Phenotypes In Zebrafish Research Articles

Esco2 regulates cx43 expression during skeletal regeneration in the zebrafish fin.

Roberts syndrome (RBS) is a rare genetic disorder characterized by craniofacial abnormalities, limb malformation, and often severe mental retardation. RBS arises from mutations in ESCO2 that encodes an acetyltransferase and modifies the cohesin subunit SMC3. Mutations in SCC2/NIPBL (encodes a cohesin loader), SMC3 or other cohesin genes (SMC1, RAD21/MCD1) give rise to a related developmental malady termed Cornelia de Lange syndrome (CdLS). RBS and CdLS exhibit overlapping phenotypes, but RBS is thought to arise through mitotic failure and limited progenitor cell proliferation while CdLS arises through transcriptional dysregulation. Here, we use the zebrafish regenerating fin model to test the mechanism through which RBS-type phenotypes arise. esco2 is up-regulated during fin regeneration and specifically within the blastema. esco2 knockdown adversely affects both tissue and bone growth in regenerating fins-consistent with a role in skeletal morphogenesis. esco2-knockdown significantly diminishes cx43/gja1 expression which encodes the gap junction connexin subunit required for cell-cell communication. cx43 mutations cause the short fin (sof(b123) ) phenotype in zebrafish and oculodentodigital dysplasia (ODDD) in humans. Importantly, miR-133-dependent cx43 overexpression rescues esco2-dependent growth defects. These results conceptually link ODDD to cohesinopathies and provide evidence that ESCO2 may play a transcriptional role critical for human development.

Phenoscape: Identifying Candidate Genes for Evolutionary Phenotypes.

Phenotypes resulting from mutations in genetic model organisms can help reveal candidate genes for evolutionarily important phenotypic changes in related taxa. Although testing candidate gene hypotheses experimentally in nonmodel organisms is typically difficult, ontology-driven information systems can help generate testable hypotheses about developmental processes in experimentally tractable organisms. Here, we tested candidate gene hypotheses suggested by expert use of the Phenoscape Knowledgebase, specifically looking for genes that are candidates responsible for evolutionarily interesting phenotypes in the ostariophysan fishes that bear resemblance to mutant phenotypes in zebrafish. For this, we searched ZFIN for genetic perturbations that result in either loss of basihyal element or loss of scales phenotypes, because these are the ancestral phenotypes observed in catfishes (Siluriformes). We tested the identified candidate genes by examining their endogenous expression patterns in the channel catfish, Ictalurus punctatus. The experimental results were consistent with the hypotheses that these features evolved through disruption in developmental pathways at, or upstream of, brpf1 and eda/edar for the ancestral losses of basihyal element and scales, respectively. These results demonstrate that ontological annotations of the phenotypic effects of genetic alterations in model organisms, when aggregated within a knowledgebase, can be used effectively to generate testable, and useful, hypotheses about evolutionary changes in morphology.

Genetic association analyses highlight biological pathways underlying mitral valve prolapse.

Non-syndromic mitral valve prolapse (MVP) is a common degenerative cardiac valvulopathy of unknown aetiology that predisposes to mitral regurgitation, heart failure and sudden death1. Previous family and pathophysiological studies suggest a complex pattern of inheritance2–5. We performed a meta-analysis of two genome-wide association studies in 1,442 cases and 2,439 controls. We identified and replicated in 1,422 cases and 6,779 controls six loci and provide functional evidence for candidate genes. We highlight LMCD1 encoding a transcription factor6, for which morpholino knockdown in zebrafish results in atrioventricular (AV) valve regurgitation. A similar zebrafish phenotype was obtained for tensin1 (TNS1), a focal adhesion protein involved in cytoskeleton organization. We also show the expression of tensin1 during valve morphogenesis and describe enlarged posterior mitral leaflets in Tns1−/− mice. This study identifies the first risk loci for MVP and suggests new mechanisms involved in mitral valve regurgitation, the most common indication for mitral valve repair7.

Innate Multigene Family Memories Are Implicated in the Viral-Survivor Zebrafish Phenotype.

