The Role of the SHB Adapter Protein in Cell Differentiation and Development

Abstract: The present study was conducted in order to assess a role of the SH2 domain-containing adapter protein SHB in development and cell differentiation.Embryonic stem (ES) cells overexpressing SHB and SHB with an inactive SH2 domain (R522K-SHB) were obtained. Microarray analysis in the SHB clone revealed altered expression of genes connected with neural cell function. The R522K-SHB clone exhibited altered expression of several transcription factors related to development. ES cells were differentiated by forming aggregates named embryoid bodies (EBs). The morphology of EBs was altered in the R522K-SHB clones, which showed fewer cavities. Expression of endodermal markers was decreased in the R522K-SHB EBs. To further investigate the role of SHB in differentiation, murine ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-) were generated. SHB deficient clones increased the expression of mesendodermal and endodermal markers and decreased expression of two receptors, VEGFR2 and FGFR1, connected with blood vessel differentiation. Similarly, blood vessels showed an altered morphology in SHB+/- and SHB-/- EBs after VEGF stimulation. SHB-/- ES cells also formed fewer blood colonies than control ES cells.Finally, the role of the SHB adapter protein in vivo was analyzed by generating a SHB deficient mouse (SHB-/-). SHB-/- animals are viable, fertile, but suffer from leukopenia and anemia. SHB-/- animals demonstrate an abnormal morphology of blood vessels in the liver and kidney. Breeding of SHB+/- animals revealed an abnormal segregation of the mutant allele with an increased number of SHB+/- animals and a decreased number of SHB-/- and SHB+/+animals. Backcross analysis of SHB+/- females with SHB+/+ males displayed an increased number of SHB+/- offspring already at the blastocyst level. Simultaneously, embryos from SHB+/- mothers show an increased malformation rate in comparison to embryos from SHB+/+ mothers.In summary, the study suggests a role of SHB in reproduction and development and in mesodermal and endodermal specification.