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Updated: Jul 19, 2025
Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis
Published on: April 1, 2022
ESCRT-dependent control of craniofacial morphogenesis with concomitant perturbation of NOTCH signaling
Viviana Hermosilla Aguayo1, Peter Martin1, Nuo Tian1
1Program in Craniofacial Biology, Institute for Human Genetics, Eli and Edythe Broad Center of Regeneration Medicine & Stem Cell Research, Dept of Orofacial Sciences and Dept of Anatomy, University of California, San Francisco, San Francisco, CA 94143, USA.
Abstract:
Craniofacial development is orchestrated by transcription factor-driven regulatory networks, epigenetic modifications, and signaling pathways. Signaling molecules and their receptors rely on endo-lysosomal trafficking to prevent accumulation on the plasma membrane. ESCRT (Endosomal Sorting Complexes Required for Transport) machinery is recruited to endosomal membranes enabling degradation of such endosomal cargoes. Studies in vitro and in invertebrate models established the requirements of the ESCRT machinery in membrane remodeling, endosomal trafficking, and lysosomal degradation of activated membrane receptors. However, investigations during vertebrate development have been scarce. By ENU-induced mutagenesis, we isolated a mouse line, Vps25ENU/ENU, carrying a hypomorphic allele of the ESCRT-II component Vps25, with craniofacial anomalies resembling features of human congenital syndromes. Here, we assessed the spatiotemporal dynamics of Vps25 and additional ESCRT-encoding genes during murine development. We show that these genes are ubiquitously expressed although enriched in discrete domains of the craniofacial complex, heart, and limbs. ESCRT-encoding genes, including Vps25, are expressed in both cranial neural crest-derived mesenchyme and epithelium. Unlike constitutive ESCRT mutants, Vps25ENU/ENU embryos display late lethality. They exhibit hypoplastic lower jaw, stunted snout, dysmorphic ear pinnae, and secondary palate clefting. Thus, we provide the first evidence for critical roles of ESCRT-II in craniofacial morphogenesis and report perturbation of NOTCH signaling in craniofacial domains of Vps25ENU/ENU embryos. Given the known roles of NOTCH signaling in the developing cranium, and notably the lower jaw, we propose that the NOTCH pathway partly mediates the craniofacial defects of Vps25ENU/ENU mouse embryos.
Insights
The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery, specifically Vps25, is crucial for craniofacial development in mice. Disruption of Vps25 leads to severe craniofacial defects and impacts NOTCH signaling.
Area of Science:
- Developmental Biology
- Molecular Biology
- Genetics
Background:
- Craniofacial development relies on complex regulatory networks, including signaling pathways and endo-lysosomal trafficking.
- The ESCRT machinery is essential for endosomal trafficking and receptor degradation, but its role in vertebrate craniofacial development is understudied.
- Previous research established ESCRT functions in vitro and in invertebrates, highlighting a gap in vertebrate developmental studies.
Purpose of the Study:
- To investigate the role of ESCRT-II component Vps25 in murine craniofacial development.
- To characterize craniofacial anomalies in a novel Vps25 hypomorphic mouse model.
- To explore the spatiotemporal expression of ESCRT-encoding genes during mouse development and their impact on signaling pathways.
Main Methods:
- Induced mutagenesis (ENU) to generate a Vps25 hypomorphic mouse line (Vps25ENU/ENU).
- Assessment of Vps25 and other ESCRT gene expression patterns during murine development.
- Phenotypic analysis of Vps25ENU/ENU embryos, focusing on craniofacial structures.
- Investigation of NOTCH signaling pathway activity in affected craniofacial domains.
Main Results:
- Vps25 and other ESCRT genes are ubiquitously expressed, with enrichment in craniofacial, cardiac, and limb domains.
- Vps25ENU/ENU embryos exhibit late lethality and significant craniofacial defects, including mandibular hypoplasia, snout shortening, ear dysmorphia, and cleft palate.
- Perturbation of NOTCH signaling was observed in the craniofacial regions of Vps25ENU/ENU embryos.
Conclusions:
- This study provides the first evidence for a critical role of ESCRT-II in vertebrate craniofacial morphogenesis.
- The Vps25ENU/ENU mouse model recapitulates features of human congenital craniofacial syndromes.
- NOTCH signaling pathway dysregulation is implicated in the craniofacial defects observed in Vps25ENU/ENU embryos, suggesting a conserved developmental mechanism.
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