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Mutations in the tetratricopeptide repeat domain 7A (TTC7A) gene are associated with severe intestinal disorders and combined immunodeficiency (CID), with poor long-term survival. This study describes the characteristics and clinical course of six patients with intestinal failure who were found to have biallelic TTC7A mutations, highlighting key management strategies for identifying these patients and improving their survival. Of the six patients included, five had multiple intestinal atresias (83%) and one had congenital enteropathy (17%). Pyloric web or atresia was present in 100% of patients. All patients had low CD3+ T-cell counts on flow cytometry, consistent with CID. Immunologic management consisted of intravenous immunoglobulin and antibiotic prophylaxis, while two patients (33%) underwent stem cell transplantation. All patients were initially dependent on parenteral nutrition, but two (33%) achieved enteral autonomy after undergoing intestinal transplantation. Patients were followed for a median of 7 years (IQR 4.75–9.25), with a long-term survival rate of 67%. The high incidence of pyloric atresia in this case series suggests that the presence of pyloric atresia, especially in the setting of other intestinal disorders, should prompt screening for CID and a genetic evaluation. Recognition of the mutation and involvement of appropriate interdisciplinary care teams are essential for optimizing survival of these complex patients.

Objective: To identify novel disease associated loci for amyotrophic lateral sclerosis (ALS), we used sequencing data and performed in vitro and in vivo experiments to demonstrate pathogenicity of mutations identified in TP73 ., Methods: We analyzed exome sequences of 87 patients with sporadic ALS and 324 controls, with confirmatory sequencing in independent ALS cohorts of >2,800 patients. For the top hit, TP73 , a regulator of apoptosis and differentiation and a binding partner and homolog of the tumor suppressor gene TP53 , we assayed mutation effects using in vitro and in vivo experiments. C2C12 myoblast differentiation assays, characterization of myotube appearance, and immunoprecipitation of p53-p73 complexes were performed in vitro. In vivo, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 targeting of zebrafish tp73 to assay motor neuron number and axon morphology., Results: Four heterozygous rare, nonsynonymous mutations in TP73 were identified in our sporadic ALS cohort. In independent ALS cohorts, we identified an additional 19 rare, deleterious variants in TP73 . Patient TP73 mutations caused abnormal differentiation and increased apoptosis in the myoblast differentiation assay, with abnormal myotube appearance. Immunoprecipitation of mutant ΔN-p73 demonstrated that patient mutations hinder the ability of ΔN-p73 to bind p53. CRISPR/Cas9 knockout of tp73 in zebrafish led to impaired motor neuron development and abnormal axonal morphology, concordant with ALS pathology., Conclusion: Together, these results strongly suggest that variants in TP73 correlate with risk for ALS and indicate a role for apoptosis in ALS disease pathology., (© 2021 American Academy of Neurology.)

Vanishing white matter disease (VWM) is a severe leukodystrophy of the central nervous system caused by mutations in subunits of the eukaryotic initiation factor 2B complex (eIF2B). Current models only partially recapitulate key disease features, and pathophysiology is poorly understood. Through development and validation of zebrafish ( Danio rerio ) models of VWM, we demonstrate that zebrafish eif2b mutants phenocopy VWM, including impaired somatic growth, early lethality, effects on myelination, loss of oligodendrocyte precursor cells, increased apoptosis in the CNS, and impaired motor swimming behavior. Expression of human EIF2B2 in the zebrafish eif2b2 mutant rescues lethality and CNS apoptosis, demonstrating conservation of function between zebrafish and human. In the mutants, intron 12 retention leads to expression of a truncated eif2b5 transcript. Expression of the truncated eif2b5 in wild-type larva impairs motor behavior and activates the ISR, suggesting that a feed-forward mechanism in VWM is a significant component of disease pathophysiology., Competing Interests: MK, HS, HS, TS, KG, DB, ES, SC, SM, EH No competing interests declared, JB Consultant: Bluebird Bio (5/2017; 10/2017; 11/2019) Calico (1/2018-1/2019) Denali therapeutics (6/2019) Enzyvant (6/2019) Neurogene (3/2020) Board of Directors wfluidx 1/2018-present Stock Orchard Therapeutics, (© 2020, Keefe et al.)

