Biology subjects

Square, T. A.

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

9 recordsLinked to original sources

bioRxiv2025

Ectodysplasin overexpression reveals spatiotemporally dynamic tooth formation competency in stickleback and zebrafish

Organ initiation is often driven by extracellular signals that activate precursor cells competent to receive and respond to the signal, yet little is known about how dynamic competency is in space and time during development. Teeth are excellent organs to study organ initiation competency because they can be activated with the addition of a single signaling ligand, Ectodysplasin (Eda). Eda, a Tumor Necrosis Factor (TNF) ligand, is a critical regulator of ectodermal organ development, including teeth, acting through TNF receptors, like Edar, to activate NF-{kappa}B signaling in tooth precursor cells. While Eda is both necessary for normal tooth formation and sufficient for ectopic tooth formation, the spatial and temporal dynamics of competency for ectopic tooth initiation and maintenance remains unknown. To investigate the role of Eda in tooth specification, we generated transgenic sticklebacks and zebrafish with heat shock-inducible Eda overexpression. We find that stickleback Eda can drive de novo tooth formation in at least eight distinct oropharyngeal and cranial domains. Both zebrafish and stickleback exhibit maximal responsiveness to Eda overexpression during the critical window of pioneer tooth initiation, highlighting that the precursors of ectopic pharyngeal teeth likely follow a similar developmental trajectory as endogenous tooth precursors. Furthermore, we observe that some induced dental fields often undergo tooth regeneration to maintain themselves, allowing them to persist for months after the cessation of transgene activation. Finally, analysis of TNF receptor expression in sticklebacks reveals that ectopic tooth formation in the pharynx correlates with Edar and Troy expression, while in the region where teeth can form on the face, only Troy expression was detected, providing a possible molecular mechanism of competency involving spatially restricted receptor expression. These findings underscore the latent developmental potential, i.e. competency, of the vertebrate dentition and provide insights into organ competency during embryonic and post-embryonic development.

developmental biology↗

bioRxiv2022

Modulation of tooth regeneration through opposing responses to Wnt and BMP signals in teleosts

Most vertebrate species undergo tooth replacement throughout adult life. This process is marked by the shedding of existing teeth and the regeneration of tooth organs. However, little is known about the genetic circuitry regulating tooth replacement. Here we tested whether fish orthologs of genes known to regulate mammalian hair regeneration have effects on tooth replacement. Using two fish species that demonstrate distinct modes of tooth regeneration, threespine stickleback (Gasterosteus aculeatus) and zebrafish (Danio rerio), we found that transgenic overexpression of four different genes changed tooth replacement rates in direction predicted by a hair regeneration model: Wnt10a and Grem2a increased tooth replacement rate, while Bmp6 and Dkk2 strongly inhibited tooth formation. Thus, similar to known roles in hair regeneration, Wnt and BMP signals promote and inhibit regeneration, respectively. Regulation of total tooth number was separable from regulation of replacement rates. RNA-seq on stickleback dental tissue showed that Bmp6 overexpression resulted in an upregulation of Wnt inhibitors. Together these data support a model where different epithelial organs like teeth and hair share genetic circuitry driving organ regeneration.

developmental biology↗

bioRxiv2022

Evolution of spatial and temporal cis-regulatory divergence between marine and freshwater sticklebacks

Cis-regulatory changes are thought to play a major role in adaptation. Threespine sticklebacks have repeatedly colonized freshwater habitats in the Northern Hemisphere, where they have evolved a suite of phenotypes that distinguish them from marine populations, including changes in physiology, behavior, and morphology. To understand the role of gene regulatory evolution in adaptive divergence, here we investigate cis-regulatory changes in gene expression between marine and freshwater ecotypes through allele-specific expression (ASE) in F1 hybrids. Surveying seven ecologically relevant tissues, including three sampled across two developmental stages, we identified cis-regulatory divergence affecting a third of genes, nearly half of which were tissue-specific. Next, we compared allele-specific expression in dental tissues at two timepoints to characterize cis-regulatory changes during development between marine and freshwater fish. Applying a genome-wide test for selection on cis-regulatory changes, we find evidence for lineage-specific selection on several processes, including the Wnt signaling pathway in dental tissues. Finally, we show that genes with ASE, particularly those that are tissue-specific, are enriched in genomic regions associated with marine-freshwater divergence, supporting an important role for cis-regulatory differences in adaptive evolution of sticklebacks. Altogether, our results provide insight into the cis-regulatory landscape of divergence between stickleback ecotypes and supports a fundamental role for cis-regulatory changes in rapid adaptation to new environments.

