Matriptase-dependent epidermal pre-neoplasm in zebrafish embryos caused by a combination of hypotonic stress and epithelial polarity defects.

Aberrantly up-regulated activity of the type II transmembrane protease Matriptase-1 has been associated with the development and progression of a range of epithelial-derived carcinomas, and a variety of signaling pathways can mediate Matriptase-dependent tumorigenic events. During mammalian carcinogenesis, gain of Matriptase activity often results from imbalanced ratios between Matriptase and its cognate transmembrane inhibitor Hai1. Similarly, in zebrafish, unrestrained Matriptase activity due to loss of hai1a results in epidermal pre-neoplasms already during embryogenesis. Here, based on our former findings of a similar tumor-suppressive role for the Na+/K+-pump beta subunit ATP1b1a, we identify epithelial polarity defects and systemic hypotonic stress as another mode of aberrant Matriptase activation in the embryonic zebrafish epidermis in vivo. In this case, however, a different oncogenic pathway is activated which contains PI3K, AKT and NFkB, rather than EGFR and PLD (as in hai1a mutants). Strikingly, epidermal pre-neoplasm is only induced when epithelial polarity defects in keratinocytes (leading to disturbed Matriptase subcellular localization) occur in combination with systemic hypotonic stress (leading to increased proteolytic activity of Matriptase). A similar combinatorial effect of hypotonicity and loss of epithelial polarity was also obtained for the activity levels of Matriptase-1 in human MCF-10A epithelial breast cells. Together, this is in line with the multi-factor concept of carcinogenesis, with the notion that such factors can even branch off from one and the same initiator (here ATP1a1b) and can converge again at the level of one and the same mediator (here Matriptase). In sum, our data point to tonicity and epithelial cell polarity as evolutionarily conserved regulators of Matriptase activity that upon de-regulation can constitute an alternative mode of Matriptase-dependent carcinogenesis in vivo.

Biallelic loss-of-function variants of ZFTRAF1 cause neurodevelopmental disorder with microcephaly and hypotonia.

PURPOSE: Neurodevelopmental disorders exhibit clinical and genetic heterogeneity, ergo manifest dysfunction in components of diverse cellular pathways; the precise pathomechanism for the majority remains elusive. METHODS: We studied 5 affected individuals from 3 unrelated families manifesting global developmental delay, postnatal microcephaly, and hypotonia. We used exome sequencing and prioritized variants that were subsequently characterized using immunofluorescence, immunoblotting, pulldown assays, and RNA sequencing. RESULTS: We identified biallelic variants in ZFTRAF1, encoding a protein of yet unknown function. Four affected individuals from 2 unrelated families segregated 2 homozygous frameshift variants in ZFTRAF1, whereas, in the third family, an intronic splice site variant was detected. We investigated ZFTRAF1 at the cellular level and signified it as a nucleocytoplasmic protein in different human cell lines. ZFTRAF1 was completely absent in the fibroblasts of 2 affected individuals. We also identified 110 interacting proteins enriched in mRNA processing and autophagy-related pathways. Based on profiling of autophagy markers, patient-derived fibroblasts show irregularities in the protein degradation process. CONCLUSION: Thus, our findings suggest that biallelic variants of ZFTRAF1 cause a severe neurodevelopmental disorder.

Interleukin-4 Receptor α Signaling in Myeloid Cells Controls Collagen Fibril Assembly in Skin Repair.

Activation of the immune response during injury is a critical early event that determines whether the outcome of tissue restoration is regeneration or replacement of the damaged tissue with a scar. The mechanisms by which immune signals control these fundamentally different regenerative pathways are largely unknown. We have demonstrated that, during skin repair in mice, interleukin-4 receptor α (IL-4Rα)-dependent macrophage activation controlled collagen fibril assembly and that this process was important for effective repair while having adverse pro-fibrotic effects. We identified Relm-α as one important player in the pathway from IL-4Rα signaling in macrophages to the induction of lysyl hydroxylase 2 (LH2), an enzyme that directs persistent pro-fibrotic collagen cross-links, in fibroblasts. Notably, Relm-ß induced LH2 in human fibroblasts, and expression of both factors was increased in lipodermatosclerosis, a condition of excessive human skin fibrosis. Collectively, our findings provide mechanistic insights into the link between type 2 immunity and initiation of pro-fibrotic pathways.

