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Abstract Gene regulatory networks specify the gene expression patterns needed for traits to develop. Differences in these networks can result in phenotypic differences between organisms. Although loss-of-function genetic screens can identify genes necessary for trait formation, gain-of-function screens can overcome genetic redundancy and identify loci whose expression is sufficient to alter trait formation. Here, we leveraged transgenic lines from the Transgenic RNAi Project at Harvard Medical school to perform both gain- and loss-of-function CRISPR/Cas9 screens for abdominal pigmentation phenotypes. We identified measurable effects on pigmentation patterns in the Drosophila melanogaster abdomen for 21 of 55 transcription factors in gain-of-function experiments and 7 of 16 tested by loss-of-function experiments. These included well-characterized pigmentation genes, such as bab1 and dsx, and transcription factors that had no known role in pigmentation, such as slp2. Finally, this screen was partially conducted by undergraduate students in a Genetics Laboratory course during the Spring semesters of 2021 and 2022. We found this screen to be a successful model for student engagement in research in an undergraduate laboratory course, that can be readily adapted to evaluate the effect of hundreds of genes on many different Drosophila traits, with minimal resources. 

Changes in gene regulation represent an important path to generate developmental differences affecting anatomical traits. Interspecific divergence in gene expression often results from changes in transcription-stimulating enhancer elements. While gene repression is crucial for precise spatiotemporal expression patterns, the relative contribution of repressive transcriptional silencers to regulatory evolution remains to be addressed. Here, we show that the Drosophila pigmentation gene ebony has mainly evolved through changes in the spatial domains of silencers patterning its abdominal expression. By precisely editing the endogenous ebony locus of D. melanogaster , we demonstrate the requirement of two redundant abdominal enhancers and three silencers that repress the redundant enhancers in a patterned manner. We observe a role for changes in these silencers in every case of ebony evolution observed to date. Our findings suggest that negative regulation by silencers likely has an under-appreciated role in gene regulatory evolution. 

Animal traits develop through the expression and action of numerous regulatory and realizator genes that comprise a gene regulatory network (GRN). For each GRN, its underlying patterns of gene expression are controlled by cis -regulatory elements (CREs) that bind activating and repressing transcription factors. These interactions drive cell-type and developmental stage-specific transcriptional activation or repression. Most GRNs remain incompletely mapped, and a major barrier to this daunting task is CRE identification. Here, we used an in silico method to identify predicted CREs (pCREs) that comprise the GRN which governs sex-specific pigmentation of Drosophila melanogaster . Through in vivo assays, we demonstrate that many pCREs activate expression in the correct cell-type and developmental stage. We employed genome editing to demonstrate that two CREs control the pupal abdomen expression of trithorax , whose function is required for the dimorphic phenotype. Surprisingly, trithorax had no detectable effect on this GRN’s key trans -regulators, but shapes the sex-specific expression of two realizator genes. Comparison of sequences orthologous to these CREs supports an evolutionary scenario where these trithorax CREs predated the origin of the dimorphic trait. Collectively, this study demonstrates how in silico approaches can shed novel insights on the GRN basis for a trait’s development and evolution.