Consequences of IFT-dynein-2 governed ciliary protein dynamics for skeletal development
Cilia represent tubulin-based antenna-like cellular structures extending from the surface of most vertebrate cells. As cilia have no protein synthesis capability, ciliary proteins are synthesized in the cytoplasm and trafficked into the cilium. Intraciliary protein transport along the axoneme is carried out by intraflagellar transport (IFT).
IFT also transports protein complexes of the cytoplasmic pool from the cilium base into the cilium and vice versa. IFT regulation is highly dynamic, enabling fast adaptation to the rapidly changing cellular needs, including assembly and disassembly of cilia, and ciliary signal transduction upon mechanical or chemical stimuli with fast-changing IFT cargo. Over the past years, we and others have identified defective components of the dynein-2 complex, representing the motor for retrograde IFT from the tip to the base as well as defective IFT components as a major cause of ciliary chondrodysplasias. However, while clearly IFT-mediated intraciliary protein dynamics are essential for cellular differentiation, maintenance, and survival, individual functions of the IFT-dynein-2-complex components and their role in mammalian development have remained largely elusive. This is primarily due to the complete loss of cilia upon IFT or dynein-2 gene knockouts, preventing the analysis of individual IFT protein functions in cilia in those models. Specifically, while is has become evident over the last decade that IFT-governed ciliary protein dynamics are crucial for skeletal development, the contribution of individual IFT components to chondrocyte cilia protein dynamics, cell identity, and cell signaling as well as cartilage extracellular matrix establishment and skeletal development has not been understood. This project will dissect individual functions of ciliary IFT/dynein-2 complex components in regulating intraciliary protein dynamics and cilia assembly/disassembly and characterize the individual role of IFT/dynein-2 components in chondrocyte differentiation and skeletal development. To achieve this, we employ hypomorphic model systems carrying human disease alleles. This will not only advance our biological understanding of ciliary protein dynamics but hopefully also provide future therapeutic entry points for ciliary chondrodysplasias.
Dynamic regulation of ciliary RNA-binding proteins in kidney epithelial cells
Polycystic kidney diseases (PKDs) are caused by mutations in genes encoding for ciliary proteins and constitute a significant manifestation of ciliopathies. Interestingly, most pathogenic mutations do not entirely abrogate ciliogenesis but rather interfere with dynamic ciliary functions such as ciliary protein trafficking and signaling or the ciliary assembly-disassembly cycle.
Recently, we identified RNA-binding proteins (RBPs) and RNA as novel components of primary cilia, that are dynamically regulated and enriched upon induction of ciliary disassembly. In our preliminary work, we could already confirm the ciliary localization for the RNA editing enzyme Adar1 and the Cell cycle associated protein 1 (Caprin-1). Besides, we detected double-stranded RNA in primary cilia. The role of RBPs and RNA in cilia dynamics is not understood. The overarching aim of the project at-hand is to understand the dynamic shuttling of RBPs and RNA between the cilium and the cytosol, and the role of RBPs and RNA within the periodic cycle of ciliary assembly and disassembly. This will be examined in the context of its impact on the organization of a complex tissue, the kidney. In particular, we will (1) characterize the dynamics of cilia-specific localization of individual RBPs and RNA, (2) investigate how ciliary RBPs and RNA modulate ciliary assembly/disassembly and signaling, and (3) aim to understand the impact of loss of ciliary RBPs on renal tissue organization.
The impact of cilium disassembly dynamics on developmental signaling
The primary cilium is a solitary, dynamic plasma membrane micro-domain that orchestrates cellular signaling cascades with important functions in organismal development. A hallmark signaling pathway in primary cilia is Hedgehog signaling, the components of which dynamically localize to the primary cilium in a signaling-dependent manner to transmit morphogen information to the rest of the cell.
