Embryogenesis and Adult Life in the Absence of Intrinsic Apoptosis Effectors BAX, BAK, and BOK.

Intrinsic apoptosis, reliant on BAX and BAK, has been postulated to be fundamental for morphogenesis, but its precise contribution to this process has not been fully explored in mammals. Our structural analysis of BOK suggests close resemblance to BAX and BAK structures. Notably, Bok-/-Bax-/-Bak-/- animals exhibited more severe defects and died earlier than Bax-/-Bak-/- mice, implying that BOK has overlapping roles with BAX and BAK during developmental cell death. By analyzing Bok-/-Bax-/-Bak-/- triple-knockout mice whose cells are incapable of undergoing intrinsic apoptosis, we identified tissues that formed well without this process. We provide evidence that necroptosis, pyroptosis, or autophagy does not substantially substitute for the loss of apoptosis. Albeit very rare, unexpected attainment of adult Bok-/-Bax-/-Bak-/- mice suggests that morphogenesis can proceed entirely without apoptosis mediated by these proteins and possibly without cell death in general.

ING4 and ING5 are essential for histone H3 lysine 14 acetylation and epicardial cell lineage development.

Inhibitor of growth 4 and 5 (ING4, ING5) are structurally similar chromatin-binding proteins in the KAT6A, KAT6B and KAT7 histone acetyltransferase protein complexes. Heterozygous mutations in the KAT6A or KAT6B gene cause human disorders with cardiac defects, but the contribution of their chromatin-adaptor proteins to development is unknown. We found that Ing5-/- mice had isolated cardiac ventricular septal defects. Ing4-/-Ing5-/- embryos failed to undergo chorioallantoic fusion and arrested in development at embryonic day 8.5, displaying loss of histone H3 lysine 14 acetylation, reduction in H3 lysine 23 acetylation levels and reduced developmental gene expression. Embryonic day 12.5 Ing4+/-Ing5-/- hearts showed a paucity of epicardial cells and epicardium-derived cells, failure of myocardium compaction, and coronary vasculature defects, accompanied by reduced expression of epicardium genes. Cell adhesion gene expression and proepicardium outgrowth were defective in the ING4- and ING5-deficient state. Our findings suggest that ING4 and ING5 are essential for heart development and promote epicardium and epicardium-derived cell fates and imply mutation of the human ING5 gene as a possible cause of isolated ventricular septal defects.

Slc35a2 mosaic knockout impacts cortical development, dendritic arborisation, and neuronal firing.

Mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE) is an important cause of drug-resistant epilepsy. A significant subset of individuals diagnosed with MOGHE display somatic mosaicism for loss-of-function variants in SLC35A2, which encodes the UDP-galactose transporter. We developed a mouse model to investigate how disruption of this transporter leads to a malformation of cortical development. We used in utero electroporation and CRISPR/Cas9 to knockout Slc35a2 in a subset of layer 2/3 cortical neuronal progenitors in the developing brains of male and female fetal mice to model mosaic expression. Mosaic Slc35a2 knockout was verified through next-generation sequencing and immunohistochemistry of GFP-labelled transfected cells. Histology of brain tissue in mosaic Slc35a2 knockout mice revealed the presence of upper layer-derived cortical neurons in the white matter. Reconstruction of single filled neurons identified altered dendritic arborisation with Slc35a2 knockout neurons having increased complexity. Whole-cell electrophysiological recordings revealed that Slc35a2 knockout neurons display reduced action potential firing, increased afterhyperpolarisation duration and reduced burst-firing when compared with control neurons. Mosaic Slc35a2 knockout mice also exhibited significantly increased epileptiform spiking and increased locomotor activity. We successfully generated a mouse model of mosaic Slc35a2 deficiency, which recapitulates features of the human phenotype, including impaired neuronal migration. We show that knockout in layer 2/3 cortical neuron progenitors is sufficient to disrupt neuronal excitability, increase epileptiform activity and cause hyperactivity in mosaic mice. Our mouse model provides an opportunity to further investigate the disease mechanisms that contribute to MOGHE and facilitate the development of precision therapies.

