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Varshney, G. K.

Publications and source records attributed to Varshney, G. K..

10 recordsLinked to original sources

A naturally evolved mutation in cavefish mc3r reshapes energy homeostasis across development in a nutrient-limited environment.

Food availability in natural habitats is often unpredictable and drives the selection of adaptive strategies in organisms to survive food scarcity. However, the genetic and molecular mechanisms that enable animals to survive under such nutritional extremes are poorly understood. Naturally adapted organisms therefore provide powerful genetic systems for uncovering the mechanisms that maintain energy homeostasis under nutritional extremes. Here, we leveraged the cavefish Astyanax mexicanus to identify a naturally occurring Thr166Met missense mutation in the melanocortin 3 receptor (mc3r) that reprograms energy homeostasis to facilitate adaptation to nutrient deprivation. To determine its functional consequences, we introduced the mutation into zebrafish mc3r using CRISPR/Cas9 genome editing. We show that adult mc3r mutants exhibit increased body size, adiposity, and hyperglycemia relative to wild-type animals, recapitulating key metabolic features of Tinaja cavefish and identifying mc3r as the first causal gene behind cavefish hyperglycemia. Investigations for early developmental consequences of the mutation revealed accelerated yolk utilization in mutant zebrafish larvae and strikingly elevated metabolic rates relative to WT, thus, uncovering a previously unrecognized role for mc3r in regulating energy expenditure. Importantly, the phenotype was also observed in Tinaja cavefish revealing a novel cavefish phenotype and establishing causal role for mc3r in elevated metabolic rate phenotype of cavefish. Together, these findings reveal that the Thr166Met variant produces distinct physiological consequences across life stages, enhanced energy storage and hyperglycemia in adulthood despite elevated larval energy expenditure. We propose that increased larval metabolism may support the energetic demands of enhanced foraging, whereas increased adiposity and blood glucose later in life provide energy reserves during periods of nutrient scarcity. More broadly, our study demonstrates how a single naturally evolved coding variant can reshape energy homeostasis across development and contribute to adaptation to nutrient-poor environments.

evolutionary biology↗

Systematic Engineering of Loss-of-Function Alleles in the Zebrafish Mitochondrial Proteome

Pathogenic variants in the 13 protein-coding genes of the mitochondrial genome underlie clinically and biochemically heterogeneous disorders. Most mtDNA-encoded genes lack defined loss-of-function (LOF) models in vivo. To address this gap, we have generated Z-Terminator, a systematic in vivo atlas of loss-of-function alleles covering all the mtDNA-encoded OXPHOS subunits in zebrafish (Danio rerio). We used mitochondrial TALE base editors to introduce premature termination codon (PTC) alleles via C-to-T transitions across Complexes I, III, IV, and V. Larvae harboring PTC alleles displayed bioenergetic defects and elevated lactate. While mtDNA mutations are associated with sensorineural hearing loss, the cellular basis has remained unclear. We show that engineered mtDNA LOF alleles directly impair hair cell function in proportion to heteroplasmy in a living vertebrate. We investigated the germline transmission and tissue-specific heteroplasmy of these LOF alleles and observed that a subset of variants was transmitted to the F1 generation and displayed distinct mutant loads across organs. These findings establish Z-Terminator as a vertebrate platform for interrogating the role of mtDNA protein-coding genes in cellular dysfunction and for elucidating the pathophysiology of mitochondrial disorders.

genetics↗

TadA-Derived Cytosine Base Editor for Precise Genome Editing in Zebrafish

CRISPR base editors are crucial for precise genome manipulation. Existing APOBEC-based cytosine base editors (CBEs), while powerful, exhibit indels and sequence context limitations, where TC-context preferences restrict effective editing of CC and GC motifs. To address these challenges, we evaluated various tRNA adenine deaminase (TadA)-derived CBEs, ultimately engineering zTadCBE that demonstrates high editing efficiency, minimized off-target effects, and an expanded targeting range. Our approach integrates beneficial mutations from TadA-based adenine base editors (ABEs) with SpRYCas9n-enhanced protospacer-adjacent motif (PAM) compatibility. Additionally, we engineered expanded-window zTadCBE variants, zTadCBE-ex1 and zTadCBE-ex2, to target wider nucleotide ranges, further increasing the versatility of this tool. To demonstrate the utility of zTadCBE variants in the functional assessment of genetic mutants, we generated a model for CDH23-associated hearing loss to validate the pathogenicity of a patient-specific variant in zebrafish. Furthermore, we induced a premature stop codon in the mediator complex gene med12 to inactivate its function using a CRISPR-STOP strategy and recapitulated patient-specific phenotypes in the founding (F0) generation. zTadCBE variants thus offer a robust set of CBEs for precise and efficient C-to-T editing in zebrafish, promising to advance the rapid functional assessment of genetic variants in vivo.

