Mouse labial-like homeobox-containing genes: structure and expression during embryogenesis
While mouse development has been well described at a morphological level, very little is known about how development is regulated. In contrast, the ease of developmental analysis in Drosophila has led to the identification of a large number of developmentally important genes. Molecular characterisation revealed that many of the genes involved specifically in determining the Drosophila body plan contain a conserved sequence called the homeobox. This sequence is highly conserved through evolution and so it can be used to isolate homologous genes in other species. In this way more than 40 homeobox- containing genes have been identified in the mouse. The high level of sequence conservation and the temporally and spatially restricted expression of the mouse genes during development indicate that they are also developmental regulators involved in conferring spatial information within the embryo. Thus, through knowledge of Drosophila development and the techniques of molecular biology, it is now possible to study mouse developmental genes in detail.In this thesis, the characterisation of two mouse homeobox -containing genes, Hox 2.9 and Hox 1.6, is presented. Sequence analysis revealed that these genes are closely related and that, among Drosophila genes, they are most similar to labial in the Antennapedia complex. They are therefore thought to have arisen by duplication of a single ancestral gene. As well as being structurally similar the genes share many features of their expression patterns. Both genes are expressed early in development (71/2 days) and, unlike other known mouse homeobox -containing genes, they are not expressed after 11 days of development. At 8 days the genes share the same anterior boundary of expression in the hindbrain and in the later embryo, with the exception of persistent Hox 2.9 expression in the hindbrain, they have the same anterioposterior restrictions. This indicates that the genes are functionally similar and also that they respond to at least some of the same signals in the embryo.A striking difference between the expression patterns of mouse labial -like genes is the unique expression of Hox 2.9 in a single segmental unit (rhombomere 4) of the hindbrain (from 81/2 days). This expression coincides perfectly with the morphological extent of rhombomere 4 and persists throughout the period that rhombomeres are visible (up to 11 days). It is therefore suggested that Hox 2.9 participates in conferring segment identity. In addition neural crest cells that arise from rhombomere 4 specifically express Hox 2.9 and this supports the idea of neural crest cells being patterned according to their position of origin in the central nervous system. Detailed analysis of the onset of segmental expression of Hox 2.9 and another segmentally expressed gene in the hindbrain, Krox 20, showed that Hox 2.9 expression becomes localised from a broad domain at 81/2 days of development, up to 6 hours before rhombomeres are clearly visible.Retinoic acid is a strong candidate for a natural morphogen in the vertebrate embryo. The effect of in vitro treatment with retinoic acid on segmentation of the mouse hindbrain and on the expression of Hox 2.9 and Krox 20 was therefore analysed. It was found that segmentation in treated embryos is abnormal and that the clear segmental localisation of expression of the two genes is not found. The hindbrain expression domains are shifted rostrally following treatment and while the expression of the two genes remains mutually exclusive there is no longer a single planar boundary between the domains. Instead there is an irregular alternation of cells expressing the two genes at the boundaryTwo differential splicing products of Hox 1.6 were isolated from the developing embryo. A comparison was made of the distribution of these transcripts, only one of which can code for a homeodomain containing protein. It was found that the relative proportion of homeodomain producing message decreases as development proceeds.