enhancers for their early mesodermal expression. Based on these results, we generated a PSM-specific Mesp1/Mesp2-null mouse by introducing a BAC clone, from which only early mesodermal Mesp1 expression is possible, into the Mesp1/Mesp2 double knockout (dKO) genetic background. This successfully rescued gastrulation defects due to the lack of the early mesoderm in the dKO mouse and we thereby obtained a PSM-specific Mesp1/Mesp2-null mouse showing a lack of segmented somites.

Mesoderm represents the evolutionary youngest germ layer and forms numerous novel tissues in bilaterian animals. Despite the established conservation of the gene regulatory networks that drive mesoderm differentiation (e.g. myogenesis), mechanisms of mesoderm specification are highly variable in distant model species. Thus, broader phylogenetic sampling is required to reveal common features of mesoderm formation across bilaterians. Here we focus on a representative of Spiralia, the marine annelid Alitta virens, whose mesoderm development is still poorly investigated on the molecular level. We characterize three novel early mesodermal markers for A. virens - Twist, Mox, and Evx - which are differentially expressed within the mesodermal lineages. The Twist mRNA is ubiquitously distributed in the fertilized egg and exhibits specific expression in endomesodermal- and ectomesodermal-founder cells at gastrulation. Twist is expressed around the blastopore and later in a segmental metameric pattern. We consider this expression to be ancestral, and in support of the enterocoelic hypothesis of mesoderm evolution. We also revealed an early pattern of the MAPK activation in A. virens that is different from the previously reported pattern in spiralians. Inhibition of the MAPK pathway by U0126 disrupts the metameric Twist and Mox expression, indicating an early requirement of the MAPK cascade for proper morphogenesis of endomesodermal tissues.

In vertebrates, the paraxial mesoderm already exhibits a complex Hox gene pattern by the time that segmentation occurs and somites are formed. The anterior boundaries of the Hox genes are always maintained at the same somite number, suggesting coordination between somite formation and Hox expression. To study this interaction, we used morpholinos to knockdown either the somitogenesis gene X-Delta-2 or the complete Hox paralogous group 1 (PG1) in Xenopus laevis. When X-Delta-2 is knocked down, Hox genes from different paralogous groups are downregulated from the beginning of their expression at gastrula stages. This effect is not via the canonical Notch pathway, as it is independent of the Notch effector Su(H). We also reveal for the first time a clear role for Hox genes in somitogenesis, as loss of PG1 gene function results in the perturbation of somite formation and downregulation of the X-Delta-2 expression in the PSM. This effect on X-Delta-2 expression is also observed during neurula stages, before the somites are formed. These results show that somitogenesis and patterning of the anteroposterior axis are closely linked via a feedback loop involving Hox genes and X-Delta-2, suggesting the existence of a coordination mechanism between somite formation and anteroposterior patterning. Such a mechanism is likely to be functional during gastrulation, before the formation of the first pair of somites, as suggested by the early X-Delta-2 regulation of the Hox genes.

The Olfactomedin family is a relatively new class of extracellular proteins. Two family members have been shown to play roles in the early development of ectodermal tissues: Noelin enhances neural crest generation in chick and Tiarin promotes dorsal neural specification in Xenopus. In this study, we introduce a novel member of the Olfactomedin family, ONT1. In the early chick embryo, ONT1 expression first appears at Hensen's node and subsequently in the axial and paraxial mesoderm. When the neural tube closes, strong expression of ONT1 is transiently found in the roof plate region from the rostral midbrain to the hindbrain. Overexpression of ONT1 in these regions prolongs the generation of neural crest cells in a manner similar to that of Noelin. Interestingly, ONT1 and Noelin have opposing effects on the expression of the migrating neural crest marker HNK-1 in the chick: they, respectively, cause suppression and ectopic induction of this marker. Differential activities among Olfactomedin-related factors are further examined in Xenopus. Microinjection of ONT1 mRNA into the Xenopus embryo expands the expression domain of the neural crest marker FoxD3 at the neurula stage whereas overexpression of Tiarin or Noelin suppresses FoxD3. ONT1 exhibits no dorsalizing effects on the Xenopus neural tube, which contrasts with the strong dorsalizing activity seen for Tiarin. Thus, distinct Olfactomedin-related factors evoke qualitatively different phenotypes even in the same experimental systems, suggesting that Olfactomedin family uses multiple response systems to mediate its signals in embryogenesis.