Inhibitors of histone acetyltransferases KAT6A/B induce senescence and arrest tumour growth.

Acetylation of histones by lysine acetyltransferases (KATs) is essential for chromatin organization and function1. Among the genes coding for the MYST family of KATs (KAT5-KAT8) are the oncogenes KAT6A (also known as MOZ) and KAT6B (also known as MORF and QKF)2,3. KAT6A has essential roles in normal haematopoietic stem cells4-6 and is the target of recurrent chromosomal translocations, causing acute myeloid leukaemia7,8. Similarly, chromosomal translocations in KAT6B have been identified in diverse cancers8. KAT6A suppresses cellular senescence through the regulation of suppressors of the CDKN2A locus9,10, a function that requires its KAT activity10. Loss of one allele of KAT6A extends the median survival of mice with MYC-induced lymphoma from 105 to 413 days11. These findings suggest that inhibition of KAT6A and KAT6B may provide a therapeutic benefit in cancer. Here we present highly potent, selective inhibitors of KAT6A and KAT6B, denoted WM-8014 and WM-1119. Biochemical and structural studies demonstrate that these compounds are reversible competitors of acetyl coenzyme A and inhibit MYST-catalysed histone acetylation. WM-8014 and WM-1119 induce cell cycle exit and cellular senescence without causing DNA damage. Senescence is INK4A/ARF-dependent and is accompanied by changes in gene expression that are typical of loss of KAT6A function. WM-8014 potentiates oncogene-induced senescence in vitro and in a zebrafish model of hepatocellular carcinoma. WM-1119, which has increased bioavailability, arrests the progression of lymphoma in mice. We anticipate that this class of inhibitors will help to accelerate the development of therapeutics that target gene transcription regulated by histone acetylation.

Control of skeletal morphogenesis by the Hippo-YAP/TAZ pathway.

The Hippo-YAP/TAZ pathway is an important regulator of tissue growth, but can also control cell fate or tissue morphogenesis. Here, we investigate the function of the Hippo pathway during the development of cartilage, which forms the majority of the skeleton. Previously, YAP was proposed to inhibit skeletal size by repressing chondrocyte proliferation and differentiation. We find that, in vitro, Yap/Taz double knockout impairs murine chondrocyte proliferation, whereas constitutively nuclear nls-YAP5SA accelerates proliferation, in line with the canonical role of this pathway in most tissues. However, in vivo, cartilage-specific knockout of Yap/Taz does not prevent chondrocyte proliferation, differentiation or skeletal growth, but rather results in various skeletal deformities including cleft palate. Cartilage-specific expression of nls-YAP5SA or knockout of Lats1/2 do not increase cartilage growth, but instead lead to catastrophic malformations resembling chondrodysplasia or achondrogenesis. Physiological YAP target genes in cartilage include Ctgf, Cyr61 and several matrix remodelling enzymes. Thus, YAP/TAZ activity controls chondrocyte proliferation in vitro, possibly reflecting a regenerative response, but is dispensable for chondrocyte proliferation in vivo, and instead functions to control cartilage morphogenesis via regulation of the extracellular matrix.

PKN2 deficiency leads both to prenatal 'congenital' cardiomyopathy and defective angiotensin II stress responses.

