mRNA localization studies suggest that murine FGF-5 plays a role in gastrulation.

During gastrulation in the mouse, the pluripotent embryonic ectoderm cells form the three primary germ layers, ectoderm, mesoderm and endoderm. Little is known about the mechanisms responsible for these processes, but evidence from previous studies in amphibians, as well as expression studies in mammals, suggest that signalling molecules of the Fibroblast Growth Factor (FGF) family may play a role in gastrulation. To determine whether this might be the case for FGF-5 in the mouse embryo, we carried out RNA in situ hybridization studies to determine when and where in the early postimplantation embryo the Fgf-5 gene is expressed. We chose to study this particular member of the FGF gene family because we had previously observed that its pattern of expression in cultures of teratocarcinoma cell aggregates is consistent with the proposal that Fgf-5 plays a role in gastrulation in vivo. The results reported here show that Fgf-5 expression increases dramatically in the pluripotent embryonic ectoderm just prior to gastrulation, is restricted to the cells forming the three primary germ layers during gastrulation, and is not detectable in any cells in the embryo once formation of the primary germ layers is virtually complete. Based on this provocative expression pattern and in light of what is known about the functions in vitro of other members of the FGF family, we hypothesize that in the mouse embryo Fgf-5 functions in an autocrine manner to stimulate the mobility of the cells that contribute to the embryonic germ layers or to render them competent to respond to other inductive or positional signals.     

Analysis of mouse Evx genes: Evx-1 displays graded expression in the primitive streak.

Two mouse genes, Evx-1 and Evx-2, each encoding a homeodomain closely related to that of the Drosophila even-skipped gene were isolated using a PCR-based strategy. The structure and sequence of these genes are described. Mapping studies localized Evx-1 to chromosome 6, near the Hox-1 gene cluster, and Evx-2 to chromosome 2, near the Hox-4 cluster. The evolutionary implications of these linkages are discussed. RNA in situ hybridization analysis of Evx expression in embryos demonstrated a striking pattern of Evx-1 expression during gastrulation, whereas Evx-2 RNA could not be detected at any stage by this technique. Evx-1 RNA is first detected shortly before the onset of gastrulation in a region of ectoderm containing cells that will soon be found in the primitive streak. This localized expression of Evx-1 provides the first molecular evidence for regional differences in the mouse embryonic ectoderm before gastrulation. Throughout gastrulation, Evx-1 expression is limited to cells near and within the streak and that expression is graded, with a posterior-to-anterior decrease in the level of RNA. Based on fate-mapping studies indicating that different types of mesoderm emerge from different regions of the primitive streak and our observation that high levels of expression are localized to the region that will give rise to extraembryonic and ventral mesoderm, we speculate that Evx-1 plays a role in the dorsoventral specification of mesodermal cell fate.     

Two distinct sources for a population of maturing axial progenitors

In mammals, the primitive streak region and its descendant, the tail bud, are the source of nascent mesoderm and spinal cord throughout axial elongation. A localised population of long-term axial progenitors has been identified in a region of the tail bud, the chordoneural hinge, but the localisation of such progenitors at earlier stages is so far untested. By studying gene expression, we have shown that a specific topological arrangement of domains persists from the streak to the tail bud, and includes an area (the node-streak border) in which ectoderm that expresses primitive streak markers overlies the prospective notochord. This arrangement persists in the chordoneural hinge. Homotopic grafts show that, as in other vertebrates, cells in the streak and node predominantly produce mesoderm, whereas those in the node-streak border and lateral to the streak additionally produce neurectoderm. Node-streak border descendants populate not only neurectoderm, somites and notochord throughout the axis, but also the chordoneural hinge. Ectoderm lateral to the embryonic day (E)8.5 streak is later recruited to the midline, where it produces somites and chordoneural hinge cells, the position of which overlaps that of border-derived cells. Therefore, the E8.5 axial progenitors that will make the tail comprise cells from two distinct sources: the border and lateral ectoderm. Furthermore, heterotopic grafts of cells from outside the border to this region also populate the chordoneural hinge. Expression of several streak- and tail bud-specific genes declines well before elongation ends, even though this late population can be successfully transplanted into earlier embryos. Therefore, at least some aspects of progenitor status are conferred by the environment and are not an intrinsic property of the cells.     

