In this study, I have presented evidence that Schwann cells and SCG glia express the class B bHLH transcription factors dHAND and eHAND. It is interesting to note that despite intense investigation by a number of research groups for several years, it is only very recently that any novel bHLH genes have been identified in glial cells. The olig genes, oligl and olig2 are expressed in the oligodendrocytes of the central nervous system (CNS). These genes were identified simultaneously by two separate groups using two different methods. One of these groups (Zhou, Q., et.al., 2000) carried out a BLAST search of the GenBank database with the bHLH domains of different families of known bHLH factors. They identified two unknown bHLH genes on a BAG clone from human chromosome 21q. They then designed degenerate primers based on these sequences and carried out PCR on mouse genomic DNA to isolate the mouse homologues of oligl and olig2. The other group (Lu, Q.R., 2000) took nestin positive neuroepithelial cells and cultured them in triiodothionine (T3) and Ciliary neurotrophic factor (CNTF) to induce differentiation, before extracting mRNA. Low stringency degenerate PCR was then carried out on cDNA transcribed from the RNA collected from the neuroepithelial cells. Bands of interest were subcloned into bluescript and used to screen a nylon membranes containing several hundered clones. Duplicate membranes were screened with PCR fragments isolated from untreated neuroepithelial cells to enable identification of sequences upregulated by T3 or CNTF. From this differential RT-PCR a 120 nucleotide fragment of oligl was identified. It is clear from the work of these two groups that these oligodendrocyte specific bHLH genes were not identified easily and unlike other genes such as neurogenin and Mashl could not be identified by simple degenerate PCR.
Until the identification of the olig genes the possibility existed that glial cell development in the CNS and PNS was not controlled by bHLH genes. This is in sharp contrast to neuronal development where different cascades of bHLH genes appear to control the development of different types of neurons. For example the
(reviewed in Anderson, 2000) and Mashl and dHAND and eHAND are involved in sympathetic neuron development (Howard et al., 2000). It could be argued that the diversity of neuronal subtypes has lead to the control of neuronal development by different sub-groups of bHLH factors, whereas the presence of class B bHLH factors is unnecessary for peripheral glial cell development as Schwann cells, satellite cells and teloglia are thought to be essentiality the same cells. The identification of oligodendrocyte specific bHLH genes in the CNS and dHAND and eHAND expression in sympathetic ganglion glia and Schwann cells means that this idea is unlikely. Furthermore, expression of the olig genes persists in the adult oligodendrocytes and the HAND genes are expressed in SCG glia post-natally, suggesting that these genes may be involved in later stages of glial development as well in early development.
Until recently, it was thought that any differences in gene expression between Schwann cells of the peripheral nerves and ganglia and satellite cells were purely due to differences in the local environment. In the indroduction to this thesis, I have discussed some of the genes which are expressed by satellite cells and not by Schwann cells or vica versa (see chapter 1 P35). It was thought that these differences were the result of the different environments to which the satellite and Schwann cells are exposed, for example, if DRG glial cells which do not express Krox 20 are taken into culture and allowed to migrate out of the ganglion, they can then be induced to express Krox 20 upon addition of certain growth factors. A recent paper however has suggested that although they arise from a common precursor, the glial cells in the ganglia show inherent differences from those in the nerve (Hagedorn et al., 2000b). In chapter 5 of this study, I attempted to discover which one of these two possibilities is correct. I did not identify any inherent differences between these glial cell populations but I did discover that the development of ganglion glial cells from immature into mature cells precedes that of the nerve glia cell by at least 24 hr. I have also shown that although peripheral nerve Schwann cells express a very low level of dHAND and eHAND mRNAs these genes are specifically expressed by satellite cells in the sympathetic ganglia and not by Schwann cells in these ganglia or by any of the glial cells in the DRG. It remains to be seen if manipulation of glial
cells in vitro can induce expression of HAND genes in all peripheral glial cells or whether the identification of the HAND genes in SCG satellite glia and Erm in DRG glia is definitive proof that there are different sub-types of glia in the PNS.