X-linked adrenoleukodystrophy (ALD) is a devastating inherited neurodegenerative disease caused by defects in the ABCD1 gene and affecting peripheral and central nervous system myelin. ABCD1 encodes a peroxisomal transmembrane protein required for very long chain fatty acid (VLCFA) metabolism. We show that zebrafish (Danio rerio) Abcd1 is highly conserved at the amino acid level with human ABCD1, and during development is expressed in homologous regions including the central nervous system and adrenal glands. We used TALENs to generate five zebrafish abcd1 mutant allele lines introducing premature stop codons in exon 1, as well as obtained an abcd1 allele from the Zebrafish Mutation Project carrying a point mutation in a splice donor site. Similar to patients with ALD, zebrafish abcd1 mutants have elevated VLCFA levels. Interestingly, we found that CNS development of the abcd1 mutants is disrupted, with hypomyelination in the spinal cord, abnormal patterning and decreased numbers of oligodendrocytes, and increased cell death. By day of life five abcd1 mutants demonstrate impaired motor function, and overall survival to adulthood of heterozygous and homozygous mutants is decreased. Expression of human ABCD1 in oligodendrocytes rescued apoptosis in the abcd1 mutant. In summary, we have established a zebrafish model of ALD that recapitulates key features of human disease pathology and which reveals novel features of underlying disease pathogenesis., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

There has been a rapid expansion in use of transgenic technologies in zebrafish. We report a novel example of transinteractions of genetic elements, or transvection. This interaction led to a novel expression pattern and illustrates a precautionary example regarding use of transgenes in zebrafish.

Tools for genetically-determined visualization of synaptic circuits and interactions are necessary to build connectomics of the vertebrate brain and to screen synaptic properties in neurological disease models. Here we develop a transgenic FingR (fibronectin intrabodies generated by mRNA display) technology for monitoring synapses in live zebrafish. We demonstrate FingR labeling of defined excitatory and inhibitory synapses, and show FingR applicability for dissecting synapse dynamics in normal and disease states. Using our system we show that chronic hypoxia, associated with neurological defects in preterm birth, affects dopaminergic neuron synapse number depending on the developmental timing of hypoxia.

Pancreatitis is caused by inflammatory injury to the exocrine pancreas, from which both humans and animal models appear to recover via regeneration of digestive enzyme-producing acinar cells. This regenerative process involves transient phases of inflammation, metaplasia, and redifferentiation, driven by cell-cell interactions between acinar cells, leukocytes, and resident fibroblasts. The NFκB signaling pathway is a critical determinant of pancreatic inflammation and metaplasia, whereas a number of developmental signals and transcription factors are devoted to promoting acinar redifferentiation after injury. Imbalances between these proinflammatory and prodifferentiation pathways contribute to chronic pancreatitis, characterized by persistent inflammation, fibrosis, and acinar dedifferentiation. Loss of acinar cell differentiation also drives pancreatic cancer initiation, providing a mechanistic link between pancreatitis and cancer risk. Unraveling the molecular bases of exocrine regeneration may identify new therapeutic targets for treatment and prevention of both of these deadly diseases.

The size of the pancreas is determined by intrinsic factors, such as the number of progenitor cells, and by extrinsic signals that control the fate and proliferation of those progenitors. Both the exocrine and endocrine compartments of the pancreas undergo dramatic expansion after birth and are capable of at least partial regeneration following injury. Whether the expansion of these lineages relies on similar mechanisms is unknown. Although we have shown that the Wnt signaling component β-catenin is selectively required in mouse embryos for the generation of exocrine acinar cells, this protein has been ascribed various functions in the postnatal pancreas, including proliferation and regeneration of islet as well as acinar cells. To address whether β-catenin remains important for the maintenance and expansion of mature acinar cells, we have established a system to follow the behavior and fate of β-catenin-deficient cells during postnatal growth and regeneration in mice. We find that β-catenin is continuously required for the establishment and maintenance of acinar cell mass, extending from embryonic specification through juvenile and adult self-renewal and regeneration. This requirement is not shared with islet cells, which proliferate and function normally in the absence of β-catenin. These results make distinct predictions for the relative role of Wnt-β-catenin signaling in the etiology of human endocrine and exocrine disease. We suggest that loss of Wnt-β-catenin activity is unlikely to drive islet dysfunction, as occurs in type 2 diabetes, but that β-catenin is likely to promote human acinar cell proliferation following injury, and might therefore contribute to the resolution of acute or chronic pancreatitis.

