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Loss-of-function mutation of ABCC9, the gene encoding the SUR2 subunit of ATP sensitive-potassium (KATP) channels, was recently associated with autosomal recessive ABCC9-related intellectual disability and myopathy syndrome (AIMS). Here we identify nine additional subjects, from seven unrelated families, harbouring different homozygous loss-of-function variants in ABCC9 and presenting with a conserved range of clinical features. All variants are predicted to result in severe truncations or in-frame deletions within SUR2, leading to the generation of non-functional SUR2-dependent KATP channels. Affected individuals show psychomotor delay and intellectual disability of variable severity, microcephaly, corpus callosum and white matter abnormalities, seizures, spasticity, short stature, muscle fatigability and weakness. Heterozygous parents do not show any conserved clinical pathology but report multiple incidences of intra-uterine fetal death, which were also observed in an eighth family included in this study. In vivo studies of abcc9 loss-of-function in zebrafish revealed an exacerbated motor response to pentylenetetrazole, a pro-convulsive drug, consistent with impaired neurodevelopment associated with an increased seizure susceptibility. Our findings define an ABCC9 loss-of-function-related phenotype, expanding the genotypic and phenotypic spectrum of AIMS and reveal novel human pathologies arising from KATP channel dysfunction., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

ABCC9-related intellectual disability and myopathy syndrome (AIMS) arises from loss-of-function (LoF) mutations in the ABCC9 gene, which encodes the SUR2 subunit of ATP-sensitive potassium (K ATP ) channels. K ATP channels are found throughout the cardiovascular system and skeletal muscle and couple cellular metabolism to excitability. AIMS individuals show fatigability, muscle spasms, and cardiac dysfunction. We found reduced exercise performance in mouse models of AIMS harboring premature stop codons in ABCC9. Given the roles of K ATP channels in all muscles, we sought to determine how myopathy arises using tissue-selective suppression of K ATP and found that LoF in skeletal muscle, specifically, underlies myopathy. In isolated muscle, SUR2 LoF results in abnormal generation of unstimulated forces, potentially explaining painful spasms in AIMS. We sought to determine whether excessive Ca 2+ influx through Ca V 1.1 channels was responsible for myopathology but found that the Ca 2+ channel blocker verapamil unexpectedly resulted in premature death of AIMS mice and that rendering Ca V 1.1 channels nonpermeable by mutation failed to reverse pathology; results which caution against the use of calcium channel blockers in AIMS., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

ATP-sensitive potassium channel (K ATP )gain- (GOF) and loss-of-function (LOF) mutations underlie human neonatal diabetes mellitus (NDM) and hyperinsulinism (HI), respectively. While transgenic mice expressing incomplete K ATP LOF do reiterate mild hyperinsulinism, K ATP knockout animals do not exhibit persistent hyperinsulinism. We have shown that islet excitability and glucose homeostasis are regulated by identical K ATP channels in zebrafish. SUR1 truncation mutation (K499X) was introduced into the abcc8 gene to explore the possibility of using zebrafish for modeling human HI. Patch-clamp analysis confirmed the complete absence of channel activity in β-cells from K499X (SUR1 -/- ) fish. No difference in random blood glucose was detected in heterozygous SUR1+/- fish nor in homozygous SUR1 -/- fish, mimicking findings in SUR1 knockout mice. Mutant fish did, however, demonstrate impaired glucose tolerance, similar to partial LOF mouse models. In paralleling features of mammalian diabetes and hyperinsulinism resulting from equivalent LOF mutations, these gene-edited animals provide valid zebrafish models of K ATP -dependent pancreatic diseases.

