Extrinsic and Intrinsic Regulation of Hematopoietic Progenitors in the Drosophila Larval Lymph Gland

Abstract

Drosophila hematopoiesis shares many similarities at the molecular and cellular level with the phylogenetically distant vertebrates. The simplicity of the model and genetic amenability provides an excellent opportunity to unravel cellular intricacies involved in the mechanisms related to blood development. Drosophila larval lymph gland, a product of definitive hematopoiesis in fly, has been studied extensively, enabling the elucidation of complex cross-talks between the distinct cell populations that the organ harbours in close proximity to each other. This treasure trove of information and similarities with vertebrate counterparts provides an excellent model to answer basic questions related to blood development. My thesis primarily dwells on the characterization of progenitors and their maintenance signals. • Objectives and Outcome Previous work on Drosophila hematopoiesis has mainly focussed on the primary lobes, owing to its prominent size and presence of three distinct zones. This organ, however, consists of two more such lobes, the secondary and the tertiary lobes. The significance of these lobes is most evident upon immune challenges such as the one against wasp parasitisation when a substantial increase in the blood pool of these lobes is observed. Studies have unravelled that these lobes contribute to extended definitive hematopoiesis in adult fruit flies during development. 1Abstract Objective 1: Interestingly, the nature of cells inhabiting these lobes was unexplored and were mainly ignored. The first objective of my thesis addresses this aspect where I carried out a detailed characterization of these lobes employing distinctive cell markers and performing functional analysis. Outcomes: 1. Lymph gland Posterior lobes harbour heterogeneous cell types. My study reveals that previously defined homogenous progenitor-like cells are actually heterogeneous, harbouring blood cells in a pre-progenitor like state. Unlike their neighbouring primary lobe counterparts, these cells are proliferative even in the late larval stages and do not differentiate until the pre-pupal stages. Upon immune challenge, these lobes proliferate extensively and precociously differentiate to release cells into the circulation, signifying the importance of these lobes at the time of immune emergency. 2. A Ubx dependent novel signalling center maintains posterior lobe progenitors. My study also identifies a unique signalling center embedded in the anterior half of the tertiary lobes. The anterior zone of the tertiary lobe is high in EBF factor Collier, expresses bonafide niche markers such as the morphogen Hedgehog, and is defined by Hox gene Ubx; all characteristics reminiscent of primary lobe niche defined by Hox gene Antp. Manipulations in this zone altered the homeostasis of both secondary and tertiary lobe progenitors in a non-cell-autonomous manner. Moreover, upon immune challenge, this zone assisted the neighbouring progenitors in orchestrating an emergency immune response. In order to maintain tissue homeostasis, the constituent cells need to interact with one another. This becomes crucial when two stem cell compartments interact to maintain tissue homeostasis. The hematopoietic niche is necessary for maintaining the blood progenitors. Previous studies have reported the existence of filopodial structures emanating from the niche. However, its role in niche and progenitor communication has never been addressed. 2Abstract Objective 2: This part of the study aimed to characterise the filopodial structures, document them, and address their functional role, if any, in progenitor maintenance. Outcome: Filopodia mediated morphogen delivery from the niche crucial for progenitor maintenance. I found that the niche employs actin-based cytoneme like filopodial structures to communicate with the neighbouring blood progenitors. Lengths of these cytonemes range from 2-40µm, and they facilitate the release of Hedgehog (Hh), the prime morphogen essential for maintaining the blood progenitors. Perturbation of cytonemes from the niche leads to an altered Hh gradient and overall reduced Hh levels within the medullary zone. This drop in the maintenance signal consequently leads to the precocious differentiation of the blood progenitor pool. Apart from intrinsic factors, tissue homeostasis can also be influenced by external cues. Circadian rhythms are daily rhythms known to orchestrate bodily functions according to the time of the day. Although several studies have implicated altered circadian rhythms to reduced immunity, its effect on blood development per se has not been explored. Objective 3: Aims to assess blood cell populations' response in response to altered external cues, which in this study is disrupted circadian rhythms. Outcome: Circadian Rhythm is critical for maintaining larval blood progenitors in Drosophila. Both cue and genetic manipulation of Circadian rhythm revealed that disruption in rhythms influences blood development in Drosophila hematopoietic organ lymph gland. Disrupted circadian rhythm upon rearing the larvae in constant light conditions leads to a precocious increase in differentiation of blood progenitors to plasmatocytes. This phenotype was a tissue extrinsic response caused by an inter- organ cross-talk between the brain that harbours core clock neurons, the ring gland, the hormone-producing organ of larvae, and the lymph gland that houses developing blood cell populations. The crosstalk seems to be facilitated by steroid hormone ecdysone that alters the cell-cycle profile of lymph gland progenitors and leads to abnormal hematopoiesis. 3Abstract • Conclusions Studies on Drosophila hematopoiesis have yielded vast amounts of valuable information on cellular processes related to blood development. They have contributed significantly to our understanding of the mechanisms and processes involved in hematopoiesis. The current work highlights the otherwise unexplored posterior lobes of the Drosophila larval hematopoietic organ. Molecular genetic study unravels a novel signalling cell center, defined by Hox gene Ubx in the anterior domain of tertiary lobes crucial for progenitor maintenance. This signaling center is analogous to the primary lobe niche, playing critical roles in normal blood development and emergency hematopoiesis. My study provides a novel instance in development where tissue organization takes place in such a way so as to optimally define locations of two functionally similar but spatially distinct signaling centers in order to maintain blood progenitors. The current work also demonstrates that apart from infections, manipulated external cues such as disrupted circadian rhythms can also lead to precocious differentiation of blood progenitors in the lymph gland. Upon experimental analyses, inter-organ crosstalk was identified that comprises brain - ring gland - lymph gland axis and was found to be essential for the blood progenitor maintenance. Clock neurons in the brain relay circadian information through pigment dispersing factor (PDF). Subsequently, the ring gland translates this information to changes in the levels of steroid hormone ecdysone. Lymph gland progenitors seem sensitive to the subtle but consistent drop in ecdysone levels and precociously differentiate. My work demonstrates how an external cue can influence organismal homeostasis via inter-organ cross-talk and is the first report to show how disrupted circadian rhythms can affect blood development. To summarize, this thesis explores and identifies several new players at both molecular and cellular levels, crucial for the proper functioning of the larval hematopoietic organ under both developmental scenarios and upon infection and cue- based conditions.