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

Abstract

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 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.

aristaless-like homeobox-3 is wound induced and promotes a low-Wnt environment required for planarian head regeneration

“…The mouse Alx3 homolog is expressed in the cephalic mesenchyme and lateral mesoderm, and is developmentally important for neural tube closure and craniofacial development ( Beverdam et al, 2001 ; Lakhwani et al, 2010 ). In zebrafish, Alx3 has a well-established role in craniofacial development: Alx3 is enriched in frontonasal neural crest cells, and loss of Alx3 results in severe neurocranium development defects ( Mitchell et al, 2021 ; Yoon et al, 2022 ). In humans, homozygous mutation of Alx3 has been associated with a form of frontonasal dysplasia known as frontorhiny ( Twigg et al, 2009 ).…”

Behavioral and transcriptomic analyses of mecp2 function in zebrafish

“…RNA-Seq Transcriptome Analysis. Embryos derived from incrosses of mecp2 Q63X homozygous adults or wildtype adults were pooled in groups of three and processed for RNA extraction at 4 dpf as previously described (81). cDNA libraries were prepared using the TruSeq stranded mRNA library preparation protocol with poly-A selection and sequenced on the Illumina HiSeq.…”

TFAP2 paralogs regulate midfacial development in part through a conserved ALX genetic pathway

“…Although much less prevalent, clefts can also occur at the midline of the medial- and upper-face region forming the forehead, nose and cheeks ( Tessier, 1976 ), generating a spectrum of midface pathologies that includes frontonasal dysplasia (MIMs: 136760, 613451, 613456) ( Farlie et al, 2016 ; Vargel et al, 2021 ). Mutations in genes encoding the ALX homeodomain-containing transcription factors ( ALX1 , ALX3 and ALX4 ) are associated with frontonasal dysplasia and produce midfacial phenotypes in vertebrate species ( Beverdam et al, 2001 ; Iyyanar et al, 2022 ; Lakhwani et al, 2010 ; Lyons et al, 2016 ; McGonnell et al, 2011 ; Mitchell et al, 2021 ; Qu et al, 1999 ; Yoon et al, 2022 ). Although ALX transcription factors regulate midfacial development, how they connect with other midface genes into regulatory nodes remains uncertain.…”