Biology subjects

Donahoe, P. K.

Publications and source records attributed to Donahoe, P. K..

7 recordsLinked to original sources

bioRxiv2026

Histone methylation activity of KMT2D is required for proliferative control of the developing lung

KMT2D, the histone methyltransferase and core component of the COMPASS/MLL4 complex, has been implicated in developmental diseases such as Kabuki Syndrome, interstitial lung disease, and congenital diaphragmatic hernia, with clear links to pediatric pulmonary disorders. Despite this, the mechanism by which KMT2D governs lung development remains unclear. Knock-in mouse models rendering, KMT2D catalytically deactivated (KMT2DKI) and reducing H3K4 methylation, have demonstrated potential in defining KMT2Ds role in pulmonary development. Our examination of the lungs of KMT2DKI mice revealed increased cellular density and impaired sacculation indicated by reduced airspace chord length, thickening of intersaccular septa, and abnormal alveolar cell differentiation. KMT2DKI mice revealed narrowed Sox2+ conducting airways and epithelial differentiation defects characterized by reduced Cc10+ club cells. Accompanying the alveolar and airway hypoplasia, blood vessel luminal area was reduced. Conversely, KMT2DKI lungs had a significantly higher proportion of proliferating cells accompanied by a dramatic expansion in Pdgfr+ mesenchymal progenitor cells. Our findings therefore suggest that KMT2D-mediated H3K4 methylation is vital to normal lung development, and its impairment results in widespread pulmonary hypoplasia and potentially pulmonary hypertension.

developmental biology↗

bioRxiv2026

Modeling patient variants of Cnot1 and Cdc42bpb results in distinct forms of congenital diaphragmatic hernia in mice

Congenital diaphragmatic hernia (CDH) is a severe congenital anomaly characterized by impairment of both diaphragm and lung development in utero. CDH presents as a spectrum of forms and severities, with diaphragm defects arising in the dorsal/posterior region typically correlating with more severe pulmonary disease and higher risk of mortality than those appearing in ventral/anterior regions. The genetic etiology underlying CDH is complex, with many genes implicated showing variable expressivity and incomplete penetrance in both human patients and mouse models. Here we present in vivo validation of two genes previously unassociated with CDH: the CDC42-interacting kinase CDC42BPB; and CNOT1, a scaffolding protein of the CCR4-NOT protein complex, critical for mRNA regulation through modifications such as deadenylation. Each gene was found to have a damaging, de novo missense variant in a recent large-scale CDH patient sequencing screen. Loss of Cdc42bpb leads to ventral diaphragmatic hernias, heart septal defects and minor lung epithelial differentiation defects in mouse embryos. Installation of the orthologous patient-specific missense variant through CRISPR/Cas9 editing leads to less severe ventral diaphragm defects. Mouse embryos with either one or two copies of the orthologous Cnot1 variant, c.1867C>T (p.R623W), develop dorsal diaphragmatic hernias with low (<50%) penetrance, and mutants showed alterations in mRNA isoform expression consistent with the molecular role of Cnot1 in RNA splicing. These results underscore the power of in vivo functional modeling to validate genes and patient-specific variants uncovered by patient sequencing, reveal two previously unrecognized genetic causes of CDH, and highlight the heterogeneity of different patient anatomic presentations.

genetics↗

bioRxiv2024

AMH protects the ovary from doxorubicin by regulating cell fate and the response to DNA damage

Anti-Mullerian hormone (AMH) protects the ovarian reserve from chemotherapy, and this effect is most pronounced with Doxorubicin (DOX). However, the mechanisms of DOX toxicity and AMH rescue in the ovary remain unclear. Herein, we characterize these mechanisms in various ovarian cell types using scRNAseq. In the mesenchyme, DOX activates the intrinsic apoptotic signaling pathway through p53 class mediators, particularly affecting theca progenitors, while co-treament with AMH halts theca differentiation and reduces apoptotic gene expression. In preantral granulosa cells, DOX upregulates the cell cycle inhibitor Cdkn1a and dysregulates Wnt signaling, which are ameliorated by AMH co-treatment. Finally, in follicles, AMH induces Id3, a protein involved in DNA repair, which is necessary to prevent the accumulation of DNA lesions marked by {gamma}-H2AX in granulosa cells. Altogether this study characterizes cell, and follicle stage-specific mechanisms of AMH protection of the ovary, offering promising new avenues for fertility preservation in cancer patients undergoing chemotherapy. HighlightsO_LIDoxorubicin treatment induces DNA damage that activates the p53 pathway in stromal and follicular cells of the ovary. C_LIO_LIAMH inhibits the proliferation and differentiation of theca and granulosa cells and promotes follicle survival following Doxorubicin insult. C_LIO_LIAMH treatment mitigates Doxorubicin-induced DNA damage in the ovary by preventing the accumulation of {gamma}-H2AX-positive unresolved foci, through increased expression of ID3, a protein involved in DNA repair. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/595356v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@19f6752org.highwire.dtl.DTLVardef@383e74org.highwire.dtl.DTLVardef@8c38f7org.highwire.dtl.DTLVardef@1efe797_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2024

