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Dietary protein absorption in neonatal mammals and fishes relies on the function of a specialized and conserved population of highly absorptive lysosome rich enterocytes (LREs). The gut microbiome has been shown to enhance absorption of nutrients, such as lipids, by intestinal epithelial cells. However, whether protein absorption is also affected by the gut microbiome is poorly understood. Here, we investigate connections between protein absorption and microbes in the zebrafish gut. Using live microscopy-based quantitative assays, we find that microbes slow the pace of protein uptake and degradation in LREs. While microbes do not affect the number of absorbing LRE cells, microbes lower the expression of endocytic and protein digestion machinery in LREs. Using transgene assisted cell isolation and single cell RNA-sequencing, we characterize all intestinal cells that take up dietary protein. We find that microbes affect expression of bacteria-sensing and metabolic pathways in LREs, and that some secretory cell types also take up protein and share components of protein uptake and digestion machinery with LREs. Using custom-formulated diets, we investigated the influence of diet and LRE activity on the gut microbiome. Impaired protein uptake activity in LREs, along with a protein-deficient diet, alters the microbial community and leads to increased abundance of bacterial genera that have the capacity to reduce protein uptake in LREs. Together, these results reveal that diet-dependent reciprocal interactions between LREs and the gut microbiome regulate protein absorption.

The rete ovarii (RO) is an appendage of the ovary that has been given little attention. Although the RO appears in drawings of the ovary in early versions of Gray's Anatomy, it disappeared from recent textbooks, and is often dismissed as a functionless vestige in the adult ovary. Using PAX8 immunostaining and confocal microscopy, we characterized the fetal development of the RO in the context of the ovary. The RO consists of three distinct regions that persist in adult life, the intraovarian rete (IOR), the extraovarian rete (EOR), and the connecting rete (CR). While the cells of the IOR appear to form solid cords within the ovary, the EOR rapidly develops into a convoluted tubular epithelium ending in a distal dilated tip. Cells of the EOR are ciliated and exhibit cellular trafficking capabilities. The CR, connecting the EOR to the IOR, gradually acquires tubular epithelial characteristics by birth. Using microinjections into the distal dilated tip of the EOR, we found that luminal contents flow towards the ovary. Mass spectrometry revealed that the EOR lumen contains secreted proteins potentially important for ovarian function. We show that the cells of the EOR are closely associated with vasculature and macrophages, and are contacted by neuronal projections, consistent with a role as a sensory appendage of the ovary. The direct proximity of the RO to the ovary and its integration with the extraovarian landscape suggest that it plays an important role in ovary development and homeostasis., Competing Interests: Competing interests The authors declare no competing or financial interests.

In bony fishes, patterning of the vertebral column, or spine, is guided by a metameric blueprint established in the notochord sheath. Notochord segmentation begins days after somitogenesis concludes and can occur in its absence. However, somite patterning defects lead to imprecise notochord segmentation, suggesting that these processes are linked. Here, we identify that interactions between the notochord and the axial musculature ensure precise spatiotemporal segmentation of the zebrafish spine. We demonstrate that myoseptum-notochord linkages drive notochord segment initiation by locally deforming the notochord extracellular matrix and recruiting focal adhesion machinery at these contact points. Irregular somite patterning alters this mechanical signaling, causing non-sequential and dysmorphic notochord segmentation, leading to altered spine development. Using a model that captures myoseptum-notochord interactions, we find that a fixed spatial interval is critical for driving sequential segment initiation. Thus, mechanical coupling of axial tissues facilitates spatiotemporal spine patterning., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background and Aims: Inflammatory bowel diseases (IBDs) are chronic inflammatory conditions influenced heavily by environmental factors. DNA methylation is a form of epigenetic regulation linking environmental stimuli to gene expression changes and inflammation. Here, we investigated how DNA methylation of the tumor necrosis factor (TNF) promoter differs between inflamed and uninflamed mucosa of IBD patients, including anti-TNF responders and nonresponders., Methods: We obtained mucosal biopsies from 200 participants (133 IBDs and 67 controls) and analyzed TNF promoter methylation using bisulfite sequencing, comparing inflamed with uninflamed segments, in addition to paired inflamed/uninflamed samples from individual patients. We conducted similar analyses on purified intestinal epithelial cells from bowel resections. We also compared TNF methylation levels of inflamed and uninflamed mucosa from a separate cohort of 15 anti-TNF responders and 17 nonresponders. Finally, we sequenced DNA methyltransferase genes to identify rare variants in IBD patients and functionally tested them using rescue experiments in a zebrafish genetic model of DNA methylation deficiency., Results: TNF promoter methylation levels were decreased in inflamed mucosa of IBD patients and correlated with disease severity. Isolated intestinal epithelial cells from inflamed tissue showed proportional decreases in TNF methylation. Anti-TNF nonresponders showed lower levels of TNF methylation than responders in uninflamed mucosa. Our sequencing analysis revealed 2 missense variants in DNA methyltransferase 1, 1 of which had reduced function in vivo., Conclusion: Our study reveals an association of TNF promoter hypomethylation with mucosal inflammation, suggesting that IBD patients may be particularly sensitive to inflammatory environmental insults affecting DNA methylation. Together, our analyses indicate that TNF promoter methylation analysis may aid in the characterization of IBD status and evaluation of anti-TNF therapy response., (© 2024 The Authors.)

