Hagerling, R. | Search bioRxiv

bioRxiv2022

Single cell, whole embryo phenotyping of pleiotropic disorders of mammalian development

Mouse models are a critical tool for studying human diseases, particularly developmental disorders, as well as for advancing our general understanding of mammalian biology. However, it has long been suspected that conventional approaches for phenotyping are insufficiently sensitive to detect subtle defects throughout the developing mouse. Here we set out to establish single cell RNA sequencing (sc-RNA-seq) of the whole embryo as a scalable platform for the systematic molecular and cellular phenotyping of mouse genetic models. We applied combinatorial indexing-based sc-RNA-seq to profile 101 embryos of 26 genotypes at embryonic stage E13.5, altogether profiling gene expression in over 1.6M nuclei. The 26 genotypes include 22 mouse mutants representing a range of anticipated severities, from established multisystem disorders to deletions of individual enhancers, as well as the 4 wildtype backgrounds on which these mutants reside. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibited changes in dozens of trajectories (e.g., the pleiotropic consequences of altering the Sox9 regulatory landscape) whereas others showed phenotypes affecting specific subsets of cells. We also identify differences between widely used wildtype strains, compare phenotyping of gain vs. loss of function mutants, and characterise deletions of topological associating domain (TAD) boundaries. Intriguingly, even among these 22 mutants, some changes are shared by heretofore unrelated models, suggesting that developmental pleiotropy might be "decomposable" through further scaling of this approach. Overall, our findings show how single cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution.

genomics↗

bioRxiv2021

Regulation and impact of cardiac lymphangiogenesis in pressure-overload-induced heart failure

RationaleLymphatics are essential for cardiac health, and insufficient lymphatic expansion (lymphangiogenesis) contributes to development of heart failure (HF) after myocardial infarction. However, the regulation and impact of lymphatics in non-ischemic cardiomyopathy induced by pressure-overload remains to be determined. ObjectiveInvestigate cardiac lymphangiogenesis following transverse aortic constriction (TAC) in adult male or female C57Bl/6J or Balb/c mice, and in patients with end-stage HF. Methods & ResultCardiac function was evaluated by echocardiography, and cardiac hypertrophy, lymphatics, inflammation, edema, and fibrosis by immunohistochemistry, flow cytometry, microgravimetry, and gene expression analysis, respectively. Treatment with neutralizing anti-VEGFR3 antibodies was applied to inhibit cardiac lymphangiogenesis in mice. The gender- and strain-dependent mouse cardiac hypertrophic response to TAC, especially increased ventricular wall stress, led to lymphatic expansion in the heart. Our experimental findings that ventricular dilation triggered cardiac lymphangiogenesis was mirrored by observations in clinical HF samples, with increased lymphatic density found in patients with dilated cardiomyopathy. Surprisingly, the striking lymphangiogenesis observed post-TAC in Balb/c mice, linked to increased cardiac Vegfc, did not suffice to resolve myocardial edema, and animals progressed to dilated cardiomyopathy and HF. Conversely, selective inhibition of the essentially Vegfd-driven capillary lymphangiogenesis observed post-TAC in male C57Bl/6J mice did not significantly aggravate cardiac edema. However, cardiac immune cell levels were increased, notably myeloid cells at 3 weeks and T lymphocytes at 8 weeks. Moreover, while the TAC-triggered development of interstitial cardiac fibrosis was unaffected by anti-VEGFR3, inhibition of lymphangiogenesis increased perivascular fibrosis and accelerated the development of left ventricular dilation and cardiac dysfunction. ConclusionsWe demonstrate for the first time that endogenous cardiac lymphangiogenesis limits pressure-overload-induced cardiac inflammation and perivascular fibrosis, thus delaying HF development. While these findings remain to be confirmed in a larger study of HF patients, we propose that under settings of pressure-overload poor cardiac lymphangiogenesis may accelerate HF development.

physiology↗