Pbx loss in cranial neural crest, unlike in epithelium, results in cleft palate only and a broader midface

Abstract

Orofacial clefting represents the most common craniofacial birth defect. Cleft lip with or without cleft palate (CL/P) is genetically distinct from cleft palate only (CPO). Numerous transcription factors (TFs) regulate normal development of the midface, comprising the premaxilla, maxilla and palatine bones, through control of basic cellular behaviors. Within the Pbx family of genes encoding Three Amino‐acid Loop Extension (TALE) homeodomain‐containing TFs, we previously established that in the mouse, Pbx1 plays a preeminent role in midfacial morphogenesis, and Pbx2 and Pbx3 execute collaborative functions in domains of coexpression. We also reported that Pbx1 loss from cephalic epithelial domains, on a Pbx2‐ or Pbx3‐deficient background, results in CL/P via disruption of a regulatory network that controls apoptosis at the seam of frontonasal and maxillary process fusion. Conversely, Pbx1 loss in cranial neural crest cell (CNCC)‐derived mesenchyme on a Pbx2‐deficient background results in CPO, a phenotype not yet characterized. In this study, we provide in‐depth analysis of PBX1 and PBX2 protein localization from early stages of midfacial morphogenesis throughout development of the secondary palate. We further establish CNCC‐specific roles of PBX TFs and describe the developmental abnormalities resulting from their loss in the murine embryonic secondary palate. Additionally, we compare and contrast the phenotypes arising from PBX1 loss in CNCC with those caused by its loss in the epithelium and show that CNCC‐specific Pbx1 deletion affects only later secondary palate morphogenesis. Moreover, CNCC mutants exhibit perturbed rostro‐caudal organization and broadening of the midfacial complex. Proliferation defects are pronounced in CNCC mutants at gestational day (E)12.5, suggesting altered proliferation of mutant palatal progenitor cells, consistent with roles of PBX factors in maintaining progenitor cell state. Although the craniofacial skeletal abnormalities in CNCC mutants do not result from overt patterning defects, osteogenesis is delayed, underscoring a critical role of PBX factors in CNCC morphogenesis and differentiation. Overall, the characterization of tissue‐specific Pbx loss‐of‐function mouse models with orofacial clefting establishes these strains as unique tools to further dissect the complexities of this congenital craniofacial malformation. This study closely links PBX TALE homeodomain proteins to the variation in maxillary shape and size that occurs in pathological settings and during evolution of midfacial morphology.

‘Building a perfect body’: control of vertebrate organogenesis by PBX-dependent regulatory networks

“…As demonstrated by loss of function (LOF) in mice, different Pbx genes play fundamental and pleiotropic roles in organogenesis. Pbx1 homozygous mutant embryos (Pbx1 −/− ) die in utero with dramatic abnormalities in multiple organs DiMartino et al 2001;Kim et al 2002;Manley et al 2004;Brendolan et al 2005;Capellini et al 2006Capellini et al , 2008Capellini et al , 2010Capellini et al , 2011aStankunas et al 2008;Ferretti et al 2011;Vitobello et al 2011;Koss et al 2012;Hurtado et al 2015;Grebbin et al 2016;Villaescusa et al 2016;Losa et al 2018;McCulley et al 2018;Welsh et al 2018). In contrast, Pbx2 −/− mice do not display detectable abnormalities , while Pbx3 −/− mutants die perinatally, from central respiratory failure ).…”

“…There is a strong association between dysregulation of PBX-directed gene networks identified in Pbx LOF mice and human congenital defects, including cleft lip/palate (CL/P) Losa et al 2018;Welsh et al 2018), congenital asplenia (Koss et al 2012), and diabetes (Kim et al 2002;Muharram et al 2005). De novo deleterious sequence variants of PBX1 were discovered recently in children affected by a new syndrome characterized by pleiotropic developmental defects that mimick the phenotypes of Pbx1 −/− embryos.…”

“…We established that compound loss of Pbx genes results in fully penetrant orofacial clefting. Depending on the combinations of Pbx mutant alleles, abnormalities of the midfacial complex in these mice manifest as unilateral CL/P, bilateral CL/P, or cleft palate only (CPO) Welsh et al 2018), providing unique models for these human birth defects. Cephalic epithelium-specific loss of Pbx1 on a Pbx2or Pbx3-deficient background results in fully penetrant CL/P, demonstrating the critical role of the epithelium in the pathogenesis of this abnormality.…”

Evolutionarily conserved regulation of embryonic fast-twitch skeletal muscle differentiation by Pbx factors

“…Because of the redundancy of, and early requirements for, Pbx proteins in mouse embryogenesis (reviewed in Selleri et al 2019), we needed to make use of conditional loss-offunction in Myf5 Cre/+ ;Pbx1 fl/fl ;Pbx2 -/compound mutants. A similar approach, using different conditional Cre lines, has previously been taken to study Pbx functions in craniofacial, spleen, and spinal cord motor neuron development and in adult neurogenesis (Ferretti et al, 2011;Grebbin et al, 2016;Hanley et al, 2016;Koss et al, 2012;Losa et al, 2018;Welsh et al, 2018).…”

Genome-Wide Epigenomic Stress Response in the European Sea Bass (Dicentrarchus Labrax, L.)

“…Anastasiadi and Piferrer [83] showed that the domestication process in D. labrax resulted notably in genome-wide methylation differences for genes involved in the nervous system and neural crest cell differentiation. In our study, GLI2 [174], PBX1 [175], DLG1 [176] and FOXJ3 [177] are DMC-related genes involved in neural crest cell proliferation and differentiation. ROBO3 is involved in the immune response, but also implied in early neurogenesis [178], as well as NPAS3 [179].…”