Expression pattern of the mouse T gene and its role in mesoderm formation.

Formation of mesoderm is a crucial event in vertebrate development, establishing many of the important features of the body. Recent studies have implicated molecules that are similar to growth factors in mesoderm formation in Xenopus, but other gene products involved in this process have yet to be identified. Genetic evidence indicates that in the mouse the T gene (Brachyury) has a role in the formation and organization of mesoderm. Mice homozygous for mutant alleles of the T gene do not generate enough mesoderm, and show severe disruption in morphogenesis of mesoderm-derived structures, in particular the notochord. The cloning of the T gene has now allowed us to examine its expression pattern. We report that T-gene expression occurs in both early stage mesoderm and its epithelial progenitor, and then becomes restricted to the notochord. This expression pattern correlates with the tissues affected in the T-gene mutant, and indicates that the T gene has a direct role in the early events of mesoderm formation and in the morphogenesis of the notochord.     

A cell autonomous function of Brachyury in T/T embryonic stem cell chimaeras

Developmental genetics has shown that the Brachyury (T) gene has a key role in mesoderm formation during gastrulation in the mouse. Homozygous embryos have a defective allantois, degenerate or absent notochord and disrupted primitive streak and node. The neural tube is kinked and somite formation interrupted. The T gene has been cloned and is expressed during the early stages of gastrulation, being restricted to the primitive streak region, nascent mesoderm and notochord. Neither the sequence of the gene nor its expression pattern define its developmental function. To study the cell autonomy of the T mutation we have isolated and genetically characterized embryonic stem cell lines and studied their behaviour in chimaeras. T/+ embryonic stem cells form normal chimaeras, whereas T/T in equilibrium with +/+ chimaeras mimic the T/T mutant phenotype. The results indicate that the T gene acts cell autonomously in the primitive streak and notochord but may activate a signalling pathway involved in the specification of other mesodermal tissues.     

Periodic notch inhibition by lunatic fringe underlies the chick segmentation clock

The segmented aspect of the vertebrate body plan first arises through the sequential formation of somites. The periodicity of somitogenesis is thought to be regulated by a molecular oscillator, the segmentation clock, which functions in presomitic mesoderm cells. This oscillator controls the periodic expression of 'cyclic genes', which are all related to the Notch pathway. The mechanism underlying this oscillator is not understood. Here we show that the protein product of the cyclic gene lunatic fringe (Lfng), which encodes a glycosyltransferase that can modify Notch activity, oscillates in the chick presomitic mesoderm. Overexpressing Lfng in the paraxial mesoderm abolishes the expression of cyclic genes including endogenous Lfng and leads to defects in segmentation. This effect on cyclic genes phenocopies inhibition of Notch signalling in the presomitic mesoderm. We therefore propose that Lfng establishes a negative feedback loop that implements periodic inhibition of Notch, which in turn controls the rhythmic expression of cyclic genes in the chick presomitic mesoderm. This feedback loop provides a molecular basis for the oscillator underlying the avian segmentation clock.     

Eomesodermin is required for mouse trophoblast development and mesoderm formation

The earliest cell fate decision in the mammalian embryo separates the extra-embryonic trophoblast lineage, which forms the fetal portion of the placenta, from the embryonic cell lineages. The body plan of the embryo proper is established only later at gastrulation, when the pluripotent epiblast gives rise to the germ layers ectoderm, mesoderm and endoderm. Here we show that the T-box gene Eomesodermin performs essential functions in both trophoblast development and gastrulation. Mouse embryos lacking Eomesodermin arrest at the blastocyst stage. Mutant trophoectoderm does not differentiate into trophoblast, indicating that Eomesodermin may be required for the development of trophoblast stem cells. In the embryo proper, Eomesodermin is essential for mesoderm formation. Although the specification of the anterior-posterior axis and the initial response to mesoderm-inducing signals is intact in mutant epiblasts, the prospective mesodermal cells are not recruited into the primitive streak. Our results indicate that Eomesodermin defines a conserved molecular pathway controlling the morphogenetic movements of germ layer formation and has acquired a new function in mammals in the differentiation of trophoblast.     

The novel Cer-like protein Caronte mediates the establishment of embryonic left-right asymmetry.

In the chick embryo, left-right asymmetric patterns of gene expression in the lateral plate mesoderm are initiated by signals located in and around Hensen's node. Here we show that Caronte (Car), a secreted protein encoded by a member of the Cerberus/Dan gene family, mediates the Sonic hedgehog (Shh)-dependent induction of left-specific genes in the lateral plate mesoderm. Car is induced by Shh and repressed by fibroblast growth factor-8 (FGF-8). Car activates the expression of Nodal by antagonizing a repressive activity of bone morphogenic proteins (BMPs). Our results define a complex network of antagonistic molecular interactions between Activin, FGF-8, Lefty-1, Nodal, BMPs and Car that cooperate to control left-right asymmetry in the chick embryo.     

