mRNA fluorescence in situ hybridization to determine overlapping gene expression in whole-mount mouse embryos.

BACKGROUND: Whole-mount in situ hybridization (ISH) is a prevalent tool to examine the spatial distribution of gene transcripts in intact embryos. Chromogenic-based methods of signal development are commonly used in mouse embryos because of their high sensitivity. Fluorescence techniques, however, offer several advantages over chromogenic methods including the ability to visualize multiple signals in a specimen at once. RESULTS: We describe a procedure for fluorescence in situ hybridization (FISH) for whole mouse embryos up to embryonic day 13.5. We show that this approach successfully produces a bright expression signal for several genes, validating the procedure in multiple tissues. Further, we show that double FISH can be used to visualize the expression of two genes in a single embryo by determining that Hoxd13 and Shh are co-expressed in both the limb bud and the hindgut. Finally, we demonstrate that FISH can be paired with confocal microscopy to take optical sections of interior regions of the embryo. CONCLUSIONS: FISH is a valid alternative to chromogenic-based ISH for visualizing gene expression in whole mouse embryos. This work provides a framework to add additional fluorescence signals in the mouse such as visualizing both mRNA and protein by pairing the procedure with immunofluorescence.

Interspecies transcriptomics identify genes that underlie disproportionate foot growth in jerboas.

Despite the great diversity of vertebrate limb proportion and our deep understanding of the genetic mechanisms that drive skeletal elongation, little is known about how individual bones reach different lengths in any species. Here, we directly compare the transcriptomes of homologous growth cartilages of the mouse (Mus musculus) and bipedal jerboa (Jaculus jaculus), the latter of which has "mouse-like" arms but extremely long metatarsals of the feet. Intersecting gene-expression differences in metatarsals and forearms of the two species revealed that about 10% of orthologous genes are associated with the disproportionately rapid elongation of neonatal jerboa feet. These include genes and enriched pathways not previously associated with endochondral elongation as well as those that might diversify skeletal proportion in addition to their known requirements for bone growth throughout the skeleton. We also identified transcription regulators that might act as "nodes" for sweeping differences in genome expression between species. Among these, Shox2, which is necessary for proximal limb elongation, has gained expression in jerboa metatarsals where it has not been detected in other vertebrates. We show that Shox2 is sufficient to increase mouse distal limb length, and a nearby putative cis-regulatory region is preferentially accessible in jerboa metatarsals. In addition to mechanisms that might directly promote growth, we found evidence that jerboa foot elongation may occur in part by de-repressing latent growth potential. The genes and pathways that we identified here provide a framework to understand the modular genetic control of skeletal growth and the remarkable malleability of vertebrate limb proportion.

Genetic interactions between Shox2 and Hox genes during the regional growth and development of the mouse limb.

The growth and development of the vertebrate limb relies on homeobox genes of the Hox and Shox families, with their independent mutation often giving dose-dependent effects. Here we investigate whether Shox2 and Hox genes function together during mouse limb development by modulating their relative dosage and examining the limb for nonadditive effects on growth. Using double mRNA fluorescence in situ hybridization (FISH) in single embryos, we first show that Shox2 and Hox genes have associated spatial expression dynamics, with Shox2 expression restricted to the proximal limb along with Hoxd9 and Hoxa11 expression, juxtaposing the distal expression of Hoxa13 and Hoxd13. By generating mice with all possible dosage combinations of mutant Shox2 alleles and HoxA/D cluster deletions, we then show that their coordinated proximal limb expression is critical to generate normally proportioned limb segments. These epistatic interactions tune limb length, where Shox2 underexpression enhances, and Shox2 overexpression suppresses, Hox-mutant phenotypes. Disruption of either Shox2 or Hox genes leads to a similar reduction in Runx2 expression in the developing humerus, suggesting their concerted action drives cartilage maturation during normal development. While we furthermore provide evidence that Hox gene function influences Shox2 expression, this regulation is limited in extent and is unlikely on its own to be a major explanation for their genetic interaction. Given the similar effect of human SHOX mutations on regional limb growth, Shox and Hox genes may generally function as genetic interaction partners during the growth and development of the proximal vertebrate limb.