Patterns of gene expression in the central nervous system are highly variable and heritable. This genetic variation among normal individuals leads to considerable structural, functional and behavioral differences. We devised a general approach to dissect genetic networks systematically across biological scale, from base pairs to behavior, using a reference population of recombinant inbred strains. We profiled gene expression using Affymetrix oligonucleotide arrays in the BXD recombinant inbred strains, for which we have extensive SNP and haplotype data. We integrated a complementary database comprising 25 years of legacy phenotypic data on these strains. Covariance among gene expression and pharmacological and behavioral traits is often highly significant, corroborates known functional relations and is often generated by common quantitative trait loci. We found that a small number of major-effect quantitative trait loci jointly modulated large sets of transcripts and classical neural phenotypes in patterns specific to each tissue. We developed new analytic and graph theoretical approaches to study shared genetic modulation of networks of traits using gene sets involved in neural synapse function as an example. We built these tools into an open web resource called WebQTL that can be used to test a broad array of hypotheses.

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The loss of HOXA13 function severely disrupts embryonic limb development. However, because embryos lacking HOXA13 die by mid-gestation, the defects present in the mutant limb could arise as a secondary consequence of failing embryonic health. In our analysis of the mutant Hoxa13 GFP allele, we identified a surviving cohort of homozygous mutants exhibiting severe limb defects including: missing phalanx elements, fusions of the carpal/tarsal elements, and significant reductions in metacarpal/metatarsal length. Characterization of prochondrogenic genes in the affected carpal/tarsal regions revealed significant reduction in Gdf5 expression, whereas Bmp2 expression was significantly elevated. Analysis of Gdf5 mRNA localization also revealed diffuse expression in the carpal/tarsal anlagen, suggesting a role for HOXA13 in the organization of the cells necessary to delineate individual carpal/tarsal elements. Together these results identify Gdf5 as a potential target gene of HOXA13 target gene and confirm a specific role for HOXA13 during appendicular skeletal development. Developmental Dynamics 239:446-457,

Mutations in Hoxa13 cause malformations of the appendicular skeleton and genitourinary tract, including digit loss, syndactyly, and hypospadias. To determine the molecular basis for these defects, the DNA sequences bound by HOXA13 were empirically determined, revealing a novel high affinity binding site. Correlating the utilization of this high affinity binding site with genes exhibiting perturbed expression in Hoxa13 mutant limbs, we identified that HOXA13 suppresses the expression of the BMP antagonist, Sostdc1. In the absence of HOXA13 function, Sostdc1 is ectopically expressed in the distal limb, causing reduced expression of BMP-activated genes and decreased SMAD phosphorylation. Limb chromatin immunoprecipitation revealed HOXA13 binding at its high affinity site in two conserved Sostdc1 regulatory sites in vivo. In vitro, HOXA13 represses gene expression through the Sostdc1 high affinity binding sites in a dosage-dependent manner. Together, these findings confirm that the high affinity HOXA13 binding site deduced by quantitative analyses is used in vivo to facilitate HOXA13 target gene regulation, providing a critical advance toward understanding the molecular basis for defects associated with the loss of HOXA13 function.

To gain insight into the coordination of gene expression profiles during forelimb and hindlimb differentiation, a transcriptome analysis of mouse embryonic autopod tissues was performed using Affymetrix Murine Gene Chips (MOE-430). Forty-four transcripts with expression differences higher than 2-fold (T test, P <0.05) were detected between forelimb and hindlimb tissues including 38 new transcripts such as Rdh10, Frzb, Tbx18, and Hip that exhibit differential limb expression. A comparison of gene expression profiles in the forelimb, hindlimb, and brain revealed 24 limb-signature genes whose expression was significantly enriched in limb autopod versus brain tissue (fold change >2, P < 0.05). Interestingly, the genes exhibiting enrichment in the developing autopod also segregated into significant fore-and hindlimbspecific clusters (P < 0.05) suggesting that by E 12.5, unique gene combinations are being used during the differentiation of each autopod type. Developmental Dynamics 234:74 -89, 2005.