ATP-sensitive potassium channels (K ATP channels) are hetero-octameric nucleotide-gated ion channels that couple cellular metabolism to excitability in various tissues. In the heart, K ATP channels are activated during ischaemia and potentially during adrenergic stimulation. In the vasculature, they are normally active at a low level, reducing vascular tone, but the ubiquitous nature of these channels leads to complex and poorly understood channelopathies as a result of gain- or loss-of-function mutations. Zebrafish (ZF) models of these channelopathies may provide insights to the link between molecular dysfunction and complex pathophysiology, but this requires understanding the tissue dependence of channel activity and subunit specificity. Thus far, direct analysis of ZF K ATP expression and functional properties has only been performed in pancreatic β-cells. Using a comprehensive combination of genetically modified fish, electrophysiology and gene expression analysis, we demonstrate that ZF cardiac myocytes (CM) and vascular smooth muscle (VSM) express functional K ATP channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. However, in contrast to mammalian cardiovascular K ATP channels, ZF channels are insensitive to potassium channel opener drugs (pinacidil, minoxidil) in both chambers of the heart and in VSM. The results provide a first characterization of the molecular properties of fish K ATP channels and validate the use of such genetically modified fish as models of human Cantú syndrome and ABCC9-related Intellectual Disability and Myopathy syndrome. KEY POINTS: Zebrafish cardiac myocytes (CM) and vascular smooth muscle (VSM) express functional K ATP channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. In contrast to mammalian cardiovascular K ATP channels, zebrafish channels are insensitive to potassium channel opener drugs (pinacidil, minoxidil) in both chambers of the heart and in VSM. We provide a first characterization of the molecular properties of fish K ATP channels and validate the use of such genetically modified fish as models of human Cantú syndrome and ABCC9-related Intellectual Disability and Myopathy syndrome., (© 2021 The Authors. The Journal of Physiology © 2021 The Physiological Society.)

Islet β-cell membrane excitability is a well-established regulator of mammalian insulin secretion, and defects in β-cell excitability are linked to multiple forms of diabetes. Evolutionary conservation of islet excitability in lower organisms is largely unexplored. Here we show that adult zebrafish islet calcium levels rise in response to elevated extracellular [glucose], with similar concentration-response relationship to mammalian β-cells. However, zebrafish islet calcium transients are nor well coupled, with a shallower glucose-dependence of cytoplasmic calcium concentration. We have also generated transgenic zebrafish that conditionally express gain-of-function mutations in ATP-sensitive K + channels (K ATP -GOF) in β-cells. Following induction, these fish become profoundly diabetic, paralleling features of mammalian diabetes resulting from equivalent mutations. K ATP -GOF fish become severely hyperglycemic, with slowed growth, and their islets lose glucose-induced calcium responses. These results indicate that, although lacking tight cell-cell coupling of intracellular Ca 2+ , adult zebrafish islets recapitulate similar excitability-driven β-cell glucose responsiveness to those in mammals, and exhibit profound susceptibility to diabetes as a result of inexcitability. While illustrating evolutionary conservation of islet excitability in lower vertebrates, these results also provide important validation of zebrafish as a suitable animal model in which to identify modulators of islet excitability and diabetes., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

The study of regeneration in an in vivo vertebrate system has the potential to reveal targetable genes and pathways that could improve our ability to heal and repair damaged tissue. We have developed a system for clonal labeling of discrete cell lineages and independently inducing gene expression under control of the heat shock promoter in the zebrafish caudal fin. Consequently we are able to test the affects of overexpressing a single gene in the context of regeneration within each of the nine different cell lineage classes that comprise the caudal fin. This can test which lineage is necessary or sufficient to provide gene function. As a first example to demonstrate this approach, we explored which lineages were competent to functionally express the kit ligand a protein as assessed by the local complementation of the mutation in the sparse-like (kitlgatc244b) background. We show that dermal fibroblast expression of kit ligand a robustly supports the rescue of melanocytes in the regenerating caudal fin. kit ligand a expression from skin and osteoblasts results in more modest and variable rescue of melanocytes, while lateral line expression was unable to complement the mutation.

The regulation of transcription and translation by specific cell types is essential to generate the cellular diversity that typifies complex multicellular organisms. Tagging and purification of ribosomal proteins has been shown to be an innovative and effective means of characterizing the ribosome bound transcriptome of highly specific cell populations in vivo. To test the feasibility of using translating ribosome affinity purification (TRAP) in zebrafish, we have generated both a ubiquitous TRAP line and a melanocyte-specific TRAP line using the native zebrafish rpl10a ribosomal protein. We have demonstrated the capacity to capture mRNA transcripts bound to ribosomes, and confirmed the expected enrichment of melanocyte specific genes and depletion of non-melanocyte genes when expressing the TRAP construct with a cell specific promoter. We have also generated a generic EGFP-rpl10a Tol2 plasmid construct (Tol2-zTRAP) that can be readily modified to target any additional cell populations with characterized promoters in zebrafish., (Copyright © 2012 Wiley Periodicals, Inc.)