NAMPT-derived NAD+ fuels PARP1 to promote skin inflammation through parthanatos cell death.

Several studies have revealed a correlation between chronic inflammation and nicotinamide adenine dinucleotide (NAD+) metabolism, but the precise mechanism involved is unknown. Here, we report that the genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis, reduced oxidative stress, inflammation, and keratinocyte DNA damage, hyperproliferation, and cell death in zebrafish models of chronic skin inflammation, while all these effects were reversed by NAD+ supplementation. Similarly, genetic and pharmacological inhibition of poly(ADP-ribose) (PAR) polymerase 1 (Parp1), overexpression of PAR glycohydrolase, inhibition of apoptosis-inducing factor 1, inhibition of NADPH oxidases, and reactive oxygen species (ROS) scavenging all phenocopied the effects of Nampt inhibition. Pharmacological inhibition of NADPH oxidases/NAMPT/PARP/AIFM1 axis decreased the expression of pathology-associated genes in human organotypic 3D skin models of psoriasis. Consistently, an aberrant induction of NAMPT and PARP activity, together with AIFM1 nuclear translocation, was observed in lesional skin from psoriasis patients. In conclusion, hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD+, mediates skin inflammation through parthanatos cell death.

TGS1 impacts snRNA 3'-end processing, ameliorates survival motor neuron-dependent neurological phenotypes in vivo and prevents neurodegeneration.

Trimethylguanosine synthase 1 (TGS1) is a highly conserved enzyme that converts the 5'-monomethylguanosine cap of small nuclear RNAs (snRNAs) to a trimethylguanosine cap. Here, we show that loss of TGS1 in Caenorhabditis elegans, Drosophila melanogaster and Danio rerio results in neurological phenotypes similar to those caused by survival motor neuron (SMN) deficiency. Importantly, expression of human TGS1 ameliorates the SMN-dependent neurological phenotypes in both flies and worms, revealing that TGS1 can partly counteract the effects of SMN deficiency. TGS1 loss in HeLa cells leads to the accumulation of immature U2 and U4atac snRNAs with long 3' tails that are often uridylated. snRNAs with defective 3' terminations also accumulate in Drosophila Tgs1 mutants. Consistent with defective snRNA maturation, TGS1 and SMN mutant cells also exhibit partially overlapping transcriptome alterations that include aberrantly spliced and readthrough transcripts. Together, these results identify a neuroprotective function for TGS1 and reinforce the view that defective snRNA maturation affects neuronal viability and function.

wnt10a is required for zebrafish median fin fold maintenance and adult unpaired fin metamorphosis.

BACKGROUND: Mutations of human WNT10A are associated with odonto-ectodermal dysplasia syndromes. Here, we present analyses of wnt10a loss-of-function mutants in the zebrafish. RESULTS: wnt10a mutant zebrafish embryos display impaired tooth development and a collapsing median fin fold (MFF). Rescue experiments show that wnt10a is essential for MFF maintenance both during embryogenesis and later metamorphosis. The MFF collapse could not be attributed to increased cell death or altered proliferation rates of MFF cell types. Rather, wnt10a mutants show reduced expression levels of dlx2a in distal-most MFF cells, followed by compromised expression of col1a1a and other extracellular matrix proteins encoding genes. Transmission electron microscopy analysis shows that although dermal MFF compartments of wnt10a mutants initially are of normal morphology, with regular collagenous actinotrichia, positioning of actinotrichia within the cleft of distal MFF cells becomes compromised, coinciding with actinotrichia shrinkage and MFF collapse. CONCLUSIONS: MFF collapse of wnt10a mutant zebrafish is likely caused by the loss of distal properties in the developing MFF, strikingly similar to the proposed molecular pathomechanisms underlying the teeth defects caused by the loss of Wnt10 in fish and mammals. In addition, it points to thus fur unknown mechanisms controlling the linear growth and stability of actinotrichia and their collagen fibrils.

The Kunitz-type serine protease inhibitor Spint2 is required for cellular cohesion, coordinated cell migration and cell survival during zebrafish hatching gland development.