genetics↗

Naa80 is required for actin N-terminal acetylation and normal hearing in zebrafish

Actin is a key component of the cytoskeleton of eukaryotic cells and is involved in numerous cellular functions. In animal cells, actins are uniquely N-terminally processed by a dedicated enzyme machinery to generate their mature acidic and acetylated forms. The final step of this maturation process involves N-terminal acetylation, a reaction catalyzed by NAA80 in humans. In human cell lines, N-terminal acetylation of actin plays a crucial role in maintaining normal cytoskeletal dynamics and cell motility. The physiological impact of actin N-terminal acetylation remains to be defined. Here, we developed a zebrafish naa80 knockout model and established that zNaa80 acetylates both muscle and non-muscle actins in vivo. Our in vitro investigation of purified zNaa80 unveiled a clear preference for acetylating N-termini derived from actins. Interestingly, zebrafish lacking actin N-terminal acetylation were viable and exhibited normal development, morphology and behaviour. In contrast, human individuals carrying pathogenic actin variants may present with hypotonia and hearing impairment. While zebrafish depleted for naa80 did not display any obvious muscle defects or abnormal muscle tissue, we found that they have abnormal inner ear development such as small otoliths and impaired response to sound stimuli. In sum, we have defined that zebrafish Naa80 N-terminally acetylates actins in vitro and in vivo and that actin N-terminal acetylation is essential for normal hearing in vivo.

biochemistry↗

High-throughput phenotypic characterization of zebrafish CRISPR mutants of DNA repair genes

A systematic knowledge of the roles of DNA repair genes at the level of the organism has been limited due to the lack of appropriate experimental techniques. Here, we generated zebrafish loss-of-function mutants for 32 DNA repair and replication genes through multiplexed CRISPR/Cas9-mediated mutagenesis. High-throughput phenotypic characterization of our mutant collection revealed that three genes (atad5a, ddb1, pcna) are essential for proper embryonic development and hematopoiesis; seven genes (apex1, atrip, ino80, mre11a, shfm1, telo2, wrn) are required for growth and development during juvenile stage and six genes (blm, brca2, fanci, rad51, rad54l, rtel1) play critical roles in sex development. Furthermore, mutation in six genes (atad5a, brca2, polk, rad51, shfm1, xrcc1) displayed hypersensitivity to DNA damage agents. Further characterization of atad5a-/- mutants demonstrate that Atad5a is required for normal brain development and hematopoiesis. Our zebrafish mutant collection provides a unique resource for understanding of the roles of DNA repair genes at the organismal level.

developmental biology↗

Cnr2 is important for Ribbon Synapse Maturation and Function in Hair Cells and Photoreceptors

The role of the cannabinoid receptor 2 (CNR2) is still poorly described in sensory epithelia. We found strong cnr2 expression in hair cells (HCs) of the inner ear and the lateral line (LL), a superficial sensory structure in fish. Next, we demonstrated that sensory synapses in HCs were severely perturbed in zebrafish larvae lacking cnr2. Appearance and distribution of presynaptic ribbons and calcium channels (Cav1.3) were profoundly altered in mutant animals. Clustering of membrane-associated guanylate kinase (MAGUK) in post-synaptic densities (PSDs) was also heavily affected, suggesting a role for cnr2 for maintaining the structure of the sensory synapse. Furthermore, vesicular trafficking in HCs was strongly perturbed suggesting a retrograde action of the endocannabinoid system (ECs) mediated via cnr2 that was modulating HC mechanotransduction. We found similar perturbations in retinal ribbon synapses. Finally, we showed that larval swimming behaviors after sound and light stimulations were significantly different in mutant animals. Thus, we propose that cnr2 is critical for the processing of sensory information in the developing larva.

