Dr. Williams, students in his lab, and collaborators recently published “Redundant and Singular Regulatory Elements Underlie the Rapidly Evolving Pigmentation of Drosophila” in the journal Molecular Biology and Evolution

The group used in silico and in vivo methods to study the DNA sequences that control the activity of fruit fly genes that are responsible for a rapidly evolving trait. The work shows how some genes are controlled by a singular regulatory sequence, while others by multiple redundant regulatory sequences. Their results reveal a trend where genes whose use has evolved experience singular-type regulation, while the genes with an unchanged usage exhibit redundant-type regulation. The authors find parallels in studies of additional traits and in different animal species, and suggest that the singular/redundant paradigm may reflect a guide rail by which animal traits can change.

Brubaker LA, Long H, Pavlus A, Williams ME, Seibert DM, Williams AV, Halfon MS, Rebeiz M, Williams TM. Redundant and Singular Regulatory Elements Underlie the Rapidly Evolving Pigmentation of Drosophila. Mol Biol Evol. 2025 Sep 4:msaf213. doi: 10.1093/molbev/msaf213. Epub ahead of print. PMID: 40905948.

• PMID: 38820091
• PMCID: PMC11373662
• DOI: 10.1093/g3journal/jkae097

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.

Keywords: Drosophila; CRISPR/Cas9; abdomen; development; gene regulation; pigmentation.

 doi: 10.1371/journal.pgen.1010653. eCollection 2023 Feb.

Michael L Weinstein ¹, Chad M Jaenke ¹, Hasiba Asma ², Matthew Spangler ¹, Katherine A Kohnen ¹, Claire C Konys ¹, Melissa E Williams ¹, Ashley V Williams ³, Mark Rebeiz ⁴, Marc S Halfon ^2 5, Thomas M Williams ^1 6

Affiliations expand

• PMID: 36795790
• PMCID: PMC9977049
• DOI: 10.1371/journal.pgen.1010653

Abstract

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.

Copyright: © 2023 Weinstein et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cédric Finet, Victoria A Kassner, Antonio B Carvalho, Henry Chung, Jonathan P Day, Stephanie Day, Emily K Delaney, Francine C De Ré, Héloïse D Dufour, Eduardo Dupim, Hiroyuki F Izumitani, Thaísa B Gautério, Jessa Justen, Toru Katoh, Artyom Kopp, Shigeyuki Koshikawa, Ben Longdon, Elgion L Loreto, Maria D S Nunes, Komal K B Raja, Mark Rebeiz, Michael G Ritchie, Gayane Saakyan, Tanya Sneddon, Machiko Teramoto, Venera Tyukmaeva, Thyago Vanderlinde, Emily E Wey, Thomas Werner, Thomas M Williams, Lizandra J Robe, Masanori J Toda, Ferdinand Marlétaz

https://doi.org/10.1093/gbe/evab179

Abstract

The vinegar fly Drosophila melanogaster is a pivotal model for invertebrate development, genetics, physiology, neuroscience, and disease. The whole family Drosophilidae, which contains over 4,400 species, offers a plethora of cases for comparative and evolutionary studies. Despite a long history of phylogenetic inference, many relationships remain unresolved among the genera, subgenera, and species groups in the Drosophilidae. To clarify these relationships, we first developed a set of new genomic markers and assembled a multilocus data set of 17 genes from 704 species of Drosophilidae. We then inferred a species tree with highly supported groups for this family. Additionally, we were able to determine the phylogenetic position of some previously unplaced species. These results establish a new framework for investigating the evolution of traits in fruit flies, as well as valuable resources for systematics.