Structural Basis of Non-Latent Signaling by the Anti-Mullerian Hormone Procomplex

Most TGF{beta} family ligands exist as procomplexes consisting of a prodomain noncovalently bound to a growth factor (GF); Whereas some prodomains confer latency, the Anti-Mullerian Hormone (AMH) prodomain maintains a remarkably high affinity for the GF yet remains active. Using single particle EM methods, we show the AMH prodomain consists of two subdomains: a vestigial TGF{beta} prodomain-like fold and a novel, helical bundle GF-binding domain, the result of an exon insertion 450 million years ago, that engages both receptor epitopes. When associated with the prodomain, the AMH GF is distorted into a strained, open conformation whose closure upon bivalent binding of AMHR2 displaces the prodomain through a conformational shift mechanism to allow for signaling.

molecular biology↗

bioRxiv2022

A tracheal aspirate-derived airway basal cell model reveals a pro-inflammatory epithelial defect in congenital diaphragmatic hernia

RationaleCongenital diaphragmatic hernia (CDH) is characterized by incomplete closure of the diaphragm and lung hypoplasia. The pathophysiology of lung defects in CDH is poorly understood. ObjectivesTo establish a translational model of human airway epithelium in CDH for pathogenic investigation and therapeutic testing. MethodsWe developed a robust methodology of epithelial progenitor derivation from tracheal aspirates of newborns. Basal stem cells (BSCs) from CDH patients and preterm and term, non-CDH controls were derived and analyzed by bulk RNA-sequencing, ATAC-sequencing, and air-liquidinterface differentiation. Lung sections from fetal human CDH samples and the nitrofen rat model of CDH were subjected to histological assessment of epithelial defects. Therapeutics to restore epithelial differentiation were evaluated in human epithelial cell culture and the nitrofen rat model of CDH. Measurements and Main ResultsTranscriptomic and epigenetic profiling of CDH and non-CDH basal stem cells reveals a disease-specific, proinflammatory signature independent of severity or hernia size. In addition, CDH basal stem cells exhibit defective epithelial differentiation in vitro that recapitulates epithelial phenotypes found in fetal human CDH lung samples and fetal tracheas of the nitrofen rat model of CDH. Furthermore, steroid treatment normalizes epithelial differentiation phenotypes of human CDH basal stem cells in vitro and in nitrofen rat tracheas in vivo. ConclusionsOur findings have identified an underlying proinflammatory signature and BSC differentiation defects as a potential therapeutic target for airway epithelial defects in CDH.

developmental biology↗

bioRxiv2022

A single cell atlas of the cycling murine ovary

The estrous cycle is regulated by rhythmic endocrine interactions of the nervous and reproductive systems, which coordinate the hormonal and ovulatory functions of the ovary. Folliculogenesis and follicle progression require the orchestrated response of a variety of cell types to allow the maturation of the follicle and its sequela, ovulation, corpus luteum (CL) formation, and ovulatory wound repair. Little is known about the cell state dynamics of the ovary during the estrous cycle, and the paracrine factors that help coordinate this process. Herein we used single-cell RNA sequencing to evaluate the transcriptome of > 34,000 cells of the adult mouse ovary and describe the transcriptional changes that occur across the normal estrous cycle and other reproductive states to build a comprehensive dynamic atlas of murine ovarian cell types and states.

developmental biology↗

bioRxiv2021

The extracellular matrix gene, Svep1, orchestrates airway patterning and the transition from lung branching morphogenesis to alveolar maturation in the mouse

Disruptions in airway branching or alveolar differentiation during lung development can lead to severe respiratory deficiencies and neonatal death. The molecular mechanisms governing branching patterning and early alveolar formation remain elusive. Loss of Svep1 function in mice results in various developmental defects, including lung hypoplasia and perinatal lethality. Our examination of the lungs of Svep1 knockout (Svep1-/-) mouse embryos, both in vivo and in vitro, revealed that Svep1 mutants exhibit an increase in the number of disorganized distal airway tips and progressively greater disruption of lung lobe morphology over time and saccular development. Svep1 interacts with FGF signaling to regulate smooth muscle differentiation and, together with Fgf9, guides airway branching patterning. Transcriptomic data from the lungs of Svep1-/- embryos revealed dysregulated gene expression affecting saccular maturation. Our findings demonstrate that Svep1 is a key extracellular matrix player shaping airway morphology and influencing alveolar fate. These insights offer potential avenues for therapeutic interventions in congenital lung disorders.

developmental biology↗