The vertebrate spine is a metameric structure composed of alternating vertebral bodies (centra) and intervertebral discs. 1 Recent studies in zebrafish have shown that the epithelial sheath surrounding the notochord differentiates into alternating cartilage-like (col2a1/col9a2+) and mineralizing (entpd5a+) segments which serve as a blueprint for centra formation. 2 , 3 , 4 , 5 This process also defines the trajectories of migrating sclerotomal cells that form the mature vertebral bodies. 4 Previous work demonstrated that notochord segmentation is typically sequential and involves the segmented activation of Notch signaling. 2 However, it is unclear how Notch is activated in an alternating and sequential fashion. Furthermore, the molecular components that define segment size, regulate segment growth, and produce sharp segment boundaries have not been identified. In this study, we uncover that a BMP signaling wave acts upstream of Notch during zebrafish notochord segmentation. Using genetically encoded reporters of BMP activity and signaling pathway components, we show that BMP signaling is dynamic as axial patterning progresses, leading to the sequential formation of mineralizing domains in the notochord sheath. Genetic manipulations reveal that type I BMP receptor activation is sufficient to ectopically trigger Notch signaling. Moreover, loss of Bmpr1ba and Bmpr1aa or Bmp3 function disrupts ordered segment formation and growth, which is recapitulated by notochord-specific overexpression of the BMP antagonist, Noggin3. Our data suggest that BMP signaling in the notochord sheath precedes Notch activation and instructs segment growth, facilitating proper spine morphogenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Wide field of view microscopy that can resolve 3D information at high speed and spatial resolution is highly desirable for studying the behaviour of freely moving model organisms. However, it is challenging to design an optical instrument that optimises all these properties simultaneously. Existing techniques typically require the acquisition of sequential image snapshots to observe large areas or measure 3D information, thus compromising on speed and throughput. Here, we present 3D-RAPID, a computational microscope based on a synchronized array of 54 cameras that can capture high-speed 3D topographic videos over an area of 135 cm2, achieving up to 230 frames per second at spatiotemporal throughputs exceeding 5 gigapixels per second. 3D-RAPID employs a 3D reconstruction algorithm that, for each synchronized snapshot, fuses all 54 images into a composite that includes a co-registered 3D height map. The self-supervised 3D reconstruction algorithm trains a neural network to map raw photometric images to 3D topography using stereo overlap redundancy and ray-propagation physics as the only supervision mechanism. The resulting reconstruction process is thus robust to generalization errors and scales to arbitrarily long videos from arbitrarily sized camera arrays. We demonstrate the broad applicability of 3D-RAPID with collections of several freely behaving organisms, including ants, fruit flies, and zebrafish larvae., Competing Interests: Competing interests RH and MH are cofounders of Ramona Optics, Inc., which is commercializing multi-camera array microscopes. MH, JP, TD, PR, VS, CBC, MZ, JPB, and GH are or were employed by Ramona Optics, Inc. during the course of this research. KCZ is a consultant for Ramona Optics, Inc. The remaining authors declare no competing interests.