The vertebrate body is built on a metameric organization which consists of a repetition of functionally equivalent units, each comprising a vertebra, its associated muscles, peripheral nerves and blood vessels. This periodic pattern is established during embryogenesis by the somitogenesis process. Somites are generated in a rhythmic fashion from the presomitic mesoderm and they subsequently differentiate to give rise to the vertebrae and skeletal muscles of the body. Somitogenesis has been very actively studied in the chick embryo since the 19th century and many of the landmark experiments that led to our current understanding of the vertebrate segmentation process have been performed in this organism. Somite formation involves an oscillator, the segmentation clock whose periodic signal is converted into the periodic array of somite boundaries by a spacing mechanism relying on a traveling threshold of FGF signaling regressing in concert with body axis extension.

We report here the isolation and characterization of a cDNA encoding zebrafish dermacan, a novel member of hyaluronan (HA)-binding proteoglycans, which was termed after its characteristic expression in the zebrafish dermal bones. The deduced protein sequence shares the typical modular elements of lecticans. Sequence comparison covering the C-terminal globular domain demonstrated that dermacan shows high homology with zebrafish versican but is distinct from any other identified lecticans. Genomic DNA analysis demonstrated that dermacan and versican were encoded by distinct genes in the zebrafish genome. The expression of dermacan is initiated in the sclerotome and cephalic paraxial mesoderm at 16 h postfertilization. During the pharyngular period, dermacan transcripts were detected in the sclerotome, tail fin bud, pharyngular arch primordial region, and otic vesicle. In the development of craniofacial bones, dermacan expression was detected typically in the opercle and dentary. These regions belong to the craniofacial dermal bones. aggrecan expression, in contrast, was observed in the elements of craniofacial cartilage bones. In the dermacan-morpholino-injected embryos, dermal bones, e.g. opercle, dentary, and branchiostegal rays, as well as axial skeleton in the trunk, showed decreased ossification. We conclude that dermacan is a novel lectican gene, and that zebrafish lectican genes have genetically diverged. In addition, our data suggest the involvement of dermacan in zebrafish dermal bone development.

Tbx6 is a member of the T-box family of transcription factor genes. Two mutant alleles of this gene establish that Tbx6 is involved in both the specification and patterning of the somites along the entire length of the embryo. The null allele, Tbx6(tm1Pa), causes abnormal patterning of the cervical somites and improper specification of more posterior paraxial mesoderm, such that it forms ectopic neural tubes. In this study, we use this allele to further investigate the mechanism of action of the Tbx6 gene and investigate possible genetic interactions. We have tested the developmental and differentiation potential of Tbx6(tm1Pa)/Tbx6(tm1Pa) cells in ectopic sites, in vitro, and in chimeras in vivo. We have also documented cell proliferation and cell death in mutant tail buds in an attempt to explain the mechanism of tail bud enlargement in the Tbx6 mutant embryos. Our results indicate specific developmental restrictions on the differentiation of posterior cells lacking Tbx6, once they have traversed the primitive streak, but no restrictions in differentiation of anterior somites, or of Tbx6 null embryonic stem (ES) cells. We further demonstrate that Tbx6 null ES cells fail to populate posterior somites in chimeric embryos. To discover whether different T-box proteins interact on the same down stream targets in areas of expression overlap, we have explored potential interactions between Tbx6 and T (Brachyury) in genetic crosses. Our results reveal that the T(Wis) mutation is epistatic to the Tbx6(tm1Pa) mutation and that there is no apparent genetic interaction. However, homozygosity for Tbx6(tm1Pa) and heterozygosity for T(Wis) mutation shows a combinatorial interaction at the phenotypic level.

