Intrinsic and extrinsic regulation of hematopoietic niche development in drosophila melanogaster

Abstract

Synopsis Introduction Stem cells are unique cells of body that have the ability to self-renew as well as differentiate into diverse cell types. About 40 years ago, it was discovered that stem cells retain their peculiar characteristics only when they are in close association with the microenvironment named as stem cell niche. A stem cell niche thus houses the stem cells wherein they receive signals that can influence their behavior. Since the niche controls the state and fate of stem and progenitor cells, it thus is a strong candidate to be targeted for therapeutics and regenerative medicine. Therefore, it is extremely important that we understand the fundamentals of a stem cell niche. For our studies, we chose to understand stem cell niche biology using Drosophila hematopoiesis as the process. Starting from transcription factors, signalling molecules and two phases of hematopoiesis, there is a high level of conservation between Drosophila hematopoiesis to its vertebrate counterparts. The definitive wave of hematopoiesis in fruit-fly takes place in the larval organ called the lymph gland. The lymph gland in a mature third instar larvae is a kidney shaped organ with the outer region consisting of differentiated hemocytes called the cortical zone (CZ), the inner core, called as Medullary Zone (MZ) houses the progenitors. The balance between differentiated and progenitor population is orchestrated by a group of 45-50 cells called Posterior Signaling Centre (PSC) that lie posterior to progenitors. PSC serves as the hematopoietic niche of the lymph gland. The elegance of lymph gland lies in the fact that all three zones are proximal to each other making it an efficient tool to unravel distinct crosstalk happening in different types of cells of the same tissue. Objectives: The primary objective of my doctoral thesis was to find out the factors necessary for maintaining the hematopoietic niche of Drosophila larvae. Towards this end, an unbiased genome wide RNAi screen was launched and various genes belonging to different classes and functions were screened by using Gal4-UAS system. Amongst this screen, a gene that was pursued for in-detail analysis was a Receptor Protein Tyrosine Phosphatase (RPTP) Leukocyte- antigen-related-like (Lar). In this study, we demonstrated a fundamental requirement of local Ifactors to integrate with the systemic signal at tissue level to maintain the homeostasis of the system. Additionally, while trying to assess the regulation of hematopoietic niche, we were able to pin down an interesting phenomenon; there is backward signalling from the progenitors, which is playing a vital role to maintain the hematopoietic niche. Outcome of this study: Genetic screen to identify the factors maintaining the hematopoietic niche of Drosophila larvae Previous studies have established, how a stem cell niche regulates and maintains the stem/progenitor cells. But the knowledge about how a stem cell niche is maintained, still lies in its infancy. To understand the basic biology of a stem cell niche, Drosophila hematopoietic organ-lymph gland was used as the model system. The physical proximity between differentiated cells, progenitors and the hematopoietic niche of lymph gland makes this organ/tissue a fantastic system to uncover the underpinnings of the hematopoietic niche. To unravel the factors maintaining the hematopoietic niche, an unbiased genome wide RNAi screen was carried out. Using the niche specific driver, Antennapedia (Antp)-Gal4, about 60 UAS based RNAi lines were screened. A temperature sensitive allele gal80ts was kept in the background of Antp- Gal4 so as to avoid any adverse effects of these genes in early embryonic stages. For the initial screening, number of niche cells was counted manually as well as using Imaris software. Niche cells were visualized by UAS-GFP construct driven by Antp-Gal4 and the niche number was further confirmed by Antp immunostaining. With the help of genetic screen, we were able to identify a variety of molecules belonging to extracellular matrix (ECM), cytoskeleton, transcription factors, phosphatases and kinases etc. playing a major role in maintaining the hematopoietic niche of Drosophila. The results of this genetic screen were divided into 3 major categories: 1. Positive Regulators: Genes whose knockdown resulted in decreased number of niche cells (<55). 2. No Changers: Genes whose knockdown did not bring a major change in the number of niche cells (40-50). 