Mutational analysis in humans and mice has demonstrated that the Ret, the product of the c-ret proto-oncogene, a member of the receptor tyrosine kinase (RTK) superfamily, is essential for development of the enteric nervous system and kidney. Despite the established role of Ret in mammalian embryogenesis, its cognate ligand(s) is currently unknown. Here we demonstrate, by using a Xenopus embryo bioassay, that glial-cell-line-derived neurotrophic factor (GDNF), a distant member of the transforming growth factor (TGF)-beta superfamily, signals through the Ret RTK. Furthermore, using explant cultures from wild-type and Ret-deficient mouse embryos, we show that normal c-ret function is necessary for GDNF signalling in the peripheral nervous system. Our data strongly suggest that Ret is a functional receptor for GDNF, and that GDNF, in addition to its potential role in the differentiation and survival of central nervous system neurons, has profound effects on kidney organogenesis and the development of the peripheral nervous system.

In Drosophila, the products of the Polycomb group (Pc‐G) of genes act as chromatin‐associated multimeric protein complexes that repress expression of homeotic genes. Vertebrate Pc‐G homologues have been identified, but the nature of the complexes they form and the mechanisms of their action are largely unknown. The Polycomb homologue M33 is implicated in mesoderm patterning in the mouse and here we show that it acts as a transcriptional repressor in transiently transfected cells. Furthermore, we have identified two murine proteins, Ring1A and Ring1B, that interact directly with the repressor domain of M33. Ring1A and Ring1B display blocks of similarity throughout their sequences, including an N‐terminal RING finger domain. However, the interaction with M33 occurs through a region at the C‐terminus. Ring1A represses transcription through sequences not involved in M33 binding. Ring1A protein co‐localizes in nuclear domains with M33 and other Pc‐G homologues, such as Bmi1. The expression of Ring1A at early stages of development is restricted to the neural tube, whereas M33 is expressed ubiquitously. Within the neural tube, Ring1A RNA is located at the rhombomere boundaries of the hindbrain. Taken together, these data suggest that Ring1A may contribute to a tissue‐specific function of Pc‐G–protein complexes during mammalian development.

The c-ret proto-oncogene, a member of the receptor tyrosine kinase gene superfamily, plays a critical role in the development of the excretory system and the enteric and autonomic nervous systems of mammalian embryos. To study the potential function of the c-ret locus in lower vertebrates, we have isolated its zebra®sh homologue, ret1 and established its expression pattern during embryogenesis. Ret1 mRNA ®rst appears during early somitogenesis in the presumptive brain, spinal cord and excretory system. Within the CNS, expression of ret1 is detected in primary motor and sensory (Rohon ± Beard) neurons. Ret1 transcripts are also expressed in subsets of neural crest cells and cranial ganglia as well as in the enteric nervous system. In the excretory system, expression is detected in the developing nephric duct and the pronephros. Our ®ndings reveal a remarkable similarity in the expression pattern of c-ret between higher and lower vertebrates, suggesting that the function of this locus has been conserved throughout vertebrate evolution. Furthermore, the conservation of ret1 expression in cell types which remain una ected by the mammalian c-ret mutations, such as motor and sensory neurons, suggests a function of this receptor in these cell lineages.

The majority of neurones and glia of the enteric nervous system (ENS) are derived from the vagal neural crest. Shortly after emigration from the neural tube, ENS progenitors invade the anterior foregut and, migrating in a rostrocaudal direction, colonise in an orderly fashion the rest of the foregut, the midgut and the hindgut. We provide evidence that activation of the receptor tyrosine kinase RET by glial cell line-derived neurotrophic factor (GDNF) is required for the directional migration of ENS progenitors towards and within the gut wall. We find that neural crest-derived cells present within foetal small intestine explants migrate towards an exogenous source of GDNF in a RET-dependent fashion. Consistent with an in vivo role of GDNF in the migration of ENS progenitors, we demonstrate that Gdnf is expressed at high levels in the gut of mouse embryos in a spatially and temporally regulated manner. Thus, during invasion of the foregut by vagal-derived neural crest cells, expression of Gdnf was restricted to the mesenchyme of the stomach, ahead of the invading NC cells. Twenty-four hours later and as the ENS progenitors were colonising the midgut,Gdnf expression was upregulated in a more posterior region —the caecum anlage. In further support of a role of endogenous GDNF in enteric neural crest cell migration, we find that in explant cultures GDNF produced by caecum is sufficient to attract NC cells residing in more anterior gut segments. In addition, two independently generated loss-of-function alleles of murine Ret, Ret.k— and miRet51, result in characteristic defects of neural crest cell migration within the developing gut. Finally, we identify phosphatidylinositol-3 kinase and the mitogen-activated protein kinase signalling pathways as playing crucial roles in the migratory response of enteric neural crest cells to GDNF.