The dorsal ectoderm of vertebrate gastrula is first specified into anterior fate by an activation signal and posteriorized by a graded transforming signal, leading to the formation of forebrain, midbrain, hindbrain and spinal cord along the anteroposterior (A-P) axis. Transplanted non-axial mesoderm rather than axial mesoderm has an ability to transform prospective anterior neural tissue into more posterior fates in zebrafish. Wnt8 is a secreted factor that is expressed in non-axial mesoderm. To investigate whether Wnt8 is the neural posteriorizing factor that acts upon neuroectoderm, we first assigned Frizzled 8c and Frizzled 9 to be functional receptors for Wnt8. We then, transplanted non-axial mesoderm into the embryos in which Wnt8 signaling is cell-autonomously blocked by the dominant-negative form of Wnt8 receptors. Non-axial mesodermal transplants in embryos in which Wnt8 signaling is cell-autonomously blocked induced the posterior neural markers as efficiently as in wild-type embryos, suggesting that Wnt8 signaling is not required in neuroectoderm for posteriorization by non-axial mesoderm. Furthermore, Wnt8 signaling, detected by nuclear localization of beta-catenin, was not activated in the posterior neuroectoderm but confined in marginal non-axial mesoderm. Finally, ubiquitous over-expression of Wnt8 does not expand neural ectoderm of posterior character in the absence of mesoderm or Nodal-dependent co-factors. We thus conclude that other factors from non-axial mesoderm may be required for patterning neuroectoderm along the A-P axis.

We report the expression of zebrafish lmo4 during the first 48 h of development. Like its murine ortholog, lmo4 is expressed in somitic mesoderm, branchial arches, otic vesicles, and limb (pectoral fin) buds. In addition, however, we report zebrafish lmo4 expression in the developing eye, cardiovascular tissue, and the neural plate and telencephalon. We demonstrate that expression in the rostral hindbrain requires acerebellar (ace/fgf8) and spiel ohne grenzen (spg/pou2) activity.

Egr1 is a highly conserved zinc finger protein which plays important roles in many aspects of vertebrate development and in the adult. The cDNA coding for zebrafish Egr1 was obtained and its expression pattern was examined during zebrafish embryogenesis using whole-mount in situ hybridization. Egr1 mRNA is first detected in adaxial cells in the presomitic mesoderm between 11 and 20 h post-fertilization (hpf), spanning the 4-24 somite stages. Later, Egr1 expression is observed only in specific brain areas, starting at 21 hpf and subsequently increasing in distinct domains of the central nervous system, e.g. in the telencephalon, diencephalon and hypothalamus. Between 24 and 48 hpf, Egr1 is expressed in specific domains of the hypothalamus, mesencephalon, tegmentum, pharynx, retina, otic vesicle and heart.

We have cloned Xenopus orthologues of the Drosophila planar cell polarity (PCP) gene Prickle. Xenopus Prickle (XPk) is expressed in tissues at the dorsal midline during gastrulation and early neurulation. XPk is later expressed in a segmental pattern in the presomitic mesoderm and then in recently formed somites. XPk is also expressed in the tailbud, pronephric duct, retina, and the otic vesicle. The complex expression pattern of XPk suggests that PCP signaling is used in a diverse array of developmental processes in vertebrate embryos.

Ets2 is a member of the ETS family of transcription factors. In order to address the developmental function of Ets2, we have examined its expression pattern in E8.5 to E13.5 embryos using RNA whole-mount in situ hybridization. In the paraxial mesoderm, Ets2 is expressed uniformly in the presomitic mesoderm and then restricted to the cells in the rostral portion of the segmenting and the next two recently formed somites. In the developing limb, Ets2 expression in the mesenchyme reflects the progressive formation of the hand or foot plate and the digital skeleton. In addition, Ets2 is expressed in the otic vesicle and its derivatives, the dorsal (posterior) root ganglia, the neuroepithelium in the dorsal part of the caudal neural tube and the inter-segmental vasculature.