In the chick, it has been shown that, in addition to the expression observed in sympathetic neurons, dHAND but not eHAND is expressed in another part of the autonomic nervous system, the enteric nervous system (Howard et a l , 1999). It is not known if dHAND expression persists in enteric neurons after birth. However it could be that in the early stages of autonomic nervous system development, expression of both HAND genes simultaneously is required, but as the cells differentiate into the different components of the autonomic nervous system expression of dHAND alone is required to specify or maintain enteric neurons while expression of eHAND alone is necessary to maintain or specify sympathetic neurons. Experiments where migratory rat neural crest cells have been transplanted into chick hosts have identified an autonomic lineage restriction in the neural crest (White and Anderson,
1999). These studies revealed that whilst E10.5 neural crest cells were capable of differentiating into sensory, sympathetic and parasympathetic neurons, enteric neural precursors isolated from the foetal gut at E14.5 were only able to generate parasympathetic neurons and enteric neurons and not sympathetic or sensory neurons. These experiments suggest that once they have migrated to the gut, the enteric precursors cells are incapable of becoming sensory or sympathetic neurons. This ‘autonomic lineage restriction’ could be imposed by dHAND.
In the future we plan to continue investigating the functions of the HAND genes in the peripheral nervous system. In particular, we intend to confirm out initial findings that eHAND is necessary for neural crest cell migration (see chapter 4 p). Further evidence in support of this theory has been provided by chimeras derived from HAND 1-null (eHAND-null) ES cells and ROSA 26 embryos (Riley et al., 2000). In these chimeras HAND 1-null cells were underrepresented in several areas of the embryos that are populated by post-migratory neural crest cells for example the branchial arches and the cardiac outflow tract. These are tissues where eHAND is known to be expressed in normal mouse embryos (Cerjesi et al., 1995). In addition, HAND 1 -null cells were excluded from the dorsal neural tube at the time when pre-
migratory neural crest cells would be present. This observation is important for at least two reasons, firstly eHAND expression has not previously been detected in pre- mi gratory neural crest cells. The fact that the ROSA 26 cells substitute for the HAND 1-null cells in the dorsal neural tube suggests that eHAND may have a function in very early neural crest cells. This supports the proposed idea that eHAND has a function in the migration of neural crest cells. Secondly, the substitution of HAND 1-null cells in the dorsal neural tube not suggests that eHAND would normally be expressed by these cells in wild-type embryos. This is an important observation since eHAND has never been detected in the neural tube by other experimental methods such as in situ hybridisation. These experiments indicate that the HAND gene products may have functional roles in the embryo despite the mRNAs being present at levels that are undetectable by in situ hybridisation. T herefore, dHAND and eHAND m ight have a function in Schw ann cell development, despite only being expressed at very low levels. These experiments will be carried out using eHAND mutant mice generated by Riley et a l , (1998). As these mice arrest at an earlier stage of development than the eHAND mice generated by Firulli et al. (1998), it may be necessary to remove the embryos from the mother and carry out whole embryo cultures for between 24-48hr before removing the neural tubes needed for the neural crest cell culture.
In chapter 5 of this study, we examined the role of HGF in early peripheral glial cell developm ent. We found that although HGF is a known mitogen for Schw ann cells (K rasnoselsky et al., 1994), in vitro, HGF will only potentiate Schwann cell precursor division in the presence of pNRG. This effect on division appears to be very specific as HGF did not enhance division in the presence of bFGF, suggesting that there is a particular interaction between pNRG and HGF. It is thought that HGF acts as an 'enhancer molecule' to enhance the response of cells to other molecules that act in a more specific manner. From the evidence so far is seems that HGF can only act as an enhancer of one particular growth factor on a specific cell type, e.g. CNTF in motor neurons and pNRG in Schwann cell precursors (see Introduction and Results, chapter 5). It is possible that other enhancer molecules exist which act in conjunction with other growth factors for example, bFGF plays an
important role in Schwann cell development and in this chapter we have shown that HGF does not potentiate the effects of bFGF on Schwann cell precursors. Perhaps another factor is able to enhance the effects of bFGF. This system of enhancers could provide an added layer of control over developmental processes.