Jesse T. Hughes, Melissa E. Williams, Mark Rebeiz, Thomas M. Williams

https://doi.org/10.1002/jez.b.23068

Abstract

Changes in gene expression are a prominent feature of morphological evolution. These changes occur to hierarchical gene regulatory networks (GRNs) of transcription factor genes that regulate the expression of trait-building differentiation genes. While changes in the expression of differentiation genes are essential to phenotypic evolution, they can be caused by mutations within cis-regulatory elements (CREs) that drive their expression (cis-evolution) or within genes for CRE-interacting transcription factors (trans-evolution). Locating these mutations remains a challenge, especially when experiments are limited to one species that possesses the ancestral or derived phenotype. We investigated CREs that control the expression of the differentiation genes tan and yellow, the expression of which evolved during the gain, modification, and loss of dimorphic pigmentation among Sophophora fruit flies. We show these CREs to be necessary components of a pigmentation GRN, as deletion from Drosophila melanogaster (derived dimorphic phenotype) resulted in lost expression and lost male-specific pigmentation. We evaluated the ability of orthologous CRE sequences to drive reporter gene expression in species with modified (Drosophila auraria), secondarily lost (Drosophila ananassae), and ancestrally absent (Drosophila willistoni) pigmentation. We show that the transgene host frequently determines CRE activity, implicating trans-evolution as a significant factor for this trait’s diversity. We validated the gain of dimorphic Bab transcription factor expression as a trans-change contributing to the dimorphic trait. Our findings suggest an amenability to change for the landscape of trans-regulators and begs for an explanation as to why this is so common compared to the evolution of differentiation gene CREs.

Original Research ARTICLE

Front. Ecol. Evol., 26 March 2020 | https://doi.org/10.3389/fevo.2020.00080

Gene Regulatory Network Homoplasy Underlies Recurrent Sexually Dimorphic Fruit Fly Pigmentation

Jesse T. Hughes¹, Melissa E. Williams¹, Rachel Johnson¹, Sumant Grover¹, Mark Rebeiz^2* and Thomas M. Williams^1,3*

1. ¹Department of Biology, University of Dayton, Dayton, OH, United States
2. ²Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, United States
3. ³The Integrative Science and Engineering Center, University of Dayton, Dayton, OH, United States

Traits that appear discontinuously along phylogenies may be explained by independent origins (homoplasy) or repeated loss (homology). While discriminating between these models is difficult, the dissection of gene regulatory networks (GRNs) which drive the development of such repeatedly occurring traits can offer a mechanistic window on this fundamental problem. The GRN responsible for the male-specific pattern of Drosophila (D.) melanogaster melanic tergite pigmentation has received considerable attention. In this system, a metabolic pathway of pigmentation enzyme genes is expressed in spatial and sex-specific (i.e., dimorphic) patterns. The dimorphic expression of several genes is regulated by the Bab transcription factors, which suppress pigmentation enzyme expression in females, by virtue of their high expression in this sex. Here, we analyzed the phylogenetic distribution of species with male-specific pigmentation and show that this dimorphism is phylogenetically widespread among fruit flies. The analysis of pigmentation enzyme gene expression in distantly related dimorphic and monomorphic species shows that dimorphism is driven by the similar deployment of a conserved metabolic pathway. However, sexually dimorphic Bab expression was found only in D. melanogaster and its close relatives. These results suggest that dimorphism evolved by parallel deployment of differentiation genes, but was derived through distinct architectures at the level of regulatory genes. This work demonstrates the interplay of constraint and flexibility within evolving GRNs, findings that may foretell the mechanisms of homoplasy more broadly.

Changes throughout a Genetic Network Mask the Contribution of Hox Gene Evolution.

Dev Biol. 2018 Sep 1;441(1):159-175. doi: 10.1016/j.ydbio.2018.07.001. Epub 2018 Jul 4.

Augmentation of a wound response element accompanies the origin of a Hox-regulated Drosophila abdominal pigmentation trait.

One primary agenda of the developmental evolution field is to elucidate molecular mechanisms governing differences in animal form. While mounting evidence has established an important role for mutations in transcription controlling cis-regulatory elements (CREs), the underlying mechanisms that translate these alterations into differences in gene expression are poorly understood. Emerging studies focused on pigmentation differences among closely related Drosophila species have provided many examples of phenotypically relevant CRE changes, and have begun to illuminate how this process works at the level of regulatory sequence function and transcription factor binding. We review recent work in this field and highlight the conceptual and technical challenges that currently await experimental attention.

Mark Rebeiz and Thomas M. Williams

Volume 19, February 2017, Pages 1–7

http://www.sciencedirect.com/science/article/pii/S2214574516301456