Repression of XMyoD expression and myogenesis by Xhairy-1 in Xenopus early embryo.

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.

Cloning and developmental expression of STAT5 in Xenopus laevis.

Transcription factors of the signal transducer and activator of transcription (STAT) family are required for cellular responses to multiple signalling molecules. After ligand binding-induced activation of cognate receptors, STAT proteins are phosphorylated, hetero- or homodimerize, and enter the nucleus. STAT dimers bind to specific DNA elements and alter the transcriptional activity of the signal-responsive genes. We report the cloning and developmental pattern of expression of XSTAT5, a Xenopus laevis member of the STAT family, closely related to the mammalian STAT5A and STAT5B. XSTAT5 is expressed maternally and zygotically. With the onset of neurulation, XSTAT5 RNA are clearly localized in the anterior neural plate and subsequently in the neural structures of the developing eye, the pineal gland and the cement gland anlage. At late tailbud stages, a faint expression is detected in a ventral location that might correspond to the ventral blood islands.

The C-terminal cytoplasmic Lys-thr-X-X-X-Trp motif in frizzled receptors mediates Wnt/beta-catenin signalling.

Frizzled receptors are components of the Wnt signalling pathway, but how they activate the canonical Wnt/beta-catenin pathway is not clear. Here we use three distinct vertebrate frizzled receptors (Xfz3, Xfz4 and Xfz7) and describe whether and how their C-terminal cytoplasmic regions transduce the Wnt/beta-catenin signal. We show that Xfz3 activates this pathway in the absence of exogenous ligands, while Xfz4 and Xfz7 interact with Xwnt5A to activate this pathway. Analysis using chimeric receptors reveals that their C-terminal cytoplasmic regions are functionally equivalent in Wnt/beta-catenin signalling. Furthermore, a conserved motif (Lys-Thr-X-X-X-Trp) located two amino acids after the seventh transmembrane domain is required for activation of the Wnt/beta-catenin pathway and for membrane relocalization and phosphorylation of Dishevelled. Frizzled receptors with point mutations affecting either of the three conserved residues are defective in Wnt/beta-catenin signalling. These findings provide functional evidence supporting a role of this conserved motif in the modulation of Wnt signalling. They are consistent with the genetic features exhibited by Drosophila Dfz3 and Caenorhabditis elegans mom-5 in which the tryptophan is substituted by a tyrosine.

Xenopus frizzled 4 is a maternal mRNA and its zygotic expression is localized to the neuroectoderm and trunk lateral plate mesoderm.

We describe the identification and expression pattern of Xenopus frizzled 4 (Xfz4) gene during early development. Xfz4 protein presents characteristic features of a frizzled family member. The mature protein sequence of Xfz4 is 93% identical to murine Mfz4. Xfz4 is a maternal mRNA, its expression level remains constant during early development. The mRNA is first localized during gastrulation to the dorsal presumptive neuroectoderm. At the end of gastrulation, Xfz4 mRNA is detected in the dorso-anterior neuroectoderm. During neurulation, Xfz4 mRNA is expressed as a band on both side of the forebrain, and in the trunk lateral plate mesoderm. As development proceeds, expression of Xfz4 mRNA in the trunk lateral plate mesoderm decreases but persists in the forebrain. It is also expressed in the posterior unsegmented somitic mesoderm from late tail-bud stage onward.

Role of fibroblast growth factor during early midbrain development in Xenopus.

