Hematopoietic stem cells (HSCs) are rare cells that arise in the embryo and sustain adult hematopoiesis. Although the functional potential of nascent HSCs is detectable by transplantation, their native contribution during development is unknown, in part due to the overlapping genesis and marker gene expression with other embryonic blood progenitors. Using single-cell transcriptomics, we define gene signatures that distinguish nascent HSCs from embryonic blood progenitors. Applying a lineage-tracing approach to selectively track HSC output in situ, we find significantly delayed lymphomyeloid contribution. An inducible HSC injury model demonstrates a negligible impact on larval lymphomyelopoiesis following HSC depletion. HSCs are not merely dormant at this developmental stage, as they showed robust regeneration after injury. Combined, our findings illuminate that nascent HSCs self-renew but display differentiation latency, while HSC-independent embryonic progenitors sustain developmental hematopoiesis. Understanding these differences could improve de novo generation and expansion of functional HSCs.

The vertebrate axial skeleton (vertebral column and ribs) is derived from embryonic structures called somites. Mechanisms of somite formation and patterning are largely conserved along the length of the body axis, but segments acquire different morphologies in part through the action of Hox transcription factors. Although Hox genes' roles in axial skeletal patterning have been extensively characterized, it is still not well understood how they interact with somite patterning pathways to regulate different vertebral morphologies. Here, we investigated the role of Hoxa-5 in after somite segmentation in chick. Hoxa-5 mRNA is expressed in posterior cervical somites, and within them is restricted mainly to a sub-domain of lateral sclerotome. RNAi-based knockdown leads to cartilage defects in lateral vertebral elements (rib homologous structures) whose morphologies vary within and outside of the Hoxa-5 expression domain. Both knockdown and misexpression suggest that Hoxa-5 acts via negative regulation of Sox-9. Further, Hoxa-5 misexpression suggests that spatial and/or temporal restriction of Hoxa-5 expression is necessary for proper vertebral morphology. Finally, the restriction of Hoxa-5 expression to lateral sclerotome, which we hypothesize is important for its patterning function, involves regulation by signaling pathways that pattern somites, Fgf-8 and Shh.

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