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

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 owing 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 are a distinct cell type with a unique cell-cycle profile and they remain multipotent for all blood cell fates. In addition, 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. This study suggests that it would be useful to characterize such intermediate populations of cells in mammalian hematopoietic systems.

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

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.

Drosophila as a Genetic Model for Hematopoiesis.

In this FlyBook chapter, we present a survey of the current literature on the development of the hematopoietic system in Drosophila The Drosophila blood system consists entirely of cells that function in innate immunity, tissue integrity, wound healing, and various forms of stress response, and are therefore functionally similar to myeloid cells in mammals. The primary cell types are specialized for phagocytic, melanization, and encapsulation functions. As in mammalian systems, multiple sites of hematopoiesis are evident in Drosophila and the mechanisms involved in this process employ many of the same molecular strategies that exemplify blood development in humans. Drosophila blood progenitors respond to internal and external stress by coopting developmental pathways that involve both local and systemic signals. An important goal of these Drosophila studies is to develop the tools and mechanisms critical to further our understanding of human hematopoiesis during homeostasis and dysfunction.

Systemic control of immune cell development by integrated carbon dioxide and hypoxia chemosensation in Drosophila.

Drosophila hemocytes are akin to mammalian myeloid blood cells that function in stress and innate immune-related responses. A multi-potent progenitor population responds to local signals and to systemic stress by expanding the number of functional blood cells. Here we show mechanisms that demonstrate an integration of environmental carbon dioxide (CO2) and oxygen (O2) inputs that initiate a cascade of signaling events, involving multiple organs, as a stress response when the levels of these two important respiratory gases fall below a threshold. The CO2 and hypoxia-sensing neurons interact at the synaptic level in the brain sending a systemic signal via the fat body to modulate differentiation of a specific class of immune cells. Our findings establish a link between environmental gas sensation and myeloid cell development in Drosophila. A similar relationship exists in humans, but the underlying mechanisms remain to be established.

Inhibition of Daughterless by Extramacrochaetae mediates Notch-induced cell proliferation.

During development, the rate of cell proliferation must be constantly monitored so that an individual tissue achieves its correct size. Mutations in genes that normally promote tissue growth often result in undersized, disorganized and non-functional organs. However, mutations in genes that encode growth inhibitors can trigger the onset of tumorigenesis and cancer. The developing eye of the fruit fly, Drosophila melanogaster, has become a premier model system for studies that are focused on identifying the molecular mechanisms that underpin growth control. Here, we examine the mechanism by which the Notch pathway, a major contributor to growth, promotes cell proliferation in the developing eye. Current models propose that the Notch pathway directly influences cell proliferation by regulating growth-promoting genes such as four-jointed, cyclin D1 and E2f1. Here, we show that, in addition to these mechanisms, some Notch signaling is devoted to blocking the growth-suppressing activity of the bHLH DNA-binding protein Daughterless (Da). We demonstrate that Notch signaling activates the expression of extramacrochaetae (emc), which encodes a helix-loop-helix (HLH) transcription factor. Emc, in turn, then forms a biochemical complex with Da. As Emc lacks a basic DNA-binding domain, the Emc-Da heterodimer cannot bind to and regulate genomic targets. One effect of Da sequestration is to relieve the repression on growth. Here, we present data supporting our model that Notch-induced cell proliferation in the developing eye is mediated in part by the activity of Emc.

Extramacrochaetae functions in dorsal-ventral patterning of Drosophila imaginal discs.

