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Signaling by the Ret receptor tyrosine kinase promotes cell movements in the Wolffian duct that give rise to the first ureteric bud tip, initiating kidney development. Although the ETS transcription factors Etv4 and Etv5 are known to be required for mouse kidney development and to act downstream of Ret, their specific functions are unclear. Here, we examine their role by analyzing the ability of Etv4 Etv5 compound mutant cells to contribute to chimeric kidneys. Etv4−/−;Etv5+/− cells show a limited distribution in the caudal Wolffian duct and ureteric bud, similar to Ret−/− cells, revealing a cell-autonomous role for Etv4 and Etv5 in the cell rearrangements promoted by Ret. By contrast, Etv4−/−;Etv5−/− cells display more severe developmental limitations, suggesting a broad role for Etv4 and Etv5 downstream of multiple signals, which are together important for Wolffian duct and ureteric bud morphogenesis.

In humans and mice, mutations in the Ret gene result in Hirschsprung's disease and renal defects. In the embryonic kidney, binding of Ret to its ligand, Gdnf, induces a program of epithelial cell remodeling that controls primary branch formation and branching morphogenesis within the kidney. Our previous studies showed that transcription factors belonging to the retinoic acid (RA) receptor family are crucial for controlling Ret expression in the ureteric bud; however, the mechanism by which retinoid-signaling acts has remained unclear. In the current study, we show that expression of a dominant-negative RA receptor in mouse ureteric bud cells abolishes Ret expression and Ret-dependent functions including ureteric bud formation and branching morphogenesis, indicating that RA-receptor signaling in ureteric bud cells is crucial for renal development. Conversely, we find that RA-receptor signaling in ureteric bud cells depends mainly on RA generated in nearby stromal cells by retinaldehyde dehydrogenase 2, an enzyme required for most fetal RA synthesis. Together, these studies suggest that renal development depends on paracrine RA signaling between stromal mesenchyme and ureteric bud cells that regulates Ret expression both during ureteric bud formation and within the developing collecting duct system.

The RET receptor tyrosine kinase plays a critical role in the development of the enteric nervous system (ENS) and the kidney. Upon glial-cell-line-derived neurotrophic factor (GDNF) stimulation, RET can activate a variety of intracellular signals, including the Ras/mitogen-activated protein kinase, phosphatidylinositol 3-kinase(PI3K)/AKT, and RAC1/JUN NH2-terminal kinase (JNK) pathways. We recently demonstrated that the RAC1/JNK pathway is regulated by serine phosphorylation at the juxtamembrane region of RET in a cAMP-dependent manner. To determine the importance of cAMP-dependent modification of the RET signal in vivo, we generated mutant mice in which serine residue 697, a putative protein kinase A (PKA) phosphorylation site, was replaced with alanine(designated S697A mice). Homozygous S697A mutant mice lacked the ENS in the distal colon, resulting from a migration defect of enteric neural crest cells(ENCCs). In vitro organ culture showed an impaired chemoattractant response of the mutant ENCCs to GDNF. JNK activation by GDNF but not ERK, AKT and SRC activation was markedly reduced in neurons derived from the mutant mice. The JNK inhibitor SP600125 and the PKA inhibitor KT5720 suppressed migration of the ENCCs in cultured guts from wild-type mice to comparable degrees. Thus,these findings indicated that cAMP-dependent modification of RET function regulates the JNK signaling responsible for proper migration of the ENCCs in the developing gut.

Axin1 and its homolog Axin2/conductin/Axil are negative regulators of the canonical Wnt pathway that suppress signal transduction by promoting degradation of β-catenin. Mice with deletion of Axin1 exhibit defects in axis determination and brain patterning during early embryonic development. We show that Axin2 is expressed in the osteogenic fronts and periosteum of developing sutures during skull morphogenesis. Targeted disruption of Axin2 in mice induces malformations of skull structures, a phenotype resembling craniosynostosis in humans. In the mutants, premature fusion of cranial sutures occurs at early postnatal stages. To elucidate the mechanism of craniosynostosis, we studied intramembranous ossification in Axin2-null mice. The calvarial osteoblast development is significantly affected by the Axin2 mutation. The Axin2mutant displays enhanced expansion of osteoprogenitors, accelerated ossification, stimulated expression of osteogenic markers and increases in mineralization. Inactivation of Axin2 promotes osteoblast proliferation and differentiation in vivo and in vitro. Furthermore, as the mammalian skull is formed from cranial skeletogenic mesenchyme, which is derived from mesoderm and neural crest, our data argue for a region-specific effect of Axin2 on neural crest dependent skeletogenesis. The craniofacial anomalies caused by the Axin2 mutation are mediated through activation of β-catenin signaling, suggesting a novel role for the Wnt pathway in skull morphogenesis.