The development of skeletal muscle in the vertebrate embryo is controlled by a transcriptional cascade that includes the four myogenic regulatory factors Myf-5, MyoD, Myogenin, and MRF4. The dynamic expression pattern of myf-5 during myogenesis is thought to be consistent with its role during early determination of the myogenic lineage. To study the factors and mechanisms, which regulate myf-5 transcription in Xenopus, we isolated a genomic DNA clone containing 4858 bp of Xmyf-5 5' flanking region. Using a transgenic reporter assay, we show here that this genomic contig is sufficient to recapitulate the dynamic stage- and tissue-specific expression pattern of Xmyf-5 from the gastrula to tail bud stages. For the primary induction of myf-5 transcription, we identify three main regulatory elements, which are responsible for (i) activation in dorsal mesoderm, (ii) activation in ventral mesoderm, and (iii) repression in midline mesoderm, respectively. Their combined activities define the two-winged expression domain of myf-5 in the preinvoluted mesoderm. Repression in midline mesoderm is mediated by a single TCF binding site located in the 5' end of the -4.8 kbp sequence, which binds XTcf-3 protein in vitro. Endogenous Wnt signaling in the lateral mesoderm is required to overcome the long-range repression through this distal TCF site, and to stimulate myf-5 transcription independently from it. The element for ventral mesoderm activation responds to Activin. Together, these results describe a regulatory mosaic of repression and activation, which defines the myf-5 expression profile in the frog gastrula.

Members of the T-box (tbx) gene family encode developmentally regulated transcription factors, several of which are implicated in human hereditary diseases. We have cloned the paralogous genes tbx15 and tbx18 in zebrafish and have characterised their expression in detail. tbx15 is expressed in paraxial head mesenchyme and its derivatives, the extraocular and jaw musculature and the posterior neurocranium. Further areas of tbx15 expression are in the anterior somitic mesoderm, in periocular mesenchyme and in the pectoral fin mesenchyme throughout larval development. Areas of strong tbx18 expression are found in the developing somitic and presomitic mesoderm, in the heart and in pectoral fin mesenchyme, as well as the ventral neuroectoderm and the developing palate. Both genes exhibit particular differences in expression compared to their murine orthologs.

DmFoxF is a novel Drosophila fork head domain factor, which is expressed in the visceral mesoderm of the embryo. Our data suggest that DmFoxF is the fly orthologue of the vertebrates FOXF1 and FOXF2 transcription factors. DmFoxF shares homology with FOXF1 and FOXF2 in its fork head domain, and it is able to specifically bind DNA sequences recognized by these vertebrate fork head factors. In stage 10–11 embryos, the DmFoxF protein is detected into the nuclei of cells of the presumptive visceral mesoderm. It localizes at the segmental cell clusters of the mesoderm, which will eventually develop to surround the midgut endoderm. DmFoxF is also expressed in the proctodeal mesoderm, which will develop into the visceral mesoderm of the hindgut.

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.

Activated Notch-Delta signalling was shown to inhibit myogenesis, but whether and how it regulates myogenic gene expression is not clear. We analyzed the implication of Xenopus hairy-1 (Xhairy-1), a member of the hairy and enhancer-of-split (E(spl)) family that may function as nuclear effector of Notch signalling pathway, in regulating XMyoD gene expression at the initial step of myogenesis. Xhairy-1 transcripts are expressed soon after mid-blastula transition and exhibits overlapping expression with Notch pathway genes such as Delta-1 in the posterior somitic mesoderm. We show that overexpression of Xhairy-1 blocks the expression of XMyoD in early gastrula ectodermal cells treated with the mesoderm-inducing factor activin, and in the mesoderm tissues of early embryos. It inhibits myogenesis and produces trunk defects at later stages. Xhairy-1 also inhibits the expression of the pan-mesodermal marker Xbra, but expression of other early mesoderm markers such as goosecoid and chordin is not affected. These effects require the basic helix-loop-helix (bHLH) domain, as well as a synergy between the central Orange domain and the C-terminus WRPW-Groucho-interacting domain. Furthermore, overexpression in ectodermal cells of Xhairy-1/VP16, in which Xhairy-1 repressor domain is replaced by the activator domain of the viral protein VP16, induces the expression of XMyoD in the absence of protein synthesis. Interestingly, Xhairy-1/VP16 does not induce the expression of Xbra and XMyf5 in the same condition. During neurulation, the expression of XMyoD induced by Xhairy-1/VP16 declines and the expression of muscle actin gene was never detected. These results suggest that Notch signalling through hairy-related genes may specifically regulate XMyoD expression at the initial step of myogenesis in vertebrates.

