Abstract
Tks5 is a scaffold protein and Src substrate involved in cell migration and matrix degradation through its essential role in invadosome formation and function. We have previously described that Tks5 is fundamental for zebrafish neural crest cell migration in vivo. In the present study, we sought to investigate the function of Tks5 in mammalian development by analyzing mice mutant for sh3pxd2a, the gene encoding Tks5. Homozygous disruption of the sh3pxd2a gene by gene-trapping in mouse resulted in neonatal death and the presence of a complete cleft of the secondary palate. Interestingly, embryonic fibroblasts from homozygous gene-trap sh3pxd2a mice lacked only the highest molecular weight band of the characteristic Tks5 triplet observed in protein extracts, leaving the lower molecular weight bands unaffected. This finding, together with the existence of two human Expressed Sequence Tags lacking the first 5 exons of SH3PXD2A, made us hypothesize about the presence of a second alternative transcription start site located in intron V. We performed 5′RACE on mouse fibroblasts and isolated a new transcript of the sh3pxd2a gene encoding a novel Tks5 isoform, that we named Tks5β. This novel isoform diverges from the long form of Tks5 in that it lacks the PX-domain, which confers affinity for phosphatidylinositol-3,4-bisphosphate. Instead, Tks5β has a short unique amino terminal sequence encoded by the newly discovered exon 6β; this exon includes a start codon located 29 bp from the 5'-end of exon 6. Tks5β mRNA is expressed in MEFs and all mouse adult tissues analyzed. Tks5β is a substrate for the Src tyrosine kinase and its expression is regulated through the proteasome degradation pathway. Together, these findings indicate the essentiality of the larger Tks5 isoform for correct mammalian development and the transcriptional complexity of the sh3pxd2a gene.
Highlights
Genetic regulation of embryonic development is a complex process that underlies and, at the same time, is controlled by, specific molecular and cellular changes exquisitely coordinated in space and time
To elucidate if the phenotypic diversity in the Tks5trap/trap mice was due to irregular efficiency of the trapping cassette or to genetic background impurity, sh3pxd2a mice were backcrossed into the C57BL/6J pure background
We note that 30% of the Tks5a/Tks5long pups produced on the mixed genetic background died in the neonatal period even though cleft palate was not evident; the reason for this would require further investigation
Summary
Genetic regulation of embryonic development is a complex process that underlies and, at the same time, is controlled by, specific molecular and cellular changes exquisitely coordinated in space and time. During the last 3 decades, the development of forward and reverse genetics through the use of model organisms such as Drosophila melanogaster, Caenorhabditis elegans, zebrafish and mouse have allowed the definition of key processes in embryo formation [1,2,3] These processes are evolutionarily conserved and have helped to elucidate the function of human orthologous genes. According to the Online Mendelian Inheritance in Man database (http://www.ncbi.nlm.nih.gov/ omim, April 2014), there are 752 congenital diseases of Mendelian nature, or suspected to have a Mendelian basis, whose molecular cause is unknown These diseases are rare in the population, there are more frequent inborn developmental errors of non-Mendelian inheritance where a polygenic origin is suspected but not determined [5,6]. They represent a substantial number of congenital conditions whose genetics have not been elucidated and that as a whole represent a considerable clinical burden to society
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