Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model

Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas approach using multiple single guide RNAs targeting simultaneously different positions in two exemplar genes (osgep or tprkb) to increase the likelihood of generating mutations on both alleles in the injected F0 generation and to achieve a similar effect as morpholinos but with the reproducibility of stable lines. This multi single guide RNA approach resulted in median likelihoods for at least one mutation on each allele of >99% and sgRNA specific insertion/deletion profiles as revealed by deep-sequencing. Immunoblot showed a significant reduction for Osgep and Tprkb proteins. For both genes, the acute multi-sgRNA knockout recapitulated the microcephaly phenotype and reduction in survival that we observed previously in stable knockout lines, though milder in the acute multi-sgRNA knockout. Finally, we quantify the degree of mutagenesis by deep sequencing, and provide a mathematical model to quantitate the chance for a biallelic loss-of-function mutation. Our findings can be generalized to acute and stable CRISPR/Cas targeting for any zebrafish gene of interest

Oxygen- and Hif1alpha-dependent regulation of skeletal muscle progenitor differentiation and skeletal muscle regeneration

Skeletal muscle progenitors, which give rise to terminally differentiated muscle, represent potential therapies for skeletal muscle disease. These progenitors reside in a low O2 environment before local blood vessels and differentiated muscle form. It has been established that low O 2 levels (hypoxia) maintain muscle precursors in an undifferentiated state in vitro, suggesting that in developing or regenerating muscle, local hypoxia may constrain progenitor differentiation until ample nutrients are available. However, this has not been formally tested. In addition, the signals linking O2 availability with progenitor differentiation are incompletely understood. For the first part of my thesis, cell culture models were used to delineate these signals. Depleting myoblasts in vitro of the Hypoxia Inducible Factors (HIFs)—the primary effectors of O2—revealed that hypoxia regulates differentiation via HIF-dependent and -independent mechanisms. We identified a key HIF-independent mechanism—AKT inactivation—concluding that multiple O2-dependent signals regulate progenitor differentiation in vitro. As the second part of my thesis, we evaluated whether these signals regulate muscle progenitors in vivo. Mouse models were generated in which Hif1α was depleted in progenitors during development and regeneration, and revealed that Hif1α is dispensable for development but constrains regeneration. These findings are consistent with a model in which O2-dependent signals maintain the undifferentiated state of muscle progenitors

Oxygen- and Hif1alpha-dependent regulation of skeletal muscle progenitor differentiation and skeletal muscle regeneration

Skeletal muscle progenitors, which give rise to terminally differentiated muscle, represent potential therapies for skeletal muscle disease. These progenitors reside in a low O2 environment before local blood vessels and differentiated muscle form. It has been established that low O 2 levels (hypoxia) maintain muscle precursors in an undifferentiated state in vitro, suggesting that in developing or regenerating muscle, local hypoxia may constrain progenitor differentiation until ample nutrients are available. However, this has not been formally tested. In addition, the signals linking O2 availability with progenitor differentiation are incompletely understood. For the first part of my thesis, cell culture models were used to delineate these signals. Depleting myoblasts in vitro of the Hypoxia Inducible Factors (HIFs)—the primary effectors of O2—revealed that hypoxia regulates differentiation via HIF-dependent and -independent mechanisms. We identified a key HIF-independent mechanism—AKT inactivation—concluding that multiple O2-dependent signals regulate progenitor differentiation in vitro. As the second part of my thesis, we evaluated whether these signals regulate muscle progenitors in vivo. Mouse models were generated in which Hif1α was depleted in progenitors during development and regeneration, and revealed that Hif1α is dispensable for development but constrains regeneration. These findings are consistent with a model in which O2-dependent signals maintain the undifferentiated state of muscle progenitors

Gene panel sequencing identifies a likely monogenic cause in 7% of 235 Pakistani families with nephrolithiasis

Nephrolithiasis (NL) affects 1 in 11 individuals worldwide and causes significant patient morbidity. We previously demonstrated a genetic cause of NL can be identified in 11-29% of pre-dominantly American and European stone formers. Pakistan, which resides within the Afro-Asian stone belt, has a high prevalence of nephrolithiasis (12%) as well as high rate of consanguinity (> 50%). We recruited 235 Pakistani subjects hospitalized for nephrolithiasis from five tertiary hospitals in the Punjab province of Pakistan. Subjects were surveyed for age of onset, NL recurrence, and family history. We conducted high-throughput exon sequencing of 30 NL disease genes and variant analysis to identify monogenic causative mutations in each subject. We detected likely causative mutations in 4 of 30 disease genes, yielding a likely molecular diagnosis in 7% (17 of 235) of NL families. Only 1 of 17 causative mutations was identified in an autosomal recessive disease gene. 10 of the 12 detected mutations were novel mutations (83%). SLC34A1 was most frequently mutated (12 of 17 solved families). We observed a higher frequency of causative mutations in subjects with a positive NL family history (13/109, 12%) versus those with a negative family history (4/120, 3%). Five missense SLC34A1 variants identified through genetic analysis demonstrated defective phosphate transport. We examined the monogenic causes of NL in a novel geographic cohort and most frequently identified dominant mutations in the sodium-phosphate transporter SLC34A1 with functional validation