Mesp1 and Mesp2 encode bHLH-type transcription factors, Mesp1 and Mesp2, respectively. The expression of both genes is observed in the nascent mesoderm, and subsequently in the rostral presomitic mesoderm. To determine the regulatory mechanism for gene expression, we attempted to identify enhancer elements by transient transgenic analysis. At least two enhancers, which are responsible for the expression of the two genes in the early mesoderm (early mesodermal enhancer, EME) and the presomitic mesoderm (PSM enhancer, PSME), and one suppressor, which is responsible for the rostrally restricted expression in the presomitic mesoderm, were identified. Deletion studies of these enhancer elements indicate that either gene may use the same enhancer for early mesoderm development, whereas both genes may utilize separate enhancers to regulate their expression in the presomitic mesoderm.

The expression pattern of the receptor tyrosine kinase gene EphB3 was examined during the early stages of chick embryogenesis, and is described in this report. In the gastrula, EphB3 is expressed in epiblast cells adjacent to and entering the anterior portion of the primitive streak; expression is extinguished once cells have ingressed. At headfold stages, EphB3 is strongly transcribed in the floor of the foregut and in anterior lateral endoderm, and is expressed in the subjacent cardiogenic mesoderm. EphB3 is transiently expressed in the lateral ectoderm, neural tube, and neural crest during these stages. Later neural expression is localized to the mesencephalon. In the somitic mesoderm, EphB3 is initially expressed in the sclerotome, but later is expressed predominantly in the dermatome. Prominent expression is also detected in the developing heart, liver, posterior ventral limb bud mesenchyme, pharyngeal arches, and head mesenchyme.

We have identified a novel frizzled gene in zebrafish, (Danio rerio): frizzled 7, highly related to mouse, chick and Xenopus fz7. No maternal expression was detected. Zygotic transcription starts at the end of gastrulation anteriorly in the presumptive neurectoderm and in the presomitic mesoderm. During somitogenesis expression is detected in developing central nervous system, including forebrain, midbrain, hindbrain and spinal cord, the lateral mesoderm and the anterior part of the forming somites. The strong sequence conservation of fz7 to the mouse, chick and Xenopus counterparts is also true for their expression pattern.

Mouse Foxc1 (previously Mf1) is a member of the conserved forkhead/winged helix transcription factor gene family. It is expressed in many mesodermal tissues including paraxial mesoderm of the trunk and head, prechondrogenic mesenchyme, branchial arches and developing kidney. Homozygous mutants die perinatally with hydrocephalus and skeletal, cardiovascular, ocular and genitourinary defects. Here, we report the cloning and expression of two zebrafish foxc1 homologues, foxc1a and foxc1b. During gastrulation and somitogenesis both genes have similar expression patterns in the hypoblast, paraxial and presomitic mesoderm, somites and trunk adaxial cells. Expression in the somites is downregulated as they differentiate, but is maintained in the sclerotome. Later, some differences in expression pattern emerge. For example, only foxc1a transcripts are detected in the pronephros primodia and in the head mesoderm around the eyes, while only foxc1b is expressed in the pharyngeal arches and pectoral fins. Early expression of foxc1a in the paraxial mesoderm is modified in chordino, swirl, somitabun, and spadetail mutants.

The homeobox gene Six4/AREC3 is a member of the vertebrate Six family of transcription factor genes. In this study we describe the cloning and expression of three zebrafish homologues of Six4/AREC3, six4.1, six4.2 and six4.3. These zebrafish Six4 proteins show high homology to the mammalian Six4/AREC3 proteins in the most C-terminal region in addition to the Six domain and homeodomain. Whole-mount in situ hybridization showed that six4.1 is expressed in the presumptive cranial placodal precursor cells, and later in the olfactory, otic and lateral line placodes. six4.2 is expressed in the presomitic mesoderm, somites and pectoral fin bud. six4.3 appears to be a unique member of the Six4 proteins and is expressed ubiquitously at gastrulation and later in the tectum.

We have identified a novel chicken gene, cMespo, which encodes a basic-helix-loop-helix (bHLH) protein with sequence homology to a subgroup of bHLH transcription factors that have been implicated in somitogenesis. cMespo transcripts are first found in the primitive streak of gastrulating chick embryos (HH stage 4) and continue to accumulate in presomitic mesoderm (PSM) until somite formation has been concluded. cMespo, however, is not expressed within somites or in tailbud mesoderm. The expression domain of cMespo in PSM largely overlaps with delta-1 but spares a region of several prospective somites at the rostral end of PSM in which c-Meso and Cek-8 are expressed.

