Zebrafish nampt-a mutants are viable despite perturbed primitive hematopoiesis.

BACKGROUND: Nicotinamide phosphoribosyltransferase (Nampt) is required for recycling NAD+ in numerous cellular contexts. Morpholino-based knockdown of zebrafish nampt-a has been shown to cause abnormal development and defective hematopoiesis concomitant with decreased NAD+ levels. However, surprisingly, nampt-a mutant zebrafish were recently found to be viable, suggesting a discrepancy between the phenotypes in knockdown and knockout conditions. Here, we address this discrepancy by directly comparing loss-of-function approaches that result in identical defective transcripts in morphants and mutants. RESULTS: Using CRISPR/Cas9-mediated mutagenesis, we generated nampt-a mutant lines that carry the same mis-spliced mRNA as nampt-a morphants. Despite reduced NAD+ levels and perturbed expression of specific blood markers, nampt-a mutants did not display obvious developmental defects and were found to be viable. In contrast, injection of nampt-a morpholinos into wild-type or mutant nampt-a embryos caused aberrant phenotypes. Moreover, nampt-a morpholinos caused additional reduction of blood-related markers in nampt-a mutants, suggesting that the defects observed in nampt-a morphants can be partially attributed to off-target effects of the morpholinos. CONCLUSIONS: Our findings show that zebrafish nampt-a mutants are viable despite reduced NAD+ levels and a perturbed hematopoietic gene expression program, indicating strong robustness of primitive hematopoiesis during early embryogenesis.

Integration of Nodal and BMP Signaling by Mutual Signaling Effector Antagonism.

Opposing sources of bone morphogenetic protein (BMP) and Nodal signaling molecules are sufficient to induce the formation of a full axis in zebrafish embryos. To address how these signals orchestrate patterning, we transplant sources of fluorescently tagged Nodal and BMP into zebrafish embryos, robustly inducing the formation of secondary axes. Nodal and BMP signal non-cell-autonomously and form similar protein gradients in this context, but the signaling range of Nodal (pSmad2) is shorter than the BMP range (pSmad5). This yields a localized region of pSmad2 activity around the Nodal source, overlapping with a broad domain of pSmad5 activity across the embryo. Cell fates induced in various regions stereotypically correlate with pSmad2-to-pSmad5 ratios and can even be induced BMP- and Nodal-independently with different ratios of constitutively active Smad2 and Smad5. Strikingly, we find that Smad2 and Smad5 antagonize each other for specific cell fates, providing a mechanism for how cells integrate and discriminate between overlapping signals during development.