Histological characterisation of the gastro-intestinal tracts of homozygous embryos revealed absence o f myenteric and submucosal plexus o f the ENS (Schuchardt et al.,
1994). This analysis was performed by immunohistochemistry and in situ hybridisation against pan neuronal and glial markers on histological sections and in vriiole-mount preparations o f embryonic guts (Schuchardt et al., 1994; Durbec et al., 1996). Initial analysis using antibodies against neurone-specific enolase and peripherin showed absence o f neurones of the myenteric plexus in the gut walls of the small and large intestines, and stomach of mutant newborn mice (Schuchardt et al., 1994). Detailed analysis, during organogenesis of the ENS, by immunostaining with sympathoadrenal linage markers (Anderson, 1993) such as tyrosine hydroxylase-TH (rate limiting enzymes in the catecholamine biosynthetic pathway), dopamine-|3-hydroxylase-D|3H c)q)ressed by enteric neuroblasts, and cRNA hybridisation against MASH-1, a transcription factor expressed in sympathetic and enteric neural crest cell population (Lo et al., 1991), and to a minor population of truncated c-ret RNA synthesised from the ablated locus (Schuchardt et al., 1994) showed failure o f enteric neuroblasts to colonise the midgut and hindgut of E l 1.5 homozygous mutant embryos (Durbec et al., 1996). However, the rudiments of the esophagus and stomach was only partially afflicted.
Lineage studies in the avian embryos have implied a lineal relationship between enteric and sympathetic nervous systems (Camathan et al., 1991). Examination o f the sympathetic nervous system o f E16.5 mutant embryos revealed complete bilateral dysplasia o f the superior cervical ganglia (SCO), the most anterior ganglion o f the sympathetic chain (Durbec et al., 1996). All adjacent ganglia from the cervical stellate to the most caudal paravertebral sympathetic ganglia and the adrenal medulla appeared normal. Using neurofilament antisera to detect postmitotic neurones and tyrosine hydroxylase (TH) antisera, Phox-2 and c-ret cRNA as sympathoblast markers, sympathetic gangliogenesis was investigated in the mutant and wild type embryos (Durbec et al., 1996). In ElO-10.5 embryos, the earliest stage in mouse embryogenesis
in which the anlage o f the sympathetic ganglia can be clearly* discemedi, no differences in the formation o f the ganglia was observed between mutant and wild-type embryos using TH and Phox-2 markers. However in E12.5 embryos, vdiilst strong immunoreactive TH^ SCG precursors were present around the dorsal aorta o f wild type and heterozygous embryos, no such immunoreactive cells were present in the homozygous mutant embryos (Durbec et al., 1996). This implies c-ret gene fimction is essential for the survival and differentiation of anlage of the SCG.
Detailed analysis o f c-ret gene expression in E9.0 - E l0.5 normal embryos showed that the SCG and enteric neuroblasts may form fi*om a common pool o f c-ret expressing cells. This common pool of cells, observed in close association with cervical branches of the dorsal aorta in E9 mouse embryos, diverge during the subsequent 1 2 hours of development into a ventral population wfiich invades the foregut mesenchyme and a more dorsal group that remain in association with the ventral side o f the dorsal aorta (Durbec et al., 1996). Vagal neural crest cells present in the mesenchyme o f the brachial arches have been shown to express c-ret (Pachnis et al., 1993). This suggested that the common pool of c-ret expressing cells present at the dorsal aorta and foregut may be of vagal crest origin. To directly test this possibility, Dil mediated lineage tracing of the vagal and trunk neural crest cells was performed by in vitro embryo culture (Durbec et al., 1996). Dil was injected into vagal and trunk neural crest cells of E8.5 embryos and the fate of the marked cells traced after 48 hours of embryo culture. Application of the tracer Dil to the dorsal aspect o f E8 . 8 embryos resulted in fluorescent cells appearing, after 48 hour embryo culture, in the vagal (X*) ganglion, foregut and midgut mesenchyme, and in the anlage o f the SCG. This indicated that, in common with the observation in the avian embryo (Camathan et al., 1991), the mammalian postotic hindbrain is an important source of precursors o f the ENS and SCG. The expression of c-ret in this population of cells appears to be an early ontogenetic marker of this linage. Furthermore, since loss-of- ftmction mutation o f the c-ret locus results in the ablation of the SCG and midgut and hindgut enteric neurones, Durbec et al. (1996) suggest this Sympatho-Enteric linage to be dependent on c-ret function.
In addition to lineage tracing of the vagal neural crest cells, Durbec et al. (1996) also Dil marked and traced the anterior trunk neural crest cell population. Labeling o f the neural crest cells posterior to the 8* somite resulted, as expected, in the appearance o f fluorescent cells in the DRG. However, labeling neural crest cells at the level of somites 6-7, resulted in fluorescent cells appearing in the anterior DRG and in foregut mesenchyme. Based on this observation, Durbec et al. (1996) hypothesise the enteric neuroblasts present in the foregut of c-ret mutant embryos may be derived from this population of trunk neural crest cells which may not be dependent on c-ret function. However, this assertion remain untested as yet.
C c-ret Fimction Essential fo r Metanephric Induction
Analysis o f gross anatomy of neonates homozygous for the loss-of-function mutation o f the c-ret locus revealed renal agenesis with either a unilateral or bilateral dysplasia o f metanephric kidneys (Schuchardt et al., 1994). 58% of homozygotes displayed complete kidney agenesis; of these some (33%) displayed a complete absence of both ureter and kidneys and others contained both ureters (5%) or only one o f the ureters (20%). The remaining 42% of homozygotes possessed incompletely developed kidney rudiments; o f these some (1 1%) possed both ureters and both kidney rudiments, others (29%) displayed unilateral ablation of one ureter and kidney rudiment and a minority (2%) of the homozygotes possed both ureters but contained only one rudimentary kidney. Histological analysis of the mutant kidney rudiments showed non-structured and reduced numbers of kidney elements, reduced branching of the ureter and incompletely formed collecting ducts.
Epithelial-mesenchymal interactions play an important role in the induction of the metanephric kidneys (Saxen, 1987). Normal kidney development involves reciprocal inductive interactions between the epithelial cells of the ureteric bud, and the mesenchymal cells of the metanephric blastema. The severity of the kidney phenotype o f c-ret null mice and the restricted expression of the gene in the epithelial cells o f the ureteric bud and nephrogenic zone (section 1.3 (D)) strongly suggest that the c-ret receptor may frinction in the transduction of a signal derived from the metanephric
mesenchyme. This signal may be essential from the induction, growth and branching o f the ureteric bud. Consistent with this suggestion, administration of recombinant GDNF, the recently identified ligand of the c-ret receptor (Treanor et al.; Jing et al.; in press), to normal kidney explants induce supernumerary branching of ureteric buds (Pichel et. al, in press). Furthermore, mice lacking functional GDNF show similar agenesis o f the metanephric kidneys and enteric nervous system as embryos | lacking functional c-ret proto-oncogene (Sanchez et al., Pichel et al., Moore et al., in press).
In addition to the expression o f the c-ret receptor, trk A, trk B, trk C neurotrophic receptors and c-ros RTKs have also been shown to be expressed in the developing metanephric kidneys (Durbeej et al., 1993).