Biology subjects

Sheth, R.

Publications and source records attributed to Sheth, R..

6 recordsLinked to original sources

bioRxiv2024

WNT signaling coordinately controls mouse limb bud outgrowth and establishment of the digit-interdigit pattern

Self-organization, such as the emergence of a pattern from a homogenous state, is a fascinating property of biological systems. Early limb bud outgrowth and patterning in mice are controlled by a robust and self-regulatory signaling system, and initiation of the periodic digit-interdigit pattern appears under control of a self-regulatory Turing system. Previous studies established the requirement of WNT and BMP signaling for both early limb bud and digit-interdigit morphogenesis, but the molecular changes underlying the transition from early limb bud signaling to the digit-interdigit patterning system remained unknown. Here, we use small molecule inhibitors to rapidly but transiently block WNT signaling to identify the early transcriptional targets that are altered during disruption and recovery of limb bud and digit development. Together, this study highlights the overarching role of WNT signaling in controlling early limb bud outgrowth and patterning, and establishment of the periodic digit-interdigit pattern. Finally, the transient WNT signaling disruption approach reveals the plasticity and robustness of these self-organizing limb bud and digit patterning systems.

developmental biology↗

bioRxiv2024

TBX3 is essential to establish the posterior boundary of anterior genes and up-regulate posterior genes with HAND2 during onset of limb bud development

During limb bud formation, axes polarities are established as evidenced by the spatially restricted expression of key regulator genes. In particular, the mutually antagonistic interaction between the GLI3 repressor and HAND2 results in the distinct and non-overlapping anterior-distal Gli3 and posterior Hand2 expression domains. This hallmarks establishment of antero-posterior (AP) limb axis polarity together with spatially restricted expression of homeodomain and other transcriptional regulators. Here, we identify TBX3 as the transcription factor to initiate AP axis polarity in mouse limb buds. ChIP-seq and differential gene expression analysis of wildtype and mutant limb buds identifies the TBX3-specific and shared TBX3-HAND2 target genes. High sensitivity fluorescent whole mount in situ hybridisation shows that the posterior expression boundaries of anterior genes are positioned by TBX3-mediated repression, which excludes anterior genes such as Gli3, Alx4, Hand1 and Irx3/5 from the posterior limb bud mesenchyme. This exclusion delineates the posterior mesenchymal territory competent to establish the Shh-expressing limb bud organiser. In turn, HAND2 is required for Shh activation and cooperates with TBX3 to up-regulate shared posterior identity target genes in early limb buds.

developmental biology↗

bioRxiv2023

Molecular pathways and cellular subsets associated with adverse clinical outcomes in overlapping immune-related myocarditis and myositis

Immune checkpoint therapies (ICTs) can induce life-threatening immune-related adverse events, including myocarditis and myositis, which are rare but often concurrent. The molecular pathways and immune subsets underlying these toxicities remain poorly understood. To address this need, we obtained heart and skeletal muscle biopsies for single-cell RNA sequencing in living patients with cancers treated with ICTs admitted to the hospital with myocarditis and /or myositis (overlapping myocarditis plus myositis, n=10; myocarditis-only, n=1) compared to ICT-exposed patients ruled out for toxicity utilized as controls (n=9) within 96 hours of clinical presentation. Analyses of 58,523 cells revealed clonally expanded CD8+ T cells with a cytotoxic phenotype expressing activation/exhaustion markers in both myocarditis and myositis. Furthermore, the analyses identified a population of tissue-resident myeloid cells expressed Fc{gamma}RIIIa, which is known to bind IgG and regulate complement activation. Immunohistochemistry of affected cardiac and skeletal muscle tissues revealed protein expression of pan-IgG and complement product C4d that were associated with the presence of high-titer serum autoantibodies against muscle antigens in a subset of patients. We further identified a population of inflammatory IL-1B+TNF+ myeloid cells specifically enriched in myocarditis and associated with greater toxicity severity and poorer clinical outcomes. These results are the first to recognize these myeloid subsets in human immune-related myocarditis and myositis tissues and nominate new targets for investigation into rational treatments to overcome these high-mortality toxicities. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/556590v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@b5895forg.highwire.dtl.DTLVardef@4dec8corg.highwire.dtl.DTLVardef@1e50cb9org.highwire.dtl.DTLVardef@a653fd_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2019

HOX13-dependent chromatin accessibility modulates the target repertoires of the HOX factors

Hox genes encode essential transcription factors that control patterning during embryonic development. Distinct combinations of nested Hox expression domains establish cell and tissue identities1-3. Consequently, spatial or temporal de-regulation of Hox genes can cause severe alterations of the body plan3. While HOX factors have very similar DNA binding motifs, their binding specificity is, in part, mediated by co-factors4-6. Yet, the interplay between HOX binding specificities and the cellular context remains largely elusive. To gain insight into this question, we took advantage of developing limbs for which the differential expression of Hox genes is well-characterized7. We show that the transcription factors HOXA13 and HOXD13 (hereafter referred as HOX13) allow another HOX factor, HOXA11, to bind loci initially assumed to be HOX13-specific. Importantly, HOXA11 is unable to bind these loci in distal limbs lacking HOX13 function indicating that HOX13 modulates HOXA11 target repertoire. In addition, we find that the HOX13 factors implement the distal limb developmental program by triggering chromatin opening, a defining property of pioneer factors8,9. Finally, single cell analysis of chromatin accessibility reveals that HOX13 factors pioneer chromatin opening in a lineage specific manner. Together, our data uncover a new mechanism underlying HOX binding specificity, whereby tissue-specific variations in the target repertoire of HOX factors rely, at least in part, on HOX13-dependent chromatin accessibility.

developmental biology↗

bioRxiv2019

Scalable and cost-effective ribonuclease-based rRNA depletion for bacterial transcriptomics

Bacterial RNA sequencing (RNA-seq) is a powerful approach for quantitatively delineating the global transcriptional profiles of microbes in order to gain deeper understanding of their physiology and function. Cost-effective bacterial RNA-seq requires efficient physical removal of ribosomal RNA (rRNA), which otherwise dominates transcriptomic reads. However, current methods to effectively deplete rRNA of diverse non-model bacterial species are lacking. Here, we describe a probe and ribonuclease based strategy for bacterial rRNA removal. We implemented the method using either chemically synthesized oligonucleotides or amplicon-based single-stranded DNA probes and validated the technique on three novel gut microbiota isolates from three distinct phyla. We further showed that different probe sets can be used on closely related species. We provide a detailed methods protocol, probe sets for >5,000 common microbes from RefSeq, and an online tool to generate custom probe libraries. This approach lays the groundwork for large-scale and cost-effective bacterial transcriptomics studies.

genomics↗

bioRxiv2018

Mutual transcriptional repression between Gli3 and Hox13 genes determines the anterior-posterior asymmetry of the autopod

In the present study we have investigated the molecular causes of the absence of digit 1 in the Hoxa13 mutant and why the absence of Hoxa13 protein, whose expression spans the entire autopod, specifically impacts the anterior-most digit. We show that in the absence of Hoxa13, the expression of Hoxd13 does not extend into the anterior mesoderm consequently leaving the presumptive territory of digit1 devoid of distal Hox expression and providing an explanation for the agenesis of digit 1. We provide compelling evidence that the lack of Hoxd13 transcription in the anterior mesoderm is due to increased Gli3R activity, in turn resulting from the loss of transcriptional repression exerted by Hoxa13 on Gli3. Our results are compatible with a mutual transcriptional repression between Gli3 and Hox13 genes that determines the anterior-posterior asymmetry of the autopod.

developmental biology↗