We have previously shown that the Kunitz-type serine protease inhibitor Spint1a, also named Hai1a, is required in the zebrafish embryonic epidermis to restrict the activity of the type II transmembrane serine protease (TTSP) Matriptase1a/St14a, thereby ensuring epidermal homeostasis. A closely related Kunitz-type inhibitor is Spint2/Hai2, which in mammals plays multiple developmental roles that are either redundant or non-redundant with those of Spint1. However, the molecular bases for these non-redundancies are not fully understood. Here, we study spint2 during zebrafish development. It is co-expressed with spint1a in multiple embryonic epithelia, including the outer/peridermal layer of the epidermis. However, unlike spint1a, spint2 expression is absent from the basal epidermal layer but present in hatching gland cells. Hatching gland cells derive from the mesendodermal prechordal plate, from where they undergo a thus far undescribed transit into, and coordinated sheet migration within, the interspace between the outer and basal layer of the epidermis to reach their final destination on the yolk sac. Hatching gland cells usually survive their degranulation that drives embryo hatching but die several days later. In spint2 mutants, cohesion among hatching gland cells and their collective intra-epidermal migration are disturbed, leading to a discontinuous organization of the gland. In addition, cells undergo precocious cell death before degranulation, so that embryos fail to hatch. Chimera analyses show that Spint2 is required in hatching gland cells, but not in the overlying periderm, their potential migration and adhesion substrate. Spint2 acts independently of all tested Matriptases, Prostasins and other described Spint1 and Spint2 mediators. However, it displays a tight genetic interaction with and acts at least partly via the cell-cell adhesion protein E-cadherin, promoting both hatching gland cell cohesiveness and survival, in line with formerly reported effects of E-cadherin during morphogenesis and cell death suppression. In contrast, no such genetic interaction was observed between Spint2 and the cell-cell adhesion molecule EpCAM, which instead interacts with Spint1a. Our data shed new light onto the mechanisms of hatching gland morphogenesis and hatching gland cell survival. In addition, they reveal developmental roles of Spint2 that are strikingly different from those of Spint1, most likely due to differences in the expression patterns and relevant target proteins.

A regulatory pathway involving retinoic acid and calcineurin demarcates and maintains joint cells and osteoblasts in regenerating fin.

During zebrafish fin regeneration, blastema cells lining the epidermis differentiate into osteoblasts and joint cells to reconstruct the segmented bony rays. We show that osteoblasts and joint cells originate from a common cell lineage, but are committed to different cell fates. Pre-osteoblasts expressing runx2a/b commit to the osteoblast lineage upon expressing sp7, whereas the strong upregulation of hoxa13a correlates with a commitment to a joint cell type. In the distal regenerate, hoxa13a, evx1 and pthlha are sequentially upregulated at regular intervals to define the newly identified presumptive joint cells. Presumptive joint cells mature into joint-forming cells, a distinct cell cluster that maintains the expression of these factors. Analysis of evx1 null mutants reveals that evx1 is acting upstream of pthlha and downstream of or in parallel with hoxa13a Calcineurin activity, potentially through the inhibition of retinoic acid signaling, regulates evx1, pthlha and hoxa13a expression during joint formation. Furthermore, retinoic acid treatment induces osteoblast differentiation in mature joint cells, leading to ectopic bone deposition in joint regions. Overall, our data reveal a novel regulatory pathway essential for joint formation in the regenerating fin.

Direct activation of chordoblasts by retinoic acid is required for segmented centra mineralization during zebrafish spine development.

Zebrafish mutants with increased retinoic acid (RA) signaling due to the loss of the RA-inactivating enzyme Cyp26b1 develop a hyper-mineralized spine with gradually fusing vertebral body precursors (centra). However, the underlying cellular mechanisms remain incompletely understood. Here, we show that cells of the notochord epithelium named chordoblasts are sensitive to RA signaling. Chordoblasts are uniformly distributed along the anteroposterior axis and initially generate the continuous collagenous notochord sheath. However, subsequently and iteratively, subsets of these cells undergo further RA-dependent differentiation steps, acquire a stellate-like shape, downregulate expression of the collagen gene col2a1a, switch on cyp26b1 expression and trigger metameric sheath mineralization. This mineralization fails to appear upon chordoblast-specific cell ablation or RA signal transduction blockade. Together, our data reveal that, despite their different developmental origins, the activities and regulation of chordoblasts are very similar to those of osteoblasts, including their RA-induced transition from osteoid-producing cells to osteoid-mineralizing ones. Furthermore, our data point to a requirement for locally controlled RA activity within the chordoblast layer in order to generate the segmented vertebral column.

Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis.

