Paths and pathways that generate cell-type heterogeneity and developmental progression in hematopoiesis

Abstract

Mechanistic studies of Drosophila lymph gland hematopoiesis are limited by the availability of cell-type-specific markers. Using a combination of bulk RNA-Seq of FACS-sorted cells, single-cell RNA-Seq, and genetic dissection, we identify new blood cell subpopulations along a developmental trajectory with multiple paths to mature cell types. This provides functional insights into key developmental processes and signaling pathways. We highlight metabolism as a driver of development, show that graded Pointed expression allows distinct roles in successive developmental steps, and that mature crystal cells specifically express an alternate isoform of Hypoxia-inducible factor (Hif/Sima). Mechanistically, the Musashi-regulated protein Numb facilitates Sima-dependent non-canonical, and inhibits canonical, Notch signaling. Broadly, we find that prior to making a fate choice, a progenitor selects between alternative, biologically relevant, transitory states allowing smooth transitions reflective of combinatorial expressions rather than stepwise binary decisions. Increasingly, this view is gaining support in mammalian hematopoiesis.

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

“…1 J,K). These data are consistent with our recent transcriptomic analysis of the LG that suggests lack of E-cad expression as a characteristic feature of the IZ and that has also identified several gene products that are uniquely representative of the IP population ( Girard et al, 2021 ).…”

“…1J,K). These data are consistent with our recent transcriptomic analysis of the LG that suggests lack of E-cad expression as a characteristic feature of the IZ and that has also identified several gene products that are uniquely representative of the IP population (Girard et al, 2021). We next characterized the cell cycle profile of these transitory cells using the Fly FUCCI system (fluorescent ubiquitination cell cycle indicator) (Zielke et al, 2011).…”

“…Importantly, we believe IPs to be a unique cell type, as their numbers can be expanded or reduced upon genetic manipulation as shown, for example, with modulation of the Ras pathway. In addition, bulk and single cell RNA-seq data obtained recently in the laboratory identifies several genes that are highly enriched within IPs when compared with their expression in all other cell types in the LG ( Girard et al, 2021 ). In future studies, these will serve well as specific IZ markers and provide further functional relevance for this population.…”

“…Upstream events that activate Ras in the IPs are currently unknown and will be of great interest for future investigation. It is possible that a canonical ligand-dependent RTK may be involved; however, other autonomous molecular mechanisms such as changes in metabolism could feed into Ras ( Girard et al, 2021 ).…”

Peeling Back the Layers of Lymph Gland Structure and Regulation

“…In line with this trend, the latest single-cell sequencing data proposed the presence of multiple cell populations with intermediate differentiation states between progenitors and plasmatocytes or crystal cells. Notably, these results also validated the early microscopic observations that suggested a gradual hemocyte differentiation in the lymph gland [ 17 , 79 , 80 ].…”