Zebrafish nampt-a mutants are viable despite perturbed primitive hematopoiesis

Area of Science:

• Developmental Biology
• Genetics
• Biochemistry

Background:

• Nicotinamide phosphoribosyltransferase (Nampt) is crucial for NAD+ recycling.
• Previous studies showed morpholino-induced nampt-a knockdown in zebrafish caused developmental and hematopoietic defects.
• Recent findings of viable nampt-a mutant zebrafish suggest a discrepancy between knockdown and knockout phenotypes.

Purpose of the Study:

• To resolve discrepancies between morpholino knockdown and genetic mutation phenotypes of zebrafish nampt-a.
• To directly compare loss-of-function approaches by generating mutants with identical defective transcripts to morphants.

Main Methods:

• CRISPR/Cas9-mediated mutagenesis to create nampt-a mutant zebrafish lines.
• Generation of mutants with identical mis-spliced mRNA transcripts as observed in morphants.
• Phenotypic analysis of mutants and morphants, including NAD+ levels and blood marker expression.

Main Results:

• Zebrafish nampt-a mutants exhibited reduced NAD+ levels and perturbed blood marker expression but remained viable without obvious developmental defects.
• Morpholino injection into wild-type or mutant embryos caused aberrant phenotypes.
• Morpholinos exacerbated blood marker reductions in nampt-a mutants, indicating off-target effects.

Conclusions:

• Zebrafish nampt-a mutants are viable despite reduced NAD+ and altered hematopoietic gene expression.
• Primitive hematopoiesis demonstrates significant robustness during early zebrafish embryogenesis.
• Morpholino-based knockdown studies may be confounded by off-target effects, necessitating careful interpretation.