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We used cDNA microarray analysis to screen for FGF target genes in Xenopus embryos treated with the FGFR1 inhibitor SU5402, and identified neurotrophin receptor homolog (NRH) as an FGF target. Causing gain of NRH function by NRH mRNA or loss of NRH function using a Morpholino antisense-oligonucleotide (Mo) led to gastrulation defects without affecting mesoderm differentiation. Depletion of NRH by the Mo perturbed the polarization of cells in the dorsal marginal zone (DMZ), thereby inhibiting the intercalation of the cells during convergent extension as well as the filopodia formation on DMZ cells. Deletion analysis showed that the carboxyl-terminal region of NRH, which includes the 'death domain,' was necessary and sufficient to rescue gastrulation defects and to induce the protrusive cell morphology. Furthermore, we found that the FGF signal was both capable of inducing filopodia in animal cap cells, where they do not normally form, and necessary for filopodia formation in DMZ cells. Finally, we demonstrated that FGF required NRH function to induce normal DMZ cell morphology. This study is the first to identify an in vivo role for FGF in the regulation of cell morphology, and we have linked this function to the control of gastrulation cell movements via NRH. Keywords: FGF signaling; Neurotrophin receptor homolog; Gastrulation; Convergent extension; Cell morphology; Xenopus

A mouse homolog of the Drosophila pair-rule gene Odd Oz (Odz4) maps to the critical region of the l7Rn3 locus on mouse chromosome 7. Here we show that Odz4 is an excellent candidate for this allelic series because (1) it spans the entire critical region, (2) the phenotypes correlate with embryonic expression, (3) the complex genetic inheritance of the alleles is consistent with complex transcriptional regulation, and (4) one allele has a mutation in a conserved amino acid. Odz4 uses five alternate promoters that encode both secreted and membrane-bound proteins. Intragenic complementation of the l7Rn3 alleles is consistent with these multiple-protein isoforms. Further, the allelic series shows that Odz4 is required to establish the anterior-posterior axis of the gastrulating mouse embryo and is necessary later for mesoderm-derived tissues such as somites, heart, and skeleton. Sequencing of RT-PCR products from five of the six alleles reveals a nonconservative amino acid change in the [l7Rn3.sup.m4] allele. This amino acid is important evolutionarily, as it is conserved to Drosophila. Together, our data indicate that Odz4 is mutated in the l7Rn3 allele series and performs roles in the mouse brain, heart, and embryonic patterning similar to those of its Drosophila counterpart.

In order to gain insights into the evolution of gastrulation mechanisms among vertebrates, we have characterized a Brachyury-related gene in a lamprey, Lampetra fluviatilis, and in a chondrichthyan, Scyliorhinus canicula. These two genes, respectively termed LfT and ScT, share with their osteichthyan counterparts prominent expression sites in the developing notochord, the tailbud, but also a transient expression in the prechordal plate, which is likely to be ancestral among vertebrates. In addition, the lamprey LfT gene is transcribed in the endoderm of the pharyngeal arches and the epiphysis, two expression sites that have not been reported thus far in gnathostomes, and, as in the chick, in the differentiating nephrotomes. Since Brachyury expression in nascent mesoderm and endoderm is highly conserved among vertebrates as well as cephalochordates, we have used this marker to identify these cell populations during gastrulation in the dogfish. The results suggest that these cells are initially present over the whole margin of the blastoderm and are displaced during gastrulation to its posterior part, which may correspond to the site of mesoderm and endoderm internalization. These data provide the first molecular characterization of gastrulation in a chondrichthyan. They indicate that gastrulation in the dogfish and in some amniotes shares striking similarities despite the phylogenetic distance between these species. This supports the hypothesis that the extensively divergent morphologies of gastrulae among vertebrates largely result from adaptations to the presence of yolk. Keywords: Brachyury; Lamprey; Dogfish; Evolution of gastrulation; Yolk; Prechordal mesoderm

Research has been conductedcon Xenopus gastrulation. The role of Wnt/Frizzled in Rac and Rho activation during this gastrulation has been investigated, and the results demonstrate that parallel activation of Rho and Rac underlines Wnt regulation of vertebrate morphogenesis.

We cloned Xenopus Strabismus (Xstbm), a homologue of the Drosophila planar cell or tissue polarity gene. Xstbm encodes four transmembrane domains in its N-terminal half and a PDZ-binding motif in its C-terminal region, a structure similar to Drosophila and mouse homologues. Xstbm is expressed strongly in the deep cells of the anterior neural plate and at lower levels in the posterior notochordal and neural regions during convergent extension. Overexpression of Xstbm inhibits convergent extension of mesodermal and neural tissues, as well as neural tube closure, without direct effects on tissue differentiation. Expression of Xstbm([DELTA]PDZ-B), which lacks the PDZ-binding region of Xstbm, inhibits convergent extension when expressed alone but rescues the effect of overexpressing Xstbm, suggesting that Xstbm([DELTA]PDZ-B) acts as a dominant negative and that both increase and decrease of Xstbm function from an optimum retards convergence and extension. Recordings show that cells expressing Xstbm or Xstbm([DELTA]PDZ-B) fail to acquire the polarized protrusive activity underlying normal cell intercalation during convergent extension of both mesodermal and neural and that this effect is population size-dependent. These results further characterize the role of Xstbm in regulating the cell polarity driving convergence and extension in Xenopus. Key Words: gastrulation; neurulation; convergence; extension; polarity; strabismus; Van Gogh; motility; PDZ-B; Xenopus.

The levels of genomic DNA methylation in vertebrate species display a wide range of developmental dynamics. Here, we show that in contrast to mice, the paternal genome of the amphibian, Xenopus laevis, is not subjected to active demethylation of 5-methyl cytosine immediately after fertilization. High levels of methylation in the DNA of both oocyte and sperm are maintained in the early embryo but progressively decline during the cleavage stages. As a result, the Xenopus genome has its lowest methylation content at the midblastula transition (MBT) and during subsequent gastrulation. Between blastula and gastrula stages, we detect a loss of methylation at individual Xenopus gene promoters (TFIIIA, Xbra, and c-Myc II) that are activated at MBT. No changes are observed in the methylation patterns of repeated sequences, genes that are inactive at MBT, or in the coding regions of individual genes. In embryos that are depleted of the maintenance methyltransferase enzyme (xDnmt1), these developmentally programmed changes in promoter methylation are disrupted, which may account for the altered patterns of gene expression that occur in these embryos. Our results suggest that DNA methylation has a role in regulating the timing of gene activation at MBT in Xenopus laevis embryos. Key Words: Xenopus; development; demethylation; xDnmt1.

