Biology subjects

Evans, T. A.

Publications and source records attributed to Evans, T. A..

11 recordsLinked to original sources

bioRxiv2025

Evolutionary conservation of midline repulsive signaling by Robo family receptors inflies and mice

The Roundabout (Robo) family is an evolutionarily conserved group of axon guidance receptors that regulate midline crossing in a wide range of animal groups by signaling midline repulsion in response to Slit ligands. However, it is not known whether Robo receptors from different species signal midline repulsion via equivalent mechanisms. To examine the evolutionary conservation of midline repulsive signaling, here we express Robo family proteins from mouse in neurons of the fruit fly, Drosophila melanogaster. We show that Robo proteins which normally function in canonical Slit-dependent midline repulsion in the mouse (mRobo1 and mRobo2) can also repel Drosophila axons from the midline in the fly embryonic ventral nerve cord, and can partially rescue the ectopic midline crossing defects caused by loss of Drosophila robo1, though less efficiently as Drosophila Robo1 itself. In contrast, mouse Robo3 isoforms (mRobo3.1 and mRobo3.2) which do not normally respond to Slit have no detectable effect on axon guidance when expressed in fly embryos. We further show that the differences in midline repulsive signaling effectiveness between fly and mouse Robos is not due to a simple difference in Slit affinity conferred by the Slit-binding Ig1 domain. Together, our results support the idea that the core signaling mechanisms employed by Robo family receptors are conserved across bilaterians, though some degree of evolutionary divergence has decreased the ability of receptors from different bilaterian clades to directly substitute for one another.

developmental biology↗

bioRxiv2024

Evolutionary conservation of midline axon guidance activity between Drosophila and Tribolium Frazzled

The regulation of midline crossing of axons is of fundamental importance for the proper development of nervous system connectivity in bilaterian animals. A number of conserved axon guidance signaling pathways coordinate to attract or repel axons at the nervous system midline to ensure the proper regulation of midline crossing. The attractive Netrin-Frazzled/DCC (Net-Fra) signaling pathway is widely conserved among bilaterians, but it is not clear whether the mechanisms by which Net and Fra promote midline crossing are also conserved. In Drosophila, Fra can promote midline crossing via Netrin-dependent and Netrin-independent mechanisms, by acting as a canonical midline attractive receptor and also through a non-canonical pathway to inhibit midline repulsion via transcriptional regulation. To examine the conservation of Fra-dependent axon guidance mechanisms among insects, in this paper we compare the midline attractive roles of the Frazzled receptor in the fruit fly (Drosophila melanogaster) and flour beetle (Tribolium castaneum) using CRISPR/Cas9-mediated gene editing. We replace the Drosophila fra gene with sequences encoding Drosophila Fra (DmFra) or Tribolium Fra (TcFra) and examine midline crossing of axons in the ventral nerve cord of embryos carrying these modified alleles. We show that Tribolium Fra can fully substitute for Drosophila Fra to promote midline crossing of axons in the embryonic nervous system, suggesting that the mechanisms by which Frazzled regulates midline axon guidance may be evolutionarily conserved within insects.

developmental biology↗

bioRxiv2023

Slit-independent guidance of longitudinal axons by Drosophila Robo3

Drosophila Robo3 is a member of the evolutionarily conserved Roundabout (Robo) receptor family and one of three Drosophila Robo paralogs. During embryonic ventral nerve cord development, Robo3 does not participate in canonical Slit-dependent midline repulsion, but instead regulates the formation of longitudinal axon pathways at specific positions along the medial-lateral axis. Longitudinal axon guidance by Robo3 is hypothesized to be Slit dependent, but this has not been directly tested. Here we create a series of Robo3 variants in which the N-terminal Ig1 domain is deleted or modified, in order to characterize the functional importance of Ig1 and Slit binding for Robo3s axon guidance activity. We show that Robo3 requires its Ig1 domain for interaction with Slit and for proper axonal localization in embryonic neurons, but deleting Ig1 from Robo3 only partially disrupts longitudinal pathway formation. Robo3 variants with modified Ig1 domains that cannot bind Slit retain proper localization and fully rescue longitudinal axon guidance. Our results indicate that Robo3 guides longitudinal axons independently of Slit, and that sequences both within and outside of Ig1 contribute to this Slit-independent activity.

