Expression pattern of the Brachyury gene in whole-mount TWis/TWis mutant embryos.

The murine Brachyury (T) gene is required in mesoderm formation. Mutants carrying different T alleles show a graded severity of defects correlated with gene dosage along the body axis. The phenotypes range from shortening of the tail to the malformation of sacral vertebrae in heterozygotes, and to disruption of trunk development and embryonic death in homozygotes. Defects include a severe disturbance of the primitive streak, an early cessation of mesoderm formation and absence of the allantois and notochord, the latter resulting in an abnormality of the neural tube and somites. The T gene is expressed in nascent mesoderm and in the notochord of wild-type embryos. Here the expression of T in whole-mount mutant embryos homozygous for the T allele TWis is described. The TWis gene product is altered, but the TWis/TWis phenotype is very similar to that of T/T embryos which lack T. In early TWis/TWis embryos T expression is normal, but ceases prematurely during early organogenesis coincident with a cessation of mesoderm formation. The archenteron/node region is disrupted and the extension of the notochord precursor comes to a halt, followed by a decrease and finally a complete loss of T gene expression in the primitive streak and the head process/notochord precursor. It appears that the primary defect of the mutant embryo is the disruption of the notochord precursor in the node region which is required for axis elongation. Thus the T gene product is directly or indirectly involved in the organization of axial development.     

Candidate genes required for embryonic development: a comparative analysis of distal mouse chromosome 14 and human chromosome 13q22

Mice homozygous for the Ednrb(s-1Acrg) deletion arrest at embryonic day 8.5 from defects associated with mesoderm development. To determine the molecular basis of this phenotype, we initiated a positional cloning of the Acrg minimal region. This region was predicted to be gene-poor by several criteria. From comparative analysis with the syntenic human locus at 13q22 and gene prediction program analysis, we found a single cluster of four genes within the 1.4-to 2-Mb contig over the Acrg minimal region that is flanked by a gene desert. We also found 130 highly conserved nonexonic sequences that were distributed over the gene cluster and desert. The four genes encode the TBC (Tre-2, BUB2, CDC16) domain-containing protein KIAA0603, the ubiquitin carboxy-terminal hydrolase L3 (UCHL3), the F-box/PDZ/LIM domain protein LMO7,and a novel gene. On the basis of their expression profile during development, all four genes are candidates for the Ednrb(s-1Acrg) embryonic lethality. Because we determined that a mutant of Uchl3 was viable, three candidate genes remain within the region.     

Experimental analysis of the mechanisms of somite morphogenesis

Earlier studies have suggested influences on somite morphogenesis by “somite-forming centers,” primitive streak regression, Hensen's node and notochord, and neural plate. Contradictions among these studies were unresolved.Our experiments resolve these conflicts and reveal roles of the primitive streak and notochord in shearing the prospective somite mesoderm into right and left halves and releasing somite-forming capabilities already present. The neural plate appears to be the principal inductor of somites.Embryo fragments containing no somite-forming centers, node, notochord, or streak nevertheless formed somites within 10 hr. Such somites disperse within the next 14–24 hr, which may explain why others failed to see them. In these fragments, an incision alongside the streak substitutes for streak regression in releasing somite formation. All such somites form simultaneously rather than in the normal anteroposterior progression. These fragments contain neural plate, but not notochord. We believe that physical attachment of somites to notochord in normal embryos stabilizes them and prevents dispersal.Pieces of epiblast were rotated 180° putting neural plate over lateral plate mesoderm regions. Somites were induced from the lateral plate by the displaced neural plate region. This is additional evidence of the powerful ability of neuroepithelium to induce somites.     

Fate maps of the primitive streak in chick and quail embryo: ingression timing of progenitor cells of each rostro-caudal axial level of somites

Developmental fates of cells emigrating from the primitive streak were traced by a fluorescent dye Dil both in chick and in quail embryos from the fully grown streak stage to 12-somite stage, focusing on the development of mesoderm and especially on the timing of ingression of each level of somitic mesoderm. The fate maps of the chick and quail streak were alike, although the chick streak was longer at all stages examined. The anterior part of the primitive streak predominantly produced somites. The thoracic and the lumbar somites were shown to begin to ingress at the 5 somite-stage and 10 somite-stage in a chick embryo, and 6 somite-stage and 9 somite-stage in a quail embryo, respectively. The posterior part of the streak served mainly as the origin of more lateral or extra embryonic mesoderm. As development proceeded, the fate of the posterior part of the streak changed from the lateral plate mesoderm to the tail bud mesoderm and then to extra embryonic, allantois mesoderm. The fate map of the primitive streak in chick and quail embryo presented here will serve as basic data for studies on mesoderm development with embryo manipulation, especially for transplantation experiments between chick and quail embryos.     