The study of melanocyte biology in the zebrafish presents a highly tractable system for understanding fundamental principles of developmental biology. Melanocytes are visible in the transparent embryo and in the mature fish following metamorphosis, a physical transformation from the larval to adult form. While early developing larval melanocytes are direct derivatives of the neural crest, the remainder of melanocytes develop from unpigmented precursors, or melanocyte stem cells (MSCs). The Tol2 transposable element has facilitated the construction of stable transgenic lines that label melanocytes. In another application, integration of Tol2 constructs makes possible clonal analysis of melanocyte and MSC lineages. Drugs that block melanin synthesis, ablate melanocytes, and block establishment of MSC populations allow the interrogation of this model system for mechanisms of adult stem cell development and regulation.

Previous research in zebrafish has demonstrated that embryonic and larval regeneration melanocytes are derived from separate lineages. The embryonic melanocytes that establish the larval pigment pattern do not require regulative melanocyte stem cell (MSC) precursors, and are termed direct-developing melanocytes. In contrast, the larval regeneration melanocytes that restore the pigment pattern after ablation develop from MSC precursors. Here, we explore whether embryonic melanocytes and MSCs share bipotent progenitors. Furthermore, we explore when fate segregation of embryonic melanocytes and MSCs occurs in zebrafish development. In order to achieve this, we develop and apply a novel lineage tracing method. We first demonstrate that Tol2-mediated genomic integration of reporter constructs from plasmids injected at the 1-2 cell stage occurs most frequently after the midblastula transition but prior to shield stage, between 3 and 6 hours post-fertilization. This previously uncharacterized timing of Tol2-mediated genomic integration establishes Tol2-mediated transposition as a means for conducting lineage tracing in zebrafish. Combining the Tol2-mediated lineage tracing strategy with a melanocyte regeneration assay previously developed in our lab, we find that embryonic melanocytes and larval regeneration melanocytes are derived from progenitors that contribute to both lineages. We estimate 50-60 such bipotent melanogenic progenitors to be present in the shield-stage embryo. Furthermore, our examination of direct-developing and MSC-restricted lineages suggests that these are segregated from bipotent precursors after the shield stage, but prior to the end of convergence and extension. Following this early fate segregation, we estimate approximately 100 embryonic melanocyte and 90 MSC-restricted lineages are generated to establish or regenerate the zebrafish larval pigment pattern, respectively. Thus, the dual strategies of direct-development and MSC-derived development are established in the early gastrula, via fate segregation of the two lineages.

Objective: To estimate allele frequencies of the hyperkalaemic periodic paralysis (HYPP), lethal white foal syndrome (LWFS), glycogen branching enzyme deficiency (GBED), hereditary equine regional dermal asthenia (HERDA), and type 1 polysaccharide storage myopathy (PSSM) genes in elite performance subgroups of American Quarter Horses (AQHs)., Design: Prospective genetic survey., Animals: 651 elite performance AQHs, 200 control AQHs, and 180 control American Paint Horses (APHs)., Procedures: Elite performance AQHs successful in 7 competitive disciplines (barrel racing, cutting, halter, racing, reining, western pleasure, and working cow horse) were geno- typed for 5 disease-causing alleles. Age-matched control AQHs and APHs were used to establish comparative whole-breed estimates of allele frequencies., Results: Highest allele frequencies among control AQHs were for type 1 PSSM (0.055) and GBED (0.054), whereas HERDA (0.021) and HYPP (0.008) were less prevalent. Control APHs uniquely harbored LWFS (0.107) and had high prevalence of HYPP (0.025), relative to AQHs. Halter horse subgroups had significantly greater allele frequencies for HYPP (0.299) and PSSM (0.155). Glycogen branching enzyme deficiency, HERDA, and PSSM were found broadly throughout subgroups; cutting subgroups were distinct for HERDA (0.142), and western pleasure subgroups were distinct for GBED (0.132). Racing and barrel racing subgroups had the lowest frequencies of the 5 disease genes., Conclusions and Clinical Relevance: Accurate estimates of disease-causing alleles in AQHs and APHs may guide use of diagnostic genetic testing, aid management of genetic diseases, and help minimize production of affected foals.

Hereditary equine regional dermal asthenia (HERDA), a degenerative skin disease that affects the Quarter Horse breed, was localized to ECA1 by homozygosity mapping. Comparative genomics allowed the development of equine gene-specific markers which were used with a set of affected horses to detect a homozygous, identical-by-descent block spanning approximately 2.5 Mb, suggesting a recent origin for the HERDA mutation. We report a mutation in cyclophilin B (PPIB) as a novel, causal candidate gene for HERDA. A c.115G>A missense mutation in PPIB alters a glycine residue that has been conserved across vertebrates. The mutation was homozygous in 64 affected horses and segregates concordant with inbreeding loops apparent in the genealogy of 11 affected horses. Screening of control Quarter Horses indicates a 3.5% carrier frequency. The development of a test that can detect affected horses prior to development of clinical signs and carriers of HERDA will allow Quarter Horse breeders to eliminate this debilitating disease.