Unipolar ingression is a common form of gastrulation in the phylum Cnidaria. Here, this process is examined in the hydrozoan Phialidium. Ultrastructural investigation confirmed that, during this process, oral cells elongate, constricting at their apical ends and/or expanding at their basal ends as they ingress. Marking studies show that most entodermal cells originate from the oral area, but a few also come from lateral, aboral-lateral, and aboral areas. Based on orientation of mitotic spindles during gastrulation, a few cells may also enter the entodermal region by delamination. Cell sorting experiments showed that, by postgastrulation, cells from aboral areas of the preingression embryo developed lower adhesive capacities than those from oral regions. Differences in adhesive capacity may allow maintenance of distinct cell layers. Postgastrulae are shorter, thinner, and more streamlined than preingression embryos. Total embryo volume and cell volume decrease, the blastocoel disappears, and 34.4% of the cell mass becomes entoderm. Activities of the ectodermal layer play an important role in gastrulation: during gastrulation, aboral cells become more columnar and oral cells less columnar. Also, cells in oral and oral-lateral regions shift toward the midline, causing oral portions of the embryo to elongate. A model of unipolar ingression is proposed. Key Words: gastrulation; unipolar; ingression; delamination; cell sorting; morphogenesis; Phialidium; Hydrozoan; Cnidarian.

Hypersensitivity to DNA damage leads to increased apoptosis during early mouse development. Greater apoptosis in early mouse development results from hypersensitivity to DNA damage. The hypersensitivity is a cell fate-dependent mechanism to ensure genome integrity in a period of very great proliferation and differentiation seen in gastrulation in mice.

A hairy-related transcriptional repressor and its coregulation of anterior and posterior mesendodermal development are discussed. Recent research has found the first model for vertebrate endoderm patterning where an early regulatory step at gastrulation, mediated by her5 controls cell contribution to the two regions jointly.

Transposition of the anterior Hoxb1 gene into the posterior HoxD complex is discussed relative to mechanisms of Hox gene colinearity. To gain insight into the ways down-regulation seen in transgene relocation in the posterior HoxD complex and to better understand the mechanisms behind colinearity, transposing the anterior Hoxb1 transgene to the back end of HoxD was undertaken. Strong repression expected was not seen in the early mesoderm domain in which the transgene was transcribed in a way like that of its endogenous counterpart. A repressive mechanism may not be implemented at the same level in all tissues. Position in the gene in a complex is not responsible entirely for time of activation.

Disruption of Fgf8, a targeted disruption which causes failure of cell migration in the gastrulating mouse embryo, is discussed. Fgf8 is a gene shown to be required for gastrulation and it has been shown that FGF8 and/or FGF4 signaling is required for cell migration away from the primitive streak.

The Wnt-inducible homeobox gene Siamois is expressed in Xenopus embryos before gastrulation and is necessary for formation of the Spemann organizer. Here we show that 5[prime]-flanking sequences of the Siamois coding region can specifically activate a heterologous reporter gene in dorsovegetal cells, thus mimicking Siamois's endogenous expression. A 245-bp DNA fragment is sufficient for activation by both Wnts and endogenous inducers. A dominant negative form of Xenopus T cell-specific factor 3 (XTCF-3) inhibited promoter activity, indicating that T cell-specific factor (TCF)/lymphocyte enhancer binding factor 1 (LEF-1) signaling is necessary for regulation of Siamois. Mutagenesis of two individual TCF sites in the -245 promoter revealed that the proximal, but not distal, site is necessary for dorsovegetal activation. These observations suggest that Siamois is directly regulated by TCFs during dorsoventral axis determination. Further deletion analysis identified a positive regulatory region that is required for dorsal activation, but not for Wnt inducibility, of the promoter. We also present evidence for autoregulation of Siamois transcription. Furthermore, the Siamois promoter was activated by Wnt signaling in 293T tissue culture cells, demonstrating that regulation of the promoter is functionally conserved.

ActRIB, a type I transmembrane serine-threonine kinase receptor, is necessary for gastrulation and egg cylinder organization in mice and seems to mediate signals necessary for those developments. ActRIB-deficient ES cell lines and mice were generated by targeting of genes. The epiblast and the extraembryonic ectoderm were disorganized in the ActRIB-/- embryos that resulted. The result was disruption and arresting of the development of the egg cylinder before gastrulation. Chimera analysis was carried out to find the function of ActRIB in mesoderm formation and gastrulation.

DRhoGEF2 has been identified as a gene that is crucial in controlling cell shape changes during gastrulation by encoding a putative guanine nucleotide exchange factor. Mesodermal and endodermal primordia are unable to invaginate without maternal DRhoGEF2 genes. The gene appears to act independent of other genes in the signalling pathway.

The sec8 gene, isolated in a gene trap screen in embryonic stem cells, is required for paraxial mesoderm formation in the mouse. Homozygous sec8 mutant embryos initiate gastrulation but are unable to progress beyond the primitive streak stage and die shortly afterward. The genomic locus and cDNA of the sec8 gene have been cloned. An open reading frame in the cDNA encodes a 971-amino-acid leucine-rich protein, similar to rat rSec8. A description of the mutant phenotype and the cloning of the gene is presented here and the results are considered in light of the possibility that the Sec8 protein is involved in secretion.