The protein kinase PKN2 is required for embryonic development and PKN2 knockout mice die as a result of failure in the expansion of mesoderm, cardiac development and neural tube closure. In the adult, cardiomyocyte PKN2 and PKN1 (in combination) are required for cardiac adaptation to pressure-overload. The specific role of PKN2 in contractile cardiomyocytes during development and its role in the adult heart remain to be fully established. We used mice with cardiomyocyte-directed knockout of PKN2 or global PKN2 haploinsufficiency to assess cardiac development and function using high resolution episcopic microscopy, MRI, micro-CT and echocardiography. Biochemical and histological changes were also assessed. Cardiomyocyte-directed PKN2 knockout embryos displayed striking abnormalities in the compact myocardium, with frequent myocardial clefts and diverticula, ventricular septal defects and abnormal heart shape. The sub-Mendelian homozygous knockout survivors developed cardiac failure. RNASeq data showed up-regulation of PKN2 in patients with dilated cardiomyopathy, suggesting an involvement in adult heart disease. Given the rarity of homozygous survivors with cardiomyocyte-specific deletion of PKN2, the requirement for PKN2 in adult mice was explored using the constitutive heterozygous PKN2 knockout. Cardiac hypertrophy resulting from hypertension induced by angiotensin II was reduced in these haploinsufficient PKN2 mice relative to wild-type littermates, with suppression of cardiomyocyte hypertrophy and cardiac fibrosis. It is concluded that cardiomyocyte PKN2 is essential for heart development and the formation of compact myocardium and is also required for cardiac hypertrophy in hypertension. Thus, PKN signalling may offer therapeutic options for managing congenital and adult heart diseases.

MOZ directs the distal-less homeobox gene expression program during craniofacial development.

Oral clefts are common birth defects. Individuals with oral clefts who have identical genetic mutations regularly present with variable penetrance and severity. Epigenetic or chromatin-mediated mechanisms are commonly invoked to explain variable penetrance. However, specific examples of these are rare. Two functional copies of the MOZ (KAT6A, MYST3) gene, encoding a MYST family lysine acetyltransferase chromatin regulator, are essential for human craniofacial development, but the molecular role of MOZ in this context is unclear. Using genetic interaction and genomic studies, we have investigated the effects of loss of MOZ on the gene expression program during mouse development. Among the more than 500 genes differentially expressed after loss of MOZ, 19 genes had previously been associated with cleft palates. These included four distal-less homeobox (DLX) transcription factor-encoding genes, Dlx1, Dlx2, Dlx3 and Dlx5 and DLX target genes (including Barx1, Gbx2, Osr2 and Sim2). MOZ occupied the Dlx5 locus and was required for normal levels of histone H3 lysine 9 acetylation. MOZ affected Dlx gene expression cell-autonomously within neural crest cells. Our study identifies a specific program by which the chromatin modifier MOZ regulates craniofacial development.

Enigma proteins regulate YAP mechanotransduction.

Human cells can sense mechanical stress acting upon integrin adhesions and respond by sending the YAP (also known as YAP1) and TAZ (also known as WWTR1) transcriptional co-activators to the nucleus to drive TEAD-dependent transcription of target genes. How integrin signaling activates YAP remains unclear. Here, we show that integrin-mediated mechanotransduction requires the Enigma and Enigma-like proteins (PDLIM7 and PDLIM5, respectively; denoted for the family of PDZ and LIM domain-containing proteins). YAP binds to PDLIM5 and PDLIM7 (hereafter PDLIM5/7) via its C-terminal PDZ-binding motif (PBM), which is essential for full nuclear localization and activity of YAP. Accordingly, silencing of PDLIM5/7 expression reduces YAP nuclear localization, tyrosine phosphorylation and transcriptional activity. The PDLIM5/7 proteins are recruited from the cytoplasm to integrin adhesions and F-actin stress fibers in response to force by binding directly to the key stress fiber component α-actinin. Thus, forces acting on integrins recruit Enigma family proteins to trigger YAP activation during mechanotransduction.This article has an associated First Person interview with the first author of the paper.

Infantile neurodegenerative disorder associated with mutations in TBCD, an essential gene in the tubulin heterodimer assembly pathway.