The formation of mesodermal tissues in the mouse embryo during gastrulation and early organogenesis

Orthotopic grafts of [3H]thymidine-labelled cells have been used to demonstrate differences in the normal fate of tissue located adjacent to and in different regions of the primitive streak of 8th day mouse embryos developing in vitro. The posterior streak produces predominantly extraembryonic mesoderm, while the middle portion gives rise to lateral mesoderm and the anterior region generates mostly paraxial mesoderm, gut and notochord. Embryonic ectoderm adjacent to the anterior part of the streak contributes mainly to paraxial mesoderm and neurectoderm. This pattern of colonization is similar to the fate map constructed in primitive-streak-stage chick embryos. Similar grafts between early-somite-stage (9th day) embryos have established that the older primitive streak continues to generate embryonic mesoderm and endoderm, but ceases to make a substantial contribution to extraembryonic mesoderm. Orthotopic grafts and specific labelling of ectodermal cells with wheat germ agglutinin conjugated to colloidal gold (WGA-Au) have been used to analyse the recruitment of cells into the paraxial mesoderm of 8th and 9th day embryos. The continuous addition of primitive-streak-derived cells to the paraxial mesoderm is confirmed and the distribution of labelled cells along the craniocaudal sequence of somites is consistent with some cell mixing occurring within the presomitic mesoderm.     

BMP signaling through ACVRI is required for left-right patterning in the early mouse embryo

Vertebrate organisms are characterized by dorsal-ventral and left-right asymmetry. The process that establishes left-right asymmetry during vertebrate development involves bone morphogenetic protein (BMP)-dependent signaling, but the molecular details of this signaling pathway remain poorly defined. This study tests the role of the BMP type I receptor ACVRI in establishing left-right asymmetry in chimeric mouse embryos. Mouse embryonic stem (ES) cells with a homozygous deletion at Acvr1 were used to generate chimeric embryos. Chimeric embryos were rescued from the gastrulation defect of Acvr1 null embryos but exhibited abnormal heart looping and embryonic turning. High mutant contribution chimeras expressed left-side markers such as nodal bilaterally in the lateral plate mesoderm (LPM), indicating that loss of ACVRI signaling leads to left isomerism. Expression of lefty1 was absent in the midline of chimeric embryos, but shh, a midline marker, was expressed normally, suggesting that, despite formation of midline, its barrier function was abolished. High-contribution chimeras also lacked asymmetric expression of nodal in the node. These data suggest that ACVRI signaling negatively regulates left-side determinants such as nodal and positively regulates lefty1. These functions maintain the midline, restrict expression of left-side markers, and are required for left-right pattern formation during embryogenesis in the mouse.     

Mesoderm patterning and somite formation during node regression: differential effects of chordin and noggin.

In Xenopus, one of the properties defining Spemann's organizer is its ability to dorsalise the mesoderm. When placed ajacent to prospective lateral/ventral mesoderm (blood, mesenchyme), the organizer causes these cells to adopt a more axial/dorsal fate (muscle). It seems likely that a similar property patterns the primitive streak of higher vertebrate embryos, but this has not yet been demonstrated clearly. Using quail/chick chimaeras and a panel of molecular markers, we show that Hensen's node (the amniote organizer) can induce posterior primitive streak (prospective lateral plate) to form somites (but not notochord) at the early neurula stage. We tested two BMP antagonists, noggin and chordin (both of which are expressed in the organizer), for their ability to generate somites and intermediate mesoderm from posterior streak, and find that noggin, but not chordin, can do this. Conversely, earlier in development, chordin can induce an ectopic primitive streak much more effectively than noggin, while neither BMP antagonist can induce neural tissue from extraembryonic epiblast. Neurulation is accompanied by regression of the node, which brings the prospective somite territory into a region expressing BMP-2, -4 and -7. One function of noggin at this stage may be to protect the prospective somite cells from the inhibitory action of BMPs. Our results suggest that the two BMP antagonists, noggin and chordin, may serve different functions during early stages of amniote development.     

Retinoic-acid signalling in node ectoderm and posterior neural plate directs left-right patterning of somitic mesoderm

Somitogenesis requires bilateral rhythmic segmentation of paraxial mesoderm along the antero-posterior axis. The location of somite segmentation depends on opposing signalling gradients of retinoic acid (generated by retinaldehyde dehydrogenase-2; Raldh2) anteriorly and fibroblast growth factor (FGF; generated by Fgf8) posteriorly. Retinoic-acid-deficient embryos exhibit somite left-right asymmetry, but it remains unclear how retinoic acid mediates left-right patterning. Here, we demonstrate that retinoic-acid signalling is uniform across the left-right axis and occurs in node ectoderm but not node mesoderm. In Raldh2(-/-) mouse embryos, ectodermal Fgf8 expression encroaches anteriorly into node ectoderm and neural plate, but its expression in presomitic mesoderm is initially unchanged. The late stages of somitogenesis were rescued in Raldh2(-/-) mouse embryos when the maternal diet was supplemented with retinoic acid until only the 6-somite stage, demonstrating that retinoic acid is only needed during node stages. A retinoic-acid-reporter transgene marking the action of maternal retinoic acid in rescued Raldh2(-/-) embryos revealed that the targets of retinoic-acid signalling during somitogenesis are the node ectoderm and the posterior neural plate, not the presomitic mesoderm. Our findings suggest that antagonism of Fgf8 expression by retinoic acid occurs in the ectoderm and that failure of this mechanism generates excessive FGF8 signalling to adjacent mesoderm, resulting initially in smaller somites and then left-right asymmetry.     