Since adaptive features such as memory were discovered in mammalian innate immunity, interest in the immunological status of primitive vertebrates after infections has grown. In this context, we used zebrafish (Danio rerio), a primitive vertebrate species suited to molecular and genetic studies to explore transcriptional memories of the immune system in long-term survivors of viral haemorrhagic septicemia virus infections. Immune-gene targeted microarrays designed in-house, multipath genes, gene set enrichment, and leading-edge analysis, reveal unexpected consistent correlations between the viral-survivor phenotype and several innate multigene families. Thus, here we describe in survivors of infections the upregulation of the multigene family of proteasome subunit macropains, zebrafish-specific novel gene sets, mitogen activated protein kinases, and epidermal growth factor. We also describe the downregulation of the multigene families of c-reactive proteins, myxovirus-induced proteins and novel immunoglobulin-type receptors. The strength of those immunological memories was reflected by the exceptional similarity of the transcriptional profiles of survivors before and after re-infection compared with primary infected fish. On the other hand, the high levels of neutralizing antibodies in the blood plasma of survivors contrasted with the depletion of transcripts specific for most cell types present in lymphoid organs. Therefore, long-term survivors maintained unexpected molecular/cellular memories of previous viral encounters by modulating the expression levels of innate multigene families as well as having specific adaptive antibodies. The implications of the so-called "trained immunity" for future research in this field are also discussed.

Abstract 15: Global RNA Splicing Regulation in Cardiac Maturation

Background: The complexity of transcriptome and proteome is contributed by alternative splicing of mRNA. Altered mRNA splicing is implicated in both development and disease. However, the change of alternative mRNA splicing during cardiomyocytes maturation is unknown, and the regulatory mechanisms remain unexplored. Methods and Results: Using deep RNA-Sequencing, we identified global alternative splicing changes associated with both cardiac development and pathological remodeling in mouse heart. Further, we identified a highly conserved splicing regulator-RBFox1 to be significantly induced during zebrafish, mouse and human cardiac maturation. RBFox1 expression was also detected in cardiomyocytes derived from both mouse and human embryonic stem cells but at much lower levels comparing to adult heart. In zebrafish embryos, inactivation of RBFox1 caused cardiomyocyte maturation defects. Expression of RBFox1 in cultured neonatal cardiomyocytes was sufficient to promote maturation by reducing fetal marker gene expression while increasing calcium handling gene expression including RyR and promoting sarcomere organization. Deep RNA-Sequencing analysis showed that RBFox1 expression promoted alternative splicing in genes involved in calcium cycling, blood vessel development and muscle contraction. Finally, we identified a highly conserved mutually exclusive alternative splicing event of transcription factor MEF2 to be a direct downstream target of RBFox1. Expression of individual MEF2 splicing variants led to different cardiac developmental phenotypes in zebrafish, indicating their different transcriptional activities. Conclusion: Our study provided the first comprehensive analysis of mRNA splicing regulation in heart during post-natal development and heart failure, and identified RBFox1 as a key regulator for alternative RNA splicing during cardiomyocytes maturation. Further exploration of RBFox1 mediated RNA splicing regulation in heart may yield novel insight to the underlying mechanisms of cardiac maturation and new approach to improve cell based therapy for heart diseases.

Pannexin 3 is required for normal progression of skeletal development in vertebrates.

The vertebrate skeletal system has various functions, including support, movement, protection, and the production of blood cells. The development of cartilage and bones, the core components of the skeletal system, is mediated by systematic inter- and intracellular communication among multiple signaling pathways in differentiating progenitors and the surrounding tissues. Recently, Pannexin (Panx) 3 has been shown to play important roles in bone development in vitro by mediating multiple signaling pathways, although its roles in vivo have not been explored. In this study, we generated and analyzed Panx3 knockout mice and examined the skeletal phenotypes of panx3 morphant zebrafish. Panx3(-/-) embryos exhibited delays in hypertrophic chondrocyte differentiation and osteoblast differentiation as well as the initiation of mineralization, resulting in shortened long bones in adulthood. The abnormal progression of hypertrophic chondrogenesis appeared to be associated with the sustained proliferation of chondrocytes, which resulted from increased intracellular cAMP levels. Similarly, osteoblast differentiation and mineralization were delayed in panx3 morphant zebrafish. Taken together, our results provide evidence of the crucial roles of Panx3 in vertebrate skeletal development in vivo.