Homozygous SMN1 loss causes spinal muscular atrophy (SMA), the most common lethal genetic childhood motor neuron disease. SMN1 encodes SMN, a ubiquitous housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. SMA-affected individuals harbor low SMN expression from one to six SMN2 copies, which is insufficient to functionally compensate for SMN1 loss. However, rarely individuals with homozygous absence of SMN1 and only three to four SMN2 copies are fully asymptomatic, suggesting protection through genetic modifier(s). Previously, we identified plastin 3 (PLS3) overexpression as an SMA protective modifier in humans and showed that SMN deficit impairs endocytosis, which is rescued by elevated PLS3 levels. Here, we identify reduction of the neuronal calcium sensor Neurocalcin delta (NCALD) as a protective SMA modifier in five asymptomatic SMN1-deleted individuals carrying only four SMN2 copies. We demonstrate that NCALD is a Ca2+-dependent negative regulator of endocytosis, as NCALD knockdown improves endocytosis in SMA models and ameliorates pharmacologically induced endocytosis defects in zebrafish. Importantly, NCALD knockdown effectively ameliorates SMA-associated pathological defects across species, including worm, zebrafish, and mouse. In conclusion, our study identifies a previously unknown protective SMA modifier in humans, demonstrates modifier impact in three different SMA animal models, and suggests a potential combinatorial therapeutic strategy to efficiently treat SMA. Since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in SMA and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies.

SSBP1 mutations in dominant optic atrophy with variable retinal degeneration.

OBJECTIVE: Autosomal dominant optic atrophy (ADOA) starts in early childhood with loss of visual acuity and color vision deficits. OPA1 mutations are responsible for the majority of cases, but in a portion of patients with a clinical diagnosis of ADOA, the cause remains unknown. This study aimed to identify novel ADOA-associated genes and explore their causality. METHODS: Linkage analysis and sequencing were performed in multigeneration families and unrelated patients to identify disease-causing variants. Functional consequences were investigated in silico and confirmed experimentally using the zebrafish model. RESULTS: We defined a new ADOA locus on 7q33-q35 and identified 3 different missense variants in SSBP1 (NM_001256510.1; c.113G>A [p.(Arg38Gln)], c.320G>A [p.(Arg107Gln)] and c.422G>A [p.(Ser141Asn)]) in affected individuals from 2 families and 2 singletons with ADOA and variable retinal degeneration. The mutated arginine residues are part of a basic patch that is essential for single-strand DNA binding. The loss of a positive charge at these positions is very likely to lower the affinity of SSBP1 for single-strand DNA. Antisense-mediated knockdown of endogenous ssbp1 messenger RNA (mRNA) in zebrafish resulted in compromised differentiation of retinal ganglion cells. A similar effect was achieved when mutated mRNAs were administered. These findings point toward an essential role of ssbp1 in retinal development and the dominant-negative nature of the identified human variants, which is consistent with the segregation pattern observed in 2 multigeneration families studied. INTERPRETATION: SSBP1 is an essential protein for mitochondrial DNA replication and maintenance. Our data have established pathogenic variants in SSBP1 as a cause of ADOA and variable retinal degeneration. ANN NEUROL 2019;86:368-383.

The Power of Human Protective Modifiers: PLS3 and CORO1C Unravel Impaired Endocytosis in Spinal Muscular Atrophy and Rescue SMA Phenotype.

Homozygous loss of SMN1 causes spinal muscular atrophy (SMA), the most common and devastating childhood genetic motor-neuron disease. The copy gene SMN2 produces only ∼10% functional SMN protein, insufficient to counteract development of SMA. In contrast, the human genetic modifier plastin 3 (PLS3), an actin-binding and -bundling protein, fully protects against SMA in SMN1-deleted individuals carrying 3-4 SMN2 copies. Here, we demonstrate that the combinatorial effect of suboptimal SMN antisense oligonucleotide treatment and PLS3 overexpression-a situation resembling the human condition in asymptomatic SMN1-deleted individuals-rescues survival (from 14 to >250 days) and motoric abilities in a severe SMA mouse model. Because PLS3 knockout in yeast impairs endocytosis, we hypothesized that disturbed endocytosis might be a key cellular mechanism underlying impaired neurotransmission and neuromuscular junction maintenance in SMA. Indeed, SMN deficit dramatically reduced endocytosis, which was restored to normal levels by PLS3 overexpression. Upon low-frequency electro-stimulation, endocytotic FM1-43 (SynaptoGreen) uptake in the presynaptic terminal of neuromuscular junctions was restored to control levels in SMA-PLS3 mice. Moreover, proteomics and biochemical analysis revealed CORO1C, another F-actin binding protein, whose direct binding to PLS3 is dependent on calcium. Similar to PLS3 overexpression, CORO1C overexpression restored fluid-phase endocytosis in SMN-knockdown cells by elevating F-actin amounts and rescued the axonal truncation and branching phenotype in Smn-depleted zebrafish. Our findings emphasize the power of genetic modifiers to unravel the cellular pathomechanisms underlying SMA and the power of combinatorial therapy based on splice correction of SMN2 and endocytosis improvement to efficiently treat SMA.