Wnt-3a mutant embryos show defects caudal to the forelimb level; somites are absent, the notochord is disrupted, and the central nervous system has a pronounced dysmorphology. Previous studies revealed that the primary defects of the mutant embryos are likely to be in the process of paraxial mesoderm formation. In this study, we analyzed the phenotype of Wnt-3a mutant embryos at early somite stages (8.0 days post coitum), when somite formation is initiated. In Wnt-3a mutants, cells which have ingressed through the primitive streak do not migrate laterally but remain under the streak and form an ectopic tubular structure. Several neural-specific molecular markers, but no paraxial mesoderm markers, are expressed in this structure, suggesting that the ectopic tube is an additional neural tube. In normal embryos, Wnt-3a is expressed in the primitive ectoderm, including the cells which are fated to give rise to the paraxial mesoderm and neurectoderm, but expression is absent in migrating mesoderm cells. These results suggest that Wnt-3a signaling may play a role in regulating paraxial mesodermal fates, at the expense of neurectodermal fates, within the primitive ectoderm of the gastrulating mouse embryo.

Communication via gap junctions provides a mechanism for the cell-cell transfer and coordination of developmental signals. The spatial restriction of gap junctions may also serve to organize cells into domains of coordinated behavior. To investigate the role of gap junctions during embryogenesis, we have characterized the expression of a member of the gap junction gene family, zebrafish connexin43.4, a homolog of connexin45 in chicken and mammals. Expression of connexin43.4 was induced in the early gastrula, coincident with the first definitive assignments of axial cell fate and the onset of the cell movements comprising convergence and extension in zebrafish. In situ hybridization and immunohistochemistry revealed that during gastrulation connexin43.4 mRNA and protein were progressively enriched in the germ ring and in the notochord primordia on the dorsal side of the embryo. Later in development connexin43.4 expression was detected in the notochord, the paraxial mesoderm, and the tail bud but was not observed after the differentiation of these tissues. In no tail mutant embryos which are defective in tail formation and proper morphogenesis of the notochord, connexin43.4 expression was absent during gastrulation from the caudal embryonic shield and notochord primordia. During somite stages in no tail embryos, connexin43.4 expression remained absent in the notochordal precursor cells and was lost in the tail bud. Thus, the no tail gene product, a transcription factor, was required for the expression of connexin43.4 in both the notochord and tail bud during morphogenesis. By microinjection of mRNA coding for a connexin43.4/green fluorescent protein fusion in the 1-cell zebrafish embryo, we showed that connexin43.4 is capable of assembling into structures reminiscent of gap junctions. The progressively restricted, developmental expression of the zebrafish connexin43.4 gene suggests that this gap junctional protein participates in the coordination of gastrulation and the formation of the notochord and tail.

We report the identification of a new recessive prenatal lethal insertional mutation, amnionless (amn). amn mutant embryos first appear abnormal during the Early Streak stage, between E6.5 and E7.0, when they initiate mesoderm production. Subsequently, the amn mutants become developmentally arrested between the Mid and Late Streak stages of gastrulation and they die and are resorbed between E9.5 and E10.5. While extraembryonic structures, including the chorion, yolk sac blood islands, and allantois appear to develop normally, the small embryonic ectoderm remains undifferentiated and generates no amnion. In addition, the embryonic mesoderm that is produced does not become organized into node, notochord, and somites and there is no morphological evidence of neural induction. Interspecific backcross and fluorescence in situ hybridization analyses map the transgene insertion, and thus the amn mutation, to the distal region of mouse chromosome 12, which has synteny with human chromosome 14q32. A gene encoding a 7.5-kb transcript has been identified at a junction between the integrated transgene and host chromosome 12 sequences that meets three criteria expected of a candidate amn gene. This gene maps to the site of transgene insertion; it is transcribed during gastrulation, and its expression is disrupted in amn mutant embryos. Nucleotide sequencing studies show that the 567 amino acid protein encoded by the 7.5-kb transcript is a member of the newly defined family of putative signal transducing proteins, TRAFs, that associate with the cytoplasmic domains of members of the tumor necrosis factor (TNF) receptor superfamily. Thus, we have named the gene encoding the 7.5-kb transcript TRAFamn. TRAFamn is identical to a recently reported protein (CD40bp, CAP-1, CRAF1, LAP1) that can bind the cytoplasmic domains of CD40 and the lymphotoxin [Beta] receptor (LT[Beta]R), both of which are known members of the TNF receptor superfamily. The implications of these findings regarding a possible role for the TNF receptor superfamily during gastrulation are discussed.

The Xenopus developmental gene DG42 is expressed during early embryonic development, between the midblastula and neurulation stages. The deduced protein sequence of Xenopus DG42 shows similarity to Rhizobium Nod C, Streptococcus Has A, and fungal chitin synthases. Previously, we found that the DG42 protein made in an in vitro transcription/translation system catalyzed synthesis of an array of chitin oligosaccharides. Here we show that cell extracts from early Xenopus and zebrafish embryos also synthesize chitooligosaccharides. cDNA fragments homologous to DG42 from zebrafish and mouse were also cloned and sequenced. Expression of these homologs was similar to that described for Xenopus based on Northern and Western blot analysis. The Xenopus anti-DG42 antibody recognized a 63-kDa protein in extracts from zebrafish embryos that followed a similar developmental expression pattern to that previously described for Xenopus. The chitin oligosaccharide synthase activity found in extracts was inactivated by a specific DG42 antibody; synthesis of hyaluronic acid (HA) was not affected under the conditions tested. Other experiments demonstrate that expression of DG42 under plasmid control in mouse 3T3 cells gives rise to chitooligosaccharide synthase activity without an increase in HA synthase level. A possible relationship between our results and those of other investigators, which show stimulation of HA synthesis by DG42 in mammalian cell culture systems, is provided by structural analyses to be published elsewhere that suggest that chitin oligosaccharides are present at the reducing ends of HA chains. Since in at least one vertebrate system hyaluronic acid formation can be inhibited by a pure chitinase, it seems possible that chitin oligosaccharides serve as primers for hyaluronic acid synthesis.

