Gene families expand by gene duplication, and resulting paralogs diverge through mutation. Functional diversification can include neofunctionalization as well as subfunctionalization of ancestral functions. In addition, redundancy in which multiple genes fulfill overlapping functions is often maintained. Here, we use the family of 40 Caenorhabditis elegans insulins to gain insight into the balance between specificity and redundancy. The insulin/insulin-like growth factor (IIS) pathway comprises a single receptor, DAF-2. To date, no single insulin-like peptide recapitulates all DAF-2-associated phenotypes, likely due to redundancy between insulin-like genes. To provide a first-level annotation of potential patterns of redundancy, we comprehensively delineate the spatiotemporal and conditional expression of all 40 insulins in living animals. We observe extensive dynamics in expression that can explain the lack of simple patterns of pairwise redundancy. We propose a model in which gene families evolve to attain differential alliances in different tissues and in response to a range of environmental stresses.

Craniosynostosis (CS) is a common congenital defect affecting more than 1/2000 infants. Infants with CS have a premature fusion of one or multiple cranial sutures resulting in restricted brain expansion. Single gene mutations account for 15-20% of cases, largely as part of a syndrome, but the majority are nonsyndromic with complex underlying genetics. Two noncoding genomic regions contributing to CS risk were previously identified by GWAS, one near BMP2 and one within BBS9. We hypothesized that the region within BBS9 contains distal regulatory elements controlling the neighboring gene encoding BMPER, a secreted modulator of BMP signaling. To identify regulatory sequences that might underlie disease risk, we surveyed conserved noncoding sequences from both risk loci identified from the GWAS for enhancer activity in transgenic Danio rerio. We identified enhancers from both regions that direct expression to skeletal tissues, consistent with the endogenous gene expression. Importantly, for each locus, we found a skeletal enhancer that also contains a sequence variant associated with CS risk. We examined the activity of each enhancer during craniofacial development and found that the BMPER-associated enhancer is active in the restricted region of cartilage closely associated with frontal bone initiation. We used an enhanced yeast one-hybrid assay to identify transcription factor interactions with several identified enhancers, implicating multiple signaling pathways in their regulation. In a targeted screen focused on risk-associated SNPs, we further identified differential binding to alternate and reference alleles. Additionally, we found that the risk allele of the BMPER enhancer directs significantly broader expression than the reference allele in transgenic zebrafish. Our findings support a specific genetic mechanism to explain the contribution of two risk loci to CS. More broadly, our combined in vivo approach is applicable to many complex genetic diseases to build a link between association studies and specific genetic mechanisms.

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