Various approaches can be applied to uncover the genetic basis of natural phenotypic variation, each with their specific strengths and limitations. Here, we use a replicated genome-wide association approach (Pool-GWAS) to fine-scale map genomic regions contributing to natural variation in female abdominal pigmentation in Drosophila melanogaster, a trait that is highly variable in natural populations and highly heritable in the laboratory. We examined abdominal pigmentation phenotypes in approximately 8000 female European D. melanogaster, isolating 1000 individuals with extreme phenotypes. We then used whole-genome Illumina sequencing to identify single nucleotide polymorphisms (SNPs) segregating in our sample, and tested these for associations with pigmentation by contrasting allele frequencies between replicate pools of light and dark individuals. We identify two small regions near the pigmentation genes tan and bric-à-brac 1, both corresponding to known cis-regulatory regions, which contain SNPs showing significant associations with pigmentation variation. While the Pool-GWAS approach suffers some limitations, its cost advantage facilitates replication and it can be applied to any non-model system with an available reference genome.

Using exome sequencing, we have identified de novo variants in MAPK8IP3 in 13 unrelated individuals presenting with an overlapping phenotype of mild to severe intellectual disability. The de novo variants comprise six missense variants, three of which are recurrent, and three truncating variants. Brain anomalies such as perisylvian polymicrogyria, cerebral or cerebellar atrophy, and hypoplasia of the corpus callosum were consistent among individuals harboring recurrent de novo missense variants. MAPK8IP3 has been shown to be involved in the retrograde axonal-transport machinery, but many of its specific functions are yet to be elucidated. Using the CRISPR-Cas9 system to target six conserved amino acid positions in Caenorhabditis elegans, we found that two of the six investigated human alterations led to a significantly elevated density of axonal lysosomes, and five variants were associated with adverse locomotion. Reverse-engineering normalized the observed adverse effects back to wild-type levels. Combining genetic, phenotypic, and functional findings, as well as the significant enrichment of de novo variants in MAPK8IP3 within our total cohort of 27,232 individuals who underwent exome sequencing, we implicate de novo variants in MAPK8IP3 as a cause of a neurodevelopmental disorder with intellectual disability and variable brain anomalies.

Our data corroborate the role of an impaired cytokinesis in the etiology of primary and syndromic microcephaly, as has been proposed by recent findings on CIT mutations. Ann Neurol 2017;82:562-577.

Hox proteins play fundamental roles in controlling morphogenetic diversity along the anterior–posterior body axis of animals by regulating distinct sets of target genes. Within their rather broad expression domains, individual Hox proteins control cell diversification and pattern formation and consequently target gene expression in a highly localized manner, sometimes even only in a single cell. To achieve this high-regulatory specificity, it has been postulated that Hox proteins co-operate with other transcription factors to activate or repress their target genes in a highly context-specific manner in vivo. However, only a few of these factors have been identified. Here, we analyze the regulation of the cell death gene reaper (rpr) by the Hox protein Deformed (Dfd) and suggest that local activation of rpr expression in the anterior part of the maxillary segment is achieved through a combinatorial interaction of Dfd with at least eight functionally diverse transcriptional regulators on a minimal enhancer. It follows that context-dependent combinations of Hox proteins and other transcription factors on small, modular Hox response elements (HREs) could be responsible for the proper spatio-temporal expression of Hox targets. Thus, a large number of transcription factors are likely to be directly involved in Hox target gene regulation in vivo.