Involvement of p21ras in Xenopus mesoderm induction.

During early vertebrate embryogenesis, mesoderm is specified by a signal emanating from prospective endoderm. This signal can respecify Xenopus prospective ectoderm as mesoderm, and can be mimicked by members of the fibroblast growth factor and transforming growth factor-beta families. In other systems, the p21c-ras proto-oncogene product has been implicated in signal transduction for various polypeptide growth factors. We report here that a dominant inhibitory ras mutant blocks the mesoderm-inducing activity of fibroblast growth factor and activin, as well as the endogenous inducing activity of prospective endoderm. A constitutively active ras mutant partially mimics these activities. These results indicate that p21ras may have a central role in the transduction of the mesoderm inductive signal. Basic fibroblast growth factor and activin have emerged as candidates for endogenous mesoderm-inducing molecules. The character of the mesoderm induced by these two factors is overlapping but distinct when assessed both by histological and molecular criteria. The signal transduction pathways used during induction by these factors are unknown. We used messenger RNA microinjection of Xenopus eggs to express a dominant inhibitory mutant ras, p21(Asn 17)Ha-ras, in cells competent to respond to inducing factors to examine the role of p21ras in this response. This mutant, which has a reduced affinity for GTP relative to GDP, blocks a variety of mitogenic signals in 3T3 fibroblasts as well as the differentiation of pheochromocytoma cells in response to nerve growth factor.     

Interaction between peptide growth factors and homoeobox genes in the establishment of antero-posterior polarity in frog embryos

The expression of the Xenopus homoeobox gene xhox3 is an early response to mesoderm induction by peptide growth factors and the level of xhox3 expression marks the antero-posterior character of the induced mesoderm. Different peptide growth factors specify different antero-posterior mesodermal cell fates as seen by the level of xhox3 expression and the capacity to induce specific secondary neural/epidermal structures. These factors and homoeobox genes thus form part of the mechanism necessary for establishing antero-posterior polarity in the frog embryo.     

Evidence that mechanisms of fin development evolved in the midline of early vertebrates

The origin of paired appendages was a major evolutionary innovation for vertebrates, marking the first step towards fin- (and later limb-) driven locomotion. The earliest vertebrate fossils lack paired fins but have well-developed median fins, suggesting that the mechanisms of fin development were assembled first in the midline. Here we show that shark median fin development involves the same genetic programs that operate in paired appendages. Using molecular markers for different cell types, we show that median fins arise predominantly from somitic (paraxial) mesoderm, whereas paired appendages develop from lateral plate mesoderm. Expression of Hoxd and Tbx18 genes, which specify paired limb positions, also delineates the positions of median fins. Proximodistal development of median fins occurs beneath an apical ectodermal ridge, the structure that controls outgrowth of paired appendages. Each median fin bud then acquires an anteroposteriorly-nested pattern of Hoxd expression similar to that which establishes skeletal polarity in limbs. Thus, despite their different embryonic origins, paired and median fins utilize a common suite of developmental mechanisms. We extended our analysis to lampreys, which diverged from the lineage leading to gnathostomes before the origin of paired appendages, and show that their median fins also develop from somites and express orthologous Hox and Tbx genes. Together these results suggest that the molecular mechanisms for fin development originated in somitic mesoderm of early vertebrates, and that the origin of paired appendages was associated with re-deployment of these mechanisms to lateral plate mesoderm.     

Jeb signals through the Alk receptor tyrosine kinase to drive visceral muscle fusion

The Drosophila melanogaster gene Anaplastic lymphoma kinase (Alk) is homologous to mammalian Alk, a member of the Alk/Ltk family of receptor tyrosine kinases (RTKs). We have previously shown that the Drosophila Alk RTK is crucial for visceral mesoderm development during early embryogenesis. Notably, observed Alk visceral mesoderm defects are highly reminiscent of the phenotype reported for the secreted molecule Jelly belly (Jeb). Here we show that Drosophila Alk is the receptor for Jeb in the developing visceral mesoderm, and that Jeb binding stimulates an Alk-driven, extracellular signal-regulated kinase-mediated signalling pathway, which results in the expression of the downstream gene duf (also known as kirre)--needed for muscle fusion. This new signal transduction pathway drives specification of the muscle founder cells, and the regulation of Duf expression by the Drosophila Alk RTK explains the visceral-mesoderm-specific muscle fusion defects observed in both Alk and jeb mutant animals.     

