Zebrafish models of alx-linked frontonasal dysplasia reveal a role for Alx1 and Alx3 in the anterior segment and vasculature of the developing eye

Biol Open. 2022 May 15;11(5):bio059189. doi: 10.1242/bio.059189. Epub 2022 Jun 7.


The cellular and genetic mechanisms that coordinate formation of facial sensory structures with surrounding skeletal and soft tissue elements remain poorly understood. Alx1, a homeobox transcription factor, is a key regulator of midfacial morphogenesis. ALX1 mutations in humans are linked to severe congenital anomalies of the facial skeleton (frontonasal dysplasia, FND) with malformation or absence of eyes and orbital contents (micro- and anophthalmia). Zebrafish with loss-of-function alx1 mutations develop with craniofacial and ocular defects of variable penetrance, likely due to compensatory upregulation in expression of a paralogous gene, alx3. Here we show that zebrafish alx1;alx3 mutants develop with highly penetrant cranial and ocular defects that resemble human ALX1-linked FND. alx1 and alx3 are expressed in anterior cranial neural crest (aCNC), which gives rise to the anterior neurocranium (ANC), anterior segment structures of the eye and vascular pericytes. Consistent with a functional requirement for alx genes in aCNC, alx1; alx3 mutants develop with nearly absent ANC and grossly aberrant hyaloid vasculature and ocular anterior segment, but normal retina. In vivo lineage labeling identified a requirement for alx1 and alx3 during aCNC migration, and transcriptomic analysis suggested oxidative stress response as a key target mechanism of this function. Oxidative stress is a hallmark of fetal alcohol toxicity, and we found increased penetrance of facial and ocular malformations in alx1 mutants exposed to ethanol, consistent with a protective role for alx1 against ethanol toxicity. Collectively, these data demonstrate a conserved role for zebrafish alx genes in controlling ocular and facial development, and a novel role in protecting these key midfacial structures from ethanol toxicity during embryogenesis. These data also reveal novel roles for alx genes in ocular anterior segment formation and vascular development and suggest that retinal deficits in alx mutants may be secondary to aberrant ocular vascularization and anterior segment defects. This study establishes robust zebrafish models for interrogating conserved genetic mechanisms that coordinate facial and ocular development, and for exploring gene--environment interactions relevant to fetal alcohol syndrome.

Keywords: ALX; Fetal alcohol syndrome; Frontonasal dysplasia; Neural crest; Ocular anterior segment.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Craniofacial Abnormalities* / genetics
  • Ethanol / metabolism
  • Face / abnormalities
  • Zebrafish*


  • Ethanol

Supplementary concepts

  • Frontonasal dysplasia