Erythropoietin, known for its role in erythroid differentiation, has been shown to be neuroprotective during brain ischaemia in adult animal models. Although high levels of erythropoietin receptor are produced in embryonic brain, the role of erythropoietin during brain development is uncertain. We now provide evidence that erythropoietin acts to stimulate neural progenitor cells and to prevent apoptosis in the embryonic brain. Mice lacking the erythropoietin receptor exhibit severe anaemia and defective cardiac development, and die at embryonic day 13.5 (E13.5). By E12.5, in addition to apoptosis in foetal liver, endocardium and myocardium, the erythropoietin receptor null mouse shows extensive apoptosis in foetal brain. Lack of erythropoietin receptor affects brain development as early as E10.5, resulting in a reduction in the number of neural progenitor cells and increased apoptosis. Corresponding in vitro cultures of cortical cells from Epor–/– mice also exhibited decreases in neuron generation compared with normal controls and increased sensitivity to low oxygen tension with no surviving neurons in Epor–/– cortical cultures after 24 hour exposure to hypoxia. The viability of primary Epor+/+ rodent embryonic cortical neurons was further increased by erythropoietin stimulation. Exposure of these cultures to hypoxia induced erythropoietin expression and a tenfold increase in erythropoietin receptor expression, increased cell survival and decreased apoptosis. Cultures of neuronal progenitor cells also exhibited a proliferative response to erythropoietin stimulation. These data demonstrate that the neuroprotective activity of erythropoietin is observed as early as E10.5 in the developing brain, and that induction of erythropoietin and its receptor by hypoxia may contribute to selective cell survival in the brain.

During kidney development, factors from the metanephric mesenchyme induce the growth and repeated branching of the ureteric bud, which gives rise to the collecting duct system and also induces nephrogenesis. One signaling pathway known to be required for this process includes the receptor tyrosine kinase RET and co-receptor GFRα-1, which are expressed in the ureteric bud, and the secreted ligand GDNF produced in the mesenchyme. To examine the role of RET signaling in ureteric bud morphogenesis, we produced transgenic mice in which the pattern of RET expression was altered, or in which a ligand-independent form of RET kinase was expressed. The Hoxb7 promoter was used to express RET throughout the ureteric bud branches, in contrast to its normal expression only at the bud tips. This caused a variable inhibition of ureteric bud growth and branching reminiscent of, but less severe than, the RET knockout phenotype. Manipulation of the level of GDNF, in vitro or in vivo, suggested that this defect was due to insufficient rather than excessive RET signaling. We propose that RET receptors expressed ectopically on ureteric bud trunk cells sequester GDNF, reducing its availability to the normal target cells at the bud tips. When crossed to RET knockout mice, the Hoxb7/RET transgene, which encoded the RET9 isoform, supported normal kidney development in some RET−/− animals, indicating that the other major isoform, RET51, is not required in this organ. Expression of a Hoxb7/RET-PTC2 transgene, encoding a ligand-independent form of RET kinase, caused the development of abnormal nodules, outside the kidney or at its periphery, containing branched epithelial tubules apparently formed by deregulated growth of the ureteric bud. This suggests that RET signaling is not only necessary but is sufficient to induce ureteric bud growth, and that the orderly, centripetal growth of the bud tips is controlled by the spatially and temporally regulated expression of GDNF and RET.

The c-ret gene encodes a receptor tyrosine kinase that is expressed in the Wolffian duct and ureteric bud of the developing excretory system. Newborn mice homozygous for a mutation in c-ret displayed renal agenesis or severe hypodysplasia, suggesting a critical role for this gene in metanephric kidney development. To investigate the embryological basis of these defects, we characterized the early development of the excretory system in mutant homozygotes, and observed a range of defects in the formation, growth and branching of the ureteric bud, which account for the spectrum of renal defects seen at birth. Co-culture of isolated ureteric buds and metanephric mesenchyme show that the primary defect is intrinsic to the ureteric bud. While the mutant bud failed to respond to induction by wild-type mesenchyme, mutant mesenchyme was competent to induce the growth and branching of the wild-type bud. Furthermore, the mutant metanephric mesenchyme displayed a normal capacity to differentiate into nephric tubules when co-cultured with embryonic spinal cord. These findings suggest a model in which c-ret encodes the receptor for a (yet to be identified) factor produced by the metanephric mesenchyme, which mediates the inductive effects of this tissue upon the ureteric bud. This factor appears to stimulate the initial evagination of the ureteric bud from the Wolffian duct, as well as its subsequent growth and branching.

c-ret encodes a tyrosine kinase receptor that is necessary for normal development of the mammalian enteric nervous system. Germline mutations in c-ret lead to congenital megacolon in humans, while a loss-of-function allele (ret.k −) causes intestinal aganglionosis in mice. Here we examine in detail the function of c-ret during neurogenesis, as well as the lineage relationships among cell populations in the enteric nervous system and the sympathetic nervous system that are dependent on c-ret function. We report that, while the intestine of newborn ret.k − mice is devoid of enteric ganglia, the esophagus and stomach are only partially affected; furthermore, the superior cervical ganglion is absent, while more posterior sympathetic ganglia and the adrenal medulla are unaffected. Analysis of mutant embryos shows that the superior cervical ganglion anlage is present at E10.5, but absent by E12.5, suggesting that c-ret is required for the survival or prolif-eration of sympathetic neuroblasts. In situ hybridization studies, as well as direct labelling of cells with DiI, indicate that a common pool of neural crest cells derived from the postotic hindbrain normally gives rise to most of the enteric nervous system and the superior cervical ganglion, and is uniquely dependent on c-ret function for normal develop-ment. We term this the sympathoenteric lineage. In contrast, a distinct sympathoadrenal lineage derived from trunk neural crest forms the more posterior sympathetic ganglia, and also contributes to the foregut enteric nervous system. Overall, our studies reveal previously unknown complexities of cell lineage and genetic control mechanisms in the developing mammalian peripheral nervous system.