ALX1-related Frontonasal Dysplasia Results From Defective Neural Crest Cell Development and Migration

Abstract

A pedigree of subjects with frontonasal dysplasia (FND) presented with bilateral oblique facial clefts and ocular phenotypes. Genome sequencing and analysis identified a L165F missense variant in the homeodomain of the transcription factor ALX1 which was imputed to be pathogenic. Induced pluripotent stem cells (iPSC) were derived from the subjects and differentiated to neural crest cells (NCC). NCC derived from ALX1 L165F/L165F iPSC were more sensitive to apoptosis, showed an elevated expression of several neural crest progenitor state markers, and exhibited impaired migration compared to wild type controls. NCC migration was also evaluated in vivo using lineage tracing in a zebrafish model, which revealed defective migration of the anterior NCC stream that contributes to the median portion of the anterior neurocranium, phenocopying the clinical presentation. Analysis of human NCC culture media revealed a change in the level of bone morphogenic proteins (BMP), with a low-level of BMP2 and a high level of BMP9. Soluble BMP2 and BMP9 antagonist treatments were able to rescue the defective migration phenotype. Taken together, these results demonstrate a mechanistic requirement of ALX1 in NCC development and migration.

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

“…We recently reported a pedigree with consanguineous parents and 13 children, four of whom were born with FND linked to a missense gene variant, l165f , in the homeodomain of alx1 ( Pini et al, 2020 ) ( Fig. 1 A).…”

“…The Aristaless-like ( alx ) gene family consists of ALX1 , ALX3 and ALX4 in human, mouse and zebrafish; ALX4 is represented by duplicated alx4a and alx4b in zebrafish ( Dee et al, 2013 ). In humans, ALX1 mutations are linked to severe frontonasal dysplasia (FND) and extreme microphthalmia, and ALX3 and ALX4 genes are associated with a phenotypic spectrum of hypertelorism and nasal-tip duplications ( Pini et al, 2020 ; Uz et al, 2010 ). These functions of alx genes are conserved in other vertebrates, but the pathogenic developmental mechanisms leading to the observed craniofacial malformations remain largely unknown ( Beverdam et al, 2001 ; Dee et al, 2013 ; Forsthoefel, 1963 ; Lakhwani et al, 2010 ; Lyons et al, 2016 ; Pini et al, 2020 ; Qu et al, 1999 ; Zhao et al, 1996 ).…”

“…In humans, ALX1 mutations are linked to severe frontonasal dysplasia (FND) and extreme microphthalmia, and ALX3 and ALX4 genes are associated with a phenotypic spectrum of hypertelorism and nasal-tip duplications ( Pini et al, 2020 ; Uz et al, 2010 ). These functions of alx genes are conserved in other vertebrates, but the pathogenic developmental mechanisms leading to the observed craniofacial malformations remain largely unknown ( Beverdam et al, 2001 ; Dee et al, 2013 ; Forsthoefel, 1963 ; Lakhwani et al, 2010 ; Lyons et al, 2016 ; Pini et al, 2020 ; Qu et al, 1999 ; Zhao et al, 1996 ). In mice, disruption of alx1 results in anencephaly, precluding analysis of its role in facial and ocular morphogenesis, while compound mutants of alx3 and alx4 present with FND-like defects ( Beverdam et al, 2001 ; Lakhwani et al, 2010 ).…”

“…Mouse alx family members, alx1 , alx3 and alx4 , are co-expressed in aCNC that gives rise to the anterior neurocranium (ANC), the primary palate ( Dee et al, 2013 ; McGonnell et al, 2011 ; Mitchell et al, 2021 ; Qu et al, 1999 ; ten Berge et al, 1998 ), anterior segment and periocular neural crest ( Ma and Lwigale, 2019 ; Sedykh et al, 2017 ). Recent reports demonstrate that zebrafish alx1 is required for regulating neural crest differentiation and migration in vitro and in vivo ( Pini et al, 2020 ) and that zebrafish alx3 controls ANC chondrocyte differentiation in zebrafish ( Mitchell et al, 2021 ). However, these studies do not address the role for alx function in the developing eye, nor do they examine the potential for compensatory interactions between alx1 and alx3 .…”

ALX1-transcribed LncRNA AC132217.4 Promotes Osteogenesis and Bone Healing via IGF-AKT Signaling in Mesenchymal Stem Cells

“…(39)(40)(41)(42)(43) In humans, missense mutations of the ALX1 gene cause type three frontonasal dysplasia (FND3), a rare congenital disorder characterized by abnormality of ocular and craniofacial components. (41,44,45) However, the role of ALX1 in MSC osteogenesis remains unclear due to prenatal lethality of ALX1 de ciency in mice. Our study reveals that ALX1 is among the highly up-regulated transcription factors in MSCs during osteogenesis.…”