Biology subjects

Martin, D. M.

Publications and source records attributed to Martin, D. M..

5 recordsLinked to original sources

bioRxiv2024

NOTCH1 S2513 is critical for the regulation of NICD levels impacting the segmentation clock in hiPSC-derived PSM cells and somitoids

The segmentation clock is a molecular oscillator that regulates the timing of somite formation in the developing vertebrate embryo. NOTCH signalling is one of the key pathways required for proper functioning of the segmentation clock. Aberrant NOTCH signalling results in developmental abnormalities such as congenital scoliosis as well as diseases such as T-cell acute lymphoblastic lymphoma (T-ALL). In this study we analyse the effects of a mutation detected in T-ALL patients on somitogenesis using human iPS derived PSM cells and somitoids. Mutation of NOTCH1 Serine 2513 into Alanine compromises the interaction of Notch intracellular domain (NICD) with the F-box protein FBXW7 and consequently increases NICD stability and NICD levels in PSM cells. Moreover, the mutation impairs several aspects of clock gene oscillations and restricts the ability of somitoids to polarise, elongate and form paired somites. The data suggest a mechanism by which post-translational modification of a key segmentation clock component plays a crucial role in vertebrate axis segmentation.

developmental biology↗

bioRxiv2022

Regulation of cellular LDL uptake by PROX1 and CHD7

An elevated level of low-density lipoprotein (LDL) in the bloodstream is a causal risk factor for atherosclerotic cardiovascular disease (ASCVD). The low-density lipoprotein receptor (LDLR) is a critical regulator of circulating LDL, and increasing LDLR activity is an effective therapeutic approach to reduce circulating LDL cholesterol levels. In this study, we characterize PROX1 and CHD7, two genes we previously identified in a genome-scale CRISPR screen as positive regulators of LDL uptake in HuH7 cells. We found that although disruption of either PROX1 or CHD7 significantly reduced LDL uptake, only PROX1 disruption significantly reduced the cellular levels of LDLR mRNA and surface-displayed LDLR protein. Consistent with a direct role for PROX1 in LDLR gene regulation, we also observed in publicly available data sets the presence of two liver-specific PROX1 binding sites near the LDLR locus, one of which colocalized with biochemical hallmarks of enhancer activity in hepatic tissue. Both PROX1 LDLR binding sites contained predicted PROX1 transcription factor binding motifs and colocalized with binding sites for HNF4, a known interactor for PROX1 and regulator of hepatic lipid metabolism and LDL uptake. In contrast to PROX1, no CHD7 binding sites were detected near the LDLR locus. Together, our results support a model in which both PROX1 and CHD7 promote cellular LDL uptake through distinct mechanisms, with PROX1 directly promoting LDLR gene expression and CHD7 functioning through an LDLR-independent pathway.

molecular biology↗

bioRxiv2022

Aberrant extracellular matrix and cardiac development in models lacking the PR-DUB component ASXL3

BackgroundClinical and research based genetic testing has uncovered genes that encode chromatin modifying complex components required for organogenesis. Covalent histone modifications play a key role in establishing transcriptional plasticity during development, required for cell fate specification, and have been implicated as a developmental mechanism that accounts for autism spectrum disorder (ASD) and CHD co-occurrence. ASXL3 has been identified as a high confidence ASD gene. ASXL3 is a component of the Polycomb Repressive Deubiquitination (PR-DUB) complex, which deubiquitinates histone H2A. However, the role of ASXL3 in cardiac development remains unknown. MethodsWe used CRISPR/Cas9 gene editing to generate clinically relevant Asxl3 frameshift alleles in a mouse model and human embryonic stem cells (hESCs). To evaluate ASXL3 function in developing hearts, we performed structural, molecular, immunostaining and histological analyses. Transcriptomic and cellular compositional changes were assessed with bulk RNA sequencing of mouse hearts and single-cell RNA sequencing (scRNA-seq) of human cardiac tissue differentiated from isogenic hESC lines. ResultsBiallelic genetic inactivation of Asxl3 leads to perinatal lethality and increased levels of histone H2A mono-ubiquitination, which are regulated by PR-DUB. Asxl3+/fs and Asxl3fs/fs mice display cardiac abnormalities including ventricular hypoplasia, septal defects, and bifid cardiac apex with variable penetrance. The presence of underdeveloped ventricles is preceded by increased progenitor proliferation in the ventricles, as determined by EdU incorporation. Differential gene expression, assessed by bulk RNA sequencing implicates extracellular matrix dysfunction as a pathogenic mechanism. This correlates with a reduction in vimentin-positive cardiac fibroblasts. scRNA-seq of cardiac cultures differentiated from human ASXL3fs/fs ESC lines exhibit altered ratios of cardiac fibroblasts and cardiomyocytes. Similar to the mouse data, genes essential for extracellular matrix composition and signaling are differentially expressed between ASXL3+/+ and ASXL3fs/fs human in vitro differentiated cardiac tissue. The observed transcriptomic changes predict diminished cell-cell signaling interactions between cardiac fibroblasts and cardiomyocyte progenitors in ASXL3 cultures. ConclusionsCollectively, our data implicates species-specific roles for ASXL3 in both human and mouse cardiac development. These results highlight the role of extracellular matrix gene programs by cardiac fibroblast during cardiomyocyte development and provide insight into mechanisms of altered cardiogenesis by autism risk genes.