Ciliation and gene expression distinguish between node and posterior notochord in the mammalian embryo

The mammalian node, the functional equivalent of the frog dorsal blastoporal lip (Spemann's organizer), was originally described by Viktor Hensen in 1876 in the rabbit embryo as a mass of cells at the anterior end of the primitive streak. Today, the term "node" is commonly used to describe a bilaminar epithelial groove presenting itself as an indentation or "pit" at the distal tip of the mouse egg cylinder, and cilia on its ventral side are held responsible for molecular laterality (left-right) determination. We find that Hensen's node in the rabbit is devoid of cilia, and that ciliated cells are restricted to the notochordal plate, which emerges from the node rostrally. In a comparative approach, we use the organizer marker gene Goosecoid (Gsc) to show that a region of densely packed epithelium-like cells at the anterior end of the primitive streak represents the node in mouse and rabbit and is covered ventrally by a hypoblast (termed "visceral endoderm" in the mouse). Expression of Nodal, a gene intricately involved in the determination of vertebrate laterality, delineates the wide plate-like posterior segment of the notochord in the rabbit and mouse, which in the latter is represented by the indentation frequently termed "the node." Similarly characteristic ciliation and nodal expression exists in Xenopus neurula embryos in the gastrocoel roof plate (GRP), i.e., at the posterior end of the notochord anterior to the blastoporal lip. Our data suggest that (1) a posterior segment of the notochord, here termed PNC (for posterior notochord), is characterized by features known to be involved in laterality determination, (2) the GRP in Xenopus is equivalent to the mammalian PNC, and (3) the mammalian node as defined by organizer gene expression is devoid of cilia and most likely not directly involved in laterality determination.     

Regionalisation of the mouse embryonic ectoderm: allocation of prospective ectodermal tissues during gastrulation

The regionalisation of cell fate in the embryonic ectoderm was studied by analyzing the distribution of graft-derived cells in the chimaeric embryo following grafting of wheat germ agglutinin--gold-labelled cells and culturing primitive-streak-stage mouse embryos. Embryonic ectoderm in the anterior region of the egg cylinder contributes to the neuroectoderm of the prosencephalon and mesencephalon. Cells in the distal lateral region give rise to the neuroectoderm of the rhombencephalon and the spinal cord. Embryonic ectoderm at the archenteron and adjacent to the middle region of the primitive streak contributes to the neuroepithelium of the spinal cord. The proximal-lateral ectoderm and the ectodermal cells adjacent to the posterior region of the primitive streak produce the surface ectoderm, the epidermal placodes and the cranial neural crest cells. Some labelled cells grafted to the anterior midline are found in the oral ectodermal lining, whereas cells from the archenteron are found in the notochord. With respect to mesodermal tissues, ectoderm at the archenteron and the distal-lateral region of the egg cylinder gives rise to rhombencephalic somitomeres, and the embryonic ectoderm adjacent to the primitive streak contributes to the somitic mesoderm and the lateral mesoderm. Based upon results of this and other grafting studies, a map of prospective ectodermal tissues in the embryonic ectoderm of the full-streak-stage mouse embryo is constructed.     

Distinct requirements for extra-embryonic and embryonic bone morphogenetic protein 4 in the formation of the node and primitive streak and coordination of left-right asymmetry in the mouse

In the mouse and chick embryo, the node plays a central role in generating left-right (LR) positional information. Using several different strategies, we provide evidence in the mouse that bone morphogenetic protein 4 (Bmp4) is required independently in two different sites for node morphogenesis and for LR patterning. Bmp4 expression in the trophoblast-derived extra-embryonic ectoderm is essential for the normal formation of the node and primitive streak. However, tetraploid chimera analysis demonstrates that Bmp4 made in epiblast-derived tissues is required for robust LR patterning, even when normal node morphology is restored. In the absence of embryonic Bmp4, the expression of left-side determinants such as Nodal and Lefty2 is absent in the left lateral plate mesoderm (LPM). Noggin-mediated inhibition of Bmp activity in cultured wild-type embryos results in suppression of Nodal expression in the LPM. Thus, unlike previous models proposed in the chick embryo in which Bmp4 suppresses left-sided gene expression, our results suggest that Bmp acts as a positive facilitator of the left-sided molecular cascade and is required for Nodal induction and maintenance in the left LPM.     