A ubiquitous feature of animal development is the formation of fluid-filled cavities or lumina, which transport gases and fluids across tissues and organs. Among different species, lumina vary drastically in size, scale, and complexity. However, all lumen formation processes share key morphogenetic principles that underly their development. Fundamentally, a lumen simply consists of epithelial cells that encapsulate a continuous internal space, and a common way of building a lumen is via opening and enlarging by filling it with fluid and/or macromolecules. Here, we discuss how polarized targeting of membrane and secreted proteins regulates lumen formation, mainly focusing on ion transporters in vertebrate model systems. We also discuss mechanistic differences observed among invertebrates and vertebrates and describe how the unique properties of the Na + /K + -ATPase and junctional proteins can promote polarization of immature epithelia to build lumina de novo in developing organs., Competing Interests: Competing interest The authors declare no competing interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

During organismal development, organs and systems are built following a genetic blueprint that produces structures capable of performing specific physiological functions. Interestingly, we have learned that the physiological activities of developing tissues also contribute to their own morphogenesis. Specifically, physiological activities such as fluid secretion and cell contractility generate hydrostatic pressure that can act as a morphogenetic force. Here, we first review the role of hydrostatic pressure in tube formation during animal development and discuss mathematical models of lumen formation. We then illustrate specific roles of the notochord as a hydrostatic scaffold in anterior-posterior axis development in chordates. Finally, we cover some examples of how fluid flows influence morphogenetic processes in other developmental contexts. Understanding how fluid forces act during development will be key for uncovering the self-organizing principles that control morphogenesis.

Polarized epithelial cells are characterized by the asymmetric distribution of proteins between apical and basolateral domains of the plasma membrane. This asymmetry is highly conserved and is fundamental to epithelial cell physiology, development, and homeostasis. How proteins are segregated for apical or basolateral delivery, a process known as sorting, has been the subject of considerable investigation for decades. Despite these efforts, the rules guiding apical sorting are poorly understood and remain controversial. Here, we consider mechanisms of apical membrane protein sorting and argue that they are largely driven by self-organization and biophysical principles. The preponderance of data to date is consistent with the idea that apical sorting is not ruled by a dedicated protein-based sorting machinery and relies instead on the concerted effects of oligomerization, phase separation of lipids and proteins in membranes, and pH-dependent glycan interactions., (© 2021 Federation of European Biochemical Societies.)

Zebrafish provide an excellent model for in vivo cell biology studies because of their amenability to live imaging. Protein visualization in zebrafish has traditionally relied on overexpression of fluorescently tagged proteins from heterologous promoters, making it difficult to recapitulate endogenous expression patterns and protein function. One way to circumvent this problem is to tag the proteins by modifying their endogenous genomic loci. Such an approach is not widely available to zebrafish researchers because of inefficient homologous recombination and the error-prone nature of targeted integration in zebrafish. Here, we report a simple approach for tagging proteins in zebrafish on their N or C termini with fluorescent proteins by inserting PCR-generated donor amplicons into non-coding regions of the corresponding genes. Using this approach, we generated endogenously tagged alleles for several genes that are crucial for epithelial biology and organ development, including the tight junction components ZO-1 and Cldn15la, the trafficking effector Rab11a, the apical polarity protein aPKC and the ECM receptor Integrin β1b. Our approach facilitates the generation of knock-in lines in zebrafish, opening the way for accurate quantitative imaging studies., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