developmental biology↗

bioRxiv2022

Chromatin remodeler Chd7 regulates photoreceptor development and outer segment length

Mutations in the chromatin remodeling factor CHD7 are the predominant cause of CHARGE syndrome, a congenital disorder that frequently includes ocular coloboma. Although CHD7 is known to be required for proper ocular morphogenesis, its role in retinal development has not been thoroughly investigated. Given that individuals with CHARGE syndrome can experience visual impairment even in the absence of coloboma, a better understanding of CHD7 function in the retina is needed. In this study, we characterized the expression pattern of Chd7 in the developing zebrafish and mouse retina and documented ocular and retinal phenotypes in Chd7 loss-of-function mutants. Zebrafish Chd7 was expressed throughout the retinal neuroepithelium when retinal progenitor cells were actively proliferating, and later in subsets of newly post-mitotic retinal cells. At stages of retinal development when most retinal cell types had terminally differentiated, Chd7 expression remained strong in the ganglion cell layer and in some cells in the inner nuclear layer. Intriguingly, strong expression of Chd7 was also observed in the outer nuclear layer where it was co-expressed with markers of post-mitotic cone and rod photoreceptors. Expression of mouse CHD7 displayed a similar pattern, including expression in the ganglion cells, subsets of inner nuclear layer cells, and in the distal outer nuclear layer as late as P15. Two different mutant chd7 zebrafish lines were characterized for ocular and retinal defects. These mutants displayed microphthalmia, reduced numbers of cone photoreceptors, and truncated rod and cone photoreceptor outer segments. Reduced cone photoreceptor number and abnormal outer segments were also observed in heterozygous Chd7 mutant mice. Taken together, our results in zebrafish and mouse reveal a conserved, previously undescribed role for Chd7 in retinal development and photoreceptor outer segment morphogenesis. Moreover, our work suggests an avenue of future investigation into the pathogenesis of visual system defects in CHARGE syndrome. HighlightsO_LIChd7 is expressed in both retinal progenitor cells and in differentiated retinal neurons, including post-mitotic rod and cone photoreceptors. C_LIO_LILoss of Chd7 results in a significant decrease in cone photoreceptors in both zebrafish and mouse. C_LIO_LICone and rod photoreceptor outer segments are truncated in chd7 mutants, suggesting a heretofore unappreciated role for Chd7 in outer segment morphogenesis. C_LI

developmental biology↗

bioRxiv2021

Single-cell lineage trajectories and chromatin regulators that initialize antiviral CD8 T cell ontogeny

Individual naive CD8 T cells activated in lymphoid organs differentiate into functionally diverse and anatomically distributed T cell phylogenies in response to intracellular microbes. During infections that resolve rapidly, including live viral vaccines1, distinct effector (TEFF) and memory (TMEM) cell populations develop that ensure long term immunity2. During chronic infections, responding cells progressively become dysfunctional and "exhaust"3. A diverse taxonomy of TEFF, TMEM and exhausted (TEX) CD8 T cell populations is known, but the initial developmental basis of this phenotypic variation remains unclear4-10. Here, we defined single-cell trajectories and identified chromatin regulators that establish antiviral CD8 T cell heterogeneity using unsupervised analyses of single-cell RNA dynamics11-13 and an in vivo RNAi screen14. Activated naive cells differentiate linearly into uncommitted effector-memory progenitor (EMP) cells, which initially branch into an analogous manifold during either acute or chronic infection. Disparate RNA velocities in single EMP cells initiate divergence into stem, circulating, and tissue-resident memory lineages that generate diverse TMEM and TEX precursor states in specific developmental orders. Interleukin-2 receptor (IL-2R) signals are essential for formation and transcriptional heterogeneity of EMP cells, and promote trajectories toward TEFF rather than TEX states. Nucleosome remodelers Smarca4 and Chd7 differentially promote transcription that delineates divergent TMEM lineages before cooperatively driving terminal TEFF cell differentiation. Thus, the lineage architecture is established by specific chromatin regulators that stabilize diverging transcription in uncommitted progenitors.

immunology↗