Biology subjects

Benthal, J. T.

Publications and source records attributed to Benthal, J. T..

5 recordsLinked to original sources

bioRxiv2025

Combinatorial multiomic analysis from a pedigree of Sox10Dom Hirschsprung mice implicates Dach1 as a modifier of Enteric Nervous System development

BackgroundHirschsprung disease (HSCR) is characterized by absence of enteric ganglia (aganglionosis) along variable lengths of the distal intestine. This disorder results from deficient colonization of fetal intestine by enteric neural crest-derived cells (ENCDCs). HSCR exhibits complex, multifactorial inheritance with penetrance and severity varying widely even within a family. SOX10 is among causal genes that predispose to aganglionosis. Yet, how gene interactions influence severity of HSCR aganglionosis is not understood. We previously used an F1-intercross strategy to map genetic modifiers of aganglionosis in the Sox10Dom HSCR mouse. Here we employ an extended pedigree mapping approach and complementary omics analyses of the developing Enteric Nervous System (ENS) to identify modifier loci that affect severity of HSCR aganglionosis. ResultsTo identify loci that modify aganglionosis extent we undertook genome-wide association study (GWAS) of an extended pedigree of Sox10Dommice on a mixed C57BL/6J x C3HeBFeJLe-a/a background. GWAS uncovered genetic modifiers of aganglionosis severity in this cohort. Within each modifier interval, we prioritize candidate genes based on gene expression in the developing ENS, proximity to open chromatin regions in ENCDCs, and presence of conserved SOX10 binding motifs. This strategy identified known genes in ENS development as well as multiple novel genes. Dach1 emerged as a top priority gene for modifying ENCDCs migration and thus influencing aganglionosis severity. ConclusionsThis study identifies genome intervals and intrinsic genes that modify Sox10Dom aganglionosis severity and that are candidate modifiers of human HSCR severity. AUTHOR SUMMARYHirschsprung disease is a complex genetic neurodevelopmental disorder that causes loss of neurons in the distal bowel. The length of gut lacking neurons, called "aganglionosis", in HSCR patients can vary widely even between affected siblings. Multiple genes are Mendelian causative for HSCR, but little is known about the gene interactions responsible for the notable variation in aganglionosis severity. In this study, we use a mouse model of HSCR to identify genomic regions, "modifiers", associated with length of aganglionosis. Genes active within these regions are then identified in RNA-sequencing and open chromatin data from progenitor cells, which form the enteric nervous system. These omics approaches identify both known and novel genes that can affect enteric neuron development and may underly HSCR severity in patients. Dach1, already known for effects on neuronal progenitor proliferation and migration in other aspects of the nervous system, emerged as the top priority gene. The findings greatly expand the gene network that influences HSCR aganglionosis.

genomics↗

bioRxiv2024

Meta-atlas of Juvenile and Adult Enteric Neuron scRNA-seq for Dataset Comparisons and Consensus on Transcriptomic Definitions of Enteric Neuron Subtypes

BackgroundThe enteric nervous system (ENS) is a complex network of interconnected ganglia within the gastrointestinal (GI) tract. Among its diverse functions, the ENS detects bowel luminal contents and coordinates the passing of stool. ENS defects predispose to GI motility disorders. Previously, distinct enteric neuron types were cataloged by dye-filling techniques, immunohistochemistry, retrograde labeling, and electrophysiology. Recent technical advances in single cell RNA-sequencing (scRNA-seq) have enabled transcriptional profiling of hundreds to millions of individual cells from the intestine. These data allow cell types to be resolved and compared to using their transcriptional profiles ("clusters") rather than relying on antibody labeling. As a result, greater diversity of enteric neuron types has been appreciated. Because each scRNA-seq study has relied on different methods for cell isolation and library generation, numbers of neuron clusters and cell types detected differs between analyses. Cell counts in each dataset are particularly important for characterization of rare cell types since small numbers of profiled cells may not sample rare cell types. Importantly, each dataset, depending on the isolation methods, may contain different proportions of cells that are not detected in other datasets. Aggregation of datasets can effectively increase the total number of cells being analyzed and can be helpful for confirming the presence of low-abundance neuron types that might be absent or observed infrequently in any single dataset. ResultsHere we briefly systematically review each Mus musculus single cell or single nucleus RNA-sequencing enteric nervous system dataset. We then reprocess and computationally integrate these select independent scRNA-seq enteric neuron datasets with the aim to identify new cell types, shared marker genes across juvenile to adult ages, dataset differences, and achieve some consensus on transcriptomic definitions of enteric neuronal subtypes. ConclusionsData aggregation generates a consensus view of enteric neuron types and improves resolution of rare neuron classes. This meta-atlas offers a deeper understanding of enteric neuron diversity and may prove useful to investigators aiming to define alterations among enteric neurons in disease states. Future studies face the challenge of connecting these deep transcriptional profiles for enteric neurons with historical classification systems.

neuroscience↗

bioRxiv2024

Single Cell Profiling in the Sox10Dom/+ Hirschsprung Mouse Implicates Hoxa6 in Enteric Neuron Lineage Allocation

