Pharyngeal morphogenesis requires fras1 - itga8 -dependent epithelial-mesenchymal interaction

Abstract

Both Fras1 and Itga8 connect mesenchymal cells to epithelia by way of an extracellular ‘Fraser protein complex’ that functions in signaling and adhesion; these proteins are vital to the development of several vertebrate organs. We previously found that zebrafish fras1 mutants have craniofacial defects, specifically, shortened symplectic cartilages and cartilage fusions that spare joint elements. During a forward mutagenesis screen, we identified a new zebrafish mutation, b1161, that we show here disrupts itga8, as confirmed using CRISPR-generated itga8 alleles. fras1 and itga8 single mutants and double mutants have similar craniofacial phenotypes, a result expected if loss of either gene disrupts function of the Fraser protein complex. Unlike fras1 mutants or other Fraser-related mutants, itga8 mutants do not show blistered tail fins. Thus, the function of the Fraser complex differs in the craniofacial skeleton and the tail fin. Focusing on the face, we find that itga8 mutants consistently show defective outpocketing of a late-forming portion of the first pharyngeal pouch, and variably express skeletal defects, matching previously characterized fras1 mutant phenotypes. In itga8 and fras1 mutants, skeletal severity varies markedly between sides, indicating that both mutants have increased developmental instability. Whereas fras1 is expressed in epithelia, we show that itga8 is expressed complementarily in facial mesenchyme. Paired with the observed phenotypic similarity, this expression indicates that the genes function in epithelial-mesenchymal interactions. Similar interactions between Fras1 and Itga8 have previously been found in mouse kidney, where these genes both regulate Nephronectin (Npnt) protein abundance. We find that zebrafish facial tissues express both npnt and the Fraser gene fibrillin2b (fbn2b), but their transcript levels do not depend on fras1 or itga8 function. Using a revertible fras1 allele, we find that the critical window for fras1 function in the craniofacial skeleton is between 1.5 and 3 days post fertilization, which coincides with the onset of fras1-dependent and itga8-dependent morphogenesis. We propose a model wherein Fras1 and Itga8 interact during late pharyngeal pouch morphogenesis to sculpt pharyngeal arches through epithelial-mesenchymal interactions, thereby stabilizing the developing craniofacial skeleton.

Transcriptome Reveals Long Non-coding RNAs and mRNAs Involved in Primary Wool Follicle Induction in Carpet Sheep Fetal Skin

“…A number of transcripts related to hair and skin development were applied to construct mRNA–mRNA and lncRNA–mRNA interaction networks. Of these, differentially expressed mRNAs were grouped and found to regulate the proliferation and differentiation of epidermal keratinocytes ( KRT1, KRT4, KRT10, KRT15 , and TP63 ), dermal-epidermal junction and basement membrane ( COL17A1, LAMA1, LAMC2, LAMC3, ITGA3, ITGA8 , and ITGB4 ), and dermal compartment ( COL5A3 and COL2A1 ) ( Pulkkinen et al, 1994 ; Kivirikko et al, 1995 ; McGrath et al, 1995 ; Mills et al, 1999 ; Imamura et al, 2000 ; Natsuga et al, 2011 ; Has et al, 2012 ; Talbot et al, 2016 ). Transcripts regulating hair placode and dermal condensation were also enriched and represented by the WNT ( WNT2B, WNT16, SFRP1, SFRP4, SERPINF1, LEF1 , and WIF1 ), EDAR ( EDAR, EDARADD , and TRAF5 ), BMP ( BMP4, BMP7, BMPER, ACVR2A, RGMA , and SOSTDC1 ), SHH ( SHH, PTCH1 , and GLI1 ), and FGF ( FGFR2 and FGF20 ) signaling pathways ( Figure 4A ).…”

Kat2a and Kat2b Acetyltransferase Activity Regulates Craniofacial Cartilage and Bone Differentiation in Zebrafish and Mice

“…These migratory and multipotent cells migrate to designated locations in the branchial arches and differentiate into either the craniofacial skeletal structures, neurons, glia of the peripheral nervous system, or melanocytes [1,4,5,9,11]. Following their arrival in the branchial arches, cNCC interact with other tissues, including the ectoderm, endoderm, and mesoderm, leading to the formation of the mandibular and maxillary prominences in mammals, and the viscerocranium and neurocranium in zebrafish [9,12,13,14,15].…”

Transgene-mediated skeletal phenotypic variation in zebrafish

“…Second, late p1 normally forms by outpocketing from the deep medial endoderm toward the outer ectoderm at just the stages that early cartilage morphogenesis is getting underway in the immediate vicinity of the pouch. Furthermore, timed heat shocks with a conditional fras1 allele revealed just when the WT gene function must be present in the endoderm for p1 to form, and for WT cartilage morphogenesis: The fras1 critical period coincides closely with late p1 outpocketing (Talbot et al, 2016). The strong suggestion from this work is that late p1 provides a key component of the embryonic environment specifically during the early period of skeletal morphogenesis.…”