Mutation in a growing spectrum of genes is known to either cause or contribute to primary or secondary microcephaly. In primary microcephaly the genetic determinants frequently involve mutations that contribute to or modulate the microtubule cytoskeleton by causing perturbations of neuronal proliferation and migration. Here we describe four patients from two unrelated families each with an infantile neurodegenerative disorder characterized by loss of developmental milestones at 9­24 months of age followed by seizures, dystonia and acquired microcephaly. The patients harboured homozygous missense mutations (A475T and A586V) in TBCD, a gene encoding one of five tubulin-specific chaperones (termed TBCA-E) that function in concert as a nanomachine required for the de novo assembly of the α/ß tubulin heterodimer. The latter is the subunit from which microtubule polymers are assembled. We found a reduced intracellular abundance of TBCD in patient fibroblasts to about 10% (in the case of A475T) or 40% (in the case of A586V) compared to age-matched wild type controls. Functional analyses of the mutant proteins revealed a partially compromised ability to participate in the heterodimer assembly pathway. We show via in utero shRNA-mediated suppression that a balanced supply of tbcd is critical for cortical cell proliferation and radial migration in the developing mouse brain. We conclude that TBCD is a novel functional contributor to the mammalian cerebral cortex development, and that the pathological mechanism resulting from the mutations we describe is likely to involve compromised interactions with one or more TBCD-interacting effectors that influence the dynamics and behaviour of the neuronal cytoskeleton.

MOZ and BMI1 play opposing roles during Hox gene activation in ES cells and in body segment identity specification in vivo.

Hox genes underlie the specification of body segment identity in the anterior-posterior axis. They are activated during gastrulation and undergo a dynamic shift from a transcriptionally repressed to an active chromatin state in a sequence that reflects their chromosomal location. Nevertheless, the precise role of chromatin modifying complexes during the initial activation phase remains unclear. In the current study, we examined the role of chromatin regulators during Hox gene activation. Using embryonic stem cell lines lacking the transcriptional activator MOZ and the polycomb-family repressor BMI1, we showed that MOZ and BMI1, respectively, promoted and repressed Hox genes during the shift from the transcriptionally repressed to the active state. Strikingly however, MOZ but not BMI1 was required to regulate Hox mRNA levels after the initial activation phase. To determine the interaction of MOZ and BMI1 in vivo, we interrogated their role in regulating Hox genes and body segment identity using Moz;Bmi1 double deficient mice. We found that the homeotic transformations and shifts in Hox gene expression boundaries observed in single Moz and Bmi1 mutant mice were rescued to a wild type identity in Moz;Bmi1 double knockout animals. Together, our findings establish that MOZ and BMI1 play opposing roles during the onset of Hox gene expression in the ES cell model and during body segment identity specification in vivo. We propose that chromatin-modifying complexes have a previously unappreciated role during the initiation phase of Hox gene expression, which is critical for the correct specification of body segment identity.

MOZ regulates B-cell progenitors and, consequently, Moz haploinsufficiency dramatically retards MYC-induced lymphoma development.

The histone acetyltransferase MOZ (MYST3, KAT6A) is the target of recurrent chromosomal translocations fusing the MOZ gene to CBP, p300, NCOA3, or TIF2 in particularly aggressive cases of acute myeloid leukemia. In this study, we report the role of wild-type MOZ in regulating B-cell progenitor proliferation and hematopoietic malignancy. In the Eµ-Myc model of aggressive pre-B/B-cell lymphoma, the loss of just one allele of Moz increased the median survival of mice by 3.9-fold. MOZ was required to maintain the proliferative capacity of B-cell progenitors, even in the presence of c-MYC overexpression, by directly maintaining the transcriptional activity of genes required for normal B-cell development. Hence, B-cell progenitor numbers were significantly reduced in Moz haploinsufficient animals. Interestingly, we find a significant overlap in genes regulated by MOZ, mixed lineage leukemia 1, and mixed lineage leukemia 1 cofactor menin. This includes Meis1, a TALE class homeobox transcription factor required for B-cell development, characteristically upregulated as a result of MLL1 translocations in leukemia. We demonstrate that MOZ localizes to the Meis1 locus in pre-B-cells and maintains Meis1 expression. Our results suggest that even partial inhibition of MOZ may reduce the proliferative capacity of MEIS1, and HOX-driven lymphoma and leukemia cells.