Fibroblast growth factor homologous factors (FHFs) have been implicated in limb and nervous system development. In this paper we describe the expression of the cFHF-4 gene during early chicken development. cFHF-4 is expressed in the paraxial mesoderm, lateral ridge, and, most prominently, in the posterior-dorsal side of the base of each limb bud. The expression pattern of cFHF-4 at the base of the limbs is not altered by tissue grafts containing the zone of polarizing activity (ZPA), by implants of Shh-expressing cells, or by implants of beads containing retinoic acid, nor does it depend on the distal growth of the limb as it is not altered in limb buds that are surgically truncated. In three chicken mutants affecting limb patterning – talpid2, limbless, and wingless – altered patterns of cFHF-4 expression are correlated with abnormal nerve plexus formation and altered patterns of limb bud innervation. Similarly, ectopic expression of cFHF-4 is correlated with a local induction of limb-like innervation patterns when beads containing FGF-2 are implanted in the flank. In these experiments, both ectopic innervation and ectopic expression of cFHF-4 in the flank were observed regardless of the size of the FGF-2-induced outgrowths. By contrast, ectopic expression of Shh and HoxD13 are seen only in the larger FGF-2-induced outgrowths. Taken together, these data suggest that cFHF-4 regulates or is coregulated with early events related to innervation at the base of the limbs.

Glycogen synthase kinase 3 (GSK-3) belongs to a highly conserved family of protein serine/threonine kinase whose members in high eukaryotes are involved in hormonal regulation, nuclear signaling, and cell fate determination. We have identified two zebrafish homologues related to mammalian GSK-3, ZGSK-3alpha and ZGSK-3beta. ZGSK-3alpha was expressed uniformly from cleavage onward, and later was found in many but not all tissues, especially in the central nervous system, spinal cord, somites and pronephric ducts. ZGSK-3beta was also transcribed maternally but the transcripts were not uniformly distributed during early cleavage stage. Most signals were concentrated in the inner part of the blastomeres. From midblastula stage onward, the ZGSK-3beta transcripts remained confined to inner parts of the deep cell layer. During shield stage, both epiblast and hypoblast expressed the transcripts. After late gastrulation, the signals were detected ubiquitously. During segmentation, prominent ZGSK-3beta signal was detected in head portion of the neural system. In the trunk, the expression was maintained in the neural tube and paraxial mesoderm and then became prominent in adaxial cells, followed by expression at the posterior region of somites. In pharyngula period ZGSK-3beta transcripts were distributed in similar regions as those of ZGSK-3alpha, namely, neural tissues of the head portion, spinal cord and somites.

The paraxial mesoderm of the neck and trunk of mouse embryos undergoes extensive morphogenesis in forming somites. Paraxial mesoderm is divided into segments, it elongates along its anterior posterior axis, and its cells organize into epithelia. Experiments were performed to determine if these processes are autonomous to the mesoderm that gives rise to the somites. Presomitic mesoderm at the tailbud stage was cultured in the presence and absence of its adjacent tissues. Somite segmentation occurred in the absence of neural tube, notochord, gut and surface ectoderm, and occurred in posterior fragments in the absence of anterior presomitic mesoderm. Mesodermal expression of Dll1 and Notch1, genes with roles in segmentation, was largely independent of other tissues, consistent with autonomous segmentation. However, surface ectoderm was found to be necessary for elongation of the mesoderm along the anterior-posterior axis and for somite epithelialization. To determine if there is specificity in the interaction between ectoderm and mesoderm, ectoderm from different sources was recombined with presomitic mesoderm. Surface ectoderm from only certain parts of the embryo supported somite epithelialization and elongation. Somite epithelialization induced by ectoderm was correlated with expression of the basic-helix-loop-helix gene Paraxis in the mesoderm. This is consistent with the genetically defined requirement for Paraxis in somite epithelialization. However, trunk ectoderm was able to induce somite epithelialization in the absence of strong Paraxis expression. We conclude that somitogenesis consists of autonomous segmentation patterned by Notch signaling and nonautonomous induction of elongation and epithelialization by surface ectoderm.