neuroscience↗

Biallelic mutation of CLRN2 causes non-syndromic hearing loss in humans

Deafness, the most frequent sensory deficit in humans, is extremely heterogenous with hundreds of genes probably involved. Clinical and genetic analyses of an extended consanguineous family with pre-lingual, moderate-to-profound autosomal recessive sensorineural hearing loss, allowed us to identify CLRN2, encoding a tetraspan protein as a new deafness gene. Homozygosity mapping followed by exome sequencing identified a 15.2 Mb locus on chromosome 4p15.32p15.1 containing a missense pathogenic variant in CLRN2 (c.494C>A, NM_001079827.2) segregating with the disease. Using in vitro RNA splicing analysis, we show that the CLRN2 c.494C>A mutation leads to two events: 1) the substitution of a highly conserved threonine (uncharged amino acid) to lysine (charged amino acid) at position 165, p.(Thr165Lys), and 2) aberrant splicing, with the retention of intron 2 resulting in a stop codon after 26 additional amino acids, p.(Gly146Lysfs*26). Expression studies and phenotyping of newly produced zebrafish and mouse models deficient for clarin 2 further confirm that clarin 2, expressed in the inner ear hair cells, is essential for normal organization and maintenance of the auditory hair bundles, and for hearing function. Together, our findings identify CLRN2 as a new deafness gene, which will impact future diagnosis and treatment for deaf patients.

genetics↗

Amyloid precursor protein-b facilitates cell adhesion during early development in zebrafish

Understanding the biological function of amyloid beta (A{beta}) precursor protein (APP) beyond its role in Alzheimers disease is emerging. Yet, its function during embryonic development is poorly understood. The zebrafish APP homologue, Appb, is strongly expressed during early development but thus far has only been studied via morpholino-mediated knockdown. Zebrafish enables analysis of cellular processes in an ontogenic context, which is limited in many other vertebrates. We characterized zebrafish carrying a homozygous mutation that introduces a premature stop in exon 2 of the appb gene. We report that appb mutants are significantly smaller until 2dpf and display perturbed enveloping layer (EVL) integrity and cell protrusions at the blastula stage.\n\nMoreover, appb mutants surviving beyond 48 hpf exhibited no behavioral defects at 6 dpf and developed into healthy and fertile adults. The expression of the app-family members, appa and aplp2, was found to be altered in appb mutants. Taken together, we show that appb orchestrates the initial development by supporting the integrity of the EVL, likely by mediating cell adhesion properties. The loss of Appb might be compensated for by other app family members to be able to implement continued normal development.

developmental biology↗

The Vertebrate Codex Gene Breaking Protein Trap Library For Genomic Discovery and Disease Modeling Applications

The zebrafish is a powerful model to explore the molecular genetics and expression of the vertebrate genome. The gene break transposon (GBT) is a unique insertional mutagen that reports the expression of the tagged member of the proteome while generating Cre-revertible genetic alleles. This 1000+ locus collection represents novel codex expression data from the illuminated mRFP protein trap, with 36% and 87% of the cloned lines showcasing to our knowledge the first described expression of these genes at day 2 and day 4 of development, respectively. Analyses of 183 molecularly characterized loci indicate a rich mix of genes involved in diverse cellular processes from cell signaling to DNA repair. The mutagenicity of the GBT cassette is very high as assessed using both forward and reverse genetic approaches. Sampling over 150 lines for visible phenotypes after 5dpf shows a similar rate of discovery of embryonic phenotypes as ENU and retroviral mutagenesis. Furthermore, five cloned insertions were in loci with previously described phenotypes; embryos homozygous for each of the corresponding GBT alleles displayed strong loss of function phenotypes comparable to published mutants using other mutagenesis strategies (ryr1b, fras1, tnnt2a, edar and hmcn1). Using molecular assessment after positional cloning, to date nearly all alleles cause at least a 99+% knockdown of the tagged gene. Interestingly, over 35% of the cloned loci represent 68 mutants in zebrafish orthologs of human disease loci, including nervous, cardiovascular, endocrine, digestive, musculoskeletal, immune and integument systems. The GBT protein trapping system enabled the construction of a comprehensive protein codex including novel expression annotation, identifying new functional roles of the vertebrate genome and generating a diverse collection of potential models of human disease.

genomics↗

MutliFRAGing: Rapid and Simultaneous Genotyping of Multiple Alleles in a Single Reaction

The powerful and simple RNA-guided CRISPR/Cas9 technology is a versatile genome editing tool that has revolutionized targeted mutagenesis. CRISPR-based genome editing has enabled large-scale functional genetic studies through the generation of gene knockouts in a variety of model organisms including zebrafish. CRISPR/Cas9 can also be used to target multiple genes simultaneously. One of the challenges associated with applying this technique to zebrafish in a high-throughput manner is the absence of a cost-effective method by which to identify mutants. To address this, we optimized the high-throughput, high-resolution fluorescent PCR-based fragment analysis method to develop MultiFRAGing, a robust and cost-effective method for genotyping of multiple targets in a single reaction. Our approach can identify indels in 4 targets from a single reaction, which represents a four-fold increase in genotyping throughput. This method can be used by any laboratory with access to capillary electrophoresis based sequencing equipment.

molecular biology↗