Genes encoding fibroblast growth factors (FGFs) are expressed in early Xenopus neurulae in the prospective midbrain-hindbrain boundary (MHB) region of the neural plate. These expression domains overlap those of XWnt-1 and XEn-2, raising the question of the role of FGF signalling in the regulation of these genes, and more generally about the function of FGF during Xenopus midbrain development. We report that explants from the prospective MHB grafted into the anterior neural plate in midneurula stage embryos induce XWnt-1 expression and, at a lower frequency, XEn-2 expression in the vicinity of the graft. Such a process is likely to involve FGF signalling. Implantation of FGF4- or FGF8-soaked beads in the prospective forebrain at neurula and tailbud stages causes the up-regulation of XWnt-1 and XEn-2 in the dorsal and lateral region of the anterior midbrain. This effect is not relayed by endogenous FGF genes since exogenous FGFs inhibit the expression of endogenous XFGF3 or XFGF8. However, consequences of grafting MHB or implanting FGF4 or FGF8 beads on tadpole brain development are different. MHB grafts induce ectopic mesencephalic structures, strongly suggesting that a region homologous to the isthmic organizer of amniotes is specified as early as the midneurula stage. In contrast, exogenous FGFs do not cause the formation of ectopic mesencephalic structures but an overgrowth of mesencephalon and diencephalon. We propose that FGF signals from the prospective MHB play a crucial role in the spatial regulation of XWnt-1 and XEn-2 expression in the posterior midbrain, but that the full organizing activity of the MHB involves other factors in combination with FGF.

Medial cell mixing during axial morphogenesis of the amphibian embryo requires cadherin function.

A truncated form of Xenopus E-cadherin (deltaE-cad) comprising the cytoplasmic and transmembrane domains was overexpressed generating a dominant negative mutation in the urodelan amphibian embryo Pleurodeles waltl. deltaE-cad mRNA and rhodamine-lysinated-dextran (RLDx) cell lineage tracer were microinjected into 32-cell stage blastomeres which contribute principally to the notochord and central nervous system. deltaE-cad expression causes defects in forebrain and hindbrain formation coupled with the development of supernumerary vesicles. Duplication of the notochord also occurs due to the retardation of medial cell intercalation with correlated duplications of spinal cord and somites. These results emphasize the role of cadherins in mediating cell-cell adhesion in early amphibian embryogenesis. They extend to Pleurodeles the observations made in Xenopus, illustrating that despite differences in morphogenetic processes, the molecular mechanisms are conserved in these two species.

Expression of Xfz3, a Xenopus frizzled family member, is restricted to the early nervous system.

Recent advances in analyzing wnt signaling have provided evidence that frizzled proteins can function as wnt receptors. We have identified Xfz3, a Xenopus frizzled family member. The amino acid sequence is 89% identical to the product of the murine gene Mfz3, and is predicted to be a serpentine receptor with seven transmembrane domains. Xfz3 is a maternal mRNA with low levels of expression until the end of gastrulation. The expression level increases significantly from neurulation onward. Whole-mount in situ hybridization analysis shows that expression of Xfz3 is highly restricted to the central nervous system. High levels of expression are detected in the anterior neural folds. Low levels of expression are also detected in the optic and otic vesicles, as well as in the pronephros anlage. In addition, Xfz3 mRNA is concentrated in a large band in the midbrain. Overexpression of Xfz3 blocks neural tube closure, resulting in embryos with either bent and strongly reduced anteroposterior axis in a dose-dependent manner. However, it does not affect gastrulation, the expression and localization of organizer-specific genes such as goosecoid, chordin and noggin. Therefore, Xfz3 is not involved in early mesodermal patterning. Injection of RNA encoding GFP-tagged Xfz3 shows that overexpressed proteins can be detected on the cell surface until at least late neurula stage, suggesting that they can exert an effect after gastrulation. Our expression data and functional analyses suggest that the Xfz3 gene product has an antagonizing activity in the morphogenesis during Xenopus development.

Identification and developmental expression of cyclin-dependent kinase 4 gene in Xenopus laevis.

Cyclin-dependent kinases (CDKs) are a family of serine/threonine protein kinases which play a pivotal role in the eucaryote cell cycle regulation. We have identified the Xenopus homologue of mammalian CDK4 (XCDK4). The protein sequence of XCDK4 has 78 and 77% overall identity to human and mouse CDK4, respectively. Northern blot analysis revealed a single transcript of approximately 4.5 kb present at various stages. XCDK4 transcripts show very dynamic expression during early development. The level of expression is higher during cleavage and gastrulation. In situ hybridization analysis revealed that the transcripts are enriched in the dorsal mesoderm at the beginning of gastrulation, then extend to both the lateral and ventral mesoderm. At the end of gastrulation, XCDK4 transcripts are mainly distributed in the blastoporal region and in the anterior neural fold. During neurulation they become restricted to optic vesicles and to neural crest cells organizing the branchial arches. As development proceeds, XCDK4 transcripts are highly expressed in the branchial arches. At late tail-bud stages, XCDK4 transcripts are also detected in ventral hematopoietic precursor cells. Therefore, this analysis clearly shows that XCDK4 has a regionalized expression during Xenopus embryogenesis.

