# ZFHX4 sets alveolar fibroblast differentiation potential of mesenchymal progenitors in the fetal lung

> **NIH NIH R03** · MASSACHUSETTS GENERAL HOSPITAL · 2024 · $164,990

## Abstract

PROJECT SUMMARY
Congenital diaphragmatic hernia (CDH) is characterized by incomplete closure of the diaphragm and lung
hypoplasia in newborns. The diaphragm defect can be surgically repaired; however, lung hypoplasia remains
the main cause of mortality and morbidity in CDH. The etiology of lung hypoplasia in CDH is multifactorial and
involves compression by herniated viscera, genetic and environmental factors. To date, over 100 CDH-
associated genes were identified in patient cohorts. However, the function of most CDH-associated genes,
including Zinc Finger Homeobox 4 (ZFHX4), in the fetal lung is unknown. In Preliminary Studies, we show
that ZFHX4 is selectively expressed in mesenchymal cells in fetal lungs of humans and mice. Functional
disruption of Zfhx4 in mice causes thickened mesenchyme, defective alveolar fibroblast differentiation, and
impaired alveologenesis, which resemble the clinical hallmarks of lung hypoplasia in CDH. Interestingly,
Zfhx4-/- mice have no diaphragmatic henia, which suggests that additional factors may cause diaphragm
defects in CDH patients with ZFHX4 variants. A lack of confounding diaphragm defects also makes Zfhx4-/-
mice an excellent model to investigate the direct role of Zfhx4 in lung mesenchyme development. We show
that Zfhx4 expression peaks at the pseudoglandular stage of lung development; however, Zfhx4-/- mice have
no lung mesenchymal phenotypes until E16.5 when Zfhx4 is no longer detectable. Supporting the impact of
early Zfhx4 expression on late mesenchyme development, transcriptome profiling of wildtype and Zfhx4-/- lung
mesenchymal cells at E16.5 identified differentiatlly expressed genes enriched in matrix and mesenchyme
developmental pathways. The observed temporal disparity between Zfhx4 expression and the phenotype in
Zfhx4-/- mice are reminiscent of two defining features of a pioneer factor -- by binding to silent gene loci early
on and by recruiting transcriptional factors and epigenetic regulators, drives later lineage specification. Based
on these preliminary findings, we hypothesize that the pioneer factor ZFHX4 accesses silent gene sites
and recruits transcriptional regulators in embryonic mesenchymal progenitors thereby orchestrating
alveolar fibroblast differentiation. Utilizing the R03 funding mechanism and leveraging available Zfhx4-/-
and Zfhx4v5 mouse lines, we propose two pilot assays to interrogate Zfhx4 regulation of lung mesenchyme
development during embryogenesis. The first assay is to investigate dynamics of Zfhx4 binding, chromatin
opening, and gene regulation in lung mesenchymal progenitors (Aim 1) and the second assay is to identify
Zfhx4-binding transcriptional regulators in lung mesenchymal progenitors (Aim 2). The results of these two
proposed assays will identify canidate mediators of Zfhx4 for future functional investigation to inform lung
mesenchyme development and pathogenesis of lung hypoplasia in CDH.

## Key facts

- **NIH application ID:** 10987257
- **Project number:** 1R03HD113887-01A1
- **Recipient organization:** MASSACHUSETTS GENERAL HOSPITAL
- **Principal Investigator:** Xingbin Ai
- **Activity code:** R03 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $164,990
- **Award type:** 1
- **Project period:** 2024-08-01 → 2026-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10987257

## Citation

> US National Institutes of Health, RePORTER application 10987257, ZFHX4 sets alveolar fibroblast differentiation potential of mesenchymal progenitors in the fetal lung (1R03HD113887-01A1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10987257. Licensed CC0.

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