Laser capture microdissection/RNA-Seq analyses of Hoxa9,10,11/Hoxd9,10,11 mutant limb development

We used a novel recombineering method to introduce frameshift mutations into the first exons of the flanking Hoxa9,10,11 and Hoxd9,10,11 genes. By interbreeding it was possibleto make mice homozygous mutant for all six genes. These mice showed an extreme shortening of the zeugopod, with only tiny cartilaginous remnants of the ulna and radius made.We used laser capture microdissection to isolate the cells of the developing mutant zeugopods. Mutant and wild type samples were compared by RNA-seq. The results define the many downstream functional pathways perturbed by the Hox mutations, including the upregulation of many genes known to inhibit chondrocyte and osteoblast differentiation, BMP signalin, and growth arrest and apoptosis. In addition we observed the downregulation of genes required for WNT signaling and chonrdocyte maturation. Overall design: RNA-seq comparison of Hoxa9,10,11/Hoxd9,10,11 mutant and wild type developing limbs in mouse.Also includes different compartments of wild type developing limb.