Strategies to interfere with Sek-1 function in the hindbrain

To analyse Sek-1 function in the hindbrain the following approaches were taken: firstly, by ectopic expression, and secondly by interfering with gene function. The first approach was based upon the observations that odd and even-numbered rhombomeres display different cellular properties, and also segmentally-restricted gene expression patterns. It was presumed that the spatial restriction of Sek^l expression may be important, and therefore ectopic expression of Sek-1 in even- numbered rhombomeres may provide insight into Sek-1 function. The second approach was to use a dominant negative mutation strategy. This approach has been used successfully for a number of receptor protein kinases, for example the PDGF receptor (Ueno et al., 1991), the insulin receptor (Levy-Toledano et al., 1994), an activin receptor in Xenopus (Hemmati-Brivanlou and Melton, 1992), a BMP receptor in Xenopus (Graff et al., 1994) and FGF receptors in both Xenopus and mouse (Amaya et al., 1991; Ueno et al., 1992; Peters et al., 1994). Many receptor kinases dimerise upon ligand binding to initiate the intracellular signal cascade through transphosphorylation and activation of the catalytic domain. A non-functional receptor lacking the kinase domain but retaining the extracellular and transmembrane domains can therefore form a dimer with a functional receptor in response to ligand binding, but disrupt the activation of the kinase domain. Over-expression of such mutant molecules ensures that the majority of endogenous receptor molecules dimerise with a non-functional receptor, such that activation of the catalytic domain of the endogenous receptor is suppressed (reviewed in Herskowitz, 1987). This strategy was employed in the hindbrain in order to specifically inactivate Sek-1 by directing expression of the mutant receptor to r3/5 (Figure 5.1).

Figure 5.1 A dominant negative mutation strategy to inactivate endogenous Sek-1.

RTK molecules dimerise upon ligand binding (L), resulting in activation of the kinase domain by phosphorylation (P). The truncated Sek-1 receptor lacking a kinase domain dimerises with a wild type receptor upon ligand binding and prevents transduction of the signal.

L: ligand bound state; P: phosphorylation.

Interfering with gene function can also be achieved through homologous recombination (gene ‘knockout’). However, until recently this technique could not be targeted to specific tissues, and therefore may result in gross defects or embryonic lethality that make it difficult to establish the role of the gene. I therefore chose to use a mutant receptor targeted specifically to the rhombomeres to ensure that the function of Sek-1 here was not masked by defects arising from a possible role earlier in development. The overlapping expression profiles of the Eph receptors in the hindbrain raises the possibility that they may function as heterodimers here. Thus, another advantage of the dominant negative mutant strategy is that the mutant receptor may interfere with more than one receptor by heterodimerisation, and give a phenotype where a gene knockout would not.

The dissection of regulatory elements of the Hox genes has lead to the identification of enhancers which can be used as reagents for targeted expression to specific rhombomeres. These are potentially advantageous over enhancers that direct expression ubiquitously in the embryo throughout development, as they allow the analysis of a mutation affecting only the hindbrain component of the Sek-1 expression profile. Regulatory elements that drive expression both earlier and more widespread in the embryo could result in more general defects arising from a possible role mediated by the earlier expression of Sek-1, for example in mesoderm.

5.2 Results

5.2.1 Assembly of constructs for expression of Sek-1 in transgenic mice

I created a dominant negative form of Sek-1 by cloning sequences encoding the truncated receptor lacking the kinase domain into the KSTART vector. The KSTART vector was designed for the insertion of cDNA clones that required the addition of a 5’ Kosak consensus sequence at the translational start site (Kozak, 1989) and 3’ translational termination sites. Three vectors were made in order to provide a translational start site and a termination codon in all three possible reading frames. A 1850 bp NcoI-BamHI Sek-1 fragment was inserted into the KSTART vector at the

Ncol site (Fig.5.2). Dissection of the regulatory elements of Hoxb-1 revealed that a 1 Kb fragment in the 5’ sequences of Hoxb-1 was sufficient to generate restricted expression in r4 and in neural crest migrating from it (Studer et al., 1994). This enhancer element drives expression of reporter genes in both a temporal and spatial pattern ideal for the ectopic expression of Sek-1 in r4 during the period that the endogenous gene is expressed. A similar dissection of regulatory elements of the Hoxb-2 gene revealed that the high level expression in r3 and r5 is imposed by an enhancer found in the 5’ sequences flanking the gene (Sham et al., 1992). This enhancer is ideal to drive expression of the truncated Sek-1 receptor in r3/5 as it directs expression in a pattern similar to that of Sek-1 both temporally and spatially. This element, a 569 bp Sau3A fragment, contains three Krox20 binding sites, but is insufficient to drive transgene expression. High levels of transgene expression are seen in r3 and r5 when the 569 bp fragment is dimerised and a further 122 bp of flanking region is added (Sham et al., 1993).

For the purpose of this study a high level of expression was required, directed to the rhombomeres by the above enhancers. Previous studies have shown that the level of expression obtained is dependent on a number of factors including the promoter, heterologous introns, enhancers and polyadenylation sequences, and these factors may vary for different genes. Therefore I prepared a number of constructs using different combinations of promoter, 3’ polyadenylation site, a heterologous intron and the Hox gene enhancer. From studies in cell culture it is known that splicing can influence the stability of mRNA in eukaryotic cells (Hamer and Leder, 1979), but in transgenic animals the situation is less clear. Whilst introns have been found to significantly increase the transcriptional efficiency of the transgene (Brinster et al., 1988 ; Choi et al., 1991), different combinations of introns can inhibit their transcription (Evans and Scarpulla, 1989). The position of the intron relative to the cDNA may also influence expression of the transgene. In previous studies highest levels of expression of one gene were achieved when the intron was placed between the promoter and the cDNA rather than 3’ of the cDNA (Palmiter et al., 1991). However, it remains unclear whether this is true universally or dependent on the promoter/enhancer/intron/gene combinations used.

More recently it has been proposed that increased levels of transcription of

Figure 5.2 Construction of a truncated Sek-1 receptor.

A 1850 bp NcoI-BamHI Sek-1 cDNA fragment encoding both the extracellular and transmembrane domains but lacking the intracellular domain, was inserted into the KSTART vector. KSTART was designed to provide a Kozak consensus sequence 5’ of the translational start site (Kozak, 1989), and a termination codon 3’ of the cDNA. Three vectors were made to ensure the correct reading frame was maintained. The Sek-1 fragment was inserted at the Ncol site, and could be retrieved as a Hpal- EcoRV fragment for insertion into transgenic constructs.

Extracellular

Transm em brane