SummaryGuided cell migration is a key mechanism for cell positioning in morphogenesis. The current model suggests that the spatially controlled activation of receptor tyrosine kinases (RTKs) by guidance cues limits Rac activity at the leading edge, which is crucial for establishing and maintaining polarized cell protrusions at the front. However, little is known about the mechanisms by which RTKs control the local activation of Rac. Here, using a multidisciplinary approach, we identify the GTP exchange factor (GEF) Vav as a key regulator of Rac activity downstream of RTKs in a developmentally regulated cell migration event, that of the Drosophila border cells (BCs). We show that elimination of the vav gene impairs BC migration. Live imaging analysis reveals that vav is required for the stabilization and maintenance of protrusions at the front of the BC cluster. In addition, activation of the PDGF/VEGF-related receptor (PVR) by its ligand the PDGF/PVF1 factor brings about activation of Vav protein by direct interaction with the intracellular domain of PVR. Finally, FRET analyses demonstrate that Vav is required in BCs for the asymmetric distribution of Rac activity at the front. Our results unravel an important role for the Vav proteins as signal transducers that couple signalling downstream of RTKs with local Rac activation during morphogenetic movements.
Several karyotypic forms have been previously described in populations of the vole species Microtus thomasi from Greece. In particular, the karyomorphs Microtus thomasi ‘thomasi’ and ‘atticus’ differ in X chromosome morphology, being acrocentric and subtelocentric, respectively. Furthermore, remarkable heterochromatin content variability has been described in sex chromosomes of both karyomorphs. Genomic DNA digestion with AluI allowed us to clone an 884 bp long repeated DNA sequence (Mth-Alu900) from the karyomorph M. thomasi ‘atticus’. This repeated DNA is AT rich and seems to be organized mainly as a dimer of the 884-bp unit, which presents three simple repeats (CAAAT, CAGAT and CAGAC) that constitute 80% of the total unit length. This repeated DNA is exclusive to M. thomasi, since it is absent from the genome of other studied Arvicolinae species. The chromosomal location of Mth-Alu900 was analyzed on M. thomasi ‘thomasi’ and M. thomasi ‘atticus’ karyomorphs, with different sex chromosome constitution. It was mainly located on the pericentromeric heterochromatin of most autosomes and X chromosomes on both karyomorphs. Results are also discussed in relation to karyotypic and sex chromosome variations in M. thomasi. To our knowledge, Mth-Alu900 constitutes a new – the third discovered so far – pericentromeric repeated DNA sequence described in Microtus species.
The chromosomal distribution of mobile genetic elements is scarcely known in Arvicolinae species, but could be of relevance to understand the origin and complex evolution of the sex chromosome heterochromatin. In this work we cloned two retrotransposon sequences, L1 and SINE-B1, from the genome of Chionomys nivalis and investigated their chromosomal distribution on several arvicoline species. Our results demonstrate first that both retroelements are the most abundant repeated DNA sequences in the genome of these species. L1 elements, in most species, are highly accumulated in the sex chromosomes compared to the autosomes. This favoured L1 insertion could have played an important role in the origin of the enlarged heterochromatic blocks existing in the sex chromosomes of some Microtus species. Also, we propose that L1 accumulation on the X heterochromatin could have been the consequence of different, independent and rapid amplification processes acting in each species. SINE elements, however, were completely lacking from the constitutive heterochromatin, either in autosomes or in the heterochromatic blocks of sex chromosomes. These data could indicate that some SINE elements are incompatible with the formation of heterochromatic complexes and hence are necessarily missing from the constitutive heterochromatin.
In the subfamily Arvicolinae (Cricetidae, Rodentia) the satellite DNA Msat-160 has been so far described in only some species from the genus Microtus and in one species from another genus, Chionomys nivalis. Here we cloned and characterized this satellite in two new arvicoline species, Microtus (Terricola) savii and Arvicola amphibius (terrestris). We have also demonstrated, by PCR and FISH, its existence in the genomes of several other species from both genera. These results suggest that Msat-160 already occurred in the common ancestor of the four genera/subgenera of Arvicolinae (Microtus, Chionomys, Arvicola, and Terricola). In Arvicola and Terricola, Msat-160 showed the basic monomer length of 160 bp, although a higher-order repeat (HORs) of 640 bp could have been probably replacing the original monomeric unit in A. a. terrestris. Msat-160 was localized by FISH mostly on the pericentromeric regions of the chromosomes, but the signal intensity and the number of carrier chromosomes varied extremely even between closely related species, resulting in a species-specific pattern of chromosomal distribution of this satellite. Such a variable pattern most likely is a consequence of a rapid amplification and contraction of particular repeats in the pericentromeric regions of chromosomes. In addition, we proposed that the rapid variation of pericentromeric repeats is strictly related to the prolific species radiation and diversification of karyotypes that characterize Arvicolinae lineage. Finally, we performed phylogenetic analysis in this group of related species based on Msat-160 that results to be in agreement with previously reported phylogenies, derived from other molecular markers.
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