P63 Mutation Impacts the Structure of Cardiopharyngeal Field Derived Muscles in Mice

The cardiopharyngeal field (CPF) includes anterior lateral mesoderm of the first heart field and the contiguous pharyngeal mesoderm. The latter gives rise to second‐heart‐field‐derived heart regions and branchiomeric head muscles. Neural crest cells (NCCs) surround and infiltrate mesoderm cells, forming connective tissue of head and heart musculature. NCCs are also involved in the septation of the cardiac outflow tract, the patterning of pharyngeal arch arteries, and in the proper development of attachments of branchiomeric muscles to skull, jaw, hyoid bone, and laryngeal cartilages. The p63 (aka Trp63 ) transcription factor is essential for the differentiation of epithelial tissue (ectoderm and endoderm) during embryogenesis. To form the correct amount of mature ectodermal, mesodermal, and NCC populations and to control the differentiation of the CPF, p63 interacts with downstream transcription factors, such as Tbx1 , Isl1, and nkx2.5 . We analyzed how the loss of p63 expression in p63 −/− mice affected the mesodermal derivatives of the CPF. We hypothesized that the loss of p63 decreases the number of mesodermal cells differentiating from the CPF, causing a smaller mesodermal cell population in the CPF. We expected to see smaller CPF derived muscles in the p63 mutant mice, as well as deficient connective tissue in head and heart musculature as well as weakened attachments of craniofacial muscles. To determine if and when certain muscular defects appear, we performed muscle antibody staining and dissected wildtype littermates and p63 −/− mice (B6.129S7‐Trp63tm2Brd/J strain) aged embryonic day (E) 12–18. We have collected minimum of five specimens for each age. We studied craniofacial, laryngeal, as well as cardiac musculature by analyzing the antibody stained specimens under the microscope and by performing microdissections under a stereomicroscope. We used 3D computed tomography scan images (taken with a uCT scanner, Skyscan 1172) to compare the craniofacial skeletons between wildtype and mutant mice. In p63 −/− mice, branchiomeric muscles such as the masseter, buccinator, and nasolabialis were properly attached to bony elements but were smaller than what was seen in the wildtype mice. The p63 −/− head is flexed and the chin is closer to the chest than in the wildtype specimens: this posture could be caused by the shortened and less dense sternocleidomastoid muscle in the p63 −/− mice. Corresponding with previous reports, our p63 −/− mice also showed several malformations (e.g., poorly developed limbs, cleft palate, cardiac abnormalities) stereotypical of this mouse mutant and which are also seen in humans with p63 mutations such as in ADULT (acro‐dermatoungual‐lacrimal‐tooth) and EEC (Ectrodactyly‐Ectodermal Dysplasia‐Cleft Lip /Palate) syndromes. Despite that p63 expression is restricted to epithelium, the described defects reveal that p63 expression is important to much earlier, and shared developmental processes, notably NCC proliferation and differentiation, that do impact tissues derived from mesoderm as well as from epithelium. Analyzing the influence of p63 on CPF differentiation will contribute to a better understanding of regulatory mechanisms during head and heart development including the role of these mechanisms in human congenital syndromes.

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