Human Limb Defects Research Articles

Thalidomide Inhibits Human iPSC Mesendoderm Differentiation by Modulating CRBN-dependent Degradation of SALL4

Exposure to thalidomide during a critical window of development results in limb defects in humans and non-human primates while mice and rats are refractory to these effects. Thalidomide-induced teratogenicity is dependent on its binding to cereblon (CRBN), the substrate receptor of the Cul4A-DDB1-CRBN-RBX1 E3 ubiquitin ligase complex. Thalidomide binding to CRBN elicits subsequent ubiquitination and proteasomal degradation of CRBN neosubstrates including SALL4, a transcription factor of which polymorphisms phenocopy thalidomide-induced limb defects in humans. Herein, thalidomide-induced degradation of SALL4 was examined in human induced pluripotent stem cells (hiPSCs) that were differentiated either to lateral plate mesoderm (LPM)-like cells, the developmental ontology of the limb bud, or definitive endoderm. Thalidomide and its immunomodulatory drug (IMiD) analogs, lenalidomide, and pomalidomide, dose-dependently inhibited hiPSC mesendoderm differentiation. Thalidomide- and IMiD-induced SALL4 degradation can be abrogated by CRBN V388I mutation or SALL4 G416A mutation in hiPSCs. Genetically modified hiPSCs expressing CRBN E377V/V388I mutant or SALL4 G416A mutant were insensitive to the inhibitory effects of thalidomide, lenalidomide, and pomalidomide on LPM differentiation while retaining sensitivity to another known limb teratogen, all-trans retinoic acid (atRA). Finally, disruption of LPM differentiation by atRA or thalidomide perturbed subsequent chondrogenic differentiation in vitro. The data here show that thalidomide, lenalidomide, and pomalidomide affect stem cell mesendoderm differentiation through CRBN-mediated degradation of SALL4 and highlight the utility of the LPM differentiation model for studying the teratogenicity of new CRBN modulating agents.

Tbx5 inhibits hedgehog signaling in determination of digit identity.

Dominant TBX5 mutation causes Holt-Oram syndrome (HOS), which is characterized by limb defects in humans, but the underlying mechanistic basis is unclear. We used a mouse model with Tbx5 conditional knockdown in Hh-receiving cells (marked by Gli1+) during E8 to E10.5, a previously established model to study atrial septum defects, which displayed polydactyly or hypodactyly. The results suggested that Tbx5 is required for digit identity in a subset of limb mesenchymal cells. Specifically, Tbx5 deletion in this cell population decreased cell apoptosis and increased the proliferation of handplate mesenchymal cells. Furthermore, Tbx5 was found to negatively regulate the Hh-signaling activity through transcriptional regulation of Ptch1, a known Hh-signaling repressor. Repression of Hh-signaling through Smo co-mutation in Tbx5 heterozygotes rescued the limb defects, thus placing Tbx5 upstream of Hh-signaling in limb defects. This work reveals an important missing component necessary for understanding not only limb development but also the molecular and genetic mechanisms underlying HOS.

Exome sequencing and CRISPR/Cas genome editing identify mutations of ZAK as a cause of limb defects in humans and mice.

The CRISPR/Cas technology enables targeted genome editing and the rapid generation of transgenic animal models for the study of human genetic disorders. Here we describe an autosomal recessive human disease in two unrelated families characterized by a split-foot defect, nail abnormalities of the hands, and hearing loss, due to mutations disrupting the SAM domain of the protein kinase ZAK. ZAK is a member of the MAPKKK family with no known role in limb development. We show that Zak is expressed in the developing limbs and that a CRISPR/Cas-mediated knockout of the two Zak isoforms is embryonically lethal in mice. In contrast, a deletion of the SAM domain induces a complex hindlimb defect associated with down-regulation of Trp63, a known split-hand/split-foot malformation disease gene. Our results identify ZAK as a key player in mammalian limb patterning and demonstrate the rapid utility of CRISPR/Cas genome editing to assign causality to human mutations in the mouse in <10 wk.

Effects of thalidomide on Fgf8, Bmp4 and Hoxa11 expression in the limb bud in Kbl:JW rabbit embryos

Thalidomide (TM) induces limb defects in humans and some animal species including rabbits. Although the mechanism of TM-induced limb defects has been investigated for a long period, the limb development-related genes expressions have not been vigorously characterized in rabbits. In this study, we investigated the Fgf8, Bmp4 and Hoxa11 expressions in TM-treated JW rabbit embryos on gestation days (GDs) 10, 11 and 12 by whole mount in situ hybridization. On GDs 10 and 11, growth retardation of the embryo was induced by TM treatment. The Fgf8 expression lengths on GDs 10 and 11 in the forelimb bud were significantly or tended to be decreased in the TM-treated embryos, which was correlated to the growth retardation and was not considered to be directly relevant to the teratogenic effect of TM in the forelimb. The TM-induced characteristic changes in the expression pattern of Hoxa11 and Bmp4 on GDs 10 and/or 11 were not noted. On GD 12, TM-induced growth retardation was not noted and the Fgf8 and Bmp4 expressions were not changed. On the contrary, Hoxa11 expression was narrowed at the anterior region, which was located on the radial side, and was not changed at the middle and posterior regions in the forelimb bud and in all regions in the hindlimb bud. Because the radius malformations were induced by TM treatment, we concluded the decrease in the Hoxa11 expression was related to the TM-induced limb defects and can be a good marker for early prediction of the teratogenic effect of TM.