One of the seminal events in the history of a tissue is the establishment of the anterior-posterior, dorsal-ventral (D/V) and proximal-distal axes. Axis formation is important for the regional specification of a tissue and allows cells along the different axes to obtain directional and positional information. Within the Drosophila retina, D/V axis formation is essential to ensure that each unit eye first adopts the proper chiral form and then rotates precisely 90° in the correct direction. These two steps are important because the photoreceptor array must be correctly aligned with the neurons of the optic lobe. Defects in chirality and/or ommatidial rotation will lead to disorganization of the photoreceptor array, misalignment of retinal and optic lobe neurons, and loss of visual acuity. Loss of the helix-loop-helix protein Extramacrochaetae (Emc) leads to defects in both ommatidial chirality and rotation. Here, we describe a new role for emc in eye development in patterning the D/V axis. We show that the juxtaposition of dorsal and ventral fated tissue in the eye leads to an enrichment of emc expression at the D/V midline. emc expression at the midline can be eliminated when D/V patterning is disrupted and can be induced in situations in which ectopic boundaries are artificially generated. We also show that emc functions downstream of Notch signaling to maintain the expression of four-jointed along the midline.

Dissection and immunostaining of imaginal discs from Drosophila melanogaster.

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Hedgehog and extramacrochaetae in the Drosophila eye: an irresistible force meets an immovable object.

During the third and final larval instar stage, thousands of pluripotent cells within the Drosophila eye imaginal disc are transformed into a near perfect neurocrystalline lattice of 800 unit eyes called ommatidia. This transformation begins with the initiation of the morphogenetic furrow at the posterior margin of the eye field. The furrow, which marks the leading edge of a wave of differentiation, passes across the epithelium transforming unpatterned and undifferentiated cells into rows of periodically spaced clusters of photoreceptor neurons. As cells enter and exit the furrow they undergo dramatic alterations in cellular architecture and gene expression, many of which are required to propel the furrow forward and for proper cell fate specification. The Decapentaplegic (Dpp) and Hedgehog (Hh) signaling pathways are required for the initiation and progression of the furrow, respectively. Consistent with a role in furrow progression, the loss of Hh pathway activity results in a "furrow stop" phenotype. In contrast, reductions in levels of the helix-loop-helix transcription factor, Extramacrochaetae (Emc), lead to the polar opposite phenotype--the furrow accelerates. Recently, we demonstrated that the furrow stop and furrow acceleration phenotypes are molecularly connected. Emc appears to serve as a brake on the furrow by dampening the activity of the Hh pathway. Loss of Emc leads to an upsurge in Hh pathway activity and a faster moving furrow. The acceleration of the furrow appears to be due to an increase in levels of the full-length isoform of Cubitus Interruptus (Ci (155)) and Suppressor of Fused [Su(fu)]. Here we will briefly review the mechanisms by which Hh drives and Emc impedes the progression of the furrow across the developing retina.

Extramacrochaetae imposes order on the Drosophila eye by refining the activity of the Hedgehog signaling gradient.

The compound eye of Drosophila melanogaster is configured by a differentiating wave, the morphogenetic furrow, that sweeps across the eye imaginal disc and transforms thousands of undifferentiated cells into a precisely ordered repetitive array of 800 ommatidia. The initiation of the furrow at the posterior margin of the epithelium and its subsequent movement across the eye field is controlled by the activity of the Hedgehog (Hh) signaling pathway. Differentiating photoreceptors that lie behind the furrow produce and secrete the Hh morphogen, which is captured by cells within the furrow itself. This leads to the stabilization of the full-length form of the zinc-finger transcription factor Cubitus interruptus (Ci(155)), the main effector of Hh signaling. Ci(155) functions as a transcriptional activator of a number of downstream targets, including decapentaplegic (dpp), a TGFß homolog. In this report, we describe a mechanism that is in place within the fly retina to limit Hh pathway activity within and ahead of the furrow. We demonstrate that the helix-loop-helix (HLH) protein Extramacrochaetae (Emc) regulates Ci(155) levels. Loss of emc leads to an increase in Ci(155) levels, nuclear migration, apical cell constriction and an acceleration of the furrow. We find that these roles are distinct from the bHLH protein Hairy (H), which we show restricts atonal (ato) expression ahead of the furrow. Secondary furrow initiation along the dorsal and ventral margins is blocked by the activity of the Wingless (Wg) pathway. We also show that Emc regulates and cooperates with Wg signaling to inhibit lateral furrow initiation.