Gain and loss of function mutations of ataxin-7 cause cilia pathology in mouse and zebrafish models

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disease characterized by progressive neuronal loss in the cerebellum and associated structures, leading to cerebellar ataxia, dysarthria and dysphagia. Additional non-cerebellar symptoms are present at variable frequencies, including visual impairment affecting 83% of patients that is caused by cone and rod photoreceptor degeneration and hearing loss concerning 24% of patients. SCA7 is caused by a toxic polyglutamine expansion in Ataxin-7, a known subunit of the transcriptional co-regulator SAGA/TFTC complex. Transcriptional alterations are observed in SCA7, however, the disease mechanism is yet unclear. Ataxin-7 is also present in the cytoplasm of neurons, where its function is unknown. Here, we show for the first time that Ataxin-7 is a bona fide component of centrosomes and is present in diverse types of mammalian cilia, including primary, motile and photoreceptor sensory cilia. We provide evidence that Ataxin-7 loss of function causes a large spectrum of ciliary-related morphological and functional phenotypes in zebrafish, and that human Ataxin-7 is able to rescue the cilia phenotypes, demonstrating that Ataxin-7 has an essential, evolutionarily conserved function in cilia. Strikingly, Ataxin-7 is progressively lost from photoreceptor cilia in SCA7 mouse models, correlating with ciliary defects and photoreceptor degeneration. Finally, we found that expression of polyQ-expanded Ataxin-7, like Ataxin-7 loss of function, leads to ciliary dysfunctions in zebrafish. Our study thus provides new insight into the function of Ataxin-7 and opens novel perspective in the understanding of SCA7 pathogenesis.

Apela Regulates Fluid Homeostasis by Binding to the APJ Receptor to Activate Gi Signaling

Apela (APJ early endogenous ligand, also known as elabela or toddler) is a recently discovered peptide hormone. Based on genetic studies in zebrafish, apela was found to be important for endoderm differentiation and heart development during embryogenesis. Although common phenotypes of apela and APJ-null zebrafish during embryonic development suggested that apela interacts with the APJ receptor, kinetics of apela binding to APJ and intracellular signaling pathways for apela remain unknown. The role of apela in adults is also uncertain. Using a chimeric apela ligand, we showed direct binding of apela to APJ with high affinity (Kd = 0.51 nm) and the ability of apelin, the known peptide ligand for APJ, to compete for apela binding. Apela, similar to apelin, acts through the inhibitory G protein pathway by inhibiting forskolin-stimulated cAMP production and by inducing ERK1/2 phosphorylation. In adult rats, apela is expressed exclusively in the kidney, unlike the wide tissue distribution of apelin. In vivo studies demonstrated the ability of apela to regulate fluid homeostasis by increasing diuresis and water intake. Dose-response studies further indicated that apela induces 2- and 5-fold higher maximal responses than apelin in ERK1/2 phosphorylation and diuresis/water intake, respectively. After designing an apela antagonist, we further demonstrated the role of endogenous ligand(s) in regulating APJ-mediated fluid homeostasis. Our results identified apela as a potent peptide hormone capable of regulating fluid homeostasis in adult kidney through coupling to the APJ-mediated Gi signaling pathway.

A point mutation of zebrafish c-cbl gene in the ring finger domain produces a phenotype mimicking human myeloproliferative disease.

