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Abstract p73 and p63 are members of the p53 family that exhibit overlapping and distinct functions in development and homeostasis. The evaluation of p73 and p63 isoform expression across human tissue can provide greater insight to the functional interactions between family members. We determined the mRNA isoform expression patterns of TP73 and TP63 across a panel of 36 human tissues and protein expression within the highest-expressing tissues. TP73 and TP63 expression significantly correlated across tissues. In tissues with concurrent mRNA expression, nuclear co-expression of both proteins was observed in a majority of cells. Using GTEx data, we quantified p73 and p63 isoform expression in human tissue and identified that the α-isoforms of TP73 and TP63 were the predominant isoform expressed in nearly all tissues. Further, we identified a previously unreported p73 mRNA product encoded by exons 4 to 14. In sum, these data provide the most comprehensive tissue-specific atlas of p73 and p63 protein and mRNA expression patterns in human and murine samples, indicating coordinate expression of these transcription factors in the majority of tissues in which they are expressed.

We report that p73 is expressed in multiciliated cells (MCCs), is required for MCC differentiation, and directly regulates transcriptional modulators of multiciliogenesis. Loss of ciliary biogenesis provides a unifying mechanism for many phenotypes observed in p73 knockout mice including hydrocephalus; hippocampal dysgenesis; sterility; and chronic inflammation/infection of lung, middle ear, and sinus. Through p73 and p63 ChIP-seq using murine tracheal cells, we identified over 100 putative p73 target genes that regulate MCC differentiation and homeostasis. We validated Foxj1, a transcriptional regulator of multiciliogenesis, and many other cilia-associated genes as direct target genes of p73 and p63. We show p73 and p63 are co-expressed in a subset of basal cells and suggest that p73 marks these cells for MCC differentiation. In summary, p73 is essential for MCC differentiation, functions as a critical regulator of a transcriptome required for MCC differentiation, and, like p63, has an essential role in development of tissues.

Summary: We report that p73 is expressed in ovarian granulosa cells and that loss of p73 leads to attenuated follicle development, ovulation, and corpus luteum formation, resulting in decreased levels of circulating progesterone and defects in mammary gland branching. Ectopic progesterone in p73-deficient mice completely rescued the mammary branching and partially rescued the ovarian follicle development defects. Performing RNA sequencing (RNA-seq) on transcripts from murine wild-type and p73-deficient antral follicles, we discovered differentially expressed genes that regulate biological adhesion programs. Through modulation of p73 expression in murine granulosa cells and transformed cell lines, followed by RNA-seq and chromatin immunoprecipitation sequencing, we discovered p73-dependent regulation of a gene set necessary for cell adhesion and migration and components of the focimatrix (focal intra-epithelial matrix), a basal lamina between granulosa cells that promotes follicle maturation. In summary, p73 is essential for ovarian folliculogenesis and functions as a key regulator of a gene network involved in cell-to-cell adhesion and migration. : Molecular Network; Functional Aspects of Cell Biology; Developmental Biology; Omics Subject Areas: Molecular Network, Functional Aspects of Cell Biology, Developmental Biology, Omics

p63 is a transcriptional regulator of ectodermal development that is required for basal cell proliferation and stem cell maintenance. p73 is a closely related p53 family member that is expressed in select p63-positive basal cells and can heterodimerize with p63. p73-/- mice lack multiciliated cells and have reduced numbers of basal epithelial cells in select tissues; however, the role of p73 in basal epithelial cells is unknown. Herein, we show that p73-deficient mice exhibit delayed wound healing despite morphologically normal-appearing skin. The delay in wound healing is accompanied by decreased proliferation and increased levels of biomarkers of the DNA damage response in basal keratinocytes at the epidermal wound edge. In wild-type mice, this same cell population exhibited increased p73 expression after wounding. Analyzing single-cell transcriptomic data, we found that p73 was expressed by epidermal and hair follicle stem cells, cell types required for wound healing. Moreover, we discovered that p73 isoforms expressed in the skin (ΔNp73) enhance p63-mediated expression of keratinocyte genes during cellular reprogramming from a mesenchymal to basal keratinocyte-like cell. We identified a set of 44 genes directly or indirectly regulated by ΔNp73 that are involved in skin development, cell junctions, cornification, proliferation, and wound healing. Our results establish a role for p73 in cutaneous wound healing through regulation of basal keratinocyte function.

In many animal species the meiosis I spindle in oocytes is anastral and lacks centrosomes. Previous studies of Drosophila oocytes failed to detect the native form of the germline-specific γ-tubulin (γTub37C) in meiosis I spindles, and genetic studies have yielded conflicting data regarding the role of γTub37C in the formation of bipolar spindles at meiosis I. Our examination of living and fixed oocytes carrying either a null allele or strong missense mutation in the γtub37C gene demonstrates a role for γTub37C in the positioning of the oocyte nucleus during late prophase, as well as in the formation and maintenance of bipolar spindles in Drosophila oocytes. Prometaphase I spindles in γtub37C mutant oocytes showed wide, non-tapered spindle poles and disrupted positioning. Additionally, chromosomes failed to align properly on the spindle and showed morphological defects. The kinetochores failed to properly co-orient and often lacked proper attachments to the microtubule bundles, suggesting that γTub37C is required to stabilize kinetochore microtubule attachments in anastral spindles. Although spindle bipolarity was sometimes achieved by metaphase I in both γtub37C mutants, the resulting chromosome masses displayed highly disrupted chromosome alignment. Therefore, our data conclusively demonstrate a role for γTub37C in both the formation of the anastral meiosis I spindle and in the proper attachment of kinetochore microtubules. Finally, multispectral imaging demonstrates the presences of native γTub37C along the length of wild-type meiosis I spindles.