Biology subjects

Dingwall, H. L.

Publications and source records attributed to Dingwall, H. L..

4 recordsLinked to original sources

bioRxiv2024

Dynamic transcriptional and epigenetic changes define postnatal tendon growth

Tendons are dynamic structures that efficiently transmit forces and enable movement. From birth, tendons undergo dramatic changes from a principally cellular tissue to a hypocellular one characterized by a dense and highly ordered extracellular matrix. During this time, tendon cells change morphology from rounded to stellate in appearance and their proliferative rates decline. There is also significant expansion and maturation of the extracellular matrix (ECM) as tendons grow in length and diameter and alter their biomechanical properties to sustain increased physical activities. Surprisingly, for such an important stage of tendon maturation, we understand very little about the transcriptional and epigenetic regulators that direct these processes. Here, we present a roadmap of genes that are differentially regulated during the early neonatal and postnatal time period. We find differentially expressed genes fall into specific transcriptional modules, representing expression increases, decreases, or gene sets undergoing dynamic changes over postnatal time. By pairing our transcriptomic data with epigenetic data, we were able to perform an integrative analysis of the datasets and further define modules with highly correlated changes in gene expression and chromatin accessibility. From this analysis, several new pathways emerge. Among them, we focus on Yap1, a transcriptional co-activator of the Hippo signaling pathway. We observe accessible regions near to differentially expressed genes, containing motifs for TEAD, the transcription factor that binds Yap to regulate transcription. Conditional loss of Yap1 at postnatal stages results in altered expression of Col1a1 and disrupted matrix organization and density, suggesting that Yap is important for refining tendon ECM maturation. Together, our analyses identify a regulator of matrix maturation and provides a rich dataset with which to interrogate transcriptional networks and pathways during this poorly understood time in tendon growth.

developmental biology↗

bioRxiv2023

A transient dermal niche and dual epidermal programs underlie sweat gland development

Eccrine glands are mammalian skin appendages indispensable for human thermoregulation. Like all skin-derived appendages, eccrine glands form from multipotent progenitors in the basal skin epidermis. It remains unclear how epidermal progenitors progressively specialize to specifically form eccrine glands, precluding efforts to regenerate these vital organs. Herein, we applied single nucleus transcriptomics to compare the expression content of wildtype, eccrine-forming mouse skin to that of mice harboring a skin-specific disruption of Engrailed 1 (En1), a transcription factor that promotes the formation of eccrine glands in both humans and mice. We identify two concurrent epidermal transcriptomes in the earliest eccrine anlagen: a predominant transcriptome that is shared with hair follicles, and a vastly underrepresented transcriptome that is En1-dependent and eccrine-specific. We demonstrate that differentiation of the eccrine anlage requires the induction of a transient and transcriptionally unique dermal niche that forms around each developing gland in humans and mice. Our study defines the transcriptional determinants underlying eccrine identity in the epidermis and uncovers the dermal niche required for eccrine developmental progression. By identifying these defining components of the eccrine developmental program, our findings set the stage for directed efforts to regenerate eccrine glands for comprehensive skin repair.

developmental biology↗

bioRxiv2022

Differential modularity of the mammalian Engrailed 1 enhancer network directs eccrine sweat gland development

Enhancers are context-specific regulators of expression that drive biological complexity and variation through the redeployment of conserved genes. An example of this is the enhancer-mediated control of the transcription of Engrailed 1 (EN1), a pleiotropic gene whose expression is required for the specification of eccrine sweat glands in mammals. We have previously identified an enhancer, ECE18 that has been highly and repeatedly derived on the human lineage to potentiate ectodermal EN1 and induce our species uniquely high eccrine gland density. Intriguingly, ECE18 quantitative activity is negligible outside of primates and ECE18 is not required for En1 regulation and eccrine gland specification in mice, raising the possibility that distinct enhancers have evolved to modulate the same trait. Here we report the identification of the ECE20 enhancer and show it has conserved functionality in mouse and human developing skin ectoderm. Unlike ECE18, knock-out of ECE20 in mice reduces ectodermal En1 and eccrine gland number. Notably, we find ECE20, but not ECE18, is also required for En1 expression in the embryonic mouse brain, demonstrating that ECE20 is a pleiotropic En1 enhancer. Finally, that ECE18 deletion does not potentiate the eccrine phenotype of ECE20 knockout mice supports the secondary incorporation of ECE18 into the regulation of this trait in primates. Our findings reveal that the mammalian En1 regulatory machinery diversified to incorporate both shared and lineage-restricted enhancers to regulate the same phenotype, and also have implications for understanding the forces that shape the robustness and evolvability of developmental traits.

evolutionary biology↗

bioRxiv2018

A robust method for RNA extraction and purification from a single adult mouse tendon

BackgroundMechanistic understanding of tendon molecular and cellular biology is crucial towards furthering our abilities to design new therapies for tendon and ligament injuries and disease. Recent transcriptomic and epigenomic studies in the field have harnessed the power of mouse genetics to reveal new insights into tendon biology. However, many mouse studies pool tendon tissues or use amplification methods to perform RNA analysis, which can significantly increase the experimental costs and limit the ability to detect changes in expression of low copy transcripts.\n\nMethodsSingle Achilles tendons were harvested from uninjured, contralateral injured, and wild type mice between 3-5 months of age, and RNA was extracted. RNA Integrity Number (RIN) and concentration were determined, and RT-qPCR gene expression analysis was performed.\n\nResultsAfter testing several RNA extraction approaches on single adult mouse Achilles tendons, we developed a protocol that was successful at obtaining high RIN and sufficient concentrations suitable for RNA analysis. We found that the RNA quality was sensitive to the time between tendon harvest and homogenization, and the RNA quality and concentration was dependent on the duration of homogenization. Using this method, we demonstrate that analysis of Scx gene expression in single mouse tendons reduces the biological variation caused by pooling tendons from multiple mice. We also show successful use of this approach to analyze Sox9 and Col1a2 gene expression changes in injured compared with uninjured control tendons.\n\nDiscussionOur work presents a robust, cost-effective, and straightforward method to extract high quality RNA from a single adult mouse Achilles tendon at sufficient amounts for RNA-seq and RT-qPCR. We show this can reduce biological variation and decrease the overall costs associated with experiments. This approach can also be applied to other skeletal tissues as well as precious human samples.

molecular biology↗