Abstract
Loss of ALX1 function causes the frontonasal dysplasia syndrome FND3, characterized by severe facial clefting and microphthalmia. Whereas the laboratory mouse has been the preeminent animal model for studying developmental mechanisms of human craniofacial birth defects, the roles of ALX1 in mouse frontonasal development have not been well characterized because the only previously reported Alx1 mutant mouse line exhibited acrania due to a genetic background-dependent failure of cranial neural tube closure. Using CRISPR/Cas9-mediated genome editing, we have generated an Alx1-deletion mouse model that recapitulates the FND craniofacial malformations, including median orofacial clefting and disruption of development of the eyes and alae nasi. In situ hybridization analysis showed that Alx1 is strongly expressed in frontonasal neural crest cells that give rise to periocular and frontonasal mesenchyme. Alx1 del/del embryos exhibited increased apoptosis of periocular mesenchyme and decreased expression of ocular developmental regulators Pitx2 and Lmxb1 in the periocular mesenchyme, followed by defective optic stalk morphogenesis. Moreover, Alx1 del/del embryos exhibited disruption of frontonasal mesenchyme identity, with loss of expression of Pax7 and concomitant ectopic expression of the jaw mesenchyme regulators Lhx6 and Lhx8 in the developing lateral nasal processes. The function of ALX1 in patterning the frontonasal mesenchyme is partly complemented by ALX4, a paralogous ALX family transcription factor whose loss-of-function causes a milder and distinctive FND. Together, these data uncover previously unknown roles of ALX1 in periocular mesenchyme development and frontonasal mesenchyme patterning, providing novel insights into the pathogenic mechanisms of ALX1-related FND.
Highlights
Frontonasal dysplasia (FND), known as median cleft face syndrome, is a group of congenital craniofacial disorders characterized by ocular hypertelorism, midline facial cleft affecting the nose and/or upper lip and palate, broad and flattened nasal bridge, notching or clefting of the nasal alae, and is sometimes associated with anterior cranium bifidum and other malformations (Wu et al, 2007; Kayserili et al, 2009; Twigg et al, 2009; Farlie et al, 2016)
ALX1 Patterns the Midface other connective tissues in the face are derived from a transient embryonic cell population called the cranial neural crest cells (CNCCs), which arise at the anterior neural plate border in human embryos in the third week of gestation, corresponding to about embryonic day (E) 8.0 in mice (Jiang et al, 2000; O’Rahilly and Müller, 2007; Yoshida et al, 2008; Zalc et al, 2021)
To further verify that the frontonasal neural crest cells migrated normally to the FNP in the Alx1del/del embryos, we analyzed the expression of known frontonasal mesenchyme markers, Alx3 and Alx4, and found that both were expressed in the frontonasal prominence in the control and Alx1del/del littermates at E9.5 (Figures 5E–H). These results indicate that early migration of cranial neural crest cells to the FNP was not overtly affected in the Alx1del/del mouse embryos, which is consistent with our finding that Alx1 expression was absent in early migrating CNCCs and was highly expressed in postmigratory periocular CNCCs and the frontonasal mesenchyme
Summary
Frontonasal dysplasia (FND), known as median cleft face syndrome, is a group of congenital craniofacial disorders characterized by ocular hypertelorism, midline facial cleft affecting the nose and/or upper lip and palate, broad and flattened nasal bridge, notching or clefting of the nasal alae, and is sometimes associated with anterior cranium bifidum and other malformations (Wu et al, 2007; Kayserili et al, 2009; Twigg et al, 2009; Farlie et al, 2016). The first group of CNCCs delaminate from the region lateral to the prospective forebrain and anterior midbrain, migrate ventrally to surround the ventral forebrain and interact with both neural and surface ectoderm to form the embryonic frontonasal prominence (FNP) by mid-fourth week of human gestation, corresponding to about E9.0 in mice (Jiang et al, 2006). The second group of CNCCs delaminate at the posterior midbrain and anterior hindbrain level and migrate ventrally to interact with the surface ectoderm to form the maxillary and mandibular processes. Formation of the intact upper lip involves extensive directional growth and subsequent fusion of the MNP, LNP, and MxP as well as merging of the nasal processes to fill the facial midline (Jiang et al, 2006). The causes of FND are complex and could result from genetic and/or environmental perturbations of CNCC migration, proliferation, survival, differentiation, or the lip fusion processes
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