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.     

Distinct functions of the major Fgf8 spliceform, Fgf8b, before and during mouse gastrulation

The vertebrate Fgf8 gene produces multiple protein isoforms by alternative splicing. Two evolutionarily conserved spliceforms, Fgf8a and Fgf8b, exhibit distinct bioactivities, with Fgf8b having a more potent inductive activity due to higher affinity for Fgf receptors. To investigate the in vivo requirement for Fgf8b, we created a splice-site mutation abolishing Fgf8b expression in mice. Analysis of this mutant has uncovered a novel function of Fgf8 signaling before the onset of gastrulation. We show that the loss of Fgf8b disrupts the induction of the brachyury gene in the pregastrular embryo and, in addition, disrupts the proper alignment of the anteroposterior axis with the shape of the embryo and the uterine axes at embryonic day (E) 6.5. Importantly, Fgf8-null embryos display the same phenotype as Fgf8b-deficient embryos at E6.5, demonstrating that signaling by Fgf8b is specifically required for development of the pregastrular embryo. By contrast, during gastrulation, Fgf8a can partially compensate for the loss of Fgf8b in mesoderm specification. We show that an increased level of Fgf8a expression, which leads to Fgf4 expression in the primitive streak, can also promote mesoderm migration in the absence of Fgf8b. Therefore, different Fgf signals may have distinct requirements for the morphogenesis and gene regulation before and during gastrulation. Importantly, our findings implicate Fgf8 in the morphogenetic process that establishes the defined relationship between the axes of the embryo and the uterus at the beginning of gastrulation, a perplexing phenomenon discovered two decades ago.     

An early marker of axial pattern in the chick embryo and its respecification by retinoic acid.

Chick Ghox 2.9 protein, a homeodomain-containing polypeptide, is first detected in the mid-gastrula stage embryo and its levels increase rapidly in the late gastrula. At this time, the initially narrow band of expression along the primitive streak expands laterally to form a shield-like domain that encompasses almost the entire posterior region of the embryo and extends anteriorly as far as Hensen's node. We have found that this expression domain co-localizes with a morphological feature that consists of a stratum of refractile, thickened mesoderm. Antibody-staining indicates that Ghox 2.9 protein is present in all cells of this mesodermal region. In contrast, expression within the ectoderm overlying the region of refractile mesoderm varies considerably. The highest levels of expression are found in ectoderm near the streak and surrounding Hensen's node, regions that recent fate mapping studies suggest that primarily destined to give rise to neurectoderm. At the definitive streak stage (Hamburger and Hamilton stage 4) the chick embryo is especially sensitive to the induction of axial malformations by retinoic acid. Four hours after the treatment of definitive streak embryos with a pulse of retinoic acid the expression of Ghox 2.9 protein is greatly elevated. This ectopic expression occurs in tissues anterior to Hensen's node, including floor plate, notochord, presumptive neural plate and lateral plate mesoderm, but does not occur in the anteriormost region of the embryo. The ectopic induction of Ghox 2.9 is strongest in ectoderm, and weaker in the underlying mesoderm. Endoderm throughout the embryo is unresponsive. At stage 11, Ghox 2.9 is normally expressed at high levels within rhombomere 4 of the developing hindbrain. In retinoic-acid-treated embryos which have developed to this stage, typical rhombomere boundaries are largely absent. Nevertheless, Ghox 2.9 is still expressed as a discrete band, but one that is widened and displaced to a more anterior position.     

Defects of the body plan of mutant embryos lacking Lim1, Otx2 or Hnf3beta activity

The orientation of the anterior-posterior (A-P) axis was examined in gastrula-stage Hnf3beta, Otx2 and Lim1 null mutant embryos that display defective axis development. In situ hybridization analysis of the expression pattern of genes associated with the posterior germ layer tissues and the primitive streak (T, Wnt3 and Fgf8) and anterior endoderm (Cer1 and Sox17) revealed that the A-P axis of mutant embryos remains aligned with the proximo-distal plane of the gastrula. Further analysis revealed that cells which express Chrd activity are either absent in Hnf3beta mutant embryos or localised in heterotopic sites in Lim1 and Otx2 null mutants. Lim1-expressing cells are present in the Hnf3beta mutant embryo albeit in heterotopic sites. In all three mutants, Gsc-expressing cells are missing from the anterior mesendoderm. These findings suggest that although some cells with organizer activity may be present in the mutant embryo, they are not properly localised and fail to contribute to the axial mesoderm of the head. By contrast, in T/T mutant embryos that display normal head fold development, the expression domains of organizer, primitive streak and anterior endoderm genes are regionalised correctly in the gastrula.