Fates of the blastomeres of the 32-cell stage Pleurodeles waltl embryo.

Cell fate of the blastomeres at the 32-cell stage in the Pleurodeles waltl embryo was analyzed by injection of rhodamine or fluorescein lysinated-dextran (RLDx or FLDx). At the tailbud stage, the progeny of each blastomere contributed to more than one germ layer with unequal distribution along the anteroposterior and dorsoventral axis. Such a regionalized positioning of the descendants of the 32-cell blastomeres was found in the neuroectoderm, the epidermis, the notochord, the somites, the lateral plate, and the endoderm, but not in the head mesenchyme, the pronephros, or the blood islands. Results of double labeling of juxtaposed blastomeres showed that cell mixing and rearrangement take place during organ formation. Results are compared with those of the 32-cell stage fate map in Xenopus and Rana and reveal the more restricted fate of 32-cell stage blastomeres in Pleurodeles germ layers.

What mechanisms drive cell migration and cell interactions in Pleurodeles?

Embryogenesis implies a strict control of cell interaction and cell migration. The spatial and temporal regulation of morphogenetic movements occurring during gastrulation is directly dependent on the early cell interactions that take place in the blastula. The newt Pleurodeles waltl is a favorable model for the study of these early morphogenetic events. The combination of orthotopic grafting and fluorescent lineage tracers has led to precise early gastrula mesoderm fate maps. It is now clear that there are no sharp boundaries between germ layers at the onset of gastrulation but rather diffuse transition zones. The coordination of cell movements during gastrulation is closely related to the establishment of dorsoventral polarity. Ventralization by U.V. irradiation or dorsalization by lithium treatment modifies the capacity for autonomous migration on the fibronectin coated substratum of marginal zone cells accordingly. It is now firmly established that mesodermal cells need to adhere to a fibrillar extracellular matrix (ECM) to undergo migration during gastrulation. Extracellular fibrils contain laminin and fibronectin (FN). Interaction of cells with ECM involves receptors of the beta 1 integrin family. A Pleurodeles homolog of the alpha v integrin subunit has been recently identified. Protein alpha v expression is restricted to the surface of mesodermal cells during gastrulation. Integrin-mediated interactions of cells with FN are essential for ECM assembly and mesodermal cell migration. Intracellular injection of antibodies to the cytoplasmic domain of beta 1 into early cleavage embryos causes inhibition of FN fibril formation. Intrablastocoelic injections of several probes including antibodies to FN or integrin alpha 5 beta 1, competitive peptides to the major cell binding site of FN or the antiadhesive protein tenascin all block mesodermal cell migration. This results in a complete arrest of gastrulation indicating that mesodermal cell migration is a major driving force in urodele gastrulation. It is now possible to approach the role of fibroblast growth factor (FGF) during cell interactions taking place in urodele embryos. Four different FGF receptors (FGFR) have been cloned in Pleurodeles. Each of them has a unique mRNA expression pattern. FGFR-1, FGFR-3, and the variant of FGFR-2 containing the IIIb exon are maternally expressed and might be involved in mesodermal induction. During gastrulation, FGFR-3 and FGFR-4 have a restricted pattern of expression, whereas FGFR-1 mRNA is nearly uniformly distributed. Splicing variants FGFR-2IIIb and FGFR-2IIIc have exclusive expression patterns during neurulation. IIIb is expressed in epidermis and IIIc in neural tissue, suggesting a function in the differentiation of ectodermal derivatives.

A truncated FGF receptor blocks neural induction by endogenous Xenopus inducers.