Retinoic acid-induced premature osteoblast-to-preosteocyte transitioning has multiple effects on calvarial development.

We have previously shown that, in human and zebrafish, hypomorphic mutations of the gene encoding the retinoic acid (RA)-metabolizing enzyme Cyp26b1 result in coronal craniosynostosis, caused by an RA-induced premature transitioning of suture osteoblasts to preosteocytes, inducing ectopic mineralization of the suture's osteoid matrix. In addition, we showed that human CYP26B1 null patients have more severe and seemingly opposite skull defects, characterized by smaller and fragmented calvaria, but the cellular basis of these defects remained largely unclear. Here, by treating juvenile zebrafish with exogenous RA or a chemical Cyp26 inhibitor in the presence or absence of osteogenic cells or bone-resorbing osteoclasts, we demonstrate that both reduced calvarial size and calvarial fragmentation are also caused by RA-induced premature osteoblast-to-preosteocyte transitioning. During calvarial growth, the resulting osteoblast deprival leads to decreased osteoid production and thereby smaller and thinner calvaria, whereas calvarial fragmentation is caused by increased osteoclast stimulation through the gained preosteocytes. Together, our data demonstrate that RA-induced osteoblast-to-preosteocyte transitioning has multiple effects on developing bone in Cyp26b1 mutants, ranging from gain to loss of bone, depending on the allelic strength, the developmental stage and the cellular context.

Re-epithelialization of cutaneous wounds in adult zebrafish combines mechanisms of wound closure in embryonic and adult mammals.

Re-epithelialization of cutaneous wounds in adult mammals takes days to complete and relies on numerous signalling cues and multiple overlapping cellular processes that take place both within the epidermis and in other participating tissues. Re-epithelialization of partial- or full-thickness skin wounds of adult zebrafish, however, is extremely rapid and largely independent of the other processes of wound healing. Live imaging after treatment with transgene-encoded or chemical inhibitors reveals that re-epithelializing keratinocytes repopulate wounds by TGF-ß- and integrin-dependent lamellipodial crawling at the leading edges of the epidermal tongue. In addition, re-epithelialization requires long-range epithelial rearrangements, involving radial intercalations, flattening and directed elongation of cells - processes that are dependent on Rho kinase, JNK and, to some extent, planar cell polarity within the epidermis. These rearrangements lead to a massive recruitment of keratinocytes from the adjacent epidermis and make re-epithelialization independent of keratinocyte proliferation and the mitogenic effect of FGF signalling, which are only required after wound closure, allowing the epidermis outside the wound to re-establish its normal thickness. Together, these results demonstrate that the adult zebrafish is a valuable in vivo model for studying and visualizing the processes involved in cutaneous wound closure, facilitating the dissection of direct from indirect and motogenic from mitogenic effects of genes and molecules affecting wound re-epithelialization.

CHP1 reduction ameliorates spinal muscular atrophy pathology by restoring calcineurin activity and endocytosis.