Objective: To assess heritability and mode of inheritance for hereditary equine regional dermal asthenia (HERDA) in Quarter Horses., Animals: 1,295 horses with Quarter Horse bloodlines, including 58 horses affected with HERDA., Procedure: Horses were classified as affected or unaffected or as undetermined when data were insufficient to assess phenotype. Pedigree data were analyzed to determine the probable mode of inheritance. Heritability was estimated by use of Bayesian statistical methods., Results: Heritability (mean+/-SD) of HERDA was estimated to be 0.38+/-0.13, with both sexes having an equal probability of being affected. Results for evaluation of the pedigrees were consistent with a single Mendelian autosomal recessive mode of inheritance., Conclusions and Clinical Relevance: HERDA in Quarter Horses is an inherited disease, and affected horses are more likely to produce affected offspring. An autosomal recessive mode of inheritance should be considered by people making breeding decisions involving Quarter Horses when a first-degree relative has been confirmed with HERDA or has produced affected offspring. In addition, breeders whose horses have produced affected offspring can reduce the likelihood of producing affected horses in the future by avoiding inbreeding.

A solution is provided for the problem of large numbers of variables in V-analysis and of large numbers of subjects in 0-analysis. The solution is that of estimating the Structure in the total set from the structures found in manageable samples of the total set. The samples are drawn randomly; structures of each are computed by existing BC TRY programs; finally the structures of the random samples are combined using new BC TRY programs. Examples of the complete analyses in two separate studies are given.

A common assertion of psychologists is that the objeotive of psychology .is to predict and control behavior. The assertion springs from the belief that psychologists will learn enough about general stimulus-raponse laws and underlying factors' of behavior to predict how a given individual will react in a given discrete stim- ulus situation, and we will be able to arrange situations in a fashion that will control what the individual does.

When persons are observed on multiple dimensions discovered by the objective procedures of the cluster analysis of variables (V-analysis), they can then be allocated to person-clusters by the equally-objective procedures of object-clustering (O-analysis). These procedures, called the Condensation Method, are completely worked out on two empirical studies. In the first study on intellectual abilities (the Holzinger Problem) 301 children are assigned to 15 O-types. In the second study on self-conceptions (the MMPI), 310 psychiatric patients and normals are allocated to 14 O-types. The means of assigning new individuals to these master types are described. All procedures are executed by computer programs of the BC TRY System, in particular the components FACS, OTYPE, and OSTAT. The logic of the methods is fully developed.

Prediction of individual differences by cluster analysis procedures is described for the case of Holzinger's 24-variable problem and the case of MMPI item-clusters. Comparisons are made between univariate, multiple regression, and person-cluster predictions. The best prediction is from person- clusters, whose score patterns are objectively isolated by BC TRY Computer System programs. The role of chance is objectively assessed by program 4CAST and not by estimation formulations.

The basic data with which psychologists commonly deal are the dichotomous responses of individuals t o stimulus presentations. The stimulus situations studied range from those presented in psychophysical brass instrument experiments .to items printed in tests of ability and personality.

Three demographic dimensions previously isolated by cluster analysis procedures (using BC TRY System computer analyses) were compared in different metropolitan areas for their stability over time and place. Also, the social areas discovered within the communities were studied for their stability over time and place. The three demographic dimensions (socio-economic independence, family life, and assimilation) accounted for the generality of 33 census tract (1940) characteristics in two communities. It was also shown that the three basic dimensions were essentially unchanged during the decade which included World War 11. The stability of these dimensions was retained even though there was considerable change in residents in each metropolitan area during the decade. The validity of cluster-search procedures is demonstrated by the stability of the three demographic dimensions.

Prediction of group differences by cluster analysis procedures is described for the case of neighborhoods (tracts) of a metropolitan area. Social areas of homogeneous neighborhoods are isolated by objective "O-analysis" procedures of the BC TRY Computer System. The predictor attributes are pre-war demographic features from which high predictions both of demographic and voting-attitudes are made up to 15 years, despite the social disruptions of a great war. Three basic dimensions, Conservatism, Territoriality, and Exclusiveness are found to predict all demographic and attitudinal characteristics of neighborhoods over a decade and a half.