We have examined the role of fibroblast growth factor (FGF) signalling in neural induction. The approach takes advantage of the fact that both noggin and the dominant negative mutant activin receptor (delta1XAR1) directly induce neural tissues in the absence of dorsal mesoderm. A truncated FGF receptor (XFD) is co-expressed with noggin or delta1XAR1 in both whole embryos and isolated animal caps. We demonstrate that inhibition of FGF signalling prevents neural induction by both factors. Furthermore, neural induction by organizers (the dorsal lip of blastopore and Hensen's node) is also blocked by inhibiting FGF signalling in ectoderm. It has been proposed that the specification of anterior neuroectoderm, including the cement gland, occurs in a sequential manner as gastrulation proceeds. We show that the specification of the most anterior neuroectoderm by noggin may occur before gastrulation and does not require FGF signalling, since both the cement gland marker XCG-1 and the anterior neural marker Otx-2 are normally expressed in ectodermal explants co-injected with noggin and XFD RNAs, but the cement gland cells are poorly differentiated. In contrast, the expression of both genes induced by CSKA.noggin, which is expressed after the mid-blastula transition, is strongly inhibited by the presence of XFD. Therefore the noggin-mediated neural induction that takes place at gastrula stages is abolished in the absence of FGF signalling. Since inhibition of FGF signalling blocks the neuralizing effect of different neural inducers that function through independent mechanisms, we propose that FGF receptor-related-signalling is required for the response to inducing signals of ectodermal cells from gastrula.

Early regionalized expression of a novel Xenopus fibroblast growth factor receptor in neuroepithelium.

A cDNA encoding a novel Xenopus fibroblast growth factor receptor, XFGFR4B, has been cloned. XFGFR4B mRNA is detected throughout embryogenesis. However, from the late gastrula stage on, XFGFR4B transcripts are expressed in two defined areas of the anterior neural plate. Interestingly, these two regions are fated to become parts of the retina, midbrain, hindbrain, and otic vesicle, all of which continue to express XFGFR4B mRNA in tailbud stage embryos and early tadpoles. Expression of XFGFR4B mRNA can be maintained at neurula stage in isolated blastula ectoderm in response to mesodermal induction by activin or neural induction by noggin, suggesting that XFGFR4B expression might be regulated by early cell interactions. Distribution of XFGFR4B mRNA suggests that XFGFR4B might serve an important function during patterning of neuroepithelium.

Tenascin expression in developing, adult and regenerating caudal spinal cord in the urodele amphibians.

Tenascin (Tn) protein and transcripts were analyzed in developing, adult and regenerating caudal spinal cord (SC) of Pleurodeles waltl. A polyclonal antibody (PAb) against Xenopus Tn and a newt Tn cDNA probe were used. In Western blots, anti-Tn PAb recognized Tn polypeptides of 200-220 kDa in tail regenerate extracts, but also the homolog of Tn/Cytotactin/J1 in brain and SC of adult newt. Immunofluorescence studies showed some reactivity around ependymoglial cells and strong labeling in the nervous tracts, in the developing as well as in the regenerating SC or adult SC. Immunogold electron microscopy revealed the presence of Tn throughout the ependymoglial cells, particularly near and along the plasma membrane of radial processes surrounding axons, especially growth cones. Tn could be more precisely found within rough endoplasmic reticulum and Golgi structures, or again in the surrounding extracellular space. This suggested that Tn was at least produced by radial glial profiles forming axonal compartments in which axons grew. Using the DNA probe for Tn, expression of Tn mRNA was also examined by Northern blot and RNAase protection analyses and by in situ hybridization, respectively. The levels of transcripts, barely detectable in adult tail, increased in regenerates from 3 days through 4-8 weeks post-amputation. In situ Tn mRNA were mainly localized in the mesenchyme, especially at the epithelial-mesenchymal interface, and in the developing cartilage, at the early regeneration stages, whereas high amounts of transcripts were seen not only at these stages, but also later, in the regenerating SC. Our main results supported the view that, in the caudal SC of newts, Tn, synthesized by radial ependymoglial cells, was similarly expressed during regeneration as well as larval development, and exhibited a sustained high accumulation level in the adult SC. On the basis of the multifunctional properties of Tn, the putative roles played by Tn as a substrate for neuronal pathfinding and boundary shaping were discussed.