Autosomal recessive spinal muscular atrophy (SMA), the leading genetic cause of infant lethality, is caused by homozygous loss of the survival motor neuron 1 (SMN1) gene. SMA disease severity inversely correlates with the number of SMN2 copies, which in contrast to SMN1, mainly produce aberrantly spliced transcripts. Recently, the first SMA therapy based on antisense oligonucleotides correcting SMN2 splicing, namely SPINRAZATM, has been approved. Nevertheless, in type I SMA-affected individuals-representing 60% of SMA patients-the elevated SMN level may still be insufficient to restore motor neuron function lifelong. Plastin 3 (PLS3) and neurocalcin delta (NCALD) are two SMN-independent protective modifiers identified in humans and proved to be effective across various SMA animal models. Both PLS3 overexpression and NCALD downregulation protect against SMA by restoring impaired endocytosis; however, the exact mechanism of this protection is largely unknown. Here, we identified calcineurin-like EF-hand protein 1 (CHP1) as a novel PLS3 interacting protein using a yeast-two-hybrid screen. Co-immunoprecipitation and pull-down assays confirmed a direct interaction between CHP1 and PLS3. Although CHP1 is ubiquitously present, it is particularly abundant in the central nervous system and at SMA-relevant sites including motor neuron growth cones and neuromuscular junctions. Strikingly, we found elevated CHP1 levels in SMA mice. Congruently, CHP1 downregulation restored impaired axonal growth in Smn-depleted NSC34 motor neuron-like cells, SMA zebrafish and primary murine SMA motor neurons. Most importantly, subcutaneous injection of low-dose SMN antisense oligonucleotide in pre-symptomatic mice doubled the survival rate of severely-affected SMA mice, while additional CHP1 reduction by genetic modification prolonged survival further by 1.6-fold. Moreover, CHP1 reduction further ameliorated SMA disease hallmarks including electrophysiological defects, smaller neuromuscular junction size, impaired maturity of neuromuscular junctions and smaller muscle fibre size compared to low-dose SMN antisense oligonucleotide alone. In NSC34 cells, Chp1 knockdown tripled macropinocytosis whereas clathrin-mediated endocytosis remained unaffected. Importantly, Chp1 knockdown restored macropinocytosis in Smn-depleted cells by elevating calcineurin phosphatase activity. CHP1 is an inhibitor of calcineurin, which collectively dephosphorylates proteins involved in endocytosis, and is therefore crucial in synaptic vesicle endocytosis. Indeed, we found marked hyperphosphorylation of dynamin 1 in SMA motor neurons, which was restored to control level by the heterozygous Chp1 mutant allele. Taken together, we show that CHP1 is a novel SMA modifier that directly interacts with PLS3, and that CHP1 reduction ameliorates SMA pathology by counteracting impaired endocytosis. Most importantly, we demonstrate that CHP1 reduction is a promising SMN-independent therapeutic target for a combinatorial SMA therapy.

p53 and TAp63 promote keratinocyte proliferation and differentiation in breeding tubercles of the zebrafish.

p63 is a multi-isoform member of the p53 family of transcription factors. There is compelling genetic evidence that ΔNp63 isoforms are needed for keratinocyte proliferation and stemness in the developing vertebrate epidermis. However, the role of TAp63 isoforms is not fully understood, and TAp63 knockout mice display normal epidermal development. Here, we show that zebrafish mutants specifically lacking TAp63 isoforms, or p53, display compromised development of breeding tubercles, epidermal appendages which according to our analyses display more advanced stratification and keratinization than regular epidermis, including continuous desquamation and renewal of superficial cells by derivatives of basal keratinocytes. Defects are further enhanced in TAp63/p53 double mutants, pointing to partially redundant roles of the two related factors. Molecular analyses, treatments with chemical inhibitors and epistasis studies further reveal the existence of a linear TAp63/p53->Notch->caspase 3 pathway required both for enhanced proliferation of keratinocytes at the base of the tubercles and their subsequent differentiation in upper layers. Together, these studies identify the zebrafish breeding tubercles as specific epidermal structures sharing crucial features with the cornified mammalian epidermis. In addition, they unravel essential roles of TAp63 and p53 to promote both keratinocyte proliferation and their terminal differentiation by promoting Notch signalling and caspase 3 activity, ensuring formation and proper homeostasis of this self-renewing stratified epithelium.

PLS3 mutations in X-linked osteoporosis with fractures.

Plastin 3 (PLS3), a protein involved in the formation of filamentous actin (F-actin) bundles, appears to be important in human bone health, on the basis of pathogenic variants in PLS3 in five families with X-linked osteoporosis and osteoporotic fractures that we report here. The bone-regulatory properties of PLS3 were supported by in vivo analyses in zebrafish. Furthermore, in an additional five families (described in less detail) referred for diagnosis or ruling out of osteogenesis imperfecta type I, a rare variant (rs140121121) in PLS3 was found. This variant was also associated with a risk of fracture among elderly heterozygous women that was two times as high as that among noncarriers, which indicates that genetic variation in PLS3 is a novel etiologic factor involved in common, multi-factorial osteoporosis.

