A Gene(s) for All-trans-Retinoic Acid-Induced Forelimb Defects Mapped and Confirmed to Murine Chromosome 11

Abstract

All-trans-retinoic acid (RA) induces various anatomical limb dysmorphologies in mice dependent on the time of exposure. During early limb development, RA induces forelimb ectrodactyly (digital absence) with varying susceptibilities for different inbred mouse strains; C57BL/6N are highly susceptible while SWV are resistant. To isolate the genetic basis of this defect, a full-genome scan was performed in 406 backcross fetuses of F 1 males to C57BL/6N females. Fetuses were exposed via a maternal injection of 75 mg of RA per kilogram of body weight on gestational day 9.25. The genome-wide analysis revealed significant linkage to a chromosome 11 locus near D11Mit39 with a maximum LOD score of 9.0 and to a chromosome 4 locus near D4Mit170. An epistatic interaction was detected between loci on chromosome 11 (D11Mit39) and chromosome 18 (D18Mit64). Linkage to the chromosome 11 locus (D11Mit39) was confirmed in RA-treated backcross fetuses of F 1 females to C57BL/6N males. Loci associated with bone density/mass in both human and mouse were previously detected in the same region, suggesting a mechanistic linkage with bone homeostasis. The human syntenic region of this locus has been previously linked to Meckel syndrome; the phenotype includes postaxial polydactyly, an ectopic digital defect hypothesized to be induced by a common molecular pathway with ectrodactyly.

Genetic and pathologic aspects of retinoic acid‐induced limb malformations in the mouse

“…Historically, the C57BL/6 and SWV strains were considered to have approximately the same number of somites at the same gestational periods during development (Kuczuk and Scott,1984). Thus, our original experimental comparisons were performed by administering retinoids to the two strains at the same time during gestation (Lee et al,2005; Collins et al,2006). However, after examining a sizable number of embryos of both strains and noticing that the limb buds of SWV mice were frequently less developed than the limb buds of C57BL/6 mice, and then counting somites on a sufficient number of embryos (Lee et al,2005), it was determined that the SWV strain was approximately 2.3 somites behind the C57 strain at E 9.25.…”

“…Thus, our original experimental comparisons were performed by administering retinoids to the two strains at the same time during gestation (Lee et al,2005; Collins et al,2006). However, after examining a sizable number of embryos of both strains and noticing that the limb buds of SWV mice were frequently less developed than the limb buds of C57BL/6 mice, and then counting somites on a sufficient number of embryos (Lee et al,2005), it was determined that the SWV strain was approximately 2.3 somites behind the C57 strain at E 9.25. Since the mouse develops a somite every 1.7 hr during somitogenesis (Goedbloed and Smits‐van Prooije,1986), the SWV embryo was approximately 4 hr behind (2.3 somites × 1.7 hr per somite) the C57 in terms of limb development.…”

“…Both conventional knockout of Sp8 (Bell et al,2003) and ectoderm‐specific knockout of Bmpr1a (Ahn et al,2001), which encodes the Bmp4 receptor, disrupted the formation of the definitive AER, demonstrating that these genes are involved in the maturation process of the AER. Dlx3 is located in the region of chromosome 11, which is associated with the strain difference in RA‐induced postaxial ectrodactyly based on the QTL analysis (Lee et al,2005) as described in the following section on a QTL procedure. Interestingly, Dlx genes were directly regulated by p63 , which had been identified as a causative gene for a variety of ectodermal dysplasias, including ectrodactyly (Radoja et al,2007; Lo Iacono et al,2008).…”

“…In our laboratory, this approach has been used for cadmium‐induced and RA‐induced forelimb defects by using backcross fetuses from the C57BL/6N and SWV mice strains (Hovland et al,2000; Lee et al,2005). For the full genome scan with RA‐induced forelimb defects, pooled F 1 (C57 female × SWV male or SWV female × C57 male) males were backcrossed with C57 females to generate backcross fetuses.…”

Integrating genetic and toxicogenomic information for determining underlying susceptibility to developmental disorders

“…Elimination of Rarg attenuates the Cyp26a1 knockout mice phenotype of caudal regression and embryonic lethality (Abu‐Abed et al,2003). These genes may underlie a synergistic association between retinoic acid and Cd, leading to the formation of ectrodactyly (Lee et al,2005). In sum, NTD gene candidates disrupted by Cd in our model may underlie NTD development or other associated phenotypic observations (e.g., embryonic mortality, other growth effects).…”