The actin cortex is involved in many biological processes and needs to be significantly remodeled during cell differentiation. Developing epithelial cells construct a dense apical actin cortex to carry out their barrier and exchange functions. The apical cortex assembles in response to three-dimensional (3D) extracellular cues, but the regulation of this process during epithelial morphogenesis remains unknown. Here, we describe the function of Smoothelin-like 2 (SMTNL2), a member of the smooth-muscle-related Smoothelin protein family, in apical cortex maturation. SMTNL2 is induced during development in multiple epithelial tissues and localizes to the apical and junctional actin cortex in intestinal and kidney epithelial cells. SMTNL2 deficiency leads to membrane herniations in the apical domain of epithelial cells, indicative of cortex abnormalities. We find that SMTNL2 binds to actin filaments and is required to slow down the turnover of apical actin. We also characterize the SMTNL2 proximal interactome and find that SMTNL2 executes its functions partly through inhibition of coronin-1B. Although coronin-1B-mediated actin dynamics are required for early morphogenesis, its sustained activity is detrimental for the mature apical shape. SMTNL2 binds to coronin-1B through its N-terminal coiled-coil region and negates its function to stabilize the apical cortex. In sum, our results unveil a mechanism for regulating actin dynamics during epithelial morphogenesis, providing critical insights on the developmental control of the cellular cortex., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The vertebrate body plan is characterized by the presence of a segmented spine along its main axis. Here, we examine the current understanding of how the axial tissues that are formed during embryonic development give rise to the adult spine and summarize recent advances in the field, largely focused on recent studies in zebrafish, with comparisons to amniotes where appropriate. We discuss recent work illuminating the genetics and biological mechanisms mediating extension and straightening of the body axis during development, and highlight open questions. We specifically focus on the processes of notochord development and cerebrospinal fluid physiology, and how defects in those processes may lead to scoliosis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The spine is a defining feature of the vertebrate body plan. However, broad differences in vertebral structures and morphogenetic strategies occur across vertebrate groups, clouding the homology between their developmental programs. Analysis of a zebrafish mutant, spondo, whose spine is dysmorphic, prompted us to reconstruct paleontological evidence, highlighting specific transitions during teleost spine evolution. Interestingly, the spondo mutant recapitulates characteristics present in basal fishes, not found in extant teleosts. Further analysis of the mutation implicated the teleost-specific notochord protein, Calymmin, as a key regulator of spine patterning in zebrafish. The mutation in cmn results in loss of notochord sheath segmentation, altering osteoblast migration to the developing spine, and increasing sensitivity to somitogenesis defects associated with congenital scoliosis in amniotes. These data suggest that signals from the notochord define the evolutionary identity of the spine and demonstrate how simple shifts in development can revert traits canalized for about 250 million years., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Epithelial cell physiology critically depends on the asymmetric distribution of channels and transporters. However, the mechanisms targeting membrane proteins to the apical surface are still poorly understood. Here, we performed a visual forward genetic screen in the zebrafish intestine and identified mutants with defective apical targeting of membrane proteins. One of these mutants, affecting the vacuolar H+-ATPase gene atp6ap1b, revealed specific requirements for luminal acidification in apical, but not basolateral, membrane protein sorting and transport. Using a low temperature block assay combined with genetic and pharmacologic perturbation of luminal pH, we monitored transport of newly synthesized membrane proteins from the TGN to apical membrane in live zebrafish. We show that vacuolar H+-ATPase activity regulates sorting of O-glycosylated proteins at the TGN, as well as Rab8-dependent post-Golgi trafficking of different classes of apical membrane proteins. Thus, luminal acidification plays distinct and specific roles in apical membrane biogenesis., (© 2020 Levic et al.)

The vertebral column or spine assembles around the notochord rod which contains a core made of large vacuolated cells. Each vacuolated cell possesses a single fluid-filled vacuole, and loss or fragmentation of these vacuoles in zebrafish leads to spine kinking. Here, we identified a mutation in the kinase gene dstyk that causes fragmentation of notochord vacuoles and a severe congenital scoliosis-like phenotype in zebrafish. Live imaging revealed that Dstyk regulates fusion of membranes with the vacuole. We find that localized disruption of notochord vacuoles causes vertebral malformation and curving of the spine axis at those sites. Accordingly, in dstyk mutants the spine curves increasingly over time as vertebral bone formation compresses the notochord asymmetrically, causing vertebral malformations and kinking of the axis. Together, our data show that notochord vacuoles function as a hydrostatic scaffold that guides symmetrical growth of vertebrae and spine formation., Competing Interests: JB, JN, KE, BP, JH, XG, SN, SM, MB No competing interests declared, DS Senior editor, eLife, (© 2020, Bagwell et al.)

Enteroendocrine cells (EECs) are specialized sensory cells in the intestinal epithelium that sense and transduce nutrient information. Consumption of dietary fat contributes to metabolic disorders, but EEC adaptations to high fat feeding were unknown. Here, we established a new experimental system to directly investigate EEC activity in vivo using a zebrafish reporter of EEC calcium signaling. Our results reveal that high fat feeding alters EEC morphology and converts them into a nutrient insensitive state that is coupled to endoplasmic reticulum (ER) stress. We called this novel adaptation 'EEC silencing'. Gnotobiotic studies revealed that germ-free zebrafish are resistant to high fat diet induced EEC silencing. High fat feeding altered gut microbiota composition including enrichment of Acinetobacter bacteria, and we identified an Acinetobacter strain sufficient to induce EEC silencing. These results establish a new mechanism by which dietary fat and gut microbiota modulate EEC nutrient sensing and signaling., Competing Interests: LY, OM, JB, MB, RL, JR No competing interests declared, (© 2019, Ye et al.)

