Probes for TDTOMATO

The cardiac vascular and perivascular niche are of major importance in homeostasis and during disease, but we lack a complete understanding of its cellular heterogeneity and alteration in response to injury as a major driver of heart failure. Using combined genetic fate tracing with confocal imaging and single-cell RNA sequencing of this niche in homeostasis and during heart failure, we unravel cell type specific transcriptomic changes in fibroblast, endothelial, pericyte and vascular smooth muscle cell subtypes. We characterize a specific fibroblast subpopulation that exists during homeostasis, acquires Thbs4 expression and expands after injury driving cardiac fibrosis, and identify the transcription factor TEAD1 as a regulator of fibroblast activation. Endothelial cells display a proliferative response after injury, which is not sustained in later remodeling, together with transcriptional changes related to hypoxia, angiogenesis, and migration. Collectively, our data provides an extensive resource of transcriptomic changes in the vascular niche in hypertrophic cardiac remodeling.

Perivascular mesenchymal cells (PMCs), which include pericytes, give rise to myofibroblasts that contribute to chronic kidney disease progression. Several PMC markers have been identified; however, PMC heterogeneity and functions are not fully understood. Here, we describe a novel subset of renal PMCs that express Meflin, a glycosylphosphatidylinositol-anchored protein that was recently identified as a marker of fibroblasts essential for cardiac tissue repair. Tracing the lineage of Meflin+ PMCs, which are found in perivascular and periglomerular areas and exhibit renin-producing potential, showed that they detach from the vasculature and proliferate under disease conditions. Although the contribution of Meflin+ PMCs to conventional α-SMA+ myofibroblasts is low, they give rise to fibroblasts with heterogeneous α-SMA expression patterns. Genetic ablation of Meflin+ PMCs in a renal fibrosis mouse model revealed their essential role in collagen production. Consistent with this, human biopsy samples showed that progressive renal diseases exhibit high Meflin expression. Furthermore, Meflin overexpression in kidney fibroblasts promoted bone morphogenetic protein 7 signals and suppressed myofibroblastic differentiation, implicating the roles of Meflin in suppressing tissue fibrosis. These findings demonstrate that Meflin marks a PMC subset that is functionally distinct from classic pericytes and myofibroblasts, highlighting the importance of elucidating PMC heterogeneity.

Sensory neurons relay gut-derived signals to the brain, yet the molecular and functional organization of distinct populations remains unclear. Here, we employed intersectional genetic manipulations to probe the feeding and glucoregulatory function of distinct sensory neurons. We reconstruct the gut innervation patterns of numerous molecularly defined vagal and spinal afferents and identify their downstream brain targets. Bidirectional chemogenetic manipulations, coupled with behavioral and circuit mapping analysis, demonstrated that gut-innervating, glucagon-like peptide 1 receptor (GLP1R)-expressing vagal afferents relay anorexigenic signals to parabrachial nucleus neurons that control meal termination. Moreover, GLP1R vagal afferent activation improves glucose tolerance, and their inhibition elevates blood glucose levels independent of food intake. In contrast, gut-innervating, GPR65-expressing vagal afferent stimulation increases hepatic glucose production and activates parabrachial neurons that control normoglycemia, but they are dispensable for feeding regulation. Thus, distinct gut-innervating sensory neurons differentially control feeding and glucoregulatory neurocircuits and may provide specific targets for metabolic control.

The glucagon gene (Gcg) encodes preproglucagon, which is cleaved to form glucagon-like peptide 1 (GLP1) and other mature signaling molecules implicated in metabolic functions. To date there are no transgenic rat models available for precise manipulation of GLP1-expressing cells in the brain and periphery.To visualize and manipulate Gcg-expressing cells in rats, CRISPR/Cas9 was used to express iCre under control of the Gcg promoter. Gcg-Cre rats were bred with tdTomato reporter rats to tag Gcg-expressing cells. Cre-dependent AAVs and RNAscope in situ hybridization were used to evaluate the specificity of iCre expression by GLP1 neurons in the caudal nucleus of the solitary tract (cNTS) and intermediate reticular nucleus (IRt), and by intestinal and pancreatic secretory cells. Food intake was assessed in heterozygous (Het) Gcg-Cre rats after chemogenetic stimulation of cNTS GLP1 neurons expressing an excitatory DREADD.While genotype has minimal effect on body weight or composition in chow-fed Gcg-Cre rats, homozygous (Homo) rats have lower plasma glucose levels. In neonatal and adult Gcg-Cre/tdTom rats, reporter-labeled cells are present in the cNTS and IRt, and in additional brain regions (e.g., basolateral amygdala, piriform cortex) that lack detectable Gcg mRNA in adults but display transient developmental or persistently low Gcg expression. Compared to wildtype (WT) rats, hindbrain Gcg mRNA and GLP1 protein in brain and plasma are markedly reduced in Homo Gcg-Cre rats. Chemogenetic stimulation of cNTS GLP1 neurons reduced overnight chow intake in males but not females, the effect in males was blocked by antagonism of central GLP1 receptors, and hypophagia was enhanced when combined with a subthreshold dose of cholecystokinin-8 to stimulate gastrointestinal vagal afferents.Gcg-Cre rats are a novel and valuable experimental tool for analyzing the development, anatomy, and function of Gcg-expressing cells in the brain and periphery. In addition, Homo Gcg-Cre rats are a unique model for assessing the role of Gcg-encoded proteins in glucose homeostasis and energy metabolism.