Controlled self-renewal and differentiation of hematopoietic stem/progenitor cells (HSPCs) are critical for vertebrate development and survival. These processes are tightly regulated by the transcription factors, signaling molecules and epigenetic factors. Impaired regulations of their function could result in hematological malignancies. Using a large-scale zebrafish N-ethyl-N-nitrosourea mutagenesis screening, we identified a line named LDD731, which presented significantly increased HSPCs in hematopoietic organs. Further analysis revealed that the cells of erythroid/myeloid lineages in definitive hematopoiesis were increased while the primitive hematopoiesis was not affected. The homozygous mutation was lethal with a median survival time around 14-15 days post fertilization. The causal mutation was located by positional cloning in the c-cbl gene, the human ortholog of which, c-CBL, is found frequently mutated in myeloproliferative neoplasms (MPN) or acute leukemia. Sequence analysis showed the mutation in LDD731 caused a histidine-to-tyrosine substitution of the amino acid codon 382 within the RING finger domain of c-Cbl. Moreover, the myeloproliferative phenotype in zebrafish seemed dependent on the Flt3 (fms-like tyrosine kinase 3) signaling, consistent with that observed in both mice and humans. Our study may shed new light on the pathogenesis of MPN and provide a useful in vivo vertebrate model of this syndrome for screening drugs.

Knockout ofRP2decreases GRK1 and rod transducin subunits and leads to photoreceptor degeneration in zebrafish

Retinitis pigmentosa (RP) affects about 1.8 million individuals worldwide. X-linked retinitis pigmentosa (XLRP) is one of the most severe forms of RP. Nearly 85% of XLRP cases are caused by mutations in the X-linked retinitis pigmentosa 2 (RP2) and RPGR. RP2 has been considered to be a GTPase activator protein for ARL3 and to play a role in the traffic of ciliary proteins. The mechanism of how RP2 mutations cause RP is still unclear. In this study, we generated an RP2 knockout zebrafish line using transcription activator-like effector nuclease technology. Progressive retinal degeneration could be observed in the mutant zebrafish. The degeneration of rods' outer segments (OSs) is predominant, followed by the degeneration of cones' OS. These phenotypes are similar to the characteristics of RP2 patients, and also partly consistent with the phenotypes of RP2 knockout mice and morpholino-mediated RP2 knockdown zebrafish. For the first time, we found RP2 deletion leads to decreased protein levels and abnormal retinal localizations of GRK1 and rod transducin subunits (GNAT1 and GNB1) in zebrafish. Furthermore, the distribution of the total farnesylated proteins in zebrafish retina is also affected by RP2 ablation. These molecular alterations observed in the RP2 knockout zebrafish might probably be responsible for the gradual loss of the photoreceptors' OSs. Our work identified the progression of retinal degeneration in RP2 knockout zebrafish, provided a foundation for revealing the pathogenesis of RP caused by RP2 mutations, and would help to develop potential therapeutics against RP in further studies.

Limb body wall complex, amniotic band sequence, or new syndrome caused by mutation in IQ Motif containing K (IQCK)?

Limb body wall complex (LBWC) and amniotic band sequence (ABS) are multiple congenital anomaly conditions with craniofacial, limb, and ventral wall defects. LBWC and ABS are considered separate entities by some, and a continuum of severity of the same condition by others. The etiology of LBWC/ABS remains unknown and multiple hypotheses have been proposed. One individual with features of LBWC and his unaffected parents were whole exome sequenced and Sanger sequenced as confirmation of the mutation. Functional studies were conducted using morpholino knockdown studies followed by human mRNA rescue experiments. Using whole exome sequencing, a de novo heterozygous mutation was found in the gene IQCK: c.667C>G; p.Q223E and confirmed by Sanger sequencing in an individual with LBWC. Morpholino knockdown of iqck mRNA in the zebrafish showed ventral defects including failure of ventral fin to develop and cardiac edema. Human wild-type IQCK mRNA rescued the zebrafish phenotype, whereas human p.Q223E IQCK mRNA did not, but worsened the phenotype of the morpholino knockdown zebrafish. This study supports a genetic etiology for LBWC/ABS, or potentially a new syndrome.

Identification and functional analysis of a SLC33A1: c.339T>G (p.Ser113Arg) variant in the original SPG42 family.