DG42 is one of the main mRNAs expressed during gastrulation in embryos of Xenopus laevis. Here we demonstrate that cells expressing this mRNA synthesize hyaluronan. The cloned DG42 cDNA was expressed in rabbit kidney (RK13) and human osteosarcoma ([tk.sup.-]) cells using a vaccinia virus system. Lysates prepared from infected cells were incubated in the presence of UDP-N-acetylglucosamine and UDP-[14C]glucuronic acid. This yielded a glycosaminoglycan with a molecular mass of about 200,000 Da. Formation of this product was only observed in the presence of both substrates. The glycosaminoglycan could be digested with testicular hyaluronidase and with Streptomyces hyaluronate lyase but not with Serratia chitinase. Hyaluronan synthase activity could also be detected in homogenates of early Xenopus embryos, and the activity was found to correlate with the expression of DG42 mRNA at different stages of development. Synthesis of hyaluronan is thus an early event after midblastula transition, indicating its importance for the ensuing cell movements in the developing embryo. Our results are at variance with a recent report (Semino, C. E. & Robbins, P. W. (1995) Proc. Natl. Acad. Sci. USA 92, 3498-3501) that DG42 codes for an enzyme that catalyzes the synthesis of chitin-like oligosaccharides.

Complete coding regions of activin receptors cActR-IIA and -IIB cDNAs were subcloned to determine whether activin can induce mesoderm in chick embryo despite the absence of the hypoblast. Expression of the receptors was detected in the primitive streak by in situ hybridization, suggesting that these genes had been activated. In vitro assay for differentiation showed that primitive streak responds to activin by differentiating into a variety of mesodermal cell types. These results show that activin activates signaling pathways during various stages of development.

Spadetail embryos mutant for snail2, a gene encoding a zinc finger protein of the Snail family, showed strongly reduced cephalic mesendodermal cell expression and a broader distribution of cells containing snail2 and no tail transcripts in the axial mesoderm. They were also shorter at the end of gastrulation. These results suggest that the spadetail mutation prevents elongation of the embryo and the convergence of the paraxial mesoderm in the trunk and the axial and lateral mesendoderm in the trunk and head at the end of gastrulation.

The chick Gnot1 homeobox gene, a member of the Not gene family, is involved in the formation of the anteroposterior axis and gastrulation process. The epiblast expressing Gnot1 during blastulation is replaced by a localized region in the Hensen's node during gastrulation. The regressing primitive streak, tail bud and notochord express Gnot1 but the neural tube floorplate does not. The increase in expression of Gnot1 in the presence of both activin and retinoic acid is more than that if they are present alone. Cycloheximide also increases the expression of Gnot1.

Cellular differentiation during gastrulation in Xenopus laevis embryos is controlled by C-cadherin, a cell-cell adhesion molecule. The introduction of a truncated C-cadherin molecule, in which the cytoplasmic tail is absent, into the dorsal involuting marginal zone causes incomplete involution and closing of the blastopore. This effect of the truncated molecule can be removed by introducing the full C-cadherin molecule. However the effect of C-cadherin is limited to the gastrulation period and does not affect the formation of mesoderm.

In situ analysis of the HOX 2.3 and HOX 2.4 genes revealed that expression begins at the posterior part of the embryo and moves toward the anterior end of the primitive streak. This pattern comprises the two-stage model for the expression of HOX. Gene expression during the second phase, which occurs at the early somite stage, is believed to be clonally transmitted. These results suggest that the first stage of HOX expression involves the mechanism that turns on the genes in the area.

Nodal is a gene expressed in the mouse node during gastrulation which may encode a molecule necessary for the differentiation of ectodermal cells into mesoderm and subsequent formation of axial structures. Nodal encodes a molecule similar to transforming growth factor beta. This molecule is first expressed at the time of mesoderm formation and then localizes in an anterior node of the primitive-streak embryo.

The gene DG42 is expressed briefly during embryogenesis of the frog genus Xenopus laevis. It is first detected at the midblastula stage then peaks at late gastrula and decays by the end of neuralation. DG42 could probably be an enzyme that synthesizes chitin oligomers during embryogenesis. It could be acting in a manner similar to the signaling oligosaccharides of plants or as a primase for hyaluronan synthesis. It may also be possible that DG42 is a hyaluronan synthase and the chitin synthase is an in vitro artifact.

Results point out that the zinc finger transcriptional activator, Churchill, functions as a switch and exhibits different roles of FGF, which is involved in the slow induction Churchill. The Churchill is expressed when cell migration stops and regulates cell ingression.

Research has been conducted on protein kinase C involved in Wnt signaling pathway. The authors report the identification of the genes encoding sigma-type protein kinase C in the Xenopus EST databases, and that this protein kinase C sigma regulates the activity and localization of Dishevelled.

To clarify the relationship between axial patterning in cnidarians and bilaterians, we have investigated the embryonic development of the hydrozoan Podocoryne carnea. The expression of Hox-like homeobox genes was analyzed by RT-PCR and in situ hybridization. Cnox1-Pc, an anterior Hox gene, is a maternal message. It is present throughout larval development, first weakly in all blastomeres and later restricted mostly to the anterior pole of the planula. Gsx, an anterior ParaHox gene, is first seen in the anterior endoderm but also extends into posterior regions. Cnox4-Pc, an orphan Hox-like gene, is expressed in the egg as a ring-shaped cloud around the germinal vesicle. After fertilization, the message remains in most animal blastomeres. When the embryo elongates in late blastula, staining is restricted to a few cells at the posterior pole where gastrulation will start. However, once gastrulation starts, the Cnox4-Pc signal disappears and is absent in later stages of larval development. Phylogenetic analysis shows that not all cnidarian Hox-like genes have recognizable orthologues in bilaterian groups. However, the expression analysis of Cnox1-Pc and Gsx correlates to some extent with the expression pattern of cognate genes of bilaterians, confirming the conservation of genes involved in organizing animal body plans and their putative common ancestral origin. Key Words: Cnidaria; embryogenesis; Hox genes; ParaHox genes; axis formation.

