Browsing by Author "Bagnat, Michel"

The formation of a single lumen during tubulogenesis is crucial for the development and function of many organs. Although 3D cell culture models have identified molecular mechanisms controlling lumen formation in vitro, their function during vertebrate organogenesis is poorly understood. In this work we used the zebrafish gut as a model to investigate single lumen formation during tubulogenesis. Previous work has shown that multiple small lumens enlarge through fluid accumulation and coalesce into a single lumen. However, since lumen formation occurs in the absence of apoptosis, other cellular processes are necessary to facilitate single lumen formation.

Using light sheet microscopy and genetic approaches we identified a distinct intermediate stage in lumen formation, characterized by two adjacent un-fused lumens. These lumens are separated by cell contacts that contain basolateral adhesion proteins. We observed that lumens arise independently from each other along the length of the gut and do not share a continuous apical surface. Resolution of this intermediate phenotype into a single, continuous lumen requires the remodeling of basolateral contacts between adjacent lumens and subsequent lumen fusion.

Furthermore, we provide insight into the genetic mechanisms regulating lumen formation through the analysis of the Hedgehog pathway. We show that lumen resolution, but not lumen opening, is impaired in smoothened (smo) mutants, indicating that fluid-driven lumen enlargement and resolution are two distinct processes. We also show that smo mutants exhibit perturbations in the Rab11 trafficking pathway, which led us to demonstrate that Rab11-mediated recycling, but not degradation, is necessary for single lumen formation. Taken together, this work demonstrates that lumen resolution is a distinct genetically-controlled process, requiring cellular rearrangement and lumen fusion events, to create a single, continuous lumen in the zebrafish gut.

The guts of neonatal mammals and stomachless fish have a limited capacity for luminal protein digestion, which allows oral acquisition of antibodies and antigens. However, this poses a challenge for dietary protein digestion and absorption during this critical developmental stage. Interestingly, it has been reported that there exist specialized intestinal cells in the ileum of suckling mammals, which are highly endocytic and possess a large lysosomal vacuole. The vacuolated enterocytes have been suggested to be responsible for protein digestion and absorption during the suckling stage. However, the molecular machinery mediating the function of the vacuolated enterocytes remained unknown. Here, we show that the vacuolated enterocytes are conserved in zebrafish as well as in suckling mammals and name them lysosome-rich enterocytes (LREs). We utilized zebrafish to investigate the molecular machinery mediating endocytic events in LREs. Using oral gavage technique and live confocal imaging, we show that LREs mediate the internalization of proteins and fluid-phase cargoes. Then, we performed RNAseq analysis on LREs and other intestine cells isolated via fluorescence-activated cell sorting (FACS). This led us to identify a conserved endocytic machinery in LREs, composed of the scavenger receptor complex Cubilin (Cubn)/Amnionless (Amn) and Dab2, that are highly enriched in LREs. By generating CRISPR/Cas9 genetic mutants of cubn, amn and dab2, we show that the Cubn/Amn receptor complex mediates protein uptake in LREs and that Dab2 mediates uptake of fluid-phase cargo as well as protein in LREs. By subjecting these genetic mutants to feeding experiments with custom diets, we show that impaired LRE uptake leads to compromised growth and survival of larval zebrafish. Moreover, impairing LRE function in suckling mice, via conditional deletion of Dab2, leads to stunted growth and severe protein malnutrition reminiscent of kwashiorkor, a devastating human malnutrition syndrome. These findings identify digestive functions and conserved molecular mechanisms in LREs that are crucial for vertebrate growth and survival.

The notochord plays critical structural and signaling roles during vertebrate development. At the center of the vertebrate notochord is a large fluid-filled organelle, the notochord vacuole. While these highly conserved intracellular structures have been described for decades, little is known about the molecular mechanisms involved in their biogenesis and maintenance. Here we show that zebrafish notochord vacuoles are specialized lysosome-related organelles whose formation and maintenance requires late endosomal trafficking regulated by the vacuole-specific Rab32a, and H+-ATPase-dependent acidification. We establish that notochord vacuoles are required for body axis elongation during embryonic development and identify a novel role for notochord vacuoles in spine morphogenesis. Thus, the vertebrate notochord plays important structural roles beyond early development.

The defining characteristic of the subphylum Vertebrata is the vertebral column, which is comprised of alternating vertebral bodies and intervertebral discs. In spite of being a highly conserved structure, the morphogenetic events that culminate in building the vertebral column remain poorly understood. In particular, patterning mechanisms underlying how segmentation of the spine is precisely established have not been examined at post-embryonic stages. For several years, vertebral column patterning was thought to hinge upon proper segmentation of the embryo, while the notochord served as a transient scaffold for the vertebral bodies and intervertebral discs. Using genetic, live-imaging, and quantitative approaches, this work illustrates that the notochord sheath in zebrafish, provides a template for osteoblast recruitment and vertebral bone formation in the developing spine. Furthermore, we show that notochord segmentation is influenced by the adjacent muscle segments and connective tissue, which may provide mechanical patterning cues. Insights from this work will better inform how adolescent idiopathic scoliosis and congenital scoliosis arise.

Fluid secretion is essential to organ development and function, yet relatively little is known about the roles of fluid secretion in vivo. Early in development, fluid secretion plays important roles during the process of lumen formation and is necessary for organ homeostasis throughout life. A human disease, cystic fibrosis (CF) is caused by loss of cystic fibrosis transmembrane conductance regulator (CFTR) function, a chloride channel and key regulator of vertebrate fluid secretion. CFTR regulates fluid secretion by governing ion transport and osmotic gradients across epithelia.

