A Recessive Embryonic Screen for Genes Regulating Hematopoietic Stem Cell and Blood Cell Generation and Function.

Abstract 2527

Poster Board II-504

Identifying the genes that regulate the development, self-renewal and differentiation of stem cells is of vital importance for understanding normal organogenesis, tailoring tissue engineering for regenerative medicine, cellular reprogramming and cancer. A forward genetic screen for aberrant long-term hematopoietic stem cells and progenitors provides an unbiased and tractable approach to finding genes responsible for stem cell homeostasis and differentiation. Here we demonstrate that chemical mutagenesis of mice combined with advances in hematopoietic stem cell reagents and genome/mapping resources can identify genes essential for mammalian stem cells and blood development. A pilot flow cytometry-based recessive screen comprehensively analyzed nine subsets of hematopoietic stem, progenitor, and red cells in over one thousand mouse embryos at embryonic day (E) 14.5 from 34 pedigrees and recovered five strains with defects in early hematopoiesis. One mutant strain (Booreana - an Australian Aboriginal name meaning white) which has excess long-term hematopoietic stem cells and platelets but reduced myelo-erythroid progenitors was outcrossed and the genetic mutation mapped and identified as a novel mis-sense mutation in the transcription factor c-Myb. The mutation in the trans-activation domain (TA) completely ablates transcriptional activation in a reporter assay which contrasts with other TA domain mutants which are partly dysfunctional[TJG1] . Moreover, the Booreana (Boo) mutation completely abrogates interaction with the transcriptional co-activator, CBP. Boo/Boo homozygous mutant mice survive into adulthood, albeit with severe anemia and massively increased platelet counts, whereas c-Myb^−/− mice die by E15.5 of development, suggesting c-Myb has essential functions in vivo which are independent of transcriptional activation. This ENU-generated mutation provides another allele of c-Myb with a phenotype in between the complete loss-of-function allele and previously identified mutant alleles from other ENU screens. ENU-generated point mutants such as the Booreana mutation can provide novel informative insights into key functional domains of proteins, and protein interactions and networks, which are missed in gene knockout mice. Other phenodeviants generated in our screen are currently being mapped and will be presented.

[TJG1]Not so in our hands, at least -– the Sandberg M303V is just as dead.