Background & AimsEnteric nervous system (ENS) development requires migration, proliferation, and appropriate neuronal diversification from progenitors to enable normal gastrointestinal (GI) motility. Sox10 deficit causes aganglionosis, modeling Hirschsprung disease, and disrupts ratios of postnatal enteric neurons in proximal ganglionated bowel. How Sox10 deficiency alters ratios of enteric neuron subtypes is unclear. Sox10s prominent expression in enteric neural crest-derived progenitors (ENCP) and lack of this gene in enteric neurons led us to examine Sox10Dom effects ENS progenitors and early differentiating enteric neurons. MethodsENS progenitors, developing neurons, and enteric glia were isolated from Sox10+/+ and Sox10Dom/+ littermates for single-cell RNA sequencing (scRNA-seq). scRNA-seq data was processed to identify cell type-specific markers, differentially expressed genes, cell fate trajectories, and gene regulatory network activity between genotypes. Hybridization chain reaction (HCR) validated expression changes detected in scRNA-seq. ResultsscRNA-seq profiles revealed three neuronal lineages emerging from cycling progenitors via two transition pathways accompanied by elevated activity of Hox gene regulatory networks (GRN) as progenitors transition to neuronal fates. Sox10Dom/+ scRNA-seq profiles exhibited a novel progenitor cluster, decreased abundance of cells in transitional states, and shifts in cell distributions between two neuronal trajectories. Hoxa6 was differentially expressed in the neuronal lineages impacted in Sox10Dom/+mutants and HCR identified altered Hoxa6 expression in early developing neurons of Sox10Dom/+ ENS. ConclusionsSox10Dom/+ mutation shifts enteric neuron types by altering neuronal trajectories during early ENS lineage segregation. Multiple neurogenic transcription factors are reduced in Sox10Dom/+ scRNA-seq profiles including multiple Hox genes. This is the first report that implicates Hox genes in lineage diversification of enteric neurons.

developmental biology↗

bioRxiv2020

Combinatorial transcriptional profiling of mouse and human enteric neurons identifies shared and disparate subtypes in situ.

AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSBACKGROUND & AIMSC_ST_ABSThe enteric nervous system (ENS) coordinates essential intestinal functions through the concerted action of diverse enteric neurons (EN). However, integrated molecular knowledge of EN subtypes is lacking. To compare human and mouse ENs, we transcriptionally profiled healthy ENS from adult humans and mice. We aimed to identify transcripts marking discrete neuron subtypes and visualize conserved EN subtypes for humans and mice in multiple bowel regions. METHODSHuman myenteric ganglia and adjacent smooth muscle were isolated by laser-capture microdissection for RNA-Seq. Ganglia-specific transcriptional profiles were identified by computationally subtracting muscle gene signatures. Nuclei from mouse myenteric neurons were isolated and subjected to single-nucleus RNA-Seq (snRNA-Seq), totaling over four billion reads and 25,208 neurons. Neuronal subtypes were defined using mouse snRNA-Seq data. Comparative informatics between human and mouse datasets identified shared EN subtype markers, which were visualized in situ using hybridization chain reaction (HCR). RESULTSSeveral EN subtypes in the duodenum, ileum, and colon are conserved between humans and mice based on orthologous gene expression. However, some EN subtype-specific genes from mice are expressed in completely distinct morphologically defined subtypes in humans. In mice, we identified several neuronal subtypes that stably express gene modules across all intestinal segments, with graded, regional expression of one or more marker genes. CONCLUSIONSOur combined transcriptional profiling of human myenteric ganglia and mouse EN provides a rich foundation for developing novel intestinal therapeutics. There is congruency among some EN subtypes, but we note multiple species differences that should be carefully considered when relating findings from mouse ENS research to human GI studies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/187211v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1b41277org.highwire.dtl.DTLVardef@12856feorg.highwire.dtl.DTLVardef@1674dadorg.highwire.dtl.DTLVardef@11ca1c8_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

neuroscience↗

bioRxiv2017

Genetic Identification of Novel Separase regulators in Caenorhabditis elegans

Separase is a highly conserved protease required for chromosome segregation. Although observations that separase also regulates membrane trafficking events have been made, it is still not clear how separase achieves this function. Here we present an extensive ENU mutagenesis suppressor screen aimed at identifying suppressors of sep-1(e2406), a temperature sensitive maternal effect embryonic lethal separase mutant. We screened nearly a million haploid genomes, and isolated sixty-eight suppressed lines. We identified fourteen independent intragenic sep-1(e2406) suppressed lines. These intragenic alleles map to seven SEP-1 residues within the N-terminus, compensating for the original mutation within the poorly conserved N-terminal domain. Interestingly, 47 of the suppressed lines have novel mutations throughout the entire coding region of the pph-5 phosphatase, indicating that this is an important regulator of separase. We also found that a mutation near the MEEVD motif of HSP-90, which binds and activates PPH-5, also rescues sep-1(e2406) mutants. Finally, we identified six potentially novel suppressor lines that fall into five complementation groups. These new alleles provide the opportunity to more exhaustively investigate the regulation and function of separase.

genetics↗