Biology subjects

Sanketi, B. D.

Publications and source records attributed to Sanketi, B. D..

3 recordsLinked to original sources

bioRxiv2023

Spatiotemporal reconstruction of the origin and assembly of smooth muscles in the intestinal villus

Intestinal smooth muscles are the workhorse of the digestive system. Inside the millions of finger-like intestinal projections called villi, strands of smooth muscle cells contract to propel absorbed dietary fats through the adjacent lymphatic vessel, called the lacteal, sending fats into the blood circulation for energy production. Despite this vital function, how villus smooth muscles form, how they assemble alongside lacteals, and how they repair throughout life remain unknown. Here we combine single-cell RNA sequencing of the mouse intestine with quantitative lineage tracing to reveal the mechanisms of formation and differentiation of villus smooth muscle cells. Within the highly regenerative villus, we uncover a local hierarchy of subepithelial fibroblast progenitors that progress to become mature smooth muscle fibers, via an intermediate contractile myofibroblast-like phenotype. This continuum persists in the adult intestine as the major source of renewal of villus smooth muscle cells during adult life. We further found that the NOTCH3-DLL4 signaling axis governs the assembly of villus smooth muscles alongside their adjacent lacteal, and we show that this is necessary for gut absorptive function. Overall, our data shed light on the genesis of a poorly defined class of intestinal smooth muscle and pave the way for new opportunities to accelerate recovery of digestive function by stimulating muscle repair.

developmental biology↗

bioRxiv2022

Inhibition of the ATM kinase rescues planarian regeneration after lethal radiation

As stem cells divide, they acquire mutations that can be passed on to daughter cells. To limit the possibility of propagating mutations, cells activate the DNA damage response (DDR) network, which dictates whether cells repair DNA or undergo apoptosis. At the helm of the DDR are three PI3-like kinases including Ataxia Telangiectasia Mutated (ATM). We report here that knockdown of ATM in planarian flatworms enables stem cells, which normally undergo apoptosis after radiation exposure, to survive lethal doses of radiation. In this context, stem cells circumvent apoptosis, replicate their DNA, and recover function using homologous recombination-mediated DNA repair. Despite radiation exposure, atm knockdown animals survive long-term and regenerate new tissues. These effects occur independently of ATMs canonical downstream effector p53. Together, our results demonstrate that ATMs primary function is to drive apoptosis, and suggest that inhibition of ATM could therefore potentially favor cell survival after radiation without adverse effects.

cell biology↗

bioRxiv2022

The Long Noncoding RNA Playrr Regulates Pitx2 Dosage and Protects Against Cardiac Arrhythmias

RationaleThe most significantly associated atrial fibrillation (AF) risk loci in humans map to a noncoding gene desert upstream of the evolutionarily conserved left-right (LR) transcription factor Pitx2, a master regulator of LR asymmetric organ development. Pitx2 dosage is fundamentally linked to the development of sinus node dysfunction (SND) and AF, the most common cardiac arrhythmia affecting adults, but the mechanistic basis for this remains obscure. We identified a conserved long noncoding RNA (lncRNA), Playrr, which is exclusively transcribed on the embryos right side, opposite to Pitx2 on the left, that participates in mutually antagonistic transcriptional regulation with Pitx2. ObjectiveThe objective of this study was to investigate a role of Playrr in regulating Pitx2 transcription and protecting against the development of cardiac rhythm disturbances. Methods and ResultsPlayrr expression in the developing heart was analyzed with RNA in situ hybridization. Playrr was expressed asymmetrically (on the right) to Pitx2 (on the left) in developing mouse embryos, including in mouse embryonic sinoatrial node cells. We utilized CRISPR/Cas9 genome editing in mice to target Playrr, generating mice lacking Playrr RNA transcript (PlayrrEx1sj allele). Using qRT-PCR we detected upregulation of the cardiac isoform, Pitx2c, during visceral organ morphogenesis in PlayrrEx1sj mutant embryos. Surface ECG (AliveCor(R)) and 24-hour telemetry ECG detected bradycardia and irregular interbeat (R-R) intervals suggestive of SND in PlayrrEx1sj mutant adults. Programmed stimulation of PlayrrEx1sj mutant adults resulted in pacing-induced AF. Within the right atrium of PlayrrEx1sj mutant hearts, Massons trichrome stain revealed increased collagen deposition indicative of fibrosis, and immunofluorescence demonstrated mis-localization of Connexin 43 in atrial cardiomyocytes. These findings suggested an altered atrial substrate in PlayrrEx1sj adult mice. Finally, transcriptomic analysis by chromatin run-on and sequencing (ChRO-seq) in atria of PlayrrEx1sj mutant mice compared to wild type controls revealed differential expression of genes involved in cell-cell adhesion and motility, fibrosis, and dysregulation of the key cardiac genes Tbx5 and Hcn1. ConclusionsAdult mice lacking functional Playrr lncRNA transcript have baseline bradyarrhythmia and increased susceptibility to AF. These cardiac phenotypes are similar to those observed in Pitx2 heterozygous mice. Interactions between Pitx2 and Playrr may provide a genetic mechanism for modulating Pitx2 dosage and susceptibility to SND and AF.

developmental biology↗