Members of the LEF-1/TCF family of transcription factors have been implicated in mediating a nuclear response to Wnt signals by association with [Beta]-catenin. Consistent with this view, mice carrying mutations in either the Wnt3a gene or in both transcription factor genes Lef1 and Tcf1 were previously found to show a similar defect in the formation of paraxial mesoderm in the gastrulating mouse embryo. In addition, mutations in the Brachyury gene, a direct transcriptional target of LEF-1, were shown to result in mesodermal defects. However, direct evidence for the role of LEF-1 and Brachyury in Wnt3a signaling has been limiting. In this study, we genetically examine the function of LEF-1 in the regulation of Brachyury expression and in signaling by Wnt3a. Analysis of the expression of Brachyury in [Lef1.sup.-/-][Tcf1.sup.-/-] mice and studies of Brachyury:lacZ transgenes containing wild type or mutated LEF-1 binding sites indicate that Lef1 is dispensable for the initiation, but is required for the maintenance of Brachyury expression. We also show that the expression of an activated form of LEF-1, containing the [Beta]-catenin activation domain fused to the amino terminus of LEF-1, can rescue a Wnt3a mutation. Together, these data provide genetic evidence that Lef1 mediates the Wnt3a signal and regulates the stable maintenance of Brachyury expression during gastrulation.

The murine T-box transcription factor Brachyury, or T, has been shown to play a crucial role in mesoderm formation during gastrulation. Embryos homozygous for the Twis allele of T lack axial mesoderm (notochord) and are deficient in paraxial mesoderm formation such that posterior somites are never formed and axial elongation fails. The Twis allele results from insertion of a transposable element into the 3' end of exon 7 of the T locus. This insertion disrupts the splice donor consensus sequence of exon 7. It has long been assumed that this disruption leads to the production of a single truncated T protein species lacking exon 8. However, analysis of the T locus transcripts produced in Twis homozygotes shows that at least five different transcripts are produced. Four of these represent alternative splicing products derived from the endogenous T exons and the fifth a chimeric transcript linking exon 7 to transposable element sequences. All five are predicted to encode different abnormal T proteins. At least seven other murine T-box genes are expressed in newly forming mesoderm (Tbx1 through Tbx6 and eomes). To further elucidate the nature of the Twis defect, potential interactions between Twis and these genes are being investigated. Similarly, possible interactions between Twis and Fgf signaling components expressed at gastruation (Fgf-3, Fgf-4, Fgf-5, FgfRI, and FgfRII) are also being studied.

The mutant line lazy mesoderm (lzme) was found in an ENU screen for recessive mutations causing morphological defects in the midgestation mouse embryo. Homozygous embryos for the lzme locus arrest during gastrulation with a characteristic morphology: the proamniotic cavity is filled with a mass of cells attached to the posterior side of the epiblast. This phenotype resembles that of mutants for two known components of the FGF pathway: Fgf8 and FGFR-1. The phenotypes of those mutations have been attributed to the failure of proper cell migration of mesodermal cells through the primitive streak. Phenotypic analysis supports the possibility that the lzme gene is a component of the FGF signaling pathway. Analysis of lzme embryos reveals that ectopic cells in the proamniotic cavity do have a mesodermal identity, as described for those of FGF pathway mutants: they have a mesenchymal morphology and they express some early mesodermal markers such as Wnt3. However, the expression profile of mesodermal markers in lzme embryos is different from that seen in Fgf8 mutants. Interestingly, some markers for the primitive streak, including T, are only weakly expressed. Thus, the lzme mutation could not only affect cell migration, but also cell identity in gastrulating embryos. Because of these differences, the analysis of the lzme phenotype could shed light on previously unidentified roles of FGF pathway in mesoderm specification, lzme maps to an interval on chromosome 5 where there are no characterized genes that affect gastrulation. Work toward the positional cloning of lzme gene is in progress.

Fate mapping experiments in the mouse reveal that the primitive streak can be divided into three functional regions. The proximal region gives rise to germ cells and the extraembryonic mesoderm of the yolk sac; the distal region generates cardiac mesoderm and node-derived axial mesendoderm; and the middle streak region produces the paraxial, intermediate, and lateral plate mesoderm of the trunk. To gain insight into the mechanisms that mediate the assembly of the primitive streak into these functional regions, we have cloned and functionally identified the disrupted gene in amnionless (amn), a recessive, embryonic lethal mutation that specifically interferes with the formation/specification of the middle primitive streak region during gastrulation. We have found that Amn encodes a novel type I transmembrane protein that is expressed exclusively in the extra-\embryonic visceral endoderm layer during gastrulation. Intriguingly, the extracellular region of the Amn protein contains a cysteine-rich domain with similarity to BMP-binding cysteine-rich domains in Chordin, its Drosophila homologue, Short gastrulation, and Procollagen IIA. These findings suggest that Amn may direct the production of middle streak-derived derived trunk mesoderm by acting in the underlying visceral endoderm to modulate a BMP signaling pathway. Two additional sites of Amn expression have been identified in the mouse: kidney proximal tubules during organogenesis and in the adult and intestinal epithelium during organogenesis. Intriguingly, all three sites of Amn expression--visceral endoderm, proximal tubule, and intestinal epithelium--represent polarized epithelia specialized for resorption, transport, and secretion. Additionally we have recently identified a Drosophila homologue of Amn that is also expressed in a fly cell type specialized for resorption and secretion. These findings indicate that Amn likely plays an evolutionarily conserved role in the development, differentiation, and/or function of these polarized epithelia. Through developmental, genetic, and biochemical strategies we seek to define the role of Amn in these specialized cells and to determine whether that role involves interactions with BMP signaling pathways.

During zebrafish gastrulation, wnt8 is required to posteriorize nascent neuroectoderm (see abstract by Lekven et al.). Loss of wnt8 function results in an expansion of the expression domains of anterior neural markers such as otx-2 and opl, with a concomitant reduction in more caudal neural genes, including the midbrain-hindbrain marker pax-2, wnt8 may posteriorize neuroectoderm by relieving the repression of posterior neural genes by the zebrafish tcf3 homologue, headless. Loss of both maternal and zygotic headless function results in a complete loss of eyes and forebrain with a concomitant expansion of midbrain-hindbrain markers, a phenotype mimicking that of zygotic wnt8 overexpression. A homologue of murine nemo-like kinase (NLK) has been implicated in a MAP-kinase-related pathway in the early Caenorhabditis elegans embryo that synergizes with Wnt/[Beta]-catenin signaling to relieve repression by a TCF-related gene called POP-1. As .a first test of whether a similar pathway may play a role in neuroectoderm patterning in zebrafish, we injected mouse NLK RNA into early zebrafish embryos. We observed a phenotype strikingly similar to that seen in headless mutant embryos, complete or partial loss of eyes and forebrain, suggesting that an NLK-like pathway may be functioning cooperatively with wnt8 to posteriorize the neuroectoderm. In addition, some injected embryos also lack notochord and have somites fused across the midline, suggesting that an NLK-mediated pathway could be cooperating with wnt8 in patterning ventrolateral mesoderm. Progress in cloning a zebrafish NLK homologue and assessing its function will be described.