Retinoic acid signalling links left-right asymmetric patterning and bilaterally symmetric somitogenesis in the zebrafish embryo

During embryogenesis, cells are spatially patterned as a result of highly coordinated and stereotyped morphogenetic events. In the vertebrate embryo, information on laterality is conveyed to the node, and subsequently to the lateral plate mesoderm, by a complex cascade of epigenetic and genetic events, eventually leading to a left-right asymmetric body plan. At the same time, the paraxial mesoderm is patterned along the anterior-posterior axis in metameric units, or somites, in a bilaterally symmetric fashion. Here we characterize a cascade of laterality information in the zebrafish embryo and show that blocking the early steps of this cascade (before it reaches the lateral plate mesoderm) results in random left-right asymmetric somitogenesis. We also uncover a mechanism mediated by retinoic acid signalling that is crucial in buffering the influence of the flow of laterality information on the left-right progression of somite formation, and thus in ensuring bilaterally symmetric somitogenesis.     

Mox2 is a component of the genetic hierarchy controlling limb muscle development.

The skeletal muscles of the limbs develop from myogenic progenitors that originate in the paraxial mesoderm and migrate into the limb-bud mesenchyme. Among the genes known to be important for muscle development in mammalian embryos are those encoding the basic helix-loop-helix (bHLH) myogenic regulatory factors (MRFs; MyoD, Myf5, myogenin and MRF4) and Pax3, a paired-type homeobox gene that is critical for the development of limb musculature. Mox1 and Mox2 are closely related homeobox genes that are expressed in overlapping patterns in the paraxial mesoderm and its derivatives. Here we show that mice homozygous for a null mutation of Mox2 have a developmental defect of the limb musculature, characterized by an overall reduction in muscle mass and elimination of specific muscles. Mox2 is not needed for the migration of myogenic precursors into the limb bud, but it is essential for normal appendicular muscle formation and for the normal regulation of myogenic genes, as demonstrated by the downregulation of Pax3 and Myf5 but not MyoD in Mox2-deficient limb buds. Our findings show that the MOX2 homeoprotein is an important regulator of vertebrate limb myogenesis.     

Mesodermal Wnt2b signalling positively regulates liver specification

Endodermal organs such as the lung, liver and pancreas emerge at precise locations along the primitive gut tube. Although several signalling pathways have been implicated in liver formation, so far no single gene has been identified that exclusively regulates liver specification. In zebrafish, the onset of liver specification is marked by the localized endodermal expression of hhex and prox1 at 22 hours post fertilization. Here we used a screen for mutations affecting endodermal organ morphogenesis to identify a unique phenotype: prometheus (prt) mutants exhibit profound, though transient, defects in liver specification. Positional cloning reveals that prt encodes a previously unidentified Wnt2b homologue. prt/wnt2bb is expressed in restricted bilateral domains in the lateral plate mesoderm directly adjacent to the liver-forming endoderm. Mosaic analyses show the requirement for Prt/Wnt2bb in the lateral plate mesoderm, in agreement with the inductive properties of Wnt signalling. Taken together, these data reveal an unexpected positive role for Wnt signalling in liver specification, and indicate a possible common theme for the localized formation of endodermal organs along the gut tube.     

Cell type-specific activation of actin genes in the early amphibian embryo

Muscle actin genes are the earliest yet described to show cell type-specific activation in amphibian embryos. Gene-specific probes show that alpha-skeletal and alpha-cardiac actin genes start to be transcribed simultaneously at the end of gastrulation, but only in those regions of the mesoderm that subsequently form embryonic muscle. Their expression provides a molecular marker for early cell determination.     

Origin of cells giving rise to mesoderm and endoderm in chick embryo.

In amniotes, all of the tissues of the adult arise from the epiblast, one of the two layers of cells present in the early embryo; the mesoderm and gut endoderm arise from an epiblast-derived structure known as the primitive streak. The monoclonal antibody HNK-1 recognizes the cells of the primitive streak in the chick embryo. Before streak formation, HNK-1 identifies cells that are randomly distributed within the epiblast. We have now used two novel ways to study cell lineage and commitment to show that the epiblast of the early chick embryo contains two distinct populations of cells with different developmental fates at a stage during which 'mesodermal induction' is believed to occur. One cell population, recognized by monoclonal antibody HNK-1, is destined to form mesoderm and endoderm; the rest of the epiblast is unable to give rise to mesoderm if this population of cells is removed.