evolutionary biology↗

bioRxiv2022

Site pleiotropy of a stickleback tooth and fin enhancer

Development and regeneration are orchestrated by gene regulatory networks that operate in part through transcriptional enhancers. Although many enhancers are pleiotropic and are active in multiple tissues, little is known about whether enhancer pleiotropy is due to 1) site pleiotropy, in which individual transcription factor binding sites (TFBS) are required for activity in multiple tissues, or 2) multiple distinct sites that regulate expression in different tissues. Here, we investigated the pleiotropy of an intronic enhancer of the stickleback Bone morphogenetic protein 6 (Bmp6) gene. This enhancer was previously shown to regulate evolved changes in tooth number and tooth regeneration, and is highly pleiotropic, with robust activity in both fins and teeth throughout embryonic, larval, and adult life. We tested the hypothesis that the pleiotropy of this enhancer is due to site pleiotropy of an evolutionarily conserved predicted Foxc1 TFBS. Transgenic analysis and site-directed mutagenesis experiments both deleting and scrambling this predicted Foxc1 TFBS revealed that the binding site is required for enhancer activity in both teeth and fins throughout embryonic, larval, and adult development. Collectively these data support a model where the pleiotropy of this Bmp6 enhancer is due to site pleiotropy and this putative binding site is required for enhancer activity in multiple anatomical sites from the embryo to the adult.

developmental biology↗

bioRxiv2021

Evolved Bmp6 enhancer alleles drive spatial shifts in gene expression during tooth development in sticklebacks

Mutations in enhancers have been shown to often underlie natural variation but the evolved differences between enhancer activity can be difficult to identify in vivo. Threespine sticklebacks (Gasterosteus aculeatus) are a robust system for studying enhancer evolution due to abundant natural genetic variation, a diversity of evolved phenotypes between ancestral marine and derived freshwater forms, and the tractability of transgenic techniques. Previous work identified a series of polymorphisms within an intronic enhancer of the Bone morphogenetic protein 6 (Bmp6) gene that are associated with evolved tooth gain, a derived increase in freshwater tooth number that arises late in development. Here we use a bicistronic reporter construct containing a genetic insulator and a pair of reciprocal two-color transgenic reporter lines to compare enhancer activity of marine and freshwater alleles of this enhancer. In older fish the two alleles drive partially overlapping expression in both mesenchyme and epithelium of developing teeth, but the freshwater enhancer drives a reduced mesenchymal domain and a larger epithelial domain relative to the marine enhancer. In younger fish these spatial shifts in enhancer activity are less pronounced. Comparing Bmp6 expression by in situ hybridization in developing teeth of marine and freshwater fish reveals similar evolved spatial shifts in gene expression. Together, these data support a model in which the polymorphisms within this enhancer underlie evolved tooth gain by shifting the spatial expression of Bmp6 during tooth development, and provide a general strategy to identify spatial differences in enhancer activity in vivo.

genetics↗

bioRxiv2020

Lamprey Lecticans Link New Vertebrate Genes to the Origin and Elaboration of Vertebrate Tissues

The evolution of vertebrates from an invertebrate chordate ancestor involved the evolution of new organs, tissues, and cell types. It was also marked by the origin and duplication of new gene families. If, and how, these morphological and genetic innovations are related is an unresolved question in vertebrate evolution. Hyaluronan is an extracellular matrix (ECM) polysaccharide important for water homeostasis and tissue structure. Vertebrates possess a novel family of hyaluronan binding proteins called Lecticans, and studies in jawed vertebrates (gnathostomes) have shown they function in many of the cells and tissues that are unique to vertebrates. This raises the possibility that the origin and/or expansion of this gene family helped drive the evolution of these vertebrate novelties. In order to better understand the evolution of the lectican gene family, and its role in the evolution of vertebrate morphological novelties, we investigated the phylogeny, genomic arrangement, and expression patterns of all lecticans in the sea lamprey (Petromyzon marinus), a jawless vertebrate. Though both P. marinus and gnathostomes have four lecticans, our phylogenetic and syntenic analyses suggest lamprey lecticans are the result of one or more cyclostome-specific duplications. Despite the independent expansion of the lamprey and gnathostome lectican families, we find highly conserved expression of lecticans in vertebrate-specific and mesenchyme-derived tissues. We also find that, unlike gnathostomes, lamprey expresses its lectican paralogs in distinct subpopulations of head skeleton precursors, potentially reflecting an ancestral diversity of skeletal tissue types. Together, these observations suggest that the ancestral pre-duplication lectican had a complex expression pattern, functioned to support mesenchymal histology, and likely played a role in the evolution of vertebrate-specific cell and tissue types.

