Biology subjects

Grubbs, B. H.

Publications and source records attributed to Grubbs, B. H..

7 recordsLinked to original sources

bioRxiv2025

Axial Nephron Fate Switching Demonstrates a Plastic System Tunable on Demand

The human nephron is a highly patterned tubular structure. It develops specialized cells that regulate bodily fluid homeostasis, blood pressure, and urine secretion throughout life. Approximately 1 million nephrons form in each kidney during embryonic and fetal development, but how they develop is poorly understood. Here we interrogate axial patterning mechanisms in the human nephron using an iPSC-derived kidney organoid system that generates hundreds of developmentally synchronized nephrons, and we compare it to in vivo human kidney development using single cell and spatial transcriptomic approaches. We show that human nephron patterning is controlled by integrated WNT/BMP/FGF signaling. Imposing a WNTON/BMPOFF state established a distal nephron identity that matures into thick ascending loop of Henle cells by endogenously activating FGF. Simultaneous suppression of FGF signaling switches cells back to a proximal cell-state, a transformation that is in itself dependent on BMP signal transduction. Our system highlights plasticity in axial nephron patterning, delineates the roles of WNT, FGF, and BMP mediated mechanisms controlling nephron patterning, and paves the way for generating nephron cells on demand.

developmental biology↗

bioRxiv2023

Defective mesenchymal Bmpr1a-mediated BMP signaling causes congenital pulmonary cysts

Abnormal lung development can cause congenital pulmonary cysts, the mechanisms of which remain largely unknown. Although the cystic lesions are believed to result directly from disrupted airway epithelial cell growth, the extent to which developmental defects in lung mesenchymal cells contribute to abnormal airway epithelial cell growth and subsequent cystic lesions has not been thoroughly examined. In the present study, we dissected the roles of BMP receptor 1a (Bmpr1a)- mediated BMP signaling in lung mesenchyme during prenatal lung development and discovered that abrogation of mesenchymal Bmpr1a disrupted normal lung branching morphogenesis, leading to the formation of prenatal pulmonary cystic lesions. Severe deficiency of airway smooth muscle cells and subepithelial elastin fibers were found in the cystic airways of the mesenchymal Bmpr1a knockout lungs. In addition, ectopic mesenchymal expression of BMP ligands and airway epithelial perturbation of the Sox2-Sox9 proximal-distal axis were detected in the mesenchymal Bmpr1a knockout lungs. However, deletion of Smad1/5, two major BMP signaling downstream effectors, from the lung mesenchyme did not phenocopy the cystic abnormalities observed in the mesenchymal Bmpr1a knockout lungs, suggesting that a Smad-independent mechanism contributes to prenatal pulmonary cystic lesions. These findings reveal for the first time the role of mesenchymal BMP signaling in lung development and a potential pathogenic mechanism underlying congenital pulmonary cysts.

developmental biology↗

bioRxiv2023

Modeling kidney development, disease, and plasticity with clonal expandable nephron progenitor cells and nephron organoids

Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here we report manipulation of p38 and YAP activity creates a synthetic niche that allows the long-term clonal expansion of primary mouse and human NPCs, and induced NPCs (iNPCs) from human pluripotent stem cells. Cultured iNPCs resemble closely primary human NPCs, generating nephron organoids with abundant distal convoluted tubule cells, which are not observed in published kidney organoids. The synthetic niche reprograms differentiated nephron cells into NPC state, recapitulating the plasticity of developing nephron in vivo. Scalability and ease of genome-editing in the cultured NPCs allow for genome-wide CRISPR screening, identi-fying novel genes associated with kidney development and disease. A rapid, efficient, and scala-ble organoid model for polycystic kidney disease was derived directly from genome-edited NPCs, and validated in drug screen. These technological platforms have broad applications to kidney development, disease, plasticity, and regeneration.

developmental biology↗

bioRxiv2023

Single nuclei multiomic analyses identify human cardiac lymphatic endothelial cells associated with coronary arteries in the epicardium

SummaryCardiac lymphatic vessels play important roles in fluid homeostasis, inflammation, disease, and regeneration of the heart. The developing cardiac lymphatics in human fetal hearts are closely associated with coronary arteries, similar to those in zebrafish hearts. We identified a population of cardiac lymphatic endothelial cells that reside in the epicardium. Single nuclei multiomic analysis of the human fetal heart revealed the plasticity and heterogeneity of the cardiac endothelium. Furthermore, we found that VEGFC is highly expressed in arterial endothelial cells, providing a molecular basis for the arterial association of cardiac lymphatic development. Using a cell-type-specific integrative analysis, we identified a novel population of cardiac lymphatic endothelial cells marked by the PROX1, the lymphangiocrine RELN, and enriched in binding motifs of ETV transcriptional factors. We report the first in vivo molecular characterization of human cardiac lymphatics and provide a valuable resource to understand fetal heart development.

developmental biology↗

bioRxiv2023

Single cell transcriptomics reveals early photoreceptor trajectories and a cancer-predisposed cone precursor state