Mesodermal expression of Moz is necessary for cardiac septum development.

Ventricular septal defects (VSDs) are the most commonly occurring congenital heart defect. They are regularly associated with complex syndromes, including DiGeorge syndrome and Holt-Oram syndrome, which are characterised by haploinsufficiency for the T-box transcription factors TBX1 and TBX5, respectively. The histone acetyltransferase monocytic leukaemia zinc finger protein, MOZ (MYST3/KAT6A), is required for the expression of the Tbx1 and Tbx5 genes. Homozygous loss of MOZ results in DiGeorge syndrome-like defects including VSD. The Moz gene is expressed in the ectodermal, mesodermal and endodermal aspects of the developing pharyngeal apparatus and heart; however it is unclear in which of these tissues MOZ is required for heart development. The role of MOZ in the activation of Tbx1 would suggest a requirement for MOZ in the mesoderm, because deletion of Tbx1 in the mesoderm causes VSDs. Here, we investigated the tissue-specific requirements for MOZ in the mesoderm. We demonstrate that Mesp1-cre-mediated deletion of Moz results in high penetrance of VSDs and overriding aorta and a significant decrease in MOZ-dependant Tbx1 and Tbx5 expression. Together, our data suggest that the molecular pathogenesis of VSDs in Moz germline mutant mice is due to loss of MOZ-dependant activation of mesodermal Tbx1 and Tbx5 expression.

Increasing histone acetylation improves sociability and restores learning and memory in KAT6B-haploinsufficient mice.

Mutations in genes encoding chromatin modifiers are enriched among mutations causing intellectual disability. The continuing development of the brain postnatally, coupled with the inherent reversibility of chromatin modifications, may afford an opportunity for therapeutic intervention following a genetic diagnosis. Development of treatments requires an understanding of protein function and models of the disease. Here, we provide a mouse model of Say-Barber-Biesecker-Young-Simpson syndrome (SBBYSS) (OMIM 603736) and demonstrate proof-of-principle efficacy of postnatal treatment. SBBYSS results from heterozygous mutations in the KAT6B (MYST4/MORF/QFK) gene and is characterized by intellectual disability and autism-like behaviors. Using human cells carrying SBBYSS-specific KAT6B mutations and Kat6b heterozygous mice (Kat6b+/-), we showed that KAT6B deficiency caused a reduction in histone H3 lysine 9 acetylation. Kat6b+/- mice displayed learning, memory, and social deficits, mirroring SBBYSS individuals. Treatment with a histone deacetylase inhibitor, valproic acid, or an acetyl donor, acetyl-carnitine (ALCAR), elevated histone acetylation levels in the human cells with SBBYSS mutations and in brain and blood cells of Kat6b+/- mice and partially reversed gene expression changes in Kat6b+/- cortical neurons. Both compounds improved sociability in Kat6b+/- mice, and ALCAR treatment restored learning and memory. These data suggest that a subset of SBBYSS individuals may benefit from postnatal therapeutic interventions.

The chromatin reader protein ING5 is required for normal hematopoietic cell numbers in the fetal liver.

ING5 is a component of KAT6A and KAT7 histone lysine acetylation protein complexes. ING5 contains a PHD domain that binds to histone H3 lysine 4 when it is trimethylated, and so functions as a 'reader' and adaptor protein. KAT6A and KAT7 function are critical for normal hematopoiesis. To examine the function of ING5 in hematopoiesis, we generated a null allele of Ing5. Mice lacking ING5 during development had decreased foetal liver cellularity, decreased numbers of hematopoietic stem cells and perturbed erythropoiesis compared to wild-type control mice. Ing5-/- pups had hypoplastic spleens. Competitive transplantation experiments using foetal liver hematopoietic cells showed that there was no defect in long-term repopulating capacity of stem cells lacking ING5, suggesting that the defects during the foetal stage were not cell intrinsic. Together, these results suggest that ING5 function is dispensable for normal hematopoiesis but may be required for timely foetal hematopoiesis in a cell-extrinsic manner.