3. Negative Regulators: Genes whose knockdown resulted in increased number of niche cells (>55). IIFor in depth analysis, Lar protein, which is a Receptor Protein Tyrosine Phosphatase was chosen as its knockdown resulted in a tremendous increase in niche cell number along with disrupted homeostasis of the lymph gland. Leukocyte-antigen-related-like (Lar) regulates insulin signalling in the hematopoietic niche to maintain the homeostasis of lymph gland. Leukocyte-antigen-related-like (Lar) is a well conserved receptor protein tyrosine phosphatase (RPTP). It is present in the nervous system of Caenorhabditis elegans, Hirudo medicinalis and Drosophila. In mammals, it is reported to be present in the basal lamina of epithelial tissues, liver, muscles and adipose cells. Interestingly, Lar was isolated from a screen of immunoglobulin family, and is homologous to leukocyte common antigens (LCA), but this/my study is one of the very first study providing insights on role of Lar in the hematopoietic system. Knockdown of Lar from the hematopoietic niche leads to a 4-5-fold increase in niche cell numbers compared to the control. Furthermore, a drastic increase in differentiated cell population with a concomitant decrease in progenitor population was also observed. Therefore, loss of Lar not only increases the niche cell proliferation but also disturbs the homeostasis of entire organ. Further analysis revealed an excessive insulin signalling in the Lar knockdown niche; confirming, an antagonistic relation between insulin and Lar in Drosophila hematopoietic niche. Moreover, an upregulated ROS was also observed in the Lar knockdown niches. Our study revealed that upregulated ROS is a consequence of hyper activated insulin signalling in the niche. Getting cues from literature, we were able to demonstrate that increased differentiation and immune response seen by lamellocyte formation in Lar knockdown niches, was a result of upregulated ROS. All the events involved in disrupting the homeostasis of the lymph gland due to loss of Lar; including increased niche cell number, increased differentiation and lamellocyte formation were an ultimate result of excessive insulin signalling in the niche. Therefore, we demonstrated that Lar in the niche regulates its exposure to insulin signalling. Broadly, we decoded a local factor (Lar) residing on the hematopoietic niche that integrates and regulates the systemic signal (insulin), thereby, monitoring the homeostasis of the entire tissue. IIIElucidating the factors from progenitors to maintain the hematopoietic niche. Our genetic screen revealed various intrinsic factors acting both positively and negatively, which are important for niche maintenance. We also demonstrated requirement of a local factor necessary to regulate the systemic signal in the hematopoietic niche, because excessive systemic signal like insulin could be disastrous for the system. From this study, it became very clear that there is a vital role of extrinsic cues in regulating niche maintenance, thereby controlling the physiology of the entire tissue. Therefore, we explored other proximal tissues to niche, which can provide it with extrinsic cues. One of those is medullary zone that houses stem cell like progenitor cells. A small RNAi screen was carried out to know whether MZ has some role in niche maintenance. Surprisingly, loss of two ECM molecules and a cell adhesion molecule from progenitors resulted in a disturbance within the niche establishing existence of a backward signal from the progenitors’ essential for niche maintenance. On further analysis it was revealed that disturbance in this backward signal not only disrupts niche number but affects its functionality. The exact signalling pathway and involvement of various factors remain to be explored. My initial findings open up a whole new arena into an otherwise unknown concept of signal/s from progenitor’s necessary for maintenance of niche cells. Conclusions Stem cell niche has always amazed the biologists with its hidden potential to retain the properties of stem cells. Grasping a sense of its capacity, it is recently being believed that stem cell niche would act as a better therapeutic target. My doctorate work apart from providing many new members important for hematopoietic niche maintenance, also try to uncover that a regulation at cellular level for systemic signals is essential. Moreover, we also introduce importance of extrinsic signals for stem cell niche maintenance. In case of Drosophila hematopoietic niche, we propose that the extrinsic signal comes from its next-door neighbours i.e. progenitor cells and it travels via ECM. With the advancement in our knowledge about stem cells, stem cell therapies became the ultimate goal for treating deadly disorders. But a majority of unsuccessful stories always implied to some missing links. Understanding the basics of stem cell niche will try to seal these gaps and open up more and better avenues into therapeutics. The various factors from the IVgenetic screen needs to explored in other stem cell-niche scenarios and in higher organisms. Moreover, biologists have always been intrigued by how cell types from the same tissue behave differently despite of having similar environment around them. This to our knowledge is the first report where we demonstrated the integration of systemic signals with local factors. Overall, this study enriches the field of Drosophila hematopoietic niche biology. We try to answer a basic question about how systemic signals are perceived differentially by different cell types of the same tissue. This study also discovers various crucial molecules for hematopoietic niche maintenance. Further, a new concept of backward signalling is put forth by this study, where surprisingly hematopoietic niche is maintained by its own progenitors, those cells that were originally housed and maintained by the niche.