Characterization of spleen colonies derived from mice with mutations at the w locus

Abstract

Mice with mutations at the W locus have a hemopoietic stem cell defect characterized by an apparent deficiency of spleen colony forming cells (CFU-S). In the present report, we provide evidence that mutant cells form colonies and we compare the characteristics of the colonies derived from mutant and normal cells. To perform the colony-derivation studies, marrow cells were transferred into lethally irradiated congenic hosts that differed from the donors in the ubiquitous genetic marker, glucose phosphate isomerase (GPI-1). Donor GPI-1 comprised over 50% of the marker in the host spleen and marrow by 12 days post injection, regardless of whether the donor was mutant or normal. To characterize the colonies, serially sectioned host spleens were examined microscopically. Colonies are present by 8 days post-transplantation regardless of donor genotype, but mutant colonies are distinctly different from normal colonies. The proportion of blast and granulocyte colonies is always greater in W/Wv than in +/+ recipients. Unlike the W/Wv donors, the +/+ donors generate primarily erythrocyte colonies at 8, 10, and 14 days and mixed colonies at 12 days post-injection. Colonies from the mutant mice are generally smaller but visible colonies do appear by 12 days. The results are consistent with the notion that the anemia in W/Wv mice is caused by the early restriction of differentiating cells to a non-erythrocyte lineage accompanied by the delayed amplification of mutant hemopoietic cells. Whether this means erythrocyte-committed cells are absent or are present but unable to respond to the appropriate cytokines is not possible to determine from the current experiments.

Heterozygous Kit Mutants with Little or No Apparent Anemia Exhibit Large Defects in Overall Hematopoietic Stem Cell Function

“…The colonies produced by the Kit mutants, including some from W-41/+, were smaller than those of the B6 controls, suggesting that CMP numbers may underestimate the Kit mutant defects. The numbers of CMP for W-41/W-41 and W-42/+ are consistent with previous reports [43,44].…”

Rescue of lethal c-KitW/W mice by erythropoietin

“…This was true for the standard dose of transplanted BM cells (2 ϫ 10 5 ) and for double the amount of progenitor cells. 26 This demonstrates that a known deficiency in c-Kit W/W HSC/progenitors has not been rescued or modified by introducing the EPO-tg. …”

Engraftment of normal stem cells in W/Wv mice assessed by a novel quantitative PCR analysis

“…They are heterozygous for two W alleles: the W allele which encodes a c-kit protein with a deletion of 78 amino acids spanning part of the transmembrane domain, and the W v allele which is characterized by a C to T substitution at position 2007 near the 3 0 end of exon 13 encoding part of the first tyrosine kinase domain of the protein (Nocka et al, 1990;Giebel et al, 1992). Although W/W v mice have normal numbers of haemopoietic stem cells (HSC), they have reduced numbers of cells capable of generating colonies in spleen colony assay (CFU-S) and of progenitor cells (Russell, 1979;Barker & Starr, 1991). Since HSC from wild-type C57BL/6J congenic mice express normal c-kit protein on the membrane, they have proliferative advantages over W/W v HSC in vivo and can be engrafted in W/W v recipients without any need of ablation (Boggs et al, 1982;Barker et al, 1988;Capel et al, 1989).…”