Activation of 2‐oxoglutarate receptor 1 (OXGR1) by α‐ketoglutarate (αKG) does not detectably stimulate Pendrin‐mediated anion exchange in Xenopus oocytes

Abstract SLC26A4/Pendrin is the major electroneutral Cl−/HCO3− exchanger of the apical membrane of the Type B intercalated cell (IC) of the connecting segment (CNT) and cortical collecting duct (CCD). Pendrin mediates both base secretion in response to systemic base load and Cl− reabsorption in response to systemic volume depletion, manifested as decreased nephron salt and water delivery to the distal nephron. Pendrin‐mediated Cl−/HCO3− exchange in the apical membrane is upregulated through stimulation of the β‐IC apical membrane G protein‐coupled receptor, 2‐oxoglutarate receptor 1 (OXGR1/GPR99), by its ligand α‐ketoglutarate (αKG). αKG is both filtered by the glomerulus and lumenally secreted by proximal tubule apical membrane organic anion transporters (OATs). OXGR1‐mediated regulation of Pendrin by αKG has been documented in transgenic mice and in isolated perfused CCD. However, aspects of the OXGR1 signaling pathway have remained little investigated since its original discovery in lymphocytes. Moreover, no ex vivo cellular system has been reported in which to study the OXGR1 signaling pathway of Type B‐IC, a cell type refractory to survival in culture in its differentiated state. As Xenopus oocytes express robust heterologous Pendrin activity, we investigated OXGR1 regulation of Pendrin in oocytes. Despite functional expression of OXGR1 in oocytes, co‐expression of Pendrin and OXGR1 failed to exhibit αKG‐sensitive stimulation of Pendrin‐mediated Cl−/anion exchange under a wide range of conditions. We conclude that Xenopus oocytes lack one or more essential molecular components or physical conditions required for OXGR1 to regulate Pendrin activity

HIF modulation of wnt signaling regulates skeletal myogenesis in vivo

Deeper insight into the molecular pathways that orchestrate skeletal myogenesis should enhance our understanding of, and ability to treat, human skeletal muscle disease. It is now widely appreciated that nutrients, such as molecular oxygen (O(2)), modulate skeletal muscle formation. During early stages of development and regeneration, skeletal muscle progenitors reside in low O(2) environments before local blood vessels and differentiated muscle form. Moreover, low O(2) availability (hypoxia) impedes progenitor-dependent myogenesis in vitro through multiple mechanisms, including activation of hypoxia inducible factor 1α (HIF1α). However, whether HIF1α regulates skeletal myogenesis in vivo is not known. Here, we explored the role of HIF1α during murine skeletal muscle development and regeneration. Our results demonstrate that HIF1α is dispensable during embryonic and fetal myogenesis. However, HIF1α negatively regulates adult muscle regeneration after ischemic injury, implying that it coordinates adult myogenesis with nutrient availability in vivo. Analyses of Hif1a mutant muscle and Hif1a-depleted muscle progenitors further suggest that HIF1α represses myogenesis through inhibition of canonical Wnt signaling. Our data provide the first evidence that HIF1α regulates skeletal myogenesis in vivo and establish a novel link between HIF and Wnt signaling in this context

Mutations in transcription factor CP2-like 1 may cause a novel syndrome with distal renal tubulopathy in humans

BACKGROUND An underlying monogenic cause of early-onset chronic kidney disease (CKD) can be detected in ∼20% of individuals. For many etiologies of CKD manifesting before 25 years of age, >200 monogenic causative genes have been identified to date, leading to the elucidation of mechanisms of renal pathogenesis. METHODS In 51 families with echogenic kidneys and CKD, we performed whole-exome sequencing to identify novel monogenic causes of CKD. RESULTS We discovered a homozygous truncating mutation in the transcription factor gene transcription factor CP2-like 1 (TFCP2L1) in an Arabic patient of consanguineous descent. The patient developed CKD by the age of 2 months and had episodes of severe hypochloremic, hyponatremic and hypokalemic alkalosis, seizures, developmental delay and hypotonia together with cataracts. We found that TFCP2L1 was localized throughout kidney development particularly in the distal nephron. Interestingly, TFCP2L1 induced the growth and development of renal tubules from rat mesenchymal cells. Conversely, the deletion of TFCP2L1 in mice was previously shown to lead to reduced expression of renal cell markers including ion transporters and cell identity proteins expressed in different segments of the distal nephron. TFCP2L1 localized to the nucleus in HEK293T cells only upon coexpression with its paralog upstream-binding protein 1 (UBP1). A TFCP2L1 mutant complementary DNA (cDNA) clone that represented the patient's mutation failed to form homo- and heterodimers with UBP1, an essential step for its transcriptional activity. CONCLUSION Here, we identified a loss-of-function TFCP2L1 mutation as a potential novel cause of CKD in childhood accompanied by a salt-losing tubulopathy