Comparative analysis of the tissue distribution of three fibroblast growth factor receptor mRNAs during amphibian morphogenesis.

We have used in situ hybridization to survey the expression pattern of three fibroblast growth factor receptor (FGFR) mRNAs (PFR-1, PFR-3 and PFR-4, which we previously identified as the amphibian Pleurodeles waltl homologs of human FGFR-1, FGFR-3 and FGFR-4, respectively) during morphogenesis. Previous work suggests that these FGFR mRNAs exhibit a distinct pattern of expression at early developmental stages. In the present study we have tested the functional activity of these receptors and shown that both FGF-1 (acidic FGF) and FGF-2 (basic FGF), but not FGF-7 (keratinocyte growth factor), can lead to their activation, suggesting that the three cDNAs encode functional receptors. Results from in situ hybridization indicate that various FGFRs are involved in various developmental events. Their involvement in these processes is both overlapping and distinct. During the differentiation of the central nervous system (CNS), PFR-1 and PFR-4 mRNAs show high levels of redundant expression, while the sites of expression of PFR-3 mRNA correlate with regions, such as the diencephalon and the rhombencephalon, undergoing important anatomic changes. The three FGFR mRNAs are distinctly expressed in the cranial ganglia, the pigmented epithelia of retina and the otic vesicles. Most significantly, we found that they are strongly expressed at cranial and branchial mesenchymal condensation sites. PFR-3 mRNA is expressed earlier in this process than PFR-1 and PFR-4 mRNAs. Furthermore PFR-3 mRNA is detected in the mesenchyme of the limb bud, while PFR-1 and PFR-4 mRNAs are found in the primordia of the skeletal elements. In addition, PFR-1 mRNA is expressed in axial mesenchyme and PFR-4 mRNA is detected in the melanophores, xanthophores and in the pronephros. These results suggest that various FGFRs may be involved in distinct developmental events including cell proliferation and differentiation. We also discuss the functional redundancy of the FGFR system during amphibian morphogenesis.

Superficial cells in the early gastrula of Rana pipiens contribute to mesodermal derivatives.

We have studied the fate of presumptive mesodermal cells in the early Rana pipiens gastrula. We labeled superficial cells of the early gastrula with 125I. We also labeled all cells in a gastrula with rhodamine-lysine-dextran cell lineage tracer and superficial cells with 125I and then grafted small pieces of the marginal zone orthotopically into unlabeled host embryos. Labeled progeny were identified in sectioned embryos at the tail bud stage. The use of double-labeled grafts allowed us to study the relative contributions by superficial and deep cells to different derivatives. We found that the notochord and somite regions are both derived from the superficial and deep portions of circumblastoporal regions. In contrast, pronephros, lateral plate, cardiac anlagen, and blood cells only arise from deep cells in circumblastoporal regions. Such observations indicate that the fate map for R. pipiens is different from that of Xenopus laevis, where mesodermal derivatives appear to be restricted to the deep layers, and from that of Pleurodeles waltl, where all mesodermal derivatives are formed both from superficial and deep layers. We also have shown that from the neurula stage there is substantial mixing between cells due to the ingression of cells in the dorsal region so that superficial labeled cells, initially located in the roof of the archenteron, contribute to ventral regions of both the somites and notochord.

Control of somitic expression of tenascin in Xenopus embryos by myogenic factors and Brachyury.