developmental biology↗

bioRxiv2022

Characterization of enhancer fragments in Drosophila robo2

Receptor proteins of the Roundabout (Robo) family regulate axon guidance decisions during nervous system development. Among the three Drosophila robo family genes (robo1, robo2, and robo3), robo2 displays a dynamic expression pattern and regulates multiple axon guidance outcomes, including preventing midline crossing in some axons, promoting midline crossing in others, forming lateral longitudinal axon pathways, and regulating motor axon guidance. The identity and location of enhancer elements regulating robo2s complex and dynamic expression pattern in different neural cell types are unknown. Here, we characterize a set of 17 transgenic lines expressing GAL4 under the control of DNA sequences derived from noncoding regions in and around robo2, to identify enhancers controlling specific aspects of robo2 expression in the embryonic ventral nerve cord. We identify individual fragments that confer expression in specific cell types where robo2 is known to function, including early pioneer neurons, midline glia, and lateral longitudinal neurons. Our results indicate that robo2s dynamic expression pattern is specified by a combination of enhancer elements that are active in different subsets of cells. We show that robo2s expression in lateral longitudinal axons represents two genetically separable subsets of neurons, and compare their axon projections with each other and with Fasciclin II (FasII), a commonly used marker of longitudinal axon pathways. In addition, we provide a general description of each fragments expression in embryonic tissues outside of the nervous system, to serve as a resource for other researchers who may be interested in robo2 expression and its functional roles outside the CNS.

developmental biology↗

bioRxiv2022

A cis-regulatory sequence of the wing selector gene, vestigial, drives the evolution of scaling relationships in Drosophila species

Scaling between specific organs and overall body size has long fascinated biologists because they are a primary mechanism through which organismal shapes evolve. Yet, the genetic mechanisms that underlie the evolution of allometries remain elusive. Here we measured wings and tibia lengths in four Drosophila species (D. melanogaster, D. simulans, D. ananassae, and D. virilis) and show that the first three of them follow a single evolutionary allometry. However, D. virilis exhibits a divergent wing-to-tibia allometry due to a dramatic underscaling of their wings with respect to their bodies compared to the other species. We asked whether the evolution of this scaling relationship could be explained by changes in a specific cis-regulatory regulatory region of the wing selector gene, vestigial (vg), whose function is broadly conserved in insects and its expression pattern determines wing size in D. melanogaster. To test this hypothesis directly, we used CRISPR/Cas9 to replace the DNA sequence of the predicted Quadrant Enhancer (vgQE) from D. virilis for the corresponding vgQE sequence in the genome of D. melanogaster. Strikingly, we discovered that D. melanogaster flies carrying the D. virilis vgQE sequence have wings that are much smaller with respect to controls, partially rescuing the wing-to-tibia ratio observed in D. virilis. Our results show that this cis-regulatory element in D. virilis contributes to the underscaling of wings in this species. This provides evidence that scaling relationships may be unconstrained and may evolve gradually through genetic variations in cis-regulatory elements. Summary statementUsing CRISPR/Cas9 replacement of a cis-regulatory element, this study suggests that changes within the vestigial Quadrant Enhancer sequence are responsible for the evolution of wing allometries in Drosophila species.

evolutionary biology↗

bioRxiv2022

Variation in TAF1 expression in female carrier induced pluripotent stem cells and human brain ontogeny has implications for adult neostriatum vulnerability in X-linked Dystonia Parkinsonism

X-linked Dystonia-Parkinsonism (XDP) is an inherited, X-linked, adult-onset movement disorder characterized by degeneration in the neostriatum. No therapeutics alter disease progression. The mechanisms underlying regional differences in degeneration and age of onset are unknown. Developing therapeutics that target XDP-related mechanisms requires a deeper understanding of how XDP-relevant features vary in health and disease. XDP is due, in part, to either a partial loss of TAF1 function and/or a SVA-driven pathological gain of function. A disease-specific SINE-VNTR-Alu (SVA) retrotransposon insertion occurs within intron 32 of TAF1, a subunit of TFIID involved in transcription initiation. While all XDP males are usually clinically affected, females are heterozygous carriers generally not manifesting the full syndrome. As a resource for disease modeling, we characterized eight iPSC lines from XDP female carrier individuals, and identified isogenic lines where one clonal iPSC line expressed the wild-type X, and the two other clonal iPSC lines expressed the XDP haplotype. Furthermore, we characterized XDP-relevant transcript expression variation in humans, and found that SVA-F expression decreases slightly after 30 years of age in the neurotypical human brain and that TAF1 is modestly decreased in the majority of female samples. Uniquely in the caudate nucleus, TAF1 expression is not sexually dymorphic and decreased after 15 years of age. These findings indicate that regional-, age- and sex-specific mechanisms regulate TAF1, highlighting the importance of disease-relevant models and postmortem tissue analysis. We propose that the decreased TAF1 expression in the adult caudate may synergize with the XDP-specific partial loss of TAF1 function in patients, thereby passing a minimum threshold of TAF1 function, and triggering degeneration in the neostriatum. Significance StatementXDP is an inherited, X-linked, adult-onset movement disorder characterized by degeneration in the neostriatum. No therapeutics alter disease progression. Developing therapeutics requires a deeper understanding of how XDP-relevant features vary in health and disease. XDP is possibly due to a partial loss of TAF1 function. While all XDP males are usually affected, females are heterozygous carriers generally not manifesting the full syndrome. As a resource for disease modeling, we characterized eight stem cell lines from XDP female carrier individuals. Furthermore, we found that, uniquely in the caudate nucleus, TAF1 expression decreases after adolescence in healthy humans. We hypothesize that the decrease of TAF1 after adolescence in human caudate, in general, may underlie the vulnerability of the adult neostriatum in XDP.