SMCHD1 has separable roles in chromatin architecture and gene silencing that could be targeted in disease.

The interplay between 3D chromatin architecture and gene silencing is incompletely understood. Here, we report a novel point mutation in the non-canonical SMC protein SMCHD1 that enhances its silencing capacity at endogenous developmental targets. Moreover, it also results in enhanced silencing at the facioscapulohumeral muscular dystrophy associated macrosatellite-array, D4Z4, resulting in enhanced repression of DUX4 encoded by this repeat. Heightened SMCHD1 silencing perturbs developmental Hox gene activation, causing a homeotic transformation in mice. Paradoxically, the mutant SMCHD1 appears to enhance insulation against other epigenetic regulators, including PRC2 and CTCF, while depleting long range chromatin interactions akin to what is observed in the absence of SMCHD1. These data suggest that SMCHD1's role in long range chromatin interactions is not directly linked to gene silencing or insulating the chromatin, refining the model for how the different levels of SMCHD1-mediated chromatin regulation interact to bring about gene silencing in normal development and disease.

CRISPR-Mediated Protein Tagging with Nanoluciferase to Investigate Native Chemokine Receptor Function and Conformational Changes.

G protein-coupled receptors are a major class of membrane receptors that mediate physiological and pathophysiological cellular signaling. Many aspects of receptor activation and signaling can be investigated using genetically encoded luminescent fusion proteins. However, the use of these biosensors in live cell systems requires the exogenous expression of the tagged protein of interest. To maintain the normal cellular context here we use CRISPR/Cas9-mediated homology-directed repair to insert luminescent tags into the endogenous genome. Using NanoLuc and bioluminescence resonance energy transfer we demonstrate fluorescent ligand binding at genome-edited chemokine receptors. We also demonstrate that split-NanoLuc complementation can be used to investigate conformational changes and internalization of CXCR4 and that recruitment of ß-arrestin2 to CXCR4 can be monitored when both proteins are natively expressed. These results show that genetically encoded luminescent biosensors can be used to investigate numerous aspects of receptor function at native expression levels.

A citizen science model for implementing statewide educational DNA barcoding.

Our aim was to develop a widely available educational program in which students conducted authentic research that met the expectations of both the scientific and educational communities. This paper describes the development and implementation of a citizen science project based on DNA barcoding of reptile specimens obtained from the Museums Victoria frozen tissue collection. The student program was run by the Gene Technology Access Centre (GTAC) and was delivered as a "one day plus one lesson" format incorporating a one-day wet laboratory workshop followed by a single lesson at school utilising online bioinformatics tools. The project leveraged the complementary resources and expertise of the research and educational partners to generate robust scientific data that could be analysed with confidence, meet the requirements of the Victorian state education curriculum, and provide participating students with an enhanced learning experience. During two 1-week stints in 2013 and 2014, 406 students mentored by 44 postgraduate university students participated in the project. Students worked mainly in pairs to process ~200 tissue samples cut from 53 curated reptile specimens representing 17 species. A total of 27 novel Cytochrome Oxidase subunit 1 (CO1) sequences were ultimately generated for 8 south-east Australian reptile species of the families Scincidae and Agamidae.

Atypical nested 22q11.2 duplications between LCR22B and LCR22D are associated with neurodevelopmental phenotypes including autism spectrum disorder with incomplete penetrance.

