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?Np63 knockout mice reveal its indispensable role as a master regulator of epithelial development and differentiation.

ABSTRACT: The transcription factor p63 is important in the development of the skin as p63-null mice exhibit striking defects in embryonic epidermal morphogenesis. Understanding the mechanisms that underlie this phenotype is complicated by the existence of multiple p63 isoforms, including TAp63 and ?Np63. To investigate the role of ?Np63 in epidermal morphogenesis we generated ?Np63 knock-in mice in which the ?Np63-specific exon is replaced by GFP. Homozygous ?Np63(gfp/gfp) animals exhibit severe developmental anomalies including truncated forelimbs and the absence of hind limbs, largely phenocopying existing knockouts in which all p63 isoforms are deleted. ?Np63-null animals show a poorly developed stratified epidermis comprising isolated clusters of disorganized epithelial cells. Despite the failure to develop a mature stratified epidermis, the patches of ?Np63-null keratinocytes are able to stratify and undergo a program of terminal differentiation. However, we observe premature expression of markers associated with terminal differentiation, which is unique to ?Np63-null animals and not evident in the skin of mice lacking all p63 isoforms. We posit that the dysregulated and accelerated keratinocyte differentiation phenotype is driven by significant alterations in the expression of key components of the Notch signaling pathway, some of which are direct transcriptional targets of ?Np63 as demonstrated by ChIP experiments. The analysis of ?Np63(gfp/gfp) knockout mice reaffirms the indispensable role of the ?N isoform of p63 in epithelial biology and confirms that ?Np63-null keratinocytes are capable of committing to an epidermal cell lineage, but are likely to suffer from diminished renewal capacity and an altered differentiation fate.

SUBMITTER: Romano RA

PROVIDER: S-EPMC3265062 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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ΔNp63 knockout mice reveal its indispensable role as a master regulator of epithelial development and differentiation.

Romano Rose-Anne RA Smalley Kirsten K Magraw Caitlin C Serna Vanida Ann VA Kurita Takeshi T Raghavan Srikala S Sinha Satrajit S

Development (Cambridge, England) 20120201 4

The transcription factor p63 is important in the development of the skin as p63-null mice exhibit striking defects in embryonic epidermal morphogenesis. Understanding the mechanisms that underlie this phenotype is complicated by the existence of multiple p63 isoforms, including TAp63 and ΔNp63. To investigate the role of ΔNp63 in epidermal morphogenesis we generated ΔNp63 knock-in mice in which the ΔNp63-specific exon is replaced by GFP. Homozygous ΔNp63(gfp/gfp) animals exhibit severe developme ...[more]

PMID: 22274697

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Project description:STUDY QUESTION:Does the upregulation of the zinc finger E-box binding homeobox 2 (ZEB2) transcription factor in human trophoblast cells lead to alterations in gene expression consistent with an epithelial-mesenchymal transition (EMT) and a consequent increase in invasiveness? SUMMARY ANSWER:Overexpression of ZEB2 results in an epithelial-mesenchymal shift in gene expression accompanied by a substantial increase in the invasive capacity of human trophoblast cells. WHAT IS KNOWN ALREADY:In-vivo results have shown that cytotrophoblast differentiation into extravillous trophoblast involves an epithelial-mesenchymal transition. The only EMT master regulatory factor which shows changes consistent with extravillous trophoblast EMT status and invasive capacity is the ZEB2 transcription factor. STUDY DESIGN, SIZE, DURATION:This study is a mechanistic investigation of the role of ZEB2 in trophoblast differentiation. We generated stable ZEB2 overexpression clones using the epithelial BeWo and JEG3 choriocarcinoma lines. Using these clones, we investigated the effects of ZEB2 overexpression on the expression of EMT-associated genes and proteins, cell morphology and invasive capability. PARTICIPANTS/MATERIALS, SETTING, METHODS:We used lentiviral transduction to overexpress ZEB2 in BeWo and JEG3 cells. Stable clones were selected based on ZEB2 expression and morphology. A PCR array of EMT-associated genes was used to probe gene expression. Protein measurements were performed by western blotting. Gain-of-function was assessed by quantitatively measuring cell invasion rates using a Transwell assay, a 3D bioprinted placenta model and the xCelligenceTM platform. MAIN RESULTS AND THE ROLE OF CHANCE:The four selected clones (2 × BeWo, 2 × JEG3, based on ZEB2 expression and morphology) all showed gene expression changes indicative of an EMT. The two clones (1 × BeWo, 1 × JEG3) showing >40-fold increase in ZEB2 expression also displayed increased ZEB2 protein; the others, with increases in ZEB2 expression <14-fold did not. The two high ZEB2-expressing clones demonstrated robust increases in invasive capacity, as assessed by three types of invasion assay. These data identify ZEB2-mediated transcription as a key mechanism transforming the epithelial-like trophoblast into cells with a mesenchymal, invasive phenotype. LARGE SCALE DATA:PCR array data have been deposited in the GEO database under accession number GSE116532. LIMITATIONS, REASONS FOR CAUTION:These are in-vitro studies using choriocarcinoma cells and so the results should be interpreted in view of these limitations. Nevertheless, the data are consistent with in-vivo findings and are replicated in two different cell lines. WIDER IMPLICATIONS OF THE FINDINGS:The combination of these data with the in-vivo findings clearly identify ZEB2-mediated EMT as the mechanism for cytotrophoblast differentiation into extravillous trophoblast. Having characterized these cellular mechanisms, it will now be possible to identify the intracellular and extracellular regulatory components which control ZEB2 and trophoblast differentiation. It will also be possible to identify the aberrant factors which alter differentiation in invasive pathologies such as preeclampsia and abnormally invasive placenta (AKA accreta, increta, percreta). STUDY FUNDING AND COMPETING INTEREST(s):Funding was provided by the Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine and Surgery at Hackensack Meridian Health, Hackensack, NJ. The 3D bioprinted placental model work done in Drs Kim and Fisher's labs was supported by the Children's National Medical Center. The xCELLigence work done in Dr Birge's lab was supported by NIH CA165077. The authors declare no competing interests.

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?Np63 knockout mice reveal its indispensable role as a master regulator of epithelial development and differentiation.

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ΔNp63 knockout mice reveal its indispensable role as a master regulator of epithelial development and differentiation.

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