Human cone photoreceptors differ from rods and serve as the retinoblastoma cell-of-origin, yet the developmental basis for their distinct behaviors is poorly understood. Here, we used deep full-length single-cell RNA-sequencing (scRNA-seq) to distinguish post-mitotic cone and rod developmental states and identify cone-specific features that contribute to retinoblastomagenesis. The analyses revealed nascent, immediately post-mitotic cone- and rod precursor populations characterized by higher THRB or NRL regulon activities, immature and maturing cone and rod precursors with concurrent cone- and rod-related gene and regulon expression, and distinct early and late cone and rod maturation states distinguished by maturation-associated declines in RAX regulon activity. Cell-state-specific gene expression features inferred based on full-length scRNA-seq were consistent with past single nucleus 3 RNA-seq analyses. Beyond the cell state characterizations, full-length scRNAseq revealed that both L/M cone and rod precursors co-expressed NRL and THRB RNAs, yet they differentially expressed functionally antagonistic NRL and THRB isoforms and prematurely terminated THRB transcripts. Moreover, early L/M cone precursors exhibited successive expression of several lncRNAs along with MYCN, which composed the seventh most L/M-cone-specific regulon, and SYK, which was implicated in the cone precursors proliferative response to RB1 loss. These findings reveal previously unresolved photoreceptor precursor states and suggest a role for early cone-precursor-intrinsic SYK expression in retinoblastoma initiation. Impact StatementFeatures acquired by early post-mitotic retinal cells underlie the distinct behaviors of rods and the cone cells of origin of retinoblastoma tumors.

developmental biology↗

bioRxiv2020

Generation of kidney ureteric bud and collecting duct organoids that recapitulate kidney branching morphogenesis

Kidney organoids model development and diseases of the nephron but not the contiguous epithelial network of the kidneys collecting duct (CD) system. Here, we report the generation of an expandable, 3D branching ureteric bud (UB) organoid culture model that can be derived from primary UB progenitors from mouse and human fetal kidneys, or generated de novo from pluripotent human stem cells. UB organoids differentiate into CD organoids in vitro, with differentiated cell types adopting spatial assemblies reflective of the adult kidney collecting system. Aggregating 3D-cultured nephron progenitor cells with UB organoids in vitro results in a reiterative process of branching morphogenesis and nephron induction, similar to kidney development. Combining efficient gene editing with the UB organoid model will facilitate an enhanced understanding of development, regeneration and diseases of the mammalian collecting system. One sentence summaryCollecting duct organoids derived from primary mouse and human ureteric bud progenitor cells and human pluripotent stem cells provide an in vitro platform for genetic dissection of development, regeneration and diseases of the mammalian collecting system.

developmental biology↗

bioRxiv2018

Developmental-stage-specific proliferation and retinoblastoma genesis in RB-deficient human but not mouse cone precursors

Most retinoblastomas initiate in response to the inactivation of the RB1 gene and loss of functional RB protein. The tumors may form without additional genomic changes and develop after a pre-malignant retinoma phase. Despite this seemingly straightforward etiology, mouse models have not recapitulated the genetic, cellular, and stage-specific features of human retinoblastoma genesis. For example, whereas human retinoblastomas appear to derive from cone photoreceptor precursors, current mouse models develop tumors that derive from other retinal cell types. To investigate the basis of the human cone-specific oncogenesis, we compared developmental-stage-specific cone precursor responses to RB loss in human and murine retina cultures and in cone-specific Rb1 knockout mice. We report that RB-depleted maturing (ARR3+) but not immature (ARR3-) human cone precursors enter the cell cycle, proliferate, and form retinoblastoma-like lesions characterized by Flexner-Wintersteiner rosettes, then form low or non-proliferative pre-malignant retinoma-like lesions with fleurettes and high p16INK4A and p130 expression, and finally form highly proliferative retinoblastoma-like masses. In contrast, in murine retina, only RB-depleted immature (Arr3-) cone precursors entered the cell cycle and they failed to progress from S to M phase. Moreover, whereas the intrinsically highly expressed MDM2 and MYCN contribute to RB-depleted maturing (ARR3+) human cone precursor proliferation, ectopic MDM2 and Mycn promoted only immature (Arr3-) murine cone precursor cell cycle entry. These findings demonstrate that developmental-stage-specific as well as speciesand cell-type-specific features sensitize to RB1 inactivation and reveal the human cone precursors capacity to model retinoblastoma initiation, proliferation, pre-malignant arrest, and tumor growth.\n\nSignificance StatementRetinoblastoma is a childhood tumor that forms in response to mutations in the RB1 gene and loss of functional RB protein. Prior studies suggested that retinoblastomas arise from cone photoreceptor precursors, whereas mouse models yield tumors deriving from other retinal cell types and lacking human retinoblastoma features. Here, we show that in cultured human retinae, retinoblastomas initiate from RB-depleted cone precursors that are in a specific maturation state and form pre-malignant \"retinomas\" prior to retinoblastoma lesions, as is believed to occur in retinoblastoma patients. In contrast, Rb-deficient mouse cone precursors of similar maturation state and supplemented with human-cone-precursor-specific oncoproteins fail to proliferate. Thus, human species-specific developmental features underlie retinoblastomagenesis and may challenge the production of accurate mouse retinoblastoma models.

cancer biology↗