However, signals can only be sensed when cells display primary cilia, which is tied to the cell cycle. Hedgehog morphogens regulate early embryonic development, including left-right axis patterning and neural tube closure with important implications for human health. Here, cells must receive signals to initiate cellular responses, followed by cilia removal to execute the initiated programs. As morphogens determine cell fates in a dose-dependent manner, signaling strength and duration are important parameters that specify signaling outputs. Yet, the relation of the dynamic removal of a cilium to the signaling response as well as the consequences of altered cilium removal on embryonic development remain largely unknown.In this project we will combine biochemical in vitro approaches with state-of-the-art developmental biology in the African Clawed Frog Xenopus laevis to study the relevance of the dynamic cilium removal for developmental signaling with a focus on Hedgehog signaling. Advanced proximity labeling technologies combined with quantitative mass spectrometry will determine the proteomic remodeling of the primary cilium during cilium removal to reveal the underlying molecular mechanisms and the consequences for cilia signaling on a molecular level. The significance of the dynamic behavior of ciliary signaling components during cilia removal for morphogenesis will be investigated in the left-right organizer and the developing neural tube in vivo. The main goals of the project are: 1) to reveal the molecular mechanisms of protein removal from cilia during cilium disassembly and how defects in this process impact cilia signaling, and 2) to understand how intraciliary Hedgehog dynamics affect cellular and tissue morphogenesis. We will thereby mechanistically resolve how alterations in cilia removal affect signaling and embryonic development across experimental systems in a comparative manner. The results will form the basis for future translational investigations focusing on human disease.
Primary cilia dynamics in pancreatic duct network development
Pancreatic cysts have been reported in a subset of ciliopathies, but the function of primary cilia in the pancreas is poorly understood, particularly in the fetus, when the cystic phenotypes are initiated. During development, ciliated progenitors of the exocrine and endocrine cells form the walls of the pancreatic duct network.
This network remodels from a mesh to a tree optimized for fluid transport, presumably in response to flow. By analogy with other systems, we hypothesize that the primary cilia on the progenitors and cilia flow sensing play an essential role in the morphogenesis of the pancreatic ducts during development. Using complementary expertise in pancreas development (Grapin-Botton) and mathematical modeling of biological hydrodynamics and signaling (Friedrich), we propose to use the pancreas as a new model to understand the role of primary cilia dynamics in tubular network formation across time-scales. At the (sub)second time scale, we will test whether cilia are flow sensors in pancreatic ducts and characterize cilia flow sensing in terms of a quantitative input-output relationships that link the magnitudes of both steady and oscillatory external fluid flow to dynamic cilia signaling, focusing primarily on calcium. We further hypothesize that flow may lead to changes in the cilia proteome, as observed for chemical signaling but so far unexplored for flow sensing. To gain mechanistic insights into the cilia proteome in pancreatic progenitors and changes in the intracilliary protein composition upon stimulation, we will use proximity labelling using NPHP3 as a bait. At the time scale of days, we will systematically investigate the proportion of cells with cilia, their length and orientation and map this morphometric data on skeletonized ductal network structures. This will enable us to establish whether cilia presence and length change with developmental time, duct diameter, and position in the pancreas duct network. Finally, we will connect the insight on ciliary mechanical sensing in the pancreas at the (sub)second scale to the physiologically relevant process of pancreas duct network remodeling during embryonic development, which takes place over days. To do so, we will monitor the flow in vivo and impose it in perfused pancreas organoids, investigating whether it triggers remodeling. Taken together, our work will offer mechanistic insights into mechanical sensing by primary cilia in the pancreas. In addition, our results will lay the foundation to unravel the implications of impaired pancreatic cilia signaling in ciliopathies.
Primary cilia dynamics in adipose tissue development and homeostasis
Primary cilia are important regulators of adipose tissue development and function. White adipose tissue (WAT), which stores energy as lipids and is vital for maintaining whole-body energy and immune homeostasis, relies on primary cilia function. This is underlined by a subset of ciliopathies, disorders that arise from cilia dysfunction, which display obesity as a cardinal feature.
The differentiation into adipocytes, termed adipogenesis, is highly orchestrated: precursor cells first commit to the adipocyte lineage and then terminally differentiate into adipocytes. Primary cilia are present on pre-adipocytes but are disassembled during proliferation and differentiation. Strikingly, loss of primary cilia formation in pre-adipocytes abolishes differentiation, underlining the importance of primary cilia function for WAT development. However, the stimuli controlling primary cilia signaling and, thereby, pre-adipocyte proliferation and differentiation are largely unknown. Macrophages, one of the major immune cell types in WAT, can influence adipogenesis and contribute to obesity-associated comorbidities. WAT-resident macrophages produce the growth factor PDGFcc, which is required for adipogenesis during development and upon diet-induced obesity. Intriguingly, PDGFs, including PDGFcc, exert their actions via the primary cilium. However, whether macrophage-derived PDGFcc in WAT acts via primary cilia on pre-adipocytes is not known. In this project, we use 2D and 3D cell culture systems in combination with transgenic mouse models to address how signals derived from macrophages affect ciliary protein dynamics and signaling in adipocyte precursors and how this shapes cell fate and function and, thereby, WAT development and homeostasis.