Using whole-exome sequencing, we surveyed all the potential pathogenic variants in an SPG42 family and found five SNPs and four indels that are shared by two patients and lie in the mapped region. Two variants, SLC33A1 p.Ser113Arg and VEPH1 p.Gln433His, cosegregated with the disease. However, VEPH1 p.Gln433His was predicted to be tolerated, thus leaving SLC33A1 p.Ser113Arg as the most plausible causal variant in this family. We found that the phosphorylated SMAD1/5/8 (P-SMAD1/5/8) and BMP receptor type 1A (BMPR1A) were substantially upregulated in fibroblasts derived from an SPG42 individual. Slc33a1 knockdown zebrafish, which exhibited defects in morphology and axon outgrowth, also showed a significant elevation in the level of P-smad1/5/8. While the phenotypes in slc33a1 knockdown zebrafish could be rescued by human wild-type SLC33A1 mRNA, this rescuing effect was diminished by coinjected mutant mRNA encoding p.Ser113Arg, indicating that p.Ser113Arg variant acts in a dominant-negative manner. Importantly, pharmacological blockade of BMPR1 activity by dorsomorphin could efficiently rescue the phenotypic defects in slc33a1 knockdown zebrafish. These results indicate that SLC33A1 can negatively regulate BMP signaling.

The Rac1 regulator ELMO controls basal body migration and docking in multiciliated cells through interaction with Ezrin

Cilia are microtubule-based organelles that are present on most cells and are required for normal tissue development and function. Defective cilia cause complex syndromes with multiple organ manifestations termed ciliopathies. A crucial step during ciliogenesis in multiciliated cells (MCCs) is the association of future basal bodies with the apical plasma membrane, followed by their correct spacing and planar orientation. Here, we report a novel role for ELMO-DOCK1, which is a bipartite guanine nucleotide exchange factor complex for the small GTPase Rac1, and for the membrane-cytoskeletal linker Ezrin, in regulating centriole/basal body migration, docking and spacing. Downregulation of each component results in ciliopathy-related phenotypes in zebrafish and disrupted ciliogenesis in Xenopus epidermal MCCs. Subcellular analysis revealed a striking impairment of basal body docking and spacing, which is likely to account for the observed phenotypes. These results are substantiated by showing a genetic interaction between elmo1 and ezrin b. Finally, we provide biochemical evidence that the ELMO-DOCK1-Rac1 complex influences Ezrin phosphorylation and thereby probably serves as an important molecular switch. Collectively, we demonstrate that the ELMO-Ezrin complex orchestrates ciliary basal body migration, docking and positioning in vivo.

Man o' war mutation in UDP-α-D-xylose synthase favors the abortive catalytic cycle and uncovers a latent potential for hexamer formation.

The man o' war (mow) phenotype in zebrafish is characterized by severe craniofacial defects due to a missense mutation in UDP-α-d-xylose synthase (UXS), an essential enzyme in proteoglycan biosynthesis. The mow mutation is located in the UXS dimer interface ∼16 Å away from the active site, suggesting an indirect effect on the enzyme mechanism. We have examined the structural and catalytic consequences of the mow mutation (R236H) in the soluble fragment of human UXS (hUXS), which shares 93% sequence identity with the zebrafish enzyme. In solution, hUXS dimers undergo a concentration-dependent association to form a tetramer. Sedimentation velocity studies show that the R236H substitution induces the formation of a new hexameric species. Using two new crystal structures of the hexamer, we show that R236H and R236A substitutions cause a local unfolding of the active site that allows for a rotation of the dimer interface necessary to form the hexamer. The disordered active sites in the R236H and R236A mutant constructs displace Y231, the essential acid/base catalyst in the UXS reaction mechanism. The loss of Y231 favors an abortive catalytic cycle in which the reaction intermediate, UDP-α-d-4-keto-xylose, is not reduced to the final product, UDP-α-d-xylose. Surprisingly, the mow-induced hexamer is almost identical to the hexamers formed by the deeply divergent UXS homologues from Staphylococcus aureus and Helicobacter pylori (21% and 16% sequence identity, respectively). The persistence of a latent hexamer-building interface in the human enzyme suggests that the ancestral UXS may have been a hexamer.

Animal model of Sar1b deficiency presents lipid absorption deficits similar to Anderson disease.