Defining the genetic pathways essential for hematopoietic stem cell (HSC) development remains a fundamental goal impacting stem cell biology and regenerative medicine. To genetically dissect HSC emergence in the aorta-gonad-mesonephros (AGM) region, we screened a collection of insertional zebrafish mutant lines for expression of the HSC marker, c-myb. Nine essential genes were identified, which were subsequently binned into categories representing their proximity to HSC induction. Using overexpression and loss-of-function studies in zebrafish, we ordered these signaling pathways with respect to each other and to the Vegf, Notch, and Runx programs. Overexpression of vegf and notch is sufficient to induce HSCs in the tbx16 mutant, despite a lack of axial vascular organization. Although embryos deficient for artery specification, such as the phospholipase C gamma-1 (plcgamma1) mutant, fail to specify HSCs, overexpression of notch or runx1 can rescue their hematopoietic defect. The most proximal HSC mutants, such as hdac1, were found to have no defect in vessel or artery formation. Further analysis demonstrated that hdac1 acts downstream of Notch signaling but upstream or in parallel to runx1 to promote AGM hematopoiesis. Together, our results establish a hierarchy of signaling programs required and sufficient for HSC emergence in the AGM.

The zebrafish has emerged as a powerful genetic model of cancer, but has been limited by the use of stable transgenic approaches to induce disease. Here, a co-injection strategy is described that capitalizes on both the numbers of embryos that can be microinjected and the ability of transgenes to segregate together and exert synergistic effects in forming tumors. Using this mosaic transgenic approach, gene pathways involved in tumor initiation and radiation sensitivity have been identified.

Embryonal rhabdomyosarcoma (ERMS) is a devastating cancer with specific features of muscle differentiation that can result from mutational activation of RAS family members. However, to date, RAS pathway activation has not been reported in a majority of ERMS patients. Here, we have created a zebrafish model of RAS-induced ERMS, in which animals develop externally visible tumors by 10 d of life. Microarray analysis and cross-species comparisons identified two conserved gene signatures found in both zebrafish and human ERMS, one associated with tumor-specific and tissue-restricted gene expression in rhabdomyosarcoma and a second comprising a novel RAS-induced gene signature. Remarkably, our analysis uncovered that RAS pathway activation is exceedingly common in human RMS. We also created a new transgenic coinjection methodology to fluorescently label distinct subpopulations of tumor cells based on muscle differentiation status. In conjunction with fluorescent activated cell sorting, cell transplantation, and limiting dilution analysis, we were able to identify the cancer stem cell in zebrafish ERMS. When coupled with gene expression studies of this cell population, we propose that the zebrafish RMS cancer stem cell shares similar self-renewal programs as those found in activated satellite cells.

RAS family members are among the most frequently mutated oncogenes in human cancers. Given the utility of zebrafish in both chemical and genetic screens, developing RAS-induced cancer models will make large-scale screens possible to understand further the molecular mechanisms underlying malignancy. We developed a heat shock-inducible Cre/Lox-mediated transgenic approach in which activated human kRASG12D can be conditionally induced within transgenic animals by heat shock treatment. Specifically, double transgenic fish Tg(B-actin-LoxP-EGFP-LoxP-kRASG12D; hsp70-Cre) developed four types of tumors and hyperplasia after heat shock of whole zebrafish embryos, including rhabdomyosarcoma, myeloproliferative disorder, intestinal hyperplasia, and malignant peripheral nerve sheath tumor. Using ex vivo heat shock and transplantation of whole kidney marrow cells from double transgenic animals, we were able to generate specifically kRASG12D-induced myeloproliferative disorder in recipient fish. This heat shock-inducible recombination approach allowed for the generation of multiple types of RAS-induced tumors and hyperplasia without characterizing tissue-specific promoters. Moreover, these tumors and hyperplasia closely resemble human diseases at both the morphologic and molecular levels.