Direct and indirect roles of Fgf3 and Fgf10 in innervation and vascularisation of the vertebrate hypothalamic neurohypophysis.

The neurohypophysis is a crucial component of the hypothalamo-pituitary axis, serving as the site of release of hypothalamic neurohormones into a plexus of hypophyseal capillaries. The growth of hypothalamic axons and capillaries to the forming neurohypophysis in embryogenesis is therefore crucial to future adult homeostasis. Using ex vivo analyses in chick and in vivo analyses in mutant and transgenic zebrafish, we show that Fgf10 and Fgf3 secreted from the forming neurohypophysis exert direct guidance effects on hypothalamic neurosecretory axons. Simultaneously, they promote hypophyseal vascularisation, exerting early direct effects on endothelial cells that are subsequently complemented by indirect effects. Together, our studies suggest a model for the integrated neurohemal wiring of the hypothalamo-neurohypophyseal axis.

Semicircular canal morphogenesis in the zebrafish inner ear requires the function of gpr126 (lauscher), an adhesion class G protein-coupled receptor gene.

Morphogenesis of the semicircular canal ducts in the vertebrate inner ear is a dramatic example of epithelial remodelling in the embryo, and failure of normal canal development results in vestibular dysfunction. In zebrafish and Xenopus, semicircular canal ducts develop when projections of epithelium, driven by extracellular matrix production, push into the otic vesicle and fuse to form pillars. We show that in the zebrafish, extracellular matrix gene expression is high during projection outgrowth and then rapidly downregulated after fusion. Enzymatic disruption of hyaluronan in the projections leads to their collapse and a failure to form pillars: as a result, the ears swell. We have cloned a zebrafish mutant, lauscher (lau), identified by its swollen ear phenotype. The primary defect in the ear is abnormal projection outgrowth and a failure of fusion to form the semicircular canal pillars. Otic expression of extracellular matrix components is highly disrupted: several genes fail to become downregulated and remain expressed at abnormally high levels into late larval stages. The lau mutations disrupt gpr126, an adhesion class G protein-coupled receptor gene. Expression of gpr126 is similar to that of sox10, an ear and neural crest marker, and is partially dependent on sox10 activity. Fusion of canal projections and downregulation of otic versican expression in a hypomorphic lau allele can be restored by cAMP agonists. We propose that Gpr126 acts through a cAMP-mediated pathway to control the outgrowth and adhesion of canal projections in the zebrafish ear via the regulation of extracellular matrix gene expression.

FAM96A is a novel pro-apoptotic tumor suppressor in gastrointestinal stromal tumors.

The ability to escape apoptosis is a hallmark of cancer-initiating cells and a key factor of resistance to oncolytic therapy. Here, we identify FAM96A as a ubiquitous, evolutionarily conserved apoptosome-activating protein and investigate its potential pro-apoptotic tumor suppressor function in gastrointestinal stromal tumors (GISTs). Interaction between FAM96A and apoptotic peptidase activating factor 1 (APAF1) was identified in yeast two-hybrid screen and further studied by deletion mutants, glutathione-S-transferase pull-down, co-immunoprecipitation and immunofluorescence. Effects of FAM96A overexpression and knock-down on apoptosis sensitivity were examined in cancer cells and zebrafish embryos. Expression of FAM96A in GISTs and histogenetically related cells including interstitial cells of Cajal (ICCs), "fibroblast-like cells" (FLCs) and ICC stem cells (ICC-SCs) was investigated by Northern blotting, reverse transcription-polymerase chain reaction, immunohistochemistry and Western immunoblotting. Tumorigenicity of GIST cells and transformed murine ICC-SCs stably transduced to re-express FAM96A was studied by xeno- and allografting into immunocompromised mice. FAM96A was found to bind APAF1 and to enhance the induction of mitochondrial apoptosis. FAM96A protein or mRNA was dramatically reduced or lost in 106 of 108 GIST samples representing three independent patient cohorts. Whereas ICCs, ICC-SCs and FLCs, the presumed normal counterparts of GIST, were found to robustly express FAM96A protein and mRNA, FAM96A expression was much reduced in tumorigenic ICC-SCs. Re-expression of FAM96A in GIST cells and transformed ICC-SCs increased apoptosis sensitivity and diminished tumorigenicity. Our data suggest FAM96A is a novel pro-apoptotic tumor suppressor that is lost during GIST tumorigenesis.