evolutionary biology↗

bioRxiv2020

Distinct tooth regeneration systems deploy a conserved battery of genes

BackgroundVertebrate teeth exhibit a wide range of regenerative systems. Many species, including most mammals, reptiles, and amphibians, form replacement teeth at a histologically distinct location called the successional dental lamina, while other species do not employ such a system. Notably, a lamina-less tooth replacement condition is found in a paraphyletic array of ray-finned fishes, such as stickleback, trout, cod, medaka, and bichir. Furthermore, the position, renewal potential, and latency times appear to vary drastically across different vertebrate tooth regeneration systems. The progenitor cells underlying tooth regeneration thus present highly divergent arrangements and potentials. Given the spectrum of regeneration systems present in vertebrates, it is unclear if morphologically divergent tooth regeneration systems deploy an overlapping battery of genes in their naive dental tissues. ResultsIn the present work, we aimed to determine whether or not tooth progenitor epithelia could be composed of a conserved cell type between vertebrate dentitions with divergent regeneration systems. To address this question, we compared the tooth regeneration processes in two ray-finned fishes: zebrafish (Danio rerio) and threespine stickleback (Gasterosteus aculeatus). These two teleost species diverged approximately 250 million years ago, and demonstrate some stark differences in dental morphology and regeneration. Here we find that the successional dental lamina in zebrafish sharply upregulates Wnt signaling and Lef1 expression during early morphogenesis stages of tooth development. Additionally, the naive zebrafish successional dental lamina expresses a battery of nine genes (Bmpr1a, Bmp6, CD34, Gli1, Igfbp5a, Lgr4, Lgr6, Nfatc1, and Pitx2). We also find that, despite the absence of a histologically distinct successional dental lamina in stickleback tooth fields, new tooth germs also sharply upregulate Wnt signaling and Lef1 expression, and additionally express the same battery of nine genes in the basalmost endodermal cell layer from which replacement tooth epithelia arise. Thus, two fish systems that either have an organized successional dental lamina (zebrafish) or lack a morphologically distinct successional dental lamina (sticklebacks) deploy similar genetic programs during tooth regeneration. ConclusionsWe propose that the expression domains described here delineate a highly conserved "successional dental epithelium" (SDE). Furthermore, a set of orthologous genes is known to mark hair follicle epithelial stem cells in mice, suggesting that regenerative systems in other epithelial appendages may utilize a related epithelial progenitor cell type, despite the highly derived nature of the resulting functional organs.

developmental biology↗

bioRxiv2020

Evolution of vertebrate gill covers via shifts in an ancient Pou3f3 enhancer

Whereas the gill chambers of extant jawless vertebrates (lampreys and hagfish) open directly into the environment, jawed vertebrates evolved skeletal appendages that promote the unidirectional flow of oxygenated water over the gills. A major anatomical difference between the two jawed vertebrate lineages is the presence of a single large gill cover in bony fishes versus separate covers for each gill chamber in cartilaginous fishes. Here we find that these divergent gill cover patterns correlate with the pharyngeal arch expression of Pou3f3 orthologs. We identify a Pou3f3 arch enhancer that is deeply conserved from cartilaginous fish through humans but undetectable in lampreys, with minor sequence differences in the bony versus cartilaginous fish enhancers driving the corresponding single versus multiple gill arch expression patterns. In zebrafish, loss of Pou3f3 gene function disrupts gill cover formation, and forced expression of Pou3f3b in the gill arches generates ectopic skeletal elements resembling the multiple gill cover pattern of cartilaginous fishes. Emergence of this Pou3f3 enhancer >430 mya and subsequent modifications may thus have contributed to the acquisition and diversification of gill covers and respiratory strategies during gnathostome evolution.

developmental biology↗

bioRxiv2019

Cooption and Specialization of Endothelin Signaling Pathways Drove Elaboration of the Neural Crest in Early Vertebrates

The neural crest (NC) is a vertebrate-specific embryonic tissue that forms an array of clade-defining adult features. A key step in the formation of these diverse derivatives is the partitioning of NC cells into subpopulations with distinct migration routes and potencies1. The evolution of these developmental modules is poorly understood. Endothelin (Edn) signaling is unique to vertebrates, and performs various functions in different NC subpopulations2-5. To better understand the evolution of NC patterning, we used CRISPR/Cas9-driven mutagenesis to disrupt Edn receptors, ligands, and Dlx transcription factors in the sea lamprey, Petromyzon marinus. Lampreys and modern gnathostomes last shared a common ancestor 500 million years ago6. Thus, comparisons between the two groups can identify deeply conserved and divergent features of vertebrate development. Using Xenopus laevis to facilitate side-by-side analyses, we show here that lamprey and gnathostomes display fundamental differences in Edn signaling function. Unlike gnathostomes, both lamprey Ednrs cooperate during oropharyngeal skeleton development. Furthermore, neither paralog regulates hand transcription factors, which are required for mandible development in gnathostomes. We also identify conserved roles for Edn signaling in dlx gene regulation, pigment cell, and heart development. Together our results illustrate the stepwise neofunctionalization and specialization of this vertebrate-specific signaling pathway, and suggest key intermediate stages in the early evolution of the NC.

developmental biology↗