Gene expression pattern of Claudin-1 during chick embryogenesis

Claudins are a family of proteins that are localized to tight junctions at the apical surface of epithelial cell layers. Over 24 family members have been identified in vertebrates. Despite being well-studied with respect to their function in tight junction selectivity and permeability, the embryonic expression patterns of most claudin family members have not been thoroughly investigated. Here, we report the cloning and expression pattern of a novel chick claudin family member that is most closely related to human claudin-1. Chick claudin-1 was expressed throughout the ectoderm of stage 4-6 chick embryos. Claudin-1 expression was particularly high in the neural epithelium and open neural tube, but decreased as the neural tube closed. High levels of claudin-1 expression were also observed in the developing otic vesicle, nasal placode, ectodermal component of the pharyngeal arches, and in the apical ectodermal ridge of the limb bud from stage 17 onwards. Claudin-1 expression was also detected in scleral papillae, feather buds and migrating primordial germ cells. Lower levels of claudin-1 expression were observed in the endoderm, the ventral pharynx, and several of its derivatives including the bronchi, developing lung epithelium, esophagus, and gut. Claudin-1 expression was detected in the nephric duct and the mesonephros, which are epithelialized derivatives of the intermediate mesoderm, but not in any other mesodermal derivates, including the heart, somites and developing muscle. With the exception of the migrating primordial germ cells and the primitive streak, all other tissues that expressed significant levels of claudin-1 were epithelialized.     

Expression of the fibroblast growth factor-5 gene in the mouse embryo.

Fibroblast growth factors (FGFs) are structurally related mitogens that can regulate the differentiation of a wide variety of cells. As a step towards elucidating the developmental roles played by one of these factors, we have used in situ hybridization methods to examine expression of the murine F gf-5 gene during embryogenesis. F gf-5 RNA was detected at seven distinct sites in the developing mouse embryo: (1) postimplantation epiblast (embryonic day 5 1/4-7 1/2), (2) lateral splanchnic mesoderm (E9 1/2-10 1/2), (3) lateral somatic mesoderm (E10 1/2-12 1/2), (4) myotomes (E10 1/2-12 1/2), (5) mastication muscle (E11 1/2-14 1/2), (6) limb mesenchyme (E12 1/2-14 1/2), and (7) acoustic ganglion (E12 1/2-14 1/2). At several of these sites, expression is spatially restricted within the tissues. We offer several hypotheses regarding the roles of FGF-5 in murine development.     

Localization of Brachyury (T) in embryonic and extraembryonic tissues during mouse gastrulation

T-box gene family members have important roles during murine embryogenesis, gastrulation, and organogenesis. Although relatively little is known about how T-box genes are regulated, published gene expression studies have revealed dynamic and specific patterns in both embryonic and extraembryonic tissues of the mouse conceptus. Mutant alleles of the T-box gene Brachyury (T) have identified roles in formation of mesoderm and its derivatives, such as somites and the allantois. However, given the cell autonomous nature of T gene activity and conflicting results of gene expression studies, it has been difficult to attribute a primary function to T in normal allantoic development. We report localization of T protein by sectional immunohistochemistry in both embryonic and extraembryonic tissues during mouse gastrulation, emphasizing T localization within the allantois. T was detected in all previously reported sites within the conceptus, including the primitive streak and its derivatives, nascent embryonic mesoderm, the node and notochord, as well as notochord-associated endoderm and posterior neurectoderm. In addition, we have clarified T within the allantois, where it was first detected in the proximal midline of the late allantoic bud (approximately 7.5 days postcoitum, dpc) and persisted within an expanded midline domain until 6-somite pairs (s; approximately 8.5 dpc). Lastly, we have discovered several novel T sites, including the developing heart, visceral endoderm, extraembryonic ectoderm, and its derivative, chorionic ectoderm. Together, these data provide a unified picture of T in the mammalian conceptus, and demonstrate T's presence in unrelated cell types and tissues in highly dynamic spatiotemporal patterns in both embryonic and extraembryonic tissues.     