Tenascin is a large glycoprotein which is expressed in a restricted pattern in the extracellular matrix (ECM) of vertebrate embryos. Tenascin interferes with cell-fibronectin interactions in vitro, and may play a role in the control of cell migration and differentiation during development. In Xenopus, tenascin immunoreactivity is first detected at the early tailbud stage in the ECM of the most anterior somite. Thereafter, it is distributed dorsally along neural crest cell migration pathways. In this paper, we report that tenascin mRNA is most abundant in dorsal mesoderm at the neurula stage and in somites at the early tailbud stage, indicating that the initial accumulation of tenascin in the ECM is due to secretion from paraxial mesoderm. To understand how tenascin expression in somitic mesoderm is controlled, we have expressed Xbra and the myogenic factors XMyoD and XMyf5 in blastula animal cap tissue. The tenascin gene is activated by all three transcription factors. Interestingly, expression of tenascin mRNA, and accumulation of the protein in the ECM, can occur without formation of muscle. Our results suggest that tenascin regionalization in early Xenopus embryos depends on tenascin RNA expression by somitic mesoderm, where it is likely to be activated by myogenic factors.

Expression of fibroblast growth factor receptor-2 splice variants is developmentally and tissue-specifically regulated in the amphibian embryo.

There is increasing evidence that distinct fibroblast growth factor receptor (FGFR) genes are involved in embryonic development and that unique expression patterns of individual FGFRs correlate with tissue-specific functions. In addition, alternative splicing of mRNA transcripts from at least two of these genes (FGFR-1 and FGFR-2) can generate receptor variants with different ligand-binding specificity. By polymerase chain reaction methods and by screening a cDNA library, we have isolated five amphibian FGFR-2 splice variants which share a high degree of identity to their human counterparts. These mRNAs are developmentally regulated and are expressed in a tissue-specific manner. In particular, two alternative exons (termed IIIb and IIIc) in the second half of the third immunoglobulin-like loop are remarkably conserved and have a distinct pattern of regulation during development. Either aFGF or bFGF can activate IIIb- or IIIc-containing receptors. In contrast, KGF only activates IIIb-containing receptors. Exon IIIb-containing receptors are maternally derived mRNAs, whereas exon IIIc-containing receptors are zygotically expressed. Furthermore, their tissue distribution pattern was mutually exclusive. From the beginning of the neurula stage onward, IIIb transcripts are expressed in the epidermis, while IIIc transcripts are activated in the neuroectoderm by neural induction. At the late tail-bud stage, in situ hybridization revealed expression of IIIc mRNA in the telencephalon and the diencephalon, as well as in the head mesenchyme condensation sites originating from the proliferating neural crest cells. IIIb mRNA was detected in the epidermis and in the epithelium of the pharynx. Our data suggest that exon IIIb-containing receptors may play a role in the development of epithelial tissues, while exon IIIc-containing receptors may play a role during neural tissue formation.

Isolation and developmental expression of the amphibian homolog of the fibroblast growth factor receptor 3.

Recent observations suggest that fibroblast growth factors (FGFs) and their receptors are involved in the control of embryogenesis. Several FGF receptor genes have been identified so far and their expression is differentially regulated. As part of a continuing effort to analyse the differential expression of FGF receptors and their potential role during amphibian development, we have isolated a Pleurodeles homolog of FGF receptor 3 (FGFR-3), which we designated PFR-3 because of its highest homology to human FGFR-3 (75% overall identity). PFR-3 is a maternally derived mRNA. While a low level of expression persists during the cleavage and gastrula stages, a significant increase in the mRNA was observed at the end of the gastrula stage. RNase protection analysis on dissected tissues showed that PFR-3 mRNA was mainly localized to the ectoderm at the early gastrula stage and then shifted to the embryonic neural tissues, whereas adult brain had decreased levels of PFR-3 mRNA expression. Consistent with the loss of FGF receptors during skeletal muscle terminal differentiation, PFR-3 as well as other FGF receptor mRNAs were undetectable in the adult skeletal muscle. However, highest levels of PFR-3 mRNA expression were found in the testis. In situ hybridization revealed strong expression in the germinal epithelium of the embryonic brain (especially the diencephalon and rhombencephalon) and neural tube, in the lens and the cranial ganglia. The epithelium of the developing gut, like the pharynx and esophagus, also prominently expressed PFR-3 mRNA. Other sites of expression were found in the liver and in the mesenchymal condensation sites of branchial arches.(ABSTRACT TRUNCATED AT 250 WORDS)