neuroscience↗

bioRxiv2021

Historical biogeography of early diverging termite lineages (Isoptera: Teletisoptera)

Termites are social cockroaches distributed throughout warm temperate and tropical ecosystems. The ancestor of modern termites (crown-Isoptera) occurred during the earliest Cretaceous, approximately 140 million years ago, suggesting that both vicariance through continental drift and overseas dispersal may have shaped the distribution of early diverging termite lineages. We reconstruct the historical biogeography of three early diverging termite families - Stolotermitidae, Hodotermitidae, and Archotermopsidae - using the nuclear rRNA genes and mitochondrial genomes of 27 samples. Our analyses confirmed the monophyly of Stolotermitidae + Hodotermitidae + Archotermopsidae (clade Teletisoptera), with Stolotermitidae diverging from a monophyletic Hodotermitidae + Archotermopsidae approximately 100.3 Ma (94.3-110.4 Ma, 95% HPD), and with Archotermopsidae paraphyletic to a monophyletic Hodotermitidae. The Oriental Archotermopsis and the Nearctic Zootermopsis diverged 50.8 Ma (40.7-61.4 Ma, 95% HPD) before land connections between the Palearctic region and North America ceased to exist. The African Hodotermes + Microhodotermes diverged from Anacanthotermes, a genus found in Africa and Asia, 32.1 Ma (24.8-39.9 Ma, 95% HPD), and the most recent common ancestor of Anacanthotermes lived 10.7 Ma (7.3-14.3 Ma, 95% HPD), suggesting that Anacanthotermes dispersed to Asia using the land bridge connecting Africa and Eurasia [~]18-20 Ma. In contrast, the common ancestors of modern Porotermes and Stolotermes lived 20.2 Ma (15.7-25.1 Ma, 95% HPD) and 26.6 Ma (18.3-35.6 Ma, 95% HPD), respectively, indicating that the presence of these genera in South America, Africa, and Australia involved over-water dispersals. Our results suggest that early diverging termite lineages acquired their current distribution through a combination of over-water dispersals and dispersal via land bridges. We clarify the classification by resolving the paraphyly of Archotermopsidae, restricting the family to Archotermopsis and Zootermopsis, and elevating Hodotermopsinae (Hodotermopsis) as Hodotermopsidae (status novum).

evolutionary biology↗

bioRxiv2021

The Slit-binding Ig1 domain is required for multiple axon guidance activities of Drosophila Robo2

Drosophila Robo2 is a member of the evolutionarily conserved Roundabout (Robo) family of axon guidance receptors. The canonical role of Robo receptors is to signal midline repulsion in response to their cognate Slit ligands, which bind to the N-terminal Ig1 domain in most Robo family members. In the Drosophila embryonic ventral nerve cord, Robo1 and Robo2 cooperate to signal Slit-dependent midline repulsion, while Robo2 also regulates the medial-lateral position of longitudinal axon pathways and acts non-autonomously to promote midline crossing of commissural axons. Although it is clear that Robo2 signals midline repulsion in response to Slit, it is less clear whether Robo2s other activities are also Slit-dependent. To determine which of Robo2s axon guidance roles depend on its Slit-binding Ig1 domain, we have used a CRISPR/Cas9-based strategy replace the endogenous robo2 gene with a robo2 variant from which the Ig1 domain has been deleted (robo2{Delta}Ig1). We compare the expression and localization of Robo2{Delta}Ig1 protein with that of full-length Robo2 in embryonic neurons in vivo, and examine its ability to substitute for Robo2 to mediate midline repulsion and lateral axon pathway formation. We find that removal of the Ig1 domain from Robo2{Delta}Ig1 disrupts both of these axon guidance activities. In addition, we find that the Ig1 domain of Robo2 is required for its proper subcellular localization in embryonic neurons, a role that is not shared by the Ig1 domain of Robo1. Finally, we report that although FasII-positive lateral axons are misguided in embryos expressing Robo2{Delta}Ig1, the axons that normally express Robo2 are correctly guided to the lateral zone, suggesting that Robo2 may guide lateral longitudinal axons through a cell non-autonomous mechanism.