Exogenous FGF-4 can suppress anterior development in the mouse embryo during neurulation and early organogenesis

Members of the fibroblast growth factor (FGF) family of peptide growth factors are widely expressed in the germ layer derivatives during gastrulation and early organogenesis of the mouse. We have investigated the effect of administering recombinant FGF-4 in the late-primitive streak stage embryo to test if the patterning of the body plan may be influenced by this growth factor. Shortly after FGF treatment the embryonic tissues up-regulated the expression of Brachyury and the RTK signaling regulator Spry2, suggesting that FGF signaling was activated as an immediate response to exogenous FGF. Concomitantly, Hesx1 expression was suppressed in the prospective anterior region of the embryo. After 24 h of in vitro development, embryos displayed a dosage-related suppression of forebrain morphogenesis, disruption of the midbrain-hindbrain partition, and inhibition of the differentiation of the embryonic mesoderm. Overall, development of the anterior-posterior axis in the late gastrula is sensitive to the delivery of exogenous FGF-4. The early response associated with the expression of Spry2 suggests that the later phenotype observed could be primarily related to an inhibition of the FGF signaling pathway.     

Evidence for involvement of activin A and bone morphogenetic protein 4 in mammalian mesoderm and hematopoietic development.

Xenopus in vitro studies have implicated both transforming growth factor beta (TGF-beta) and fibroblast growth factor (FGF) families in mesoderm induction. Although members of both families are present during mouse mesoderm formation, there is little evidence for their functional role in mesoderm induction. We show that mouse embryonic stem cells, which resemble primitive ectoderm, can differentiate to mesoderm in vitro in a chemically defined medium (CDM) in the absence of fetal bovine serum. In CDM, this differentiation is responsive to TGF-beta family members in a concentration-dependent manner, with activin A mediating the formation of dorsoanterior-like mesoderm and bone morphogenetic protein 4 mediating the formation of ventral mesoderm, including hematopoietic precursors. These effects are not observed in CDM alone or when TGF-beta 1, -beta 2, or -beta 3, acid FGF, or basic FGF is added individually to CDM. In vivo, at day 6.5 of mouse development, activin beta A RNA is detectable in the decidua and bone morphogenetic protein 4 RNA is detectable in the egg cylinder. Together, our data strongly implicate the TGF-beta family in mammalian mesoderm development and hematopoietic cell formation.     

Essential role of heparan sulfate 2-O-sulfotransferase in chick limb bud patterning and development

The interactions of heparan sulfate (HS) with heparin-binding growth factors, such as fibroblast growth factors (FGFs), depend greatly on the chain structures. O-Sulfations at various positions on the chain are major factors determining HS structure; therefore, O-sulfation patterns may play a crucial role in controlling the developmental and morphogenetic processes of various tissues and organs by spatiotemporally regulating the activities of heparin-binding growth factors. In a previous study, we found that HS-2-O-sulfotransferase is strongly expressed throughout the mesoderm of chick limb buds during the early stages of development. Here we show that inhibition of HS-2-O-sulfotransferase in the prospective limb region by small inhibitory RNA resulted in the truncation of limb buds and reduced Fgf-8 expression in the apical ectodermal ridge. The treatment also reduced Fgf-10 expression in the mesenchyme. Moreover 2-O-sulfated HS, normally abundant in the basement membranes and mesoderm under ectoderm in limb buds, was significantly reduced in the treated buds. Phosphorylation levels of ERK and Akt were up-regulated in such truncated buds. Thus, we have shown for the first time that 2-O-sulfation of HS is essential for the FGF signaling required for limb bud development and outgrowth.     

Ventral axial organs regulate expression of myotomal Fgf-8 that influences rib development