BACKGROUND: Chromosome 22q11.2 is susceptible to genomic rearrangements and the most frequently reported involve deletions and duplications between low copy repeats LCR22A to LCR22D. Atypical nested deletions and duplications are rarer and can provide a valuable opportunity to investigate the dosage effects of a smaller subset of genes within the 22q11.2 genomic disorder region. METHODS: We describe thirteen individuals from six families, each with atypical nested duplications within the central 22q11.2 region between LCR22B and LCR22D. We then compared the molecular and clinical data for patients from this study and the few reported atypical duplication cases, to the cases with larger typical duplications between LCR22A and LCR22D. Further, we analyzed genes with the nested region to identify candidates highly enriched in human brain tissues. RESULTS: We observed that atypical nested duplications are heterogeneous in size, often familial, and associated with incomplete penetrance and highly variable clinical expressivity. We found that the nested atypical duplications are a possible risk factor for neurodevelopmental phenotypes, particularly for autism spectrum disorder (ASD), speech and language delay, and behavioral abnormalities. In addition, we analyzed genes within the nested region between LCR22B and LCR22D to identify nine genes (ZNF74, KLHL22, MED15, PI4KA, SERPIND1, CRKL, AIFM3, SLC7A4, and BCRP2) with enriched expression in the nervous system, each with unique spatiotemporal patterns in fetal and adult brain tissues. Interestingly, PI4KA is prominently expressed in the brain, and this gene is included either partially or completely in all of our subjects. CONCLUSION: Our findings confirm variable expressivity and incomplete penetrance for atypical nested 22q11.2 duplications and identify genes such as PI4KA to be directly relevant to brain development and disorder. We conclude that further work is needed to elucidate the basis of variable neurodevelopmental phenotypes and to exclude the presence of a second disorder. Our findings contribute to the genotype-phenotype data for atypical nested 22q11.2 duplications, with implications for genetic counseling.

YAP drives cutaneous squamous cell carcinoma formation and progression.

Squamous cell carcinoma (SCC) can progress to malignant metastatic cancer, including an aggressive subtype known as spindle cell carcinoma (spSCC). spSCC formation involves epithelial-to-mesenchymal transition (EMT), yet the molecular basis of this event remains unknown. The transcriptional co-activator YAP undergoes recurrent amplification in human SCC and overexpression of YAP drives SCC formation in mice. Here, we show that human spSCC tumours also feature strong nuclear localisation of YAP and overexpression of activated YAP (NLS-YAP-5SA) with Keratin-5 (K5-CreERt) is sufficient to induce rapid formation of both SCC and spSCC in mice. spSCC tumours arise at sites of epithelial scratch wounding, where tumour-initiating epithelial cells undergo EMT to generate spSCC. Expression of the EMT transcription factor ZEB1 arises upon wounding and is a defining characteristic of spSCC in mice and humans. Thus, the wound healing response synergises with YAP to drive metaplastic transformation of SCC to spSCC.

Subtle Changes in the Levels of BCL-2 Proteins Cause Severe Craniofacial Abnormalities.

Apoptotic cell death removes unwanted cells and is regulated by interactions between pro-survival and pro-apoptotic members of the BCL-2 protein family. The regulation of apoptosis is thought to be crucial for normal embryonic development. Accordingly, complete loss of pro-survival MCL-1 or BCL-XL (BCL2L1) causes embryonic lethality. However, it is not known whether minor reductions in pro-survival proteins could cause developmental abnormalities. We explored the rate-limiting roles of MCL-1 and BCL-XL in development and show that combined loss of single alleles of Mcl-1 and Bcl-x causes neonatal lethality. Mcl-1+/-;Bcl-x+/- mice display craniofacial anomalies, but additional loss of a single allele of pro-apoptotic Bim (Bcl2l11) restores normal development. These findings demonstrate that the control of cell survival during embryogenesis is finely balanced and suggest that some human craniofacial defects, for which causes are currently unknown, may be due to subtle imbalances between pro-survival and pro-apoptotic BCL-2 family members.