Genetic inversion in mast cell-deficient (Wsh) mice interrupts corin and manifests as hematopoietic and cardiac aberrancy

Abstract

Mast cells participate in pathophysiological processes that range from antimicrobial defense to anaphylaxis and inflammatory arthritis. Much of the groundwork for the understanding of mast cells was established in mice that lacked mast cells through defects in either stem cell factor or its receptor, Kit. Among available strains, C57BL/6-Kit(W-sh) (W(sh)) mice are experimentally advantageous because of their background strain and fertility. However, the genetic inversion responsible for the W(sh) phenotype remains poorly defined, and its effects beyond the mast cell have been incompletely characterized. We report that W(sh) animals exhibit splenomegaly with expanded myeloid and megakaryocyte populations. Hematopoietic abnormalities extend to the bone marrow and are reflected by neutrophilia and thrombocytosis. In contrast, mast cell-deficient WBB6F1-Kit(W)/Kit(W-v) (W/W(v)) mice display mild neutropenia, but no changes in circulating platelet numbers. To help define the basis for the W(sh) phenotype, a "DNA walking" strategy was used to identify the precise location of the 3' breakpoint, which was found to reside 67.5 kb upstream of Kit. The 5' breakpoint disrupts corin, a cardiac protease responsible for the activation of atrial natriuretic peptide. Consistent with this result, transcription of full-length corin is ablated and W(sh) mice develop symptoms of cardiomegaly. Studies performed using mast cell-deficient strains must consider the capacity of associated abnormalities to either expose or compensate for the missing mast cell lineage.

Figure 1

W^sh animals exhibit gross and microscopic splenic abnormalities. A: Representative spleens from 12 week male B6 and matching W^sh mice showing variable enlargement of W^sh spleens even among littermates. B: Quantitation of spleen mass from age-matched mast cell-deficient mice and controls, n = 10 (WBB6 and W/W^v), n = 22 (B6), n = 23 (W^sh). C: H&E staining of B6 and W^sh spleens from 12-week-old mice (magnification = original ×100). Note the clear separation of white pulp and red pulp in B6 that is lost in W^sh, along with the appearance of large multinucleated cells. This phenotype is observed typically in larger W^sh spleens, while others may show relatively normal histology. D: High power image of W^sh spleen (magnification = original ×400) demonstrating large multinucleated megakaryocytes (open arrowheads) and an abundance of neutrophil-lineage forms (solid arrowheads).

Figure 2

W/W^v and W^sh mice display contrasting myeloid aberrancy in spleen. Spleens from mast-cell deficient animals and age-matched controls were disaggregated and analyzed by cytofluorimetry and histomorphometry. Cytofluorimetric data reported are the percentage of viable, CD45+ cells in individual mice (n = 10 to 26 W/W^v and WBB6 mice and n = 13 to 29 W^sh and B6 mice pooled from 2 to 7 independent experiments). A: CD11b (component of Mac-1). B: Gr-1. C: F4/80. D: Megakaryocyte quantitation by histomorphometry (B6 3.0 ± 0.7 vs. W^sh 66.3 ± 11.4 cells/10x hpf, n = 13 male mice aged 10 to 14 weeks/group pooled from three experiments). E: Representative dot plots of viable CD45+ splenocytes showing two Gr-1+CD11b+ populations defined by expression level of these markers. F: Quantitation of Gr-1/CD11b-expressing populations.

Figure 3

W^sh animals exhibit an elevated proportion of cells expressing functional Kit. A: Surface expression of Kit in n = 15 to 17 W/W^v and WBB6 mice and n = 22 W^sh and B6 mice pooled from 3 to 6 independent experiments. B: Quantitation of splenic mast cell progenitor populations in W/W^v, WBB6, W^sh, and B6 mice. Note that W^sh mice demonstrate a fourfold higher frequency per 10⁶ mononuclear cells as compared with B6 and a 20-fold excess compared to W/W^v (n = 5 to 7 mice per group pooled from 2 separate experiments with similar results). MNC, mononuclear cells.

Figure 4

Bone marrow is neutropenic in W/W^v and neutrophilic in W^sh mice. Cytofluorimetric analysis of CD45+ bone marrow populations in W/Wv, WBB6, W^sh, and B6 mice (n = 10 to 26 W/W^v and WBB6 mice and n = 9–22 W^sh and B6 mice pooled from 2 to 6 independent experiments). A: CD11b. B: Gr-1. C: F4/80. D: Cells co-expressing Gr-1 and CD11b at an intermediate level. E: Cells co-expressing Gr-1 and CD11b at a high level. F: C-kit.

Figure 5

W^sh mice exhibit circulating neutrophilia and thrombocytosis, while W/W^v mice are mildly neutropenic. Blood from cardiac puncture was analyzed by automated cytometer to obtain complete blood count and leukocyte differential. A: Absolute neutrophil count. B: Platelet count by automated cytometer demonstrating marked elevation in W^sh mice. Data shown represent individual mice pooled from at least 5 independent experiments (n = 15 to 28 mice/group).

Figure 6

Precise definition of the W^sh inversion mutation. A: Products from PCR of Wt/Wt, Wt/W^sh and W^sh/W^sh genomic DNA using primers flanking the sites of the 3′ breakpoint in the wild-type allele (Wt = C57BL/6J) or W^sh allele. B: Schematic representation (not to scale) of W^sh inversion on chromosome 5 showing only corin and Kit (at least 21 genes intervene). Arrows point to sites of 5′ and 3′ breakpoints on W and consequent inversion on W^sh. Gray nucleotides belong to inverted corin, underlined and italicized bases are insertions and black nucleotides are bases upstream of Kit unaffected by inversion. C: Products from genomic PCR using primers designed to corin regions immediately upstream of the 5′ breakpoint. Two regions of intron 5 (Int 5A and Int 5B) and most of exon 6 (Exo 6) were assayed alongside controls for the Wt and W^sh alleles. D: Quantitative PCR analyses of transcripts for various corin exon pairs were performed using RNA prepared from wild-type (n = 5) or W^sh (n = 6) atria. Data were normalized to a single wild-type data set and the mean and SD are shown. ND, not detected. E: Heart weight relative to total body weight from wild-type (n = 9) and W^sh (n = 9) male mice aged 7 to 8 weeks pooled from 2 experiments.