Four genes – deltaA, deltaB, deltaC and deltaD – coding for homologues of the Notch ligand Delta have been discovered in zebrafish ( Haddon et al., 1998b ). We report here the cDNA sequence and expression pattern of deltaC. Its closest relatives are deltaB and Xenopus X-Delta-2. Unlike deltaA, deltaB, and deltaD, deltaC is not expressed in the majority of nascent primary neurons; but it is strongly expressed in the early retina, where it precedes other delta genes. It is also expressed in cranial ganglia, in sensory epithelia including ear and lateral line, and in scattered epidermal cells. In the mesoderm, expression is visible by 50% epiboly; it is seen subsequently in the tail bud, in stripes in the presomitic mesoderm and in the posterior half of each somite. There is expression also in notochord, blood vessels and pronephros.

mab-21 has been identified as a critical component required for sensory organ identity establishment in Caenorhabditis elegans. [Chow, K.L., Emmons, S.W., 1994. Development 120, 2579–2592; Chow, E.L., Hall, D.H., Emmons, S.W., 1995. Development 121, 3615-3625]. Human and mouse homologs of this gene have been isolated and their transcripts are predominantly detected in the eye and cerebellum [Margolis, R.L., Stine, O.C., McInnis, M.G., et al., 1996. Hum. Mol. Genet 5, 607–616; Mariani, M., Corradi, A., Baldessari, D., et al., 1998. Mech. Dev. 79, 131–135. We report here the expression profile of a second murine mab-21 homolog, Mab21l2 [Wong, R.L.Y., Wong, H.T., Chow, K.L., 1999. Cyto. Cell Genet., [in press]. Whole mount in situ hybridization data from embryonic day 8.5 to day 15 revealed that Mab21l2 expression patterns partially overlapped with that of Mab21l1. In addition, its strong expression in the mid- and hindbrain, otic vesicle, optic vesicle, maxillary and mandibular process, paraxial mesoderm, dorsal midline, limb bud and developing digits suggest that Mab21l2 has more diverse functions in vertebrate development.

We report here the expression pattern of Nkx3.1, a murine homolog of Drosophila bagpipe, in different stages of embryos and in neonates. Nkx3.1 was expressed in paraxial mesoderm at day 7.5 p.c., in the somite by day 8.0 p.c. and in a subset of vascular smooth muscle cells by day 9.5 p.c. In later stage embryos and neonates, Nkx3.1 was expressed in subpopulations of epithelial cells, particularly in epithelial outgrowths. Nkx3.1 was also expressed in restricted regions of the central nervous system.

We describe the expression pattern of cSix4, a chick homologue of the murine Six4/AREC3 gene. cSix4 transcripts are detected at gastrula stages in the blastoderm surrounding the developing axial midline. As the neural plate begins to form cSix4 mRNA is detected in a crescent-shaped band, which surrounds the anterior developing neural plate and corresponds to the presumptive placode region. This expression is maintained in all the placodes (olfactory, optic, neural and otic) as they develop but with different characteristics. Further, abundant expression of cSix4 was localised to the paraxial mesoderm and the entire developing somites, becoming restricted first to their dorsal portion, then to the dermomyotome and finally to the myotome. cSix4 expression is maintained in the developing and adult muscular tissue. Additional sites of cSix4 expression are the presumptive and developing limb buds, the notochord, trigeminal ganglia, cells of the spinal cord, particularly the motor neurones, the dorsal root ganglia, the neural retina, as well as the epithelial component of the developing kidney.

We describe a novel chick WD-protein, cSWiP-1, expressed in somitic mesoderm and developing limb buds as well as in other embryonic structures where Hedgehog signalling has been shown to play a role. Using embryonic manipulations we show that in somites cSWiP-1 expression integrates two signals originating from structures adjacent to the segmental mesoderm: a positive signal from the notochord and a negative signal from intermediate and/or lateral mesoderm. In explant cultures of somitic mesoderm, Shh protein induces cSWiP-1, while a blocking antibody to Shh inhibits the induction of cSWiP-1 by the notochord. These results show that the positive signal from the notochord is mediated by Shh. We also show that in limb buds cSWiP-1 is upregulated by ectopic Shh. This occurs in about the same time period as upregulation of BMP2, placing cSWiP-1 among the earliest markers for the change of limb pattern caused by ectopic Shh. We also describe a human homologue of cSWiP-1 and a mouse gene, mSWiP-2, that is more distantly related to SWiP-1, suggesting that SWiP-1 belongs to a novel subfamily of WD-proteins.