To identify the developmental requirements for cftr function, we generated cftr mutant zebrafish using transcription activator like effector nucleases (TALENs). In cftr mutant zebrafish, we observed defects in the specification of left-right (LR) asymmetry. In the zebrafish, LR asymmetry is specified in part by directional fluid flow within a ciliated structure, Kupffer's vesicle (KV). Using live imaging of several transgenic markers in KV, we determined that lumen expansion is impaired in cftr mutants, which prevents directional fluid flow necessary for KV function. To examine cftr expression, we generated bacterial artificial chromosome (BAC) transgenic zebrafish expressing fluorescent Cftr fusion proteins under the control of the cftr promoter. These transgenes express Cftr within the KV epithelium and the protein localizes to the apical membrane. These transgenes rescue the KV function and the specification of LR asymmetry. These studies reveal a new role for cftr during KV morphogenesis and function in the zebrafish.

In the zebrafish pancreas, we found that loss of cftr function leads to defects reminiscent of CF including destruction of exocrine tissue and changes in islet morphology. Additionally, we observed exocrine pancreatic destruction by 3 weeks post fertilization (wpf). Analysis of cftr BAC expression in the adult and larval zebrafish pancreata revealed that cftr is expressed specifically within the ducts, localized to the apical membrane throughout life. Adult cftr mutant pancreata developed substantial degeneration of exocrine tissue and experienced reduced growth rates. In contrast, we found that cftr is not necessary for the specification or initial development of the larval pancreas. Exocrine and endocrine tissues developed similarly in WT and cftr mutant larvae. These results indicate that cftr-dependent fluid secretion is important for maintenance of the zebrafish pancreas. Altogether, these studies of cftr function in KV and the pancreas demonstrate that fluid secretion is an essential component of lumen morphogenesis and organ function.

Vertebrates are distinguished by the presence of a segmented spine that supports the body axis and facilitates movement. The establishment of alternating domains of vertebral centra and intervertebral discs is a complex biological phenomenon. Recent studies in teleost fish demonstrate that the epithelial sheath of the notochord segments to provide positional information for the development of vertebral bone. The studies performed for this dissertation uncover specific components of the gene regulatory network guiding notochord segmentation. Genetic manipulations and live confocal imaging of transgenic zebrafish demonstrate that BMP activity triggers sheath cell differentiation and regulates the lateral expansion of notochord segments. Moreover, the importance of notochord segmentation during the development and evolution of the spine is highlighted by a unique extracellular matrix mutant in which notochord patterning is lost. Without a segmented notochord framework, sclerotomal osteoblasts alter their migratory trajectories and solely rely on paraxial mesoderm patterning to form centra structures. The resulting mode of spine morphogenesis shares commonalities with basal gnathostome species, suggesting that notochord signals prompted specific morphological transitions during spine evolution.

The notochord is a defining feature of our phylum Chordata and has critical roles in human development that are highly conserved in vertebrates. The notochord functions as a hydrostatic scaffold to provide structural rigidity needed for anterior-posterior axis elongation and later for proper spine development. The notochord’s mechanical properties depend on its unique structure. In zebrafish, the notochord consists of a core of giant vacuolated cells surrounded by an epithelial -like sheath. Previous research from our lab has shown that during early development, the notochord vacuole rapidly accumulates fluid and expands within the inelastic notochord sheath. In this work we first investigated the molecular processes by which large vacuolated cells of the notochord maintain integrity while being subjected to a significant amount of stress. We determined that caveolae play a mechanoprotective role in the zebrafish notochord and are crucial in preserving notochord integrity. Upon loss of caveolae, the vacuolated cell collapses at discrete positions under the mechanical strain of locomotion then sheath cells invade the inner notochord and differentiate into vacuolated cells thereby restoring notochord function and allowing normal spine development. Findings from our caveolae work next allowed us to investigate the arrangement of vacuolated cells within the zebrafish notochord. During notochord morphogenesis, the vacuolated cells in wild-type zebrafish arrange themselves in a staircase pattern. However, in both caveolae and vacuole mutants, this pattern is disrupted. We investigated the basis of this pattern and found that it can be described by simple physical principles. We modeled the arrangement of vacuolated cells using a system composed of silicone tubing and sodium polyacrylate jelly beads demonstrating that what we observe in vivo can be described by the theory developed for the packing of spheres in cylinders. We determined that the organization of vacuolated cells within the zebrafish notochord is controlled by the density of fluid filled vacuoles and the diameter of the notochord tube. Lastly, based on our finding that sheath cells of the notochord can form de novo vacuoles, we wanted to identify key factors contributing to notochord vacuole biogenesis and integrity. We used a two-pronged transcriptomics and proteomics approach to identify proteins involved in de novo vacuole formation. We find that loss of a protein previously linked to lysosome related organelle function, Lyst, leads to fragmentation of notochord vacuoles and impaired axis elongation. Interestingly, upon injury of the notochord, sheath cells fail to form a fully inflated vacuole and continue to grow outside of notochord boundaries, forming a tumor-like mass. The tumor-like mass appears very similar to a rare tumor type called chordoma, which is characterized by overgrowth of intervertebral disc tissue. This work suggests that Lyst is important for notochord vacuole biogenesis in zebrafish and may play an important role in chordoma formation. Our work has elucidated novel mechanisms of cell surface integrity and has shown how proper vacuolated cell inflation leads to a structurally intact notochord. Additionally, we have demonstrated the remarkable regenerative capacity of the zebrafish notochord and identified potential regulators of both vacuole biogenesis and chordoma formation.