Anderson disease (ANDD) or chylomicron retention disease (CMRD) is a rare, hereditary lipid malabsorption syndrome associated with mutations in the SAR1B gene that is characterized by failure to thrive and hypocholesterolemia. Although the SAR1B structure has been resolved and its role in formation of coat protein II (COPII)-coated carriers is well established, little is known about the requirement for SAR1B during embryogenesis. To address this question, we have developed a zebrafish model of Sar1b deficiency based on antisense oligonucleotide knockdown. We show that zebrafish sar1b is highly conserved among vertebrates; broadly expressed during development; and enriched in the digestive tract organs, brain, and craniofacial skeleton. Consistent with ANDD symptoms of chylomicron retention, we found that dietary lipids in Sar1b-deficient embryos accumulate in enterocytes. Transgenic expression analysis revealed that Sar1b is required for growth of exocrine pancreas and liver. Furthermore, we found abnormal differentiation and maturation of craniofacial cartilage associated with defects in procollagen II secretion and absence of select, neuroD-positive neurons of the midbrain and hindbrain. The model presented here will help to systematically dissect developmental roles of Sar1b and to discover molecular and cellular mechanisms leading to organ-specific ANDD pathology. Key messages: Sar1b depletion phenotype in zebrafish resembles Anderson disease deficits. Sar1b deficiency results in multi-organ developmental deficits. Sar1b is required for dietary cholesterol uptake into enterocytes.

Reverse Genetic Screening Reveals Poor Correlation between Morpholino-Induced and Mutant Phenotypes in Zebrafish

The widespread availability of programmable site-specific nucleases now enables targeted gene disruption in the zebrafish. In this study, we applied site-specific nucleases to generate zebrafish lines bearing individual mutations in more than 20 genes. We found that mutations in only a small proportion of genes caused defects in embryogenesis. Moreover, mutants for ten different genes failed to recapitulate published Morpholino-induced phenotypes (morphants). The absence of phenotypes in mutant embryos was not likely due to maternal effects or failure to eliminate gene function. Consistently, a comparison of published morphant defects with the Sanger Zebrafish Mutation Project revealed that approximately 80% of morphant phenotypes were not observed in mutant embryos, similar to our mutant collection. Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses.

The Rac1 regulator ELMO controls basal body migration and docking in multiciliated cells through interaction with Ezrin.

Cilia are microtubule-based organelles that are present on most cells and are required for normal tissue development and function. Defective cilia cause complex syndromes with multiple organ manifestations termed ciliopathies. A crucial step during ciliogenesis in multiciliated cells (MCCs) is the association of future basal bodies with the apical plasma membrane, followed by their correct spacing and planar orientation. Here, we report a novel role for ELMO-DOCK1, which is a bipartite guanine nucleotide exchange factor complex for the small GTPase Rac1, and for the membrane-cytoskeletal linker Ezrin, in regulating centriole/basal body migration, docking and spacing. Downregulation of each component results in ciliopathy-related phenotypes in zebrafish and disrupted ciliogenesis in Xenopus epidermal MCCs. Subcellular analysis revealed a striking impairment of basal body docking and spacing, which is likely to account for the observed phenotypes. These results are substantiated by showing a genetic interaction between elmo1 and ezrin b. Finally, we provide biochemical evidence that the ELMO-DOCK1-Rac1 complex influences Ezrin phosphorylation and thereby probably serves as an important molecular switch. Collectively, we demonstrate that the ELMO-Ezrin complex orchestrates ciliary basal body migration, docking and positioning in vivo.

TNF-mediated inflammation represses GATA1 and activates p38 MAP kinase in RPS19-deficient hematopoietic progenitors

AbstractDiamond-Blackfan anemia (DBA) is an inherited disorder characterized by defects in erythropoiesis, congenital abnormalities, and predisposition to cancer. Approximately 25% of DBA patients have a mutation in RPS19, which encodes a component of the 40S ribosomal subunit. Upregulation of p53 contributes to the pathogenesis of DBA, but the link between ribosomal protein mutations and erythropoietic defects is not well understood. We found that RPS19 deficiency in hematopoietic progenitor cells leads to decreased GATA1 expression in the erythroid progenitor population and p53-dependent upregulation of tumor necrosis factor-α (TNF-α) in nonerythroid cells. The decrease in GATA1 expression was mediated, at least in part, by activation of p38 MAPK in erythroid cells and rescued by inhibition of TNF-α or p53. The anemia phenotype in rps19-deficient zebrafish was reversed by treatment with the TNF-α inhibitor etanercept. Our data reveal that RPS19 deficiency leads to inflammation, p53-dependent increase in TNF-α, activation of p38 MAPK, and decreased GATA1 expression, suggesting a novel mechanism for the erythroid defects observed in DBA.