developmental biology↗

bioRxiv2020

Conserved and divergent aspects of Robo receptor signaling and regulation between Drosophila Robo1 and C. elegans SAX-3

The evolutionarily conserved Roundabout (Robo) family of axon guidance receptors control midline crossing of axons in response to the midline repellant ligand Slit in bilaterian animals including insects, nematodes, and vertebrates. Despite this strong evolutionary conservation, it is unclear whether the signaling mechanism(s) downstream of Robo receptors are similarly conserved. To directly compare midline repulsive signaling in Robo family members from different species, here we use a transgenic approach to express the Robo family receptor SAX-3 from the nematode Caenorhabditis elegans in neurons of the fruit fly, Drosophila melanogaster. We examine SAX-3s ability to repel Drosophila axons from the Slit-expressing midline in gain of function assays, and test SAX-3s ability to substitute for Drosophila Robo1 during fly embryonic development in genetic rescue experiments. We show that C. elegans SAX-3 is properly translated and localized to neuronal axons when expressed in the Drosophila embryonic CNS, and that SAX-3 can signal midline repulsion in Drosophila embryonic neurons, although not as efficiently as Drosophila Robo1. Using a series of Robo1/SAX-3 chimeras, we show that the SAX-3 cytoplasmic domain can signal midline repulsion to the same extent as Robo1 when combined with the Robo1 ectodomain. We show that SAX-3 is not subject to endosomal sorting by the negative regulator Commissureless (Comm) in Drosophila neurons in vivo, and that peri-membrane and ectodomain sequences are both required for Comm sorting of Drosophila Robo1.

developmental biology↗

bioRxiv2020

Genome assembly of a Mesoamerica-derived Phaseolus lunatus cultivar enables the identification and comparative genomic analysis of a complex resistance locus

Lima bean, Phaseolus lunatus, is closely related to common bean and is high in fiber and protein, with a low glycemic index. Lima bean is widely grown in the state of Delaware, where late summer and early fall weather are conducive to pod production. The same weather conditions also promote diseases such as pod rot and downy mildew, the latter of which has caused previous epidemics. A better understanding of the genes underlying resistance to this and other pathogens is needed to keep this industry thriving in the region. Our current study sought to sequence, assemble and annotate a commercially available cultivar called Bridgeton, which could then serve as a reference genome, a basis of comparison to other Phaseolus taxa and a resource for identification of potential resistance genes. Combined efforts of sequencing, linkage and comparative mapping analysis resulted in a 625 Mb genome assembly, as well as a better understanding of a locus underlying resistance to the current downy mildew race in the field.

genomics↗

bioRxiv2020

Minimal structural elements required for midline repulsive signaling and regulation of Drosophila Robo1

The Roundabout (Robo) family of axon guidance receptors has a conserved ectodomain arrangement of five immunoglobulin-like (Ig) domains plus three fibronectin (Fn) repeats. Based on the strong evolutionary conservation of this domain structure among Robo receptors, as well as in vitro structural and domain-domain interaction studies of Robo family members, this ectodomain arrangement is predicted to be important for Robo receptor signaling in response to Slit ligands. Here, we define the minimal ectodomain structure required for Slit binding and midline repulsive signaling in vivo by Drosophila Robo1. We find that the majority of the Robo1 ectodomain is dispensable for both Slit binding and repulsive signaling. We show that a significant level of midline repulsive signaling activity is retained when all Robo1 ectodomain elements apart from Ig1 are deleted, and that the combination of Ig1 plus one additional ectodomain element (Ig2, Ig5, or Fn3) is sufficient to restore midline repulsion to wild type levels. Further, we find that deleting four out of five Robo1 Ig domains ({Delta}Ig2-5) does not affect negative regulation of Robo1 by Commissureless (Comm) or Robo2, while variants lacking all three fibronectin repeats ({Delta}Fn1-3 and {Delta}Ig2-Fn3) are insensitive to regulation by both Comm and Robo2, signifying a novel regulatory role for Robo1s Fn repeats. Our results provide an in vivo perspective on the importance of the conserved 5+3 ectodomain structure of Robo receptors, and suggest that specific biochemical properties and/or ectodomain structural conformations observed in vitro for domains other than Ig1 may have limited significance for in vivo signaling in the context of midline repulsion.

developmental biology↗