The LIM-homeodomain protein, Lim1, plays multiple roles during embryonic development including dorsal/anterior patterning and kidney formation. In Xenopus laevis, early expression of Xlim1 is localized to the organizer region of the gastrula embryo. A variety of experiments indicate that Xlim1 is a transcriptional activator and that it is required for head formation. To probe further the role of Xlim1 during embryonic development, we employed depletion studies using DEED antisense oligonucleotides. First, we investigated possible functional redundancy between Xlim1 and the closely related LIM-homeodomain protein, Xlim5. Xlim14 antisense oligonucleotide-injected embryos showed a marked reduction in Xlim1 mRNA and lacked head structures. This phenotype could be rescued efficiently by injection of zebra fish lim1 mRNA and almost as efficiently with Xlim5 mRNA. These results imply functional redundancy between Xlim1 and Xlim5. Second, we analyzed in more detail the role of Xlim1 in the organizer during gastrulation. In Xlim1-depleted embryos, organizer genes such as goosecoid and chordin were still expressed in a temporally correct manner. However, cells expressing these markers, which normally involute toward the animal pole, remained restricted to the region in the vicinity of dorsal lip. Thus involution of the dorsal mesoderm was greatly impeded in Xlim1-depleted embryos. These observations suggest that Xlim1 may be required for the proper involution of axial mesoderm during gastrulation.

Morphogenetic cell behaviors include coordinate cell divisions, coordinate cell shape changes, and stereotyped patterns of cell migration and intercalation. During embryogenesis, a group of cells (or a group of cell nuclei within a syncytium) that transiently express a common morphogenetic cell behavior can be viewed as constituting a morphogenetic domain. Although the cell behaviors that are expressed in morphogenetic domains are clearly linked to the expression of early-acting gene networks, it is unclear whether these morphogenetic cell behaviors are activated in strict conjunction with restrictions of cell fate. The yolk syncytial layer (YSL) of zebrafish embryos (a syncytial extraembryonic endodermal layer) provides a unique system to determine whether morphogenetic domains can be expressed independently of cell fate restrictions. The YSL is generated during the midblastula period and persists through larval development. However, the YSL syncytium does not contribute cells (or nuclei) to adult zebrafish tissues. Moreover, nuclei within the zebrafish YSL become postmitotic shortly after the midblastula period. Thus, the YSL does not represent a cell progenitor domain. The YSL, however, does express a number of spatially distinct morphogenetic domains, which are clearly visible in the highly patterned movements of YSL nuclei during gastrulation. YSL nuclei movements are organized along the AP and DV axes and display convergent-extension behavior in the axial region of the zebrafish yolk cell. These movements of the YSL nuclei, as well as macroscopic shape changes of the zebrafish yolk cell, are homologous to a number of morphogenetic behaviors that are regionally expressed within the vegetal endodermal cell mass of gastrulating Xenopus embryos. These homologous morphogenetic behaviors suggest that specification of morphogenetic domains and cell progenitor domains are parallel, but separable, outputs of early-acting gene networks.

In the mouse, embryological and genetic studies have indicated that two spatially distinct signalling centres, the anterior visceral endoderm and the node and its derivatives, are required for the correct patterning of the anterior neural ectoderm. The divergent homeobox gene Hex is expressed in the anterior visceral endoderm, in the node (transiently), and in the anterior definitive endoderm. Other sites of Hex expression include the liver and thyroid primordia and the endothelial cell precursors. We have used transgenic analysis to map the cis-acting regulatory elements controlling Hex expression during early mouse development. A 4.2-kb upstream region is important for Hex expression in the endothelial cell precursors, liver, and thyroid, and a 633-bp intronic fragment is both necessary and sufficient for Hex expression in the anterior visceral endoderm and the anterior definitive endoderm. These same regions drive expression in homologous structures in Xenopus laevis, indicating conservation of these regulatory regions in vertebrates. Analysis of the anterior visceral endoderm/ anterior definitive endoderm enhancer identifies a repressor region that is required to downregulate Hex expression in the node once the anterior definitive endoderm has formed. This analysis also reveals that the initiation of Hex expression in the anterior visceral endoderm and axial mesendoderm requires common elements, but maintenance of expression is regulated independently in these tissues. Key Words: Hex; homeobox; endoderm; AVE; node; gastrulation.

The NUP98 gene encodes precursor proteins that generate two nucleoplasmically oriented nucleoporins, NUP98 and NUP96. By using gene targeting, we have selectively disrupted the murine NUP98 protein, leaving intact the expression and localization of NUP96. We show that NUP98 is essential for mouse gastrulation, a developmental stage that is associated with rapid cell proliferation, but dispensable for basal cell growth. NUP98-/- cells had an intact nuclear envelope with a normal number of embedded nuclear pore complexes. Typically, NUP98-deficient cells contained on average approximately 5-fold more cytoplasmic annulate lamellae than control cells. We found that a set of cytoplasmically oriented nucleoporins, including NUP358, NUP214, NUP88, and p62, assembled inefficiently into nuclear pores of NUP98-/- cells. Instead, these nucleoporins were prominently associated with the annulate lamellae. By contrast, a group of nucleoplasmically oriented nucleoporins, including NUP153, NUP50, NUP96, and NUP93, had no affinity for annulate lamellae and assembled normally into nuclear pores. Mutant pores were significantly impaired in transport receptor-mediated docking of proteins with a nuclear localization signal or M9 import signal and showed weak nuclear import of such substrates. In contrast, the ability of mutant pores to import ribosomal protein L23a and spliceosome protein U1A appeared intact. These observations show that NUP98 disruption selectively impairs discrete protein import pathways and support the idea that transport of distinct import complexes through the nuclear pore complex is mediated by specific subsets of nucleoporins.