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, how dietary protein is absorbed during critical developmental stages when the gut is still immature is unknown. Here, we show that specialized intestinal cells, which we call lysosome-rich enterocytes (LREs), internalize dietary protein via receptor-mediated and fluid-phase endocytosis for intracellular digestion and trans-cellular transport. In LREs, we identify a conserved endocytic machinery, composed of the scavenger receptor complex Cubilin/Amnionless and Dab2, that is required for protein uptake by LREs and for 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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The notochord is a conserved axial structure that in vertebrates serves as a hydrostatic scaffold for embryonic axis elongation and, later on, for proper spine assembly. It consists of a core of large fluid-filled vacuolated cells surrounded by an epithelial sheath that is encased in extracellular matrix. During morphogenesis, the vacuolated cells inflate their vacuole and arrange in a stereotypical staircase pattern. We investigated the origin of this pattern and found that it can be achieved purely by simple physical principles. We are able to model the arrangement of vacuolated cells within the zebrafish notochord using a physical model composed of silicone tubes and water-absorbing polymer beads. The biological structure and the physical model can be accurately described by the theory developed for the packing of spheres and foams in cylinders. Our experiments with physical models and numerical simulations generated several predictions on key features of notochord organization that we documented and tested experimentally in zebrafish. Altogether, our data reveal that the organization of the vertebrate notochord is governed by the density of the osmotically swelling vacuolated cells and the aspect ratio of the notochord rod. We therefore conclude that self-organization underlies morphogenesis of the vertebrate notochord.This article is part of the Theo Murphy meeting issue on 'Mechanics of development'., (© 2018 The Author(s).)

The spine is a segmented axial structure made of alternating vertebral bodies (centra) and intervertebral discs (IVDs) assembled around the notochord. Here, we show that, prior to centra formation, the outer epithelial cell layer of the zebrafish notochord, the sheath, segments into alternating domains corresponding to the prospective centra and IVD areas. This process occurs sequentially in an anteroposterior direction via the activation of Notch signaling in alternating segments of the sheath, which transition from cartilaginous to mineralizing domains. Subsequently, osteoblasts are recruited to the mineralized domains of the notochord sheath to form mature centra. Tissue-specific manipulation of Notch signaling in sheath cells produces notochord segmentation defects that are mirrored in the spine. Together, our findings demonstrate that notochord sheath segmentation provides a template for vertebral patterning in the zebrafish spine., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cell migration is essential for morphogenesis, organ formation, and homeostasis, with relevance for clinical conditions. The migration of primordial germ cells (PGCs) is a useful model for studying this process in the context of the developing embryo. Zebrafish PGC migration depends on the formation of cellular protrusions in form of blebs, a type of protrusion found in various cell types. Here we report on the mechanisms allowing the inflation of the membrane during bleb formation. We show that the rapid expansion of the protrusion depends on membrane invaginations that are localized preferentially at the cell front. The formation of these invaginations requires the function of Cdc42, and their unfolding allows bleb inflation and dynamic cell-shape changes performed by migrating cells. Inhibiting the formation and release of the invaginations strongly interfered with bleb formation, cell motility, and the ability of the cells to reach their target., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The notochord, a conserved axial structure required for embryonic axis elongation and spine development, consists of giant vacuolated cells surrounded by an epithelial sheath [1-3]. During morphogenesis, vacuolated cells maintain their structural integrity despite being under constant mechanical stress [4]. We hypothesized that the high density of caveolae present in vacuolated cells [5, 6] could buffer mechanical tension. Caveolae are 50- to 80-nm membrane invaginations lined by cage-like polygonal structures [7, 8] formed by caveolin 1 (Cav1) or Cav3 and one of the cavin proteins [6, 9-11]. Recent in vitro work has shown that plasma membrane caveolae constitute a membrane reservoir that can buffer mechanical stresses such as stretching or osmotic swelling [12]. Moreover, mechanical integrity of vascular and muscle cells is partly dependent on caveolae [13-15]. However, the in vivo mechano-protective roles of caveolae have only begun to be explored. Using zebrafish mutants for cav1, cav3, and cavin1b, we show that caveolae are essential for notochord integrity. Upon loss of caveola function, vacuolated cells collapse 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. Our data further indicate that nucleotides released by dying vacuolated cells promote sheath cell vacuolization and trans-differentiation. This work reveals a novel structural role for caveolae in vertebrates and provides unique insights into the mechanisms that safeguard notochord and spine development., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Somatosensory information from the periphery is routed to the spinal cord through centrally-projecting sensory axons that cross into the central nervous system (CNS) via the dorsal root entry zone (DREZ). The glial cells that ensheath these axons ensure rapid propagation of this information. Despite the importance of this glial-axon arrangement, how this afferent nerve is assembled during development is unknown. Using in vