# Regulation of Craniofacial Development by ALX Transcription Factors

> **NIH NIH R01** · CINCINNATI CHILDRENS HOSP MED CTR · 2021 · $663,200

## Abstract

Abstract
 Frontonasal dysplasia (FND), also known as median cleft face syndrome, is a major class of craniofacial
birth defects that profoundly impact the form and function of the face. FND patients require multiple corrective
surgeries and often suffer life-long impairment. Whilst most FND cases occur sporadically with unknown
etiology, loss-of-function mutations in each of the three ALX family genes, ALX1, ALX3, and ALX4, have been
identified as the genetic causes for autosomal recessive FND, with disruption of ALX1 associated with severe
facial clefting and extreme microphthalmia in FND3 patients while mutations in ALX3 and ALX4 resulted in
milder but clinically distinctive frontonasal malformations. Little is known about how ALX transcription factors
regulate craniofacial development, and the overall molecular mechanism controlling frontonasal development
is poorly understood. In preliminary studies, we have generated Alx1 mutant mice using CRISPR-mediated
genome editing and found that they recapitulated the FND3 phenotypes, including reduced frontonasal bones
and cartilages, cleft palate, and microphthalmia. We found that Alx1-/- embryos exhibited ectopic neuroglial
differentiation and reduction in ectomesenchymal gene expression in the frontonasal prominence. Moreover,
Alx1-/-Alx4-/- double mutant mouse embryos exhibited increased ectopic cranial ganglia and much severer
frontonasal deficiency than Alx1-/- mutants. Previous studies in multiple animal model systems revealed that
the Twist1 transcription factor, whose expression is activated in cranial neural crest cells at the onset of
migration, is critical for ectomesenchyme specification. Remarkably, while the molecular mechanism acting
downstream of Twist1 in promoting ectomesenchymal fate is still unclear, Twist1-/- mouse embryos failed to
activate the expression of all three Alx genes in cranial neural crest cells. Our finding of ectopic neuroglial
differentiation in the frontonasal regions of Alx1-/- and Alx1-/-Alx4-/- embryos suggests that Twist1 and the ALX
transcription factors act in the same molecular network to regulate cranial neural crest fate determination
between the ectomesenchymal and neuroglial lineages. The specific aims of this research project are to
determine the cellular and molecular mechanisms mediating ALX transcription factor function in frontonasal
development and to unravel and reconstruct the gene regulatory network consisting of Twist1 and ALX
transcription factors regulating cranial neural crest differentiation. Results from these studies will fill a
longstanding critical gap in craniofacial developmental biology and lead to new improvements in molecular
diagnosis and treatment/care of a large number of craniofacial disorders.

## Key facts

- **NIH application ID:** 10259802
- **Project number:** 5R01DE029417-02
- **Recipient organization:** CINCINNATI CHILDRENS HOSP MED CTR
- **Principal Investigator:** RULANG JIANG
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $663,200
- **Award type:** 5
- **Project period:** 2020-09-09 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10259802

## Citation

> US National Institutes of Health, RePORTER application 10259802, Regulation of Craniofacial Development by ALX Transcription Factors (5R01DE029417-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10259802. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*