Probes for FN1

Fibronectin (Fn1) is an evolutionarily conserved extracellular matrix glycoprotein essential for embryonic development. Global deletion of Fn1 leads to mid-gestation lethality from cardiovascular defects. However, severe morphogenetic defects that occur early in embryogenesis in these embryos precluded assigning a direct role for Fn1 in cardiovascular development. We noticed that Fn1 is expressed in strikingly non-uniform patterns during mouse embryogenesis, and that its expression is particularly enriched in the pharyngeal region corresponding with the pharyngeal arches 3, 4, and 6. This region bears a special importance for the developing cardiovascular system, and we hypothesized that the localized enrichment of Fn1 in the pharyngeal region may be essential for cardiovascular morphogenesis. To test this hypothesis, we ablated Fn1 using the Isl1Cre knock-in strain of mice. Deletion of Fn1 using the Isl1Cre strain resulted in defective formation of the 4th pharyngeal arch arteries (PAAs), aberrant development of the cardiac outflow tract (OFT), and ventricular septum defects. To determine the cell types responding to Fn1 signaling during cardiovascular development, we deleted a major Fn1 receptor, integrin α5 using the Isl1Cre strain, and observed the same spectrum of abnormalities seen in the Fn1 conditional mutants. Additional conditional mutagenesis studies designed to ablate integrin α5 in distinct cell types within the Isl1+ tissues and their derivatives, suggested that the expression of integrin α5 in the pharyngeal arch mesoderm, endothelium, surface ectoderm and the neural crest were not required for PAA formation. Our studies suggest that an (as yet unknown) integrin α5-dependent signal extrinsic to the pharyngeal endothelium mediates the formation of the 4th PAAs.

The chemical and mechanical properties of extracellular matrices (ECMs) modulate diverse aspects of cellular fates; however, how regional heterogeneity in ECM composition regulates developmental programs is not well understood. We discovered that fibronectin 1 (Fn1) is expressed in strikingly non-uniform patterns during mouse development, suggesting that regionalized synthesis of the ECM plays cell-specific regulatory roles during embryogenesis. To test this hypothesis, we ablated Fn1 in the neural crest (NC), a population of multi-potent progenitors expressing high levels of Fn1. We found that Fn1 synthesized by the NC mediated morphogenesis of the aortic arch artery and differentiation of NC cells into vascular smooth muscle cells (VSMCs) by regulating Notch signaling. We show that NC Fn1 signals in an NC cell-autonomous manner through integrin α5β1 expressed by the NC, leading to activation of Notch and differentiation of VSMCs. Our data demonstrate an essential role of the localized synthesis of Fn1 in cardiovascular development and spatial regulation of Notch signaling.

The mammalian hippocampus, comprised of serially connected subfields, participates in diverse behavioral and cognitive functions. It has been postulated that parallel circuitry embedded within hippocampal subfields may underlie such functional diversity. We sought to identify, delineate, and manipulate this putatively parallel architecture in the dorsal subiculum, the primary output subfield of the dorsal hippocampus. Population and single-cell RNA-seq revealed that the subiculum can be divided into two spatially adjacent subregions associated with prominent differences in pyramidal cell gene expression. Pyramidal cells occupying these two regions differed in their long-range inputs, local wiring, projection targets, and electrophysiological properties. Leveraging gene-expression differences across these regions, we use genetically restricted neuronal silencing to show that these regions differentially contribute to spatial working memory. This work provides a coherent molecular-, cellular-, circuit-, and behavioral-level demonstration that the hippocampus embeds structurally and functionally dissociable streams within its serial architecture.

In the hippocampus, the classical pyramidal cell type of the subiculum acts as a primary output, conveying hippocampal signals to a diverse suite of downstream regions. Accumulating evidence suggests that the subiculum pyramidal cell population may actually be comprised of discrete subclasses. Here, we investigated the extent and organizational principles governing pyramidal cell heterogeneity throughout the mouse subiculum. Using single-cell RNA-seq, we find that the subiculum pyramidal cell population can be deconstructed into eight separable subclasses. These subclasses were mapped onto abutting spatial domains, ultimately producing a complex laminar and columnar organization with heterogeneity across classical dorsal-ventral, proximal-distal, and superficial-deep axes. We further show that these transcriptomically defined subclasses correspond to differential protein products and can be associated with specific projection targets. This work deconstructs the complex landscape of subiculum pyramidal cells into spatially segregated subclasses that may be observed, controlled, and interpreted in future experiments.

Late-delayed radiation-induced brain injury (RIBI) is a major adverse effect of fractionated whole-brain irradiation (fWBI). Characterized by progressive cognitive dysfunction, and associated cerebrovascular and white matter injury, RIBI deleteriously affects quality of life for cancer patients. Despite extensive morphological characterization of the injury, the pathogenesis is unclear, thus limiting the development of effective therapeutics. We previously reported that RIBI is associated with increased gene expression of the extracellular matrix (ECM) protein fibronectin (FN1). We hypothesized that fibronectin contributes to perivascular ECM, which may impair diffusion to the dependent parenchyma, thus contributing to the observed cognitive decline. The goal of this study was to determine the localization of fibronectin in RIBI and further characterize the composition of perivascular ECM, as well as identify the cell of origin for FN1 by in situ hybridization. Briefly, fibronectin localized to the vascular basement membrane of morphologically normal blood vessels from control comparators and animals receiving fWBI, and to the perivascular space of edematous and fibrotic vascular phenotypes of animals receiving fWBI. Additional mild diffuse parenchymal staining in areas of vascular injury suggested blood-brain-barrier disruption and plasma fibronectin extravasation. Perivascular ECM lacked amyloid and contained lesser amounts of collagens I and IV, which localized to the basement membrane. These changes occurred in the absence of alterations in microvascular area fraction or microvessel density. Fibronectin transcripts were rarely expressed in control comparators, and were most strongly induced within cerebrovascular endothelial and vascular smooth muscle cells after fWBI. Our results demonstrate that fibronectin is produced by cerebrovascular endothelial and smooth muscle cells in late-delayed RIBI and contributes to perivascular ECM, which we postulate may contribute to diffusion barrier formation. We propose that pathways that antagonize fibronectin deposition and matrix assembly or enhance degradation may serve as potential therapeutic targets in RIBI.