Abstract IA05: Fanconi's anemia: Emerging therapeutic opportunities

Abstract Fanconi's anemia (FA) is a genetically heterogeneous inherited disorder characterized by bone marrow failure, birth defects and cancer susceptibility. At the cellular level, FA features hypersensitivity to DNA cross-linking agents, cell cycle abnormalities and abnormal responses to cytokines. Therapy for the hematopoietic defects of FA has consisted of supportive care, stem cell transplantation and administration of androgens such as oxymetholone. Unfortunately, even successfully transplanted FA patients have a very high risk for subsequent solid tumors, especially squamous cell carcinomas of oropharyngeal and urogenital areas. The pharmacological treatment of FA has been unchanged for several decades and for this reason our group has embarked on systematic search for small molecule therapeutics that could benefit FA patients. This effort has been made possible by the cloning of 16 FA genes and biochemical insights into the function of FA proteins. Mouse models, zebra fish models and FA patient cell lines, including IPSC are available for screening and validation. The specific goals of small molecule interventions are to delay or prevent hematopoietic failure and also to delay or prevent carcinogenesis. To date we have shown that the superoxide dismutase mimetic drug Tempol can significantly delay tumor formation in FA mice. Similarly, resveratrol clearly improved hematopoiesis in the same model. Medium throughput screens have revealed numerous new molecules which ameliorate the FA phenotypes of cells, mice and zebrafish respectively. Using genetic approaches we also have identified the p53 pathway as a therapeutic target in FA. These therapeutic opportunities and progress in FA small molecule screening will be discussed. Citation Format: Markus Grompe, John Postlethwait, Alan D'Andrea, Susan Olson, Qingshuo Zhang, Grover Bagby. Fanconi's anemia: Emerging therapeutic opportunities. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr IA05. doi:10.1158/1538-7445.CANSUSC14-IA05

Human Kidney Disease-causing INF2 Mutations Perturb Rho/Dia Signaling in the Glomerulus

Mutations in Inverted Formin 2 (INF2), a diaphanous formin family protein that regulates actin cytoskeleton dynamics, cause focal segmental glomerulosclerosis (FSGS) and Charcot–Marie–Tooth Disease (CMT) in humans. In addition to directly remodeling actin filaments in vitro, we have shown that INF2 regulates intracellular actin dynamics and actin dependent cellular behavior by opposing Rhoa/Dia signaling. As a step towards a better understanding of the human kidney disease, we wanted to explore the relevance of these findings to the in vivo situation. We used dose dependent knockdown of INF2 to first define an in vivo model and establish an overt glomerular phenotype in zebrafish. This simple assay was validated by rescue with wild type INF2 confirming the specificity of the findings. The edema, podocyte dysfunction, and an altered glomerular filtration barrier observed in the zebrafish pronephros correlate with mistrafficking of glomerular slit diaphragm proteins, defective slit-diaphragm signaling, and disinhibited diaphanous formin (mDia) activity. In contrast to wild-type human INF2, INF2 mutants associated with kidney disease fail to rescue the zINF2 morphant phenotype. Of particular interest, this INF2 knockdown phenotype is also rescued by loss of either RhoA or Dia2. This simple assay allows the demonstration that INF2 functions, at least in part, to modulate Dia-mediated Rho signaling, and that disease causing mutations specifically impair this regulatory function. These data support a model in which disease-associated diaphanous inhibitory domain (DID) mutants in INF2 interfere with its binding to and inhibition of Dia, leading to uncontrolled Rho/Dia signaling and perturbed actin dynamics. Methods to fine tune Rho signaling in the glomerulus may lead to new approaches to therapy in humans.