Primary cilia dynamics in determining neural progenitor cell maintenance in brain development
The primary cilium is critical for mammalian brain development. For example, its dysfunction can cause congenital microcephaly, a neurodevelopmental disorder in which the neural progenitor cell (NPC) pool is depleted. During mammalian neocortex development, the self-renewing NPCs expand their population via symmetric divisions regulated by an orchestrated cilium assembly and disassembly program.
However, how NPCs accomplish timely cilium disassembly and how cilium dynamics determine NPC fate, regulate signaling, and maintain NPCs remains unknown. NPCs dynamically assemble and disassemble primary cilia, which is tightly correlated with cell-cycle exit (G1-G0) and re-entry (G1-S to M), respectively. In turn, a delay or failure in cilium disassembly acts as a brake, retaining cells in G0/G1 and transiently preventing cell cycle progression. This could be a rate-limiting step in regulating NPCs’ cell cycle progression and fate in the developing brain. We hypothesize that the accurate recruitment of cilium disassembly components at the ciliary base ensures a timely cilium disassembly, which, in turn, regulates neuroepithelium development. In this project, we will first study the dynamic localization of cilium disassembly components in NPCs and manipulate them to explore the consequences of a delayed cilium disassembly on NPC fate. Second, we will dissect the altered signaling dynamics due to delayed disassembly, mainly focusing on platelet-derived growth factor (PDGF) signaling. Finally, we aim to identify how controlled cilium dynamics regulate NPC maintenance in brain tissue organization in human brain organoids. Our project will reveal molecular insights into how primary cilia dynamics control neuroepithelium organization.
Primary cilia dynamics in retinal pigment epithelium development, homeostasis, and function
The retinal pigment epithelium is a prime target to understand the molecular mechanisms of how temporal and spatial regulation of cilia dynamics control epithelial organization. This monolayer of highly specialized, tightly connected polarized cells located between the neural retina and the vascular choroid is essential for vision. Similar to other epithelia, the primary cilium in the RPE is a highly dynamic organelle, that regulates many signaling pathways controlling RPE specification, development, and differentiation.
Of these, the WNT signaling pathway seems to be one of the most prominent. To what extent the precise regulation of cilium assembly and disassembly dynamics influence RPE development, maturation, or function via regulation of WNT signaling is unknown. To address these questions, we propose the following three specific objectives. 1. Investigate the molecular mechanisms of RPE maturation and function via cilia-mediated WNT signaling in vitro. 2. Compare cilia ablation vs. altered ciliary signaling in RPE cells in vivo and analyze the consequences for retina and visual function. 3. Characterize cilia assembly and disassembly in real-time explant tissue cultures. Our particular focus is to distinguish the relevance between ciliation vs. ciliary signaling and explore how cilium assembly and disassembly can influence RPE maturation and function. Using well-established RPE cell models (in vitro) and RPE-specific cilia mouse mutants (in vivo) and we will combine gene knockout/silencing and pharmacology to inhibit ciliation and manipulate ciliary signaling. In each of these systems we will manipulate three ciliary proteins (BBS8, IFT20, IFT88) to either modulate ciliary signaling or ablate the cilium. Our three aims are interrelated but not co-dependent. Upon completing the first two aims, we will discover whether the primary cilium assembly/disassembly dynamics are key in controlling RPE differentiation and maturation and which ciliary signaling pathways dynamically regulate this process. By comparing the phenotype of these we can distinguish between ciliation vs. ciliary signaling effects. In the third aim we will establish a platform for further downstream analyses. Insights into how primary cilia dynamics regulate RPE organization is not only crucial for vision research but can also be applied to other epithelial tissues.