The mouse Otx2 gene is a homeobox transcription factor required as early as gastrulation for the proper development of the head. We compared gene expression profiles in wild-type and [Otx2.sub.-/-] 6.5 days postcoitum embryos by using a serial analysis of gene expression assay adapted to microdissected structures. Among a broader list, the study of six genes found to be differentially expressed allows defining a role for Otx2 in the orchestration of cell movements leading to the adequate organization of the embryo before gastrulation.

The homeobox gene Rpx/Hesx1 is expressed during gastrulation in the anterior endoderm, prechordal plate and the cephalic neural plate. At later stages of development, its expression is restricted to Rathke's pouch, the primordium of the pituitary gland, and is no longer detectable in the neuroectoderm. The importance of Rpx for embryonic development has been shown by targeted deletion of the Rpx gene. Most Rpx -/- mice die between E10.5 and E12.5 with many developmental defects in the forebrain, pituitary and eye. The expression pattern of the Rpx gene suggested that at least two stages of Rpx regulation, an early stage which controls expression in the anterior endoderm and neural plate and a later stage which controls expression in the Rathke's pouch. There are several homeobox genes that are candidates for regulating Rpx during these different stages of development. In particular, there are several LIM homeobox genes which are co-expressed with Rpx. LIM1 and Lmx1b are expressed in anterior visceral endoderm and anterior neural plate during gastrulation, respectively. Lmx1b and LIM3/Lhx3 are both expressed in Rathke's pouch. In Lim1-/- embryos, the expression of Rpx is not detectable, and Lhx3-/embryos fail to maintain Rpx expression in Rathke's pouch. To test the function of these LIM proteins, co-transfection experiments were done. Lim1, Lmx1 and Lim3 all can positively regulate the Rpx promoter, and serial deletions and mutations have delineated LIM binding sites in the 390bp upstream sequence necessary for activity. The importance of these binding sites was confirmed by gel shift analysis. Xanf, the Rpx homologue in Xenopus, has a similar expression pattern as the mouse Rpx gene, so its regulation is thought to be similar to that of Rpx. To test if the regulatory elements of the Rpx gene are functionally conserved, a lacZ reporter construct sufficient for proper expression of Rpx in mice was microinjected into Xenopus embryos at the 4-cell stage. The expression pattern of lacZ in the frog neural plate and pituitary primordium is similar to that in transgenic mice. This suggests that the regulatory elements of Rpx are accurately recognized in Xenopus. This system will be used to examine conserved regulatory elements between species.

Eph receptors and their cell-surface-bound ligands, the ephrins, are known to regulate axon guidance in the developing brain, modulate vascular remodeling and play an important role in somite formation. The eph/ephrin signaling system is proposed to mediate repulsive cell contact-dependent interactions that results in the proper maintenance of discrete domains essential for pattern formation in embryonic development. We are interested in the bi-directional signaling properties of the class B ephrins and their role in the developing zebrafish embryo. The ephrin B ligands are transmembrane proteins capable of transducing signals in the ligand cell, in addition to initiating a signal in the receptor cell. We found that zebrafish ephrin B proteins are tyrosine phosphorylated in vivo, as early as mid-gastulation, suggesting that bi-directional signaling is required for embryonic development. To investigate this further, we have identified 4 members of the class B ephrins in the zebrafish. Each ligand is expressed in discrete domains as the embryo develops. Ephrin B ligands are expressed in the early gastrula, in the germ ring and shield, suggesting essential roles in early development, perhaps in co-ordinating cellular movements during gastrulation. We examined the functional roles of these proteins by injection of mRNA, encoding dominant negative forms of ephrin B proteins. Interruption of eph signaling resulted in dramatic phenotypes indicative of gastrulation problems. Our analysis demonstrates that disruption of eph signaling perturbs prechordal plate movement during gastrulation. In addition to eph/ephrin roles in later developmental events, our study points to an important early role for eph signaling. This study provides further evidence to support an essential role for bi-directional signaling of ephrin B proteins.

The zebrafish gastrulation mutant trilobite(tri) exhibits severe defects in the migration of hindbrain motor neurons. In the wild type hindbrain, facial (nVII) and glossopharyngeal (nIX) motor neurons are induced in rhombomeres 4 and 6, respectively, and subsequently migrate caudally into r6 and r7 (nVII), and r7 (niX). In all three trialleles examined, while normal numbers of motor neurons are induced, nVII motor neurons are found exclusively in r4, and niX-like motor neurons are found exclusively in r6, suggesting strongly that caudal migration of these neuronal populations is abolished. Migration of other cell populations such as neural crest cells, adaxial muscle cells, lateral line primordial cells, as well as axon guidance of several neuronal populations are all unaffected in trimutants, demonstrating the specificity of the neuron migration defect. Furthermore, the induction and patterning of other hindbrain neurons, and the expression of several rhombomere-specific genes are unaffected in trimutants, indicating that overall patterning of the hind-brain is normal. Interestingly, nVII and nIX neurons migrate normally in the gastrulation mutant knypek, suggesting that the trilobiteneuron migration defect does not arise non-specifically from aberrant gastrulation-associated cell movements. Current experiments include genetic mosaic analysis to determine whether trifunctions cell-autonomously during motor neuron migration, and time lapse microscopy to analyze defective migratory behavior in mutants. Our studies suggest that trilobiterepresents a genetic tool to analyze neuronal migration in the vertebrate hindbrain.

EGF-CFC genes encode a novel class of extracellular, membrane-associated proteins, notably playing an important role during vertebrate gastrulation. Whereas the two cysteine-rich domains that characterize these proteins, namely the extracellular EGF-like and the CFC domain, are known to be encoded by two evolutionarily conserved exons, it is generally assumed, based on a weak primary sequence identity, that the remaining parts of the protein are different among vertebrates, suggesting that known members of EGF-CFC family do not represent true orthologs. Here, by characterizing the full cDNA and genomic sequences of a new EGF-CFC gene in chick, and by comparing them with their counterpart in human (CRIPTO), mouse (cripto and cryptic), Xenopus (FRL-1) and zebrafish (one-eyed pinhead), we show that all EGF-CFC genes share an identical genomic structure over the entire coding region. Not only are the central two exons EGF-like and CFC conserved, but also the total number of exons, their size, intron phase, and correlation with discrete modules of the protein are conserved, in particular those predicted to allow the EGF-CFC motif to be membrane-associated. Therefore, despite apparent divergence between their 5' and 3'-terminal exons, all known EGF-CFC genes are structurally orthologous. We named this novel ortholog in bird, chick-cripto. We report the mRNA distribution of chick-cripto, which starts in the epiblast of the gastrula, with a pattern similar to EGF-CFC genes of other organisms. Supported by NIH Grant no. NS 18112 and an ARC postdoctoral fellowship (France).

