Rump white inversion in the mouse disrupts dipeptidyl aminopeptidase-like protein 6 and causes dysregulation of Kit expression

Abstract

The mouse rump white (Rw) mutation causes a pigmentation defect in heterozygotes and embryonic lethality in homozygotes. At embryonic day (E) 7.5, Rw/Rw embryos are retarded in growth, fail to complete neurulation and die around E 9.5. The Rw mutation is associated with a chromosomal inversion spanning 30 cM of the proximal portion of mouse chromosome 5. The Rw embryonic lethality is complemented by the W19H deletion, which spans the distal boundary of the Rw inversion, suggesting that the Rw lethality is not caused by the disruption of a gene at the distal end of the inversion. Here, we report the molecular characterization of sequences disrupted by both inversion breakpoints. These studies indicate that the distal breakpoint of the inversion is associated with ectopic Kit expression and therefore may be responsible for the dominant pigmentation defect in Rw/+ mice; whereas the recessive lethality of Rw is probably due to the disruption of the gene encoding dipeptidyl aminopeptidase-like protein 6, Dpp6 [Wada, K., Yokotani, N., Hunter, C., Doi, K., Wenthold, R. J. & Shimasaki, S. (1992) Proc. Natl. Acad. Sci. USA 89, 197-201] located at the proximal inversion breakpoint.

Figure 1

PFGE analysis of the chromosomal rearrangements associated with the Rw and Ph mutations. (A) Map positions of the loci Pdgfra, Kit, D5Buc2, D5Buc3, D5Buc4, and D5Buc5 relative to the chromosomal rearrangements associated with Ph and Rw. The probes D5Buc2 and D5Buc3 are described previously (14, 26), and the probes D5Buc4 and D5Buc5 are unique probes from cosmids cRBH4 and cRBH5, respectively, as described in the text. (B) PFGE analysis of the Rw mutation: C57BL/6J (lane 1), C3H/He (lane 2), and Rw/+ (lane 3) DNA digested with BssHII hybridized with Pdgfra, D5Buc4, and D5Buc5. (C) PFGE analysis of the Rw mutation: C57BL/6J (lane 1), C3H/He (lane 2), and Rw/+ (lane 3) DNA digested with NotI hybridized with Pdgfra, D5Buc4, and D5Buc5. (D) PFGE analysis of the Ph mutation. C57BL/6J (lane 1), C3H/He (lane 2), and Ph/+ (lane 3) DNA digested with BssHII hybridized with Pdgfra, D5Buc2, D5Buc4, and D5Buc5. The sizes (in kb) of DNA fragments are shown on the right.

Figure 2

In situ hybridization of Kit to wild-type and Rw+/+W^19H embryos. The expression of Kit in the otic vesicle (ot) in wild-type (A and B) and the absence of Kit expression in Rw+/+W^19H (E and F). Ectopic expression of Kit is seen in Rw+/+W^19H embryos in the heart (h) (G and H). The expression of Kit in a parasagital section of the surface ectoderm (se) in a wild-type E 10.5 embryo (C and D) and the absence of or reduced expression in transverse sections of surface ectoderm (se, see region denoted by black arrow) from a Rw+/+W^19H embryo (I and J). Note that the plane of section in the wild-type (C) is equivalent to the region denoted by the black arrows in the Rw+/+W^19H section (I). Ectopic expression is seen in Rw+/+W^19H embryos in the dermatome (dm) (I and J) and the dorsal region of the neural tube (nt) (see white arrow head in J).

Figure 3

Genomic structure of the proximal Rw inversion breakpoints. (A) Wild-type and Rw chromosomal maps give the approximate map positions of genes based on previous studies (14, 22, 31). The Rw inversion is represented by a cross-hatched box. Cosmid cAB1 was isolated by screening a genomic library of Rw/+ DNA with a unique probe from cRBH5 (D5Buc4) as described in the text. Chromosomal distances are not represented on scale. The restriction map of cAB1 is shown. Dashed lines indicate the sequenced region. Solid lines below cAB1 give the approximate positions of Dpp6 exons found by sequencing and grail2 analysis. Arrow shows the positions of the inversion breakpoint. (B) Sequence analysis of the embryonic Dpp6 cDNA (GenBank accession no. AF092507). Shown are the cDNA sequence and the deduced amino acid sequence. The putative intracellular domain is boxed and the transmembrane domain double underlined. The first 20 amino acids are unique in the embryonic DPP6 isoform. The conserved residues, which form the putative catalytic site in nonclassical serine peptidases, are gray shaded, and the aspartic residue, which is replaced by a serine residue shown to be essential for peptidase activity in DPP4, is boxed (32). The location of the proximal Rw inversion breakpoint, which eliminates the C-terminal portion of DPP6, is indicated by an arrowhead.

Figure 4

Expression analysis of Dpp6 on the Rw and +/+ chromosome. (A) Northern blot analysis of Dpp6 in +/+ and Rw/+ mice. The Dpp6 cDNA probe detects 4.4 kb and 3.6 kb transcripts in the brain of +/+ and of Rw/+. A transcript of 3.4 kb also was detected in the testes of +/+ and of Rw/+. Total RNA was isolated from adult tissue. RNA loading was assessed by hybridization with β actin and a phosphorimager was used to quantitate and compare levels of expression in +/+ and Rw/+ RNA samples. (B) Analysis of Dpp6 expression in embryonic RNA samples. The probe for Dpp6 detects a 4.4 kb and a 3.6 kb transcripts in 11- to 17-day mouse embryos. A transcript of 3.9 kb was detected in 7-day mouse embryos. The Northern filter (CLONTECH) contains 2 μg of polyadenlyated RNA from E 7, E 11, E 15, and E 17 mouse embryos. RNA loading was assessed by hybridization with β actin.