title: Inactivation of Numb and Numblike in spermatogonial stem cells by cell-permeant Cre recombinase.
abstract: Spermatogonial stem cells (SSC) ensure continuous production of mammalian male gametes. In rodents, the SSC are Asingle spermatogonia (As). Gene loss and gain-of-function mutations have provided some clues into SSC function, but genetic dissection of SSC physiology has not yet been accomplished. The adaptor protein Numb is an evolutionarily conserved protein originally implicated in the control of the fate of sibling cells. Mice homozygous for deficient Numb die before embryonic day 11.5, hampering the analysis of its inactivation in postnatal male germline. Here, we have developed an experimental strategy to conditionally inactivate Numb and its homolog Numblike in the postnatal germline by in vitro delivery of cell-permeant Cre recombinase. Cre-transduced SSC isolated from wild-type mice retained their ability to self-renew and to differentiate in vivo, as shown by their ability to give rise to normal spermatogenic colonies when transplanted in recipient testes. Cre-transduced SSC from conditional mutant mouse line were able to colonize recipient testes upon transplantation. Inactivation of either Numb or Numblike in SSC did not impair the development of normal donor-derived spermatogenesis. However, compared to single-null SSC, double-null SSC generated shorter colonies in which the germ cells failed to differentiate beyond the round spermatid stage, underscoring the essential roles of both Numb and Numblike in spermatogenesis. We demonstrate the feasibility of gene inactivation in adult SSC by ex vivo Cre delivery. This provides a means to analyze the function of genes that operate on a cell-autonomous basis or those that are coupled to signals that SSC receive from bystander cells.
authors.affiliation: Fondazione Pasteur Cenci Bolognetti, Department of Histology and Medical Embryology La Sapienza University of Rome, Via Antonio Scarpa, No. 14, 00161 Roma, Italia.
authors.forename: Laura
authors.initials: L
authors.lastname: Grisanti
authors.forename: Serena
authors.initials: S
authors.lastname: Corallini
authors.forename: Stefania
authors.initials: S
authors.lastname: Fera
authors.forename: Barbara
authors.initials: B
authors.lastname: Muciaccia
authors.forename: Mario
authors.initials: M
authors.lastname: Stefanini
authors.forename: Walter
authors.initials: W
authors.lastname: Witke
authors.forename: Elena
authors.initials: E
authors.lastname: Vicini
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology*; D020868:Gene Silencing / Q000502:physiology*; D019426:Integrases / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000235:genetics*; D013093:Spermatogonia / Q000166:cytology* / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013737:Testis / Q000166:cytology / Q000502:physiology; D014161:Transduction, Genetic
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C106293:Numb protein, mouse; C106292:Numbl protein, mouse; C045073:Cre recombinase; D019426:Integrases