The Wnt gene family is large, and new members are continually being added. Because existing phylogenetic arrangements of these genes are out-of-date, we have constructed a parsimony-tree for the Wnt family based on all of the 82 full-length sequences currently available. Sequences from the cephalochordate amphioxus, the closest living invertebrate relative to the vertebrates, are especially useful in comprehensive gene trees because they tend to mark the base of vertebrate diversifications. Thus, AmphiWnt4, AmphiWnt7, AmphiWnt8, and AmphiWnt11 fall at the base of vertebrate Wnt4, Wnt7, Wnt8, and Wnt11 subfamilies, respectively. However, AmphiWnt1 has an anomalous position, clustering outside of the Wnt1 subfamily relatively close to arthropod Wnt1 genes. Similarly, expression of amphioxus Wnt genes, except for AmphiWnt1, tends to be like that of their respective homologs. Thus, AmphiWnt7 is primarily expressed in the CNS, like its vertebrate counterparts. AmphiWnt1 transcription, however, cannot be detected in the CNS like vertebrate Wnt1 genes, but is located around the blastopore like the early expression of Drosophila Wnt1/wingless around the amnioproctodeal invagination. Taken together with expression of homologs of other genes such as brachyury and caudal around the blastopore and in the hindgut of both amphioxus and higher chordates, these data support the unexpected homology of the chordate blastopore and the amnioproctodeal invagination of Drosophila.

T (Brachyury), the prototype T-box transcription factor, is critical for the genesis and maintenance of axial and paraxial mesoderm during gastrulation. We unexpectedly found that chicken T (Ch-T) was expressed in the limb by Northern and in situ hybridization. Ch-T was already expressed at stage 15/16 in the limb field lateral plate and later in both forelimb and hindlimb mesoderm just below the AER. FGF signals have been implicated in limb initiation, outgrowth and patterning. It has been proposed that a relay of successive FGF signals from the midline to periphery initiates limb bud formation. In Xenopus, T (Xbra) can induce FGF expression as part of a positive feedback loop with eFGF. To determine whether T may be a component of the FGF signaling cascade regulating limb development, we evaluated early limb bud stage in T mutants (E10-10.5) and found that the expression of FGFs were altered. Retroviral misexpression of Ch-T in the chick hind limb region produced several related phenotypes including digit 1 duplication, ectopic cartilage condensations and expanded soft tissue consistent with augmented AER function. Our results suggest that T may participate in regulating limb outgrowth and patterning through FGF pathways.

The studies described here sought to identify and characterize genes involved in the gastrulation and morphogenetic movements that occur during sea urchin embryogenesis. An orthologue of the T-box family transcription factor, Brachyury, was cloned through a candidate gene approach. Brachyury (T) is the founding member of this T-box transcription factor family and has been implicated in gastrulation movements in Xenopus, zebrafish, and mouse embryogenesis. Polyclonal serum was generated to LvBrac in order to characterize protein expression. LvBrac initially appears at mesenchyme blastula stage in two distinct regions with embryonic expression perduring until pluteus stage. Vegetally, LvBrac expression is in endoderm and lies circumferentially around the blastopore. This torus-shaped area of LvBrac expression remains constant in size as endoderm cells express LvBrac upon moving into that circumference and cease LvBrac expression as they leave the circumference. Vegetal expression remains around the anus through pluteus stage. The second domain of LvBrac expression first appears broadly in the oral ectoderm at mesenchyme blastula stage and at later embryonic stages is refined to just the stomodael opening. Vegetal LvBrac expression depends on autonomous [beta]-catenin signaling in macromeres and does not require micromere or veg2-inductive signals. It was then determined that LvBrac is necessary for the morphogenetic movements occurring in both expression regions. A dominant-interfering construct was generated by fusing the DNA binding domain of LvBrac to the transcriptional repression module of the Drosophila Engrailed gene in order to perturb gene function. Microinjection of mRNA encoding this LvBrac-EN construct resulted in a block in gastrulation movements but not expression of endoderm and mesoderm marker genes. Furthermore, injection of LvBrac-EN into one of two blastomeres resulted in normal gastrulation movements of tissues derived from the injected blastomere, indicating that LvBrac downstream function may be nonautonomous during sea urchin gastrulation. Key Words: Brachyury (T); sea urchin; gastrulation; morphogenesis.

Evx genes are widely used in animal development. In vertebrates they are crucial in gastrulation, neurogenesis, appendage development and tailbud formation, whilst in protostomes they are involved in gastrulation and neurogenesis, as well as segmentation at least in Drosophila. We have cloned the Evx genes of amphioxus (Branchiostoma floridae), and analysed their expression to understand how the functions of Evx have evolved between invertebrates and vertebrates, and in particular at the origin of chordates and during their subsequent evolution. Amphioxus has two Evx genes (AmphiEvxA and AmphiEvxB) which are genomically linked. AmphiEvxA is prototypical to the vertebrate Evx1 and Evx2 genes with respect to its sequence and expression, whilst AmphiEvxB is very divergent. Mapping the expression of AmphiEvxA onto a phylogeny shows that a role in gastrulation, dorsal--ventral patterning and neurogenesis is probably retained throughout bilaterian animals. AmphiEvxA expression during tailbud development implies a role for Evx throughout the chordates in this process, whilst lack of expression at the homologous region to the vertebrate Midbrain-Hindbrain Boundary (MHB) is consistent with the elaboration of the full organiser properties of this region being a vertebrate innovation. Key Words: Amphioxus; Evx genes; chordates; gastrulation; neurogenesis; Midbrain--Hindbrain Boundary; tailbud development.