Intermediate Progenitor cells provide a transition between hematopoietic progenitors and their differentiated descendants

Abstract

Genetic and genomic analysis in Drosophila suggests that hematopoietic progenitors likely transition into terminal fates via intermediate progenitors (IPs) with some characteristics of either, but perhaps maintaining IP-specific markers. In the past, IPs have not been directly visualized and investigated due to lack of appropriate genetic tools. Here we report a split-GAL4 construct, CHIZ-GAL4, that identifies IPs as cells physically juxtaposed between true progenitors and differentiating hemocytes. IPs comprise a distinct cell type with a unique cell-cycle profile and they remain multipotent for all blood cell fates. Additionally, through their dynamic control of the Notch ligand, Serrate, IPs specify the fate of direct neighbors. The Ras pathway controls the number of IP cells and promotes their transition into differentiating cells. The split-GAL4 strategy is amenable for adoption in mammalian systems and would be invaluable in assigning trajectories that stem and progenitor populations follow as they develop into mature blood cells.

Paths and Pathways that Generate Cell-Type Heterogeneity and Developmental Progression in Hematopoiesis

“…Since the loss of Pnt in the MZ blocks entry into IZ, its even higher expression in the IZ suggests a different and additional role in this zone. This IZ function is explored in some detail in a companion paper, where we demonstrate that loss of Pnt in the IZ prevents these cells from exiting their transitional state (Spratford et al 2020). Together with the data presented here, we conclude that Pnt is required for both entry into and exit from the IZ.…”

“… (K-M’’) Manipulation of JNK pathway using CHIZ-GAL4 , UAS-mGFP (green) that marks IZ cells (Spratford et al 2020). Immunolocalization of MMP1 is shown in magenta.…”

“…Indeed, using a genetic background that allows simultaneous visualization of three different reporters, we detect by direct observation, a significant number of dome MESO positive, but Tep4 and Hml double negative, cells in wild-type 3rd instar larval lymph glands (Figure 4G). These are not IZ cells as they do not express an Intermediate zone specific-Gal4 driver ( CHIZ-GAL4 , Spratford et al 2020; Figure 4H). Taken together, these data identify a post- Tep4 and pre-IZ population of dome -expressing cells within the MZ, and show that Pnt functions in this group.…”

“…The Drosophila lines from our lab stock were used in this study as follows: dome MESO >GFP Hml Δ -DsRed , lz-GAL4 , dome MESO -GFP.nls Hml Δ -DsRed.nls , CHIZ-GAL4 UAS-mGFP (IZ-specific GAL4, (Spratford et al 2020)), Tep4>GFP Hml Δ -DsRed , Tep4-QF2 (first used in this study), Hml>2xEGFP , and UAS-Notch ACT . The following lines were obtained from the Bloomington Drosophila Stock Center (BDSC): lz-Gal4 UAS-mGFP (#6314), UAS-pnt RNAi (#35038), UAS-hep ACT (#9306), UAS-mihep (#35210), UAS-numb RNAi (#35045), LexAop2-6XmCherry (#52271), UAS-Notch RNAi (#7077), Msi-GFP MiMIC protein trap (#61750), 10XQUAS-6XmCherry (#52269).…”

“…Blood progenitors, marked by dome and Tep4 , reside in the medullary zone (MZ) while differentiating hemocytes (expressing plasmatocyte and CC markers) occupy the cortical zone (CZ) (Jung et al 2005). A third zone named the intermediate zone (IZ) is the site of the intermediate progenitors (IPs) (Krzemien et al 2010) that is explored in detail in this, and in a companion paper (Spratford et al 2020). Prior work describing the zonation of the lymph gland also noted a small population of cells near the dorsal vessel with characteristics of a pre-progenitor (Jung et al 2005; Dey et al 2016; Tiwari et al 2020), but the rest of the MZ was considered to be fairly homogeneous.…”