Fgf-8 encodes a secreted signaling molecule mediating key roles in embryonic patterning. This study analyzes the expression pattern, regulation, and function of this growth factor in the paraxial mesoderm of the avian embryo. In the mature somite, expression of Fgf-8 is restricted to a subpopulation of myotome cells, comprising most, but not all, epaxial and hypaxial muscle precursors. Following ablation of the notochord and floor plate, Fgf-8 expression is not activated in the somites, in either the epaxial or the hypaxial domain, while ablation of the dorsal neural tube does not affect Fgf-8 expression in paraxial mesoderm. Contrary to the view that hypaxial muscle precursors are independent of regulatory influences from axial structures, these findings provide the first evidence for a regulatory influence of ventral, but not dorsal axial structures on the hypaxial muscle domain. Sonic hedgehog can substitute for the ventral neural tube and notochord in the initiation of Fgf-8 expression in the myotome. It is also shown that Fgf-8 protein leads to an increase in sclerotomal cell proliferation and enhances rib cartilage development in mature somites, whereas inhibition of Fgf signaling by SU 5402 causes deletions in developing ribs. These observations demonstrate: (1) a regulatory influence of the ventral axial organs on the hypaxial muscle compartment; (2) regulation of epaxial and hypaxial expression of Fgf-8 by Sonic hedgehog; and (3) independent regulation of Fgf-8 and MyoD in the hypaxial myotome by ventral axial organs. It is postulated that the notochord and ventral neural tube influence hypaxial expression of Fgf-8 in the myotome and that, in turn, Fgf-8 has a functional role in rib formation.     

Signaling by FGF4 and FGF8 is required for axial elongation of the mouse embryo

Fibroblast growth factor (FGF) signaling has been shown to play critical roles in vertebrate segmentation and elongation of the embryonic axis. Neither the exact roles of FGF signaling, nor the identity of the FGF ligands involved in these processes, has been conclusively determined. Fgf8 is required for cell migration away from the primitive streak when gastrulation initiates, but previous studies have shown that drastically reducing the level of FGF8 later in gastrulation has no apparent effect on somitogenesis or elongation of the embryo. In this study, we demonstrate that loss of both Fgf8 and Fgf4 expression during late gastrulation resulted in a dramatic skeletal phenotype. Thoracic vertebrae and ribs had abnormal morphology, lumbar and sacral vertebrae were malformed or completely absent, and no tail vertebrae were present. The expression of Wnt3a in the tail and the amount of nascent mesoderm expressing Brachyury were both severely reduced. Expression of genes in the NOTCH signaling pathway involved in segmentation was significantly affected, and somite formation ceased after the production of about 15-20 somites. Defects seen in the mutants appear to result from a failure to produce sufficient paraxial mesoderm, rather than a failure of mesoderm precursors to migrate away from the primitive streak. Although the epiblast prematurely decreases in size, we did not detect evidence of a change in the proliferation rate of cells in the tail region or excessive apoptosis of epiblast or mesoderm cells. We propose that FGF4 and FGF8 are required to maintain a population of progenitor cells in the epiblast that generates mesoderm and contributes to the stem cell population that is incorporated in the tailbud and required for axial elongation of the mouse embryo after gastrulation.     

TCreERT2, a Transgenic Mouse Line for Temporal Control of Cre-Mediated Recombination in Lineages Emerging from the Primitive Streak or Tail Bud

The study of axis extension and somitogenesis has been greatly advanced through the use of genetic tools such as the TCre mouse line. In this line, Cre is controlled by a fragment of the T (Brachyury) promoter that is active in progenitor cells that reside within the primitive streak and tail bud and which give rise to lineages emerging from these tissues as the embryonic axis extends. However, because TCre-mediated recombination occurs early in development, gene inactivation can result in an axis truncation that precludes the study of gene function in later or more posterior tissues. To address this limitation, we have generated an inducible TCre transgenic mouse line, called TCreERT2, that provides temporal control, through tamoxifen administration, in all cells emerging from the primitive streak or tail bud throughout development. TCreERT2 activity is mostly silent in the absence of tamoxifen and, in its presence, results in near complete recombination of emerging mesoderm from E7.5 through E13.5. We demonstrate the utility of the TCreERT2 line for determining rate of posterior axis extension and somite formation, thus providing the first in vivo tool for such measurements. To test the usefulness of TCreERT2 for genetic manipulation, we demonstrate that an early deletion of ß-Catenin via TCreERT2 induction phenocopies the TCre-mediated deletion of ß-Catenin defect, whereas a later induction bypasses this early phenotype and produces a similar defect in more caudal tissues. TCreERT2 provides a useful and novel tool for the control of gene expression of emerging embryonic lineages throughout development.