Abstract:  In recent years, there has been a move to identify novel regulators of myeloid differentiation, aiming to improve human health and provide new targets for future therapeutics. Previous research demonstrated that Grna was required for the development of neutrophil and macrophage populations (Campbell et al., Blood Adv. 2021). We now aim to develop a molecular mechanism to explain the role of Grna in myeloid differentiation.Bulk RNA-sequencing of zebrafish hematopoietic tissue (kidney marrow) was utilized to identify candidate pathways. For the targeted knockdown of jak1 and stat3 gene expression, we used translation-blocking morpholinos and a previously published mutant line; to rescue mutants, we used a constitutively active Stat3 construct (Bromberg et al., 1999). The recently published CUT&RUN protocol (Barakat et al.; Zebrafish, 2022) was utilized to test potential Stat3 binding sites within the untranslated regions of grna.  Transcriptomic comparison of grna-/- and grna+/+ kidneys revealed downregulation of the Jak1-Stat3 pathway in grna-/- tissue. Knockdown of jak1 and stat3 decreased the number of neutrophils and macrophages, similar to the grna-/- embryos phenotype. Conversely, the introduction of constitutively active Stat3 was able to rescue these populations within grna-/- embryos. Furthermore, analysis of predicted Stat3 binding sites on grna indicated several valid sites.Based on current data, we hypothesize that Grna acts on myeloid differentiation by promoting Jak1-Stat3 signaling with a feedback loop between Grna and Stat3. Further research on this signaling pathway includes searching for potential receptors that mediate Grna-Jak1 interaction. This model provides the potential for use of Grna as a therapeutic modulator of Stat3 expression. 

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