REGLAMENTO NACIONAL DE EDIFICACIONES

3.1 Introduction

When the study described in this thesis was first conceived our knowledge of the role of polypeptide growth factors in sensory neuron development was limited. From the early work of Rita Levi-Montalcini (reviewed by Levi-Montalcini and Angeletti, 1968) it was clear that NGF, the prototypical neurotrophin, promoted the survival of sensory and sympathetic neurons of the peripheral nervous system. Experiments which assessed the survival and neurite outgrowth of explants and dissociated cells in culture demonstrated that sympathetic and sensory DRG neurons responded to NGF treatment. Furthermore, in vivo immune deprivation studies provided definitive proof that NGF was essential for the survival of a subset of neural crest derived sensory and sympathetic neurons.

The discovery of BDNF (Barde et al., 1982; Leibrock et al., 1989; Emfors et al., 1990) revealed a second member of the neurotrophin family which also supported the survival of a subset of neural crest-derived sensory DRG neurons and, in addition, neurons within particular cranial sensory ganglia, for example the placode-derived nodose ganglion, which were unresponsive to NGF (Lindsay et al., 1985). Whereas sympathetic neurons survived in culture in the presence of NGF, they were unresponsive to BDNF (Lindsay et al., 1985). Thus a picture was emerging that individual neurotrophins supported the survival of distinct subsets of neurons within the peripheral nervous system. Soon afterwards several groups cloned NT-3 (Hohn et al. 1990; Maisonpierre et al., 1990; Rosenthal et al., 1990) and NT-4/5 (Hallbook et al., 1991; Berkemeier et al., 1991; Ip et al., 1992) from a number of species by employing the polymerase chain reaction using primers which were complementary to regions identified as conserved between NGF and BDNF.

The isolation and cloning of trkA (Martin-Zanca et al., 1990), trkB (Klein et al., 1989) and trkC (Lamballe et al., 1991), the high affinity receptors for NGF, BDNF and NT-4/5, and NT-3 respectively, paved the way to identify at the single cell level neurotrophin responsive cells. Using in situ hybridisation it was demonstrated that trkA, trkB and trkC

were expressed in principally distinct subsets of DRG neurons and that these neurons were, based upon size, likely to subserve different functions (Mu et al., 1993; McMahon

in the neurotrophins or their high affinity receptors (Conover et ah, 1995; Crowley et al.,

1994; Emfors et a l, 1994a; Emfors et al., 1994b; Farinas et al., 1994; Jones et al., 1994; Klein et al., 1993; Klein et al., 1994; Liu et al., 1995; Smeyne et al., 1994).

Ret is an orphan receptor tyrosine kinase. Northem blot and in situ hybridisation analysis had demonstrated that c-ret is expressed during mouse embryogenesis with a spatial and temporal pattem suggestive of a role for this receptor in the development of particular cell lineages within the nervous system (see Introduction). The generation of mice homozygous for a loss-of-function mutation in the c-ret gene {ret-k~ mice) showed definitively that signalling through the Ret receptor is essential for the development of the embryonic kidney, the enteric nervous system (Schuchardt et al., 1994) and the superior cervical ganglion (Vassilis Pachnis, personal communication).

The use of radiolabelled RNA probes to determine c-ret mRNA distribution had suggested that only a subset of DRG and cranial sensory ganglion neurons expressed this proto­ oncogene. Moreover, c-ret was expressed early in sensory ganglion development suggesting that signalling through this receptor may play a role in the proliferation, migration, differentiation and/or survival of sensory neuron precursors prior to target cell innervation; hybridisation signal was observed over neural crest cells, migrating from rhombomere 4 of the hindbrain, which contribute to neurons and glia of the facial ganglion; in addition, c-ret signal was observed over migrating tmnk neural crest cells which coalesce to form the DRG and the sympathetic ganglia (Pachnis et al., 1993).

Gross anatomical analysis of ret-kr DRG suggested that there was no major perturbation in their development. However, this cursary analysis did not determine whether a subset of c-ref-positive neurons were absent from ret-k' DRG. There were many unanswered questions regarding the role of Ret in sensory ganglion development, such as: is c-ret

expressed by a particular functional subset of sensory neurons and if so was signalling through Ret essential for the development of these neurons?

With in situ hybridisation using digoxygenin labelled RNA probes, which allow single cell resolution of label within even very small cells (Dent et al., 1993), it would be possible to answer some of these questions. By comparing c-ret and trk gene expression within individual DRG neurons I could establish whether c-ret was expressed by a particular functional subset of sensory neurons. In this chapter I begin by describing the pattem of c- ret and trk gene expression in wildtype mouse thoracic DRG.

This study was carried out in the mouse since we had mouse genomic and cDNA clones of

this stage the DRG neurons have grown to a size which would enable serial section reconstruction within individual cells. Also, the bulk of naturally occurring neuronal death is complete by this time so that the populations sampled should largely represent those of mature animals. Finally, I also wished to study DRG from mice homozygous for a loss- of-function mutation in c-ret and since these animals die within 24 hours of birth, PO is the latest time point at which analysis could be carried out.

3.2 The murine embryonic and neonatal pattern of c-ret expression within