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Biology subjects

Petree, C.

Publications and source records attributed to Petree, C..

5 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↗

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↗