RET is a member of the receptor tyrosine kinase (RTK) superfamily, which can transduce signalling by glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) in cultured cells. In order to determine whether in addition to being sufficient, RET is also necessary for signalling by these growth factors, we studied the response to GDNF and NTN of primary neuronal cultures (peripheral sensory and central dopaminergic neurons) derived from wild-type and RET-deficient mice. Our experiments show that absence of a functional RET receptor abrogates the biological responses of neuronal cells to both GDNF and NTN. Despite the established role of the RET signal transduction pathway in the development of the mammalian enteric nervous system (ENS), very little is known regarding its cellular mechanism(s) of action. Here, we have studied the effects of GDNF and NTN on cultures of neural crest (NC)-derived cells isolated from the gut of rat embryos. Our findings suggest that GDNF and NTN promote the survival of enteric neurons as well as the survival, proliferation and differentiation of multipotential ENS progenitors present in the gut of E12.5-13.5 rat embryos. However, the effects of these growth factors are stage-specific, since similar ENS cultures established from later stage embryos (E14. 5–15.5), show markedly diminished response to GDNF and NTN. To examine whether the in vitro effects of RET activation reflect the in vivo function(s) of this receptor, the extent of programmed cell death was examined in the gut of wild-type and RET-deficient mouse embryos by TUNEL histochemistry. Our experiments show that a subpopulation of enteric NC undergoes apoptotic cell death specifically in the foregut of embryos lacking the RET receptor. We suggest that normal function of the RET RTK is required in vivo during early stages of ENS histogenesis for the survival of undifferentiated enteric NC and their derivatives.

The products of the Polycomb group (PcG) of genes act as transcriptional repressors involved in the maintenance of homeotic gene expression patterns throughout development, from flies to mice. Biochemical and molecular evidence suggests that the mouse Ring1A gene is a member of the PcG of genes. However, genetic evidence is needed to establish PcG function for Ring1A, since contrary to all other murine PcG genes, there is no known Drosophila PcG gene encoding a homolog of the Ring1A protein. To study Ring1A function we have generated a mouse line lacking Ring1A and mouse lines overexpressing Ring1A. Both Ring1A(−/−)and Ring1A(+/−) mice show anterior transformations and other abnormalities of the axial skeleton, which indicates an unusual sensitivity of axial skeleton patterning to Ring1A gene dosage. Ectopic expression of Ring1A also results in dose-dependent anterior transformations of vertebral identity, many of which, interestingly, are shared by Ring1A(−/−) mice. In contrast, the alterations of Hox gene expression observed in both type of mutant mice are subtle and involve a reduced number of Hox genes. Taken together, these results provide genetic evidence for a PcG function of the mouse Ring1A gene.

The enteric nervous system of vertebrates is derived from neural crest cells that invade the gut wall and generate a highly organised network of enteric ganglia. Among the genes that play an important role in ENS development is c-Ret, mutations of which result in failure of formation of enteric ganglia (intestinal aganglionosis). To further understand the development of the mammalian ENS in general and the mechanism of action of the RET RTK in particular, we have developed and used an organotypic culture system of mouse fetal gut. At the stage of culture initiation, the gut is partially populated by undifferentiated ENS progenitors, but culture for several days results in extensive neuronal and glial differentiation. Using this organ culture system, we have compared the development of the ENS in wild-type and RET-deficient gut and showed that the aganglionic phenotype observed in vivo is consistently reproduced under the in vitro culture conditions. Microinjection of RET+ cells isolated from E11.5 mouse bowel into wild-type or RET-deficient aganglionic gut in organ culture, results in extensive repopulation of their wall by exogenously derived neurons and glia. Finally, using a similar approach, we demonstrate that single RET+ cells introduced into the wall of wild-type gut generate both cell lineages of the ENS, i.e. neurons and glia. Our data show the NC-derived RET+ population of fetal gut in mammalian embryos consists of multipotential progenitors capable of colonising efficiently both wild-type and RET-deficient aganglionic bowel in organ culture.

The expression of transgene loci in mammals often occurs in a heterocellular fashion resulting in variegated patterns of expression. We have examined the effect of chromosomal integration site, copy number, and transcriptionally activating sequences on the variegation of a keratin 5-lacZ (K5Z) construct in the stratified epithelia of transgenic mice. lacZ expression in these mice is always mosaic, and the β-gal activity per cell is usually higher in the lines with a higher proportion of expressing cells. Similar constructs, in which cDNAs were exchanged by lacZ sequences, showed no variegation. Also, when a strongly active, nonvariegating construct was coinjected with K5Z, most transgenic lines showed an almost homogeneous lacZ expression. The comparison of transgene arrays of different copies inserted at the same locus (obtained by using a lox/Cre system) showed that the reduction of copy number does not lead to an increase in the proportion of cells that express the transgene. Finally, in most of the variegating or nonexpressing lines the transgenes were located both at intermediate positions and at peritelomeric regions in the long chromosome arms. These findings suggest that the probability and efficiency of expression of K5Z genes depend on both long range chromosomal influences and on sequences in the transgene array.