We have isolated a novel gene from Xenopus, called Mespo, which encodes a protein containing a basic helix-loop-helix (bHLH) motif characteristic of a family of transcriptional activators. Mespo expression begins at the gastrula stage and continues throughout tailbud stages; expression occurs in the presomitic mesoderm and the posterior tailbud. Mespo has high similarity to a subfamily of bHLH transcription factors involved in segmentation of the presomitic paraxial mesoderm.

Signaling molecules belonging to the Fibroblast growth factor (Fgf) family are necessary for directing bud outgrowth during tracheal development in Drosophila and lung development in mouse. A potential inhibitor of the Fgf signaling pathway, called Sprouty, has been identified in Drosophila. We have identified three potential mouse homologues ofsprouty. One of them, called Sprouty4, exhibits a very restricted expression pattern. At 8.0 dpc (days post coitum) Sprouty4 is strongly expressed in the primitive streak region. At 9.5 and 10.5 dpc, Sprouty4 is expressed in the nasal placode, the maxillary and mandibular processes, the otic vesicule, the second branchial arch, in the progress region of the limb buds and the presomitic mesoderm. Sprouty4 expression is also detected in the lateral region of the somites. In the developing lung, Sprouty4 is expressed broadly in the distal mesenchyme. © 1999 Elsevier Science Ireland Ltd. All rights reserved.

The formation of somites involves the subdivision of segmented presomitic mesoderm into segmentally arranged somite blocks. In mice and chicks, the basic-Helix-Loop-Helix (bHLH) gene, paraxis, is involved in this process. Here, we report the isolation of a zebrafish homologue of paraxis, par1. par1 is expressed in presomitic paraxial mesoderm from late gastrula stages, and expression is maintained in ventrolateral cells after somite formation. In spt− embryos, par1 expression is both delayed and severely reduced whereas in flh− embryos, ectopic transcripts are detected in axial mesoderm. Spatial regulation of par1 expression within the somites is affected in several mutants with defects in axial midline tissues.

We have examined the expression pattern of the GBX2 gene during chicken embryogenesis. First transcripts are found in the epiblast of a HH st. 3+ embryo. With the onset of neurogenesis, transcripts mark the posterior neuroectoderm. Later on, expression is detectable in the isthmic region, the hindbrain and the neural tube. We show that GBX2 transcripts, as well as the protein, mark the presumptive hindbrain region. After establishment of the brain vesicles GBX2 transcripts were also detected in distinct domains of the diencephalon. In addition to neural sites of expression, GBX2 was found in several domains including the otic vesicle, the somitic mesoderm, the lateral foregut endoderm, the ventral limb bud ectoderm and in the feather buds.

Gene knock-out and knock-in strategies are employed to investigate the function of MesP1. MesP1 belongs to the same family of bHLH transcription factors as MesP2. The early expression pattern observed in the early mesoderm at the onset of gastrulation is restricted to Mesp1, while the later expression pattern in the anterior presomitic mesoderm during somitogenesis is almost the same for Mesp1 as for Mesp2. Homozygous Mesp1 null mice exhibited growth retardation after 7.5 dpc and died before 10.5 dpc with many developmental defects. The function of MesP1 during somitogenesis was not clearly revealed because of their early death and the possible compensation by MesP2. In order to examine the functions of MesP1 during somitogenesis, we replaced the Mesp2 gene with Mesp1 cDNA, using a gene knock-in strategy. The introduced Mesp1 cDNA could rescue the defects caused by Mesp2 deficiency in a dosage-dependent manner. Mice which lacked Mesp2 expression but had four copies of the Mesp1 gene survived into the adulthood and were fertile. The skeletal defects and the reduction in expression of Notch1, Notch2 and FGFR-1 previously observed in Mesp2 null mice were almost completely rescued by the introduced MesP1. Thus, it is concluded that the functions of MesP1 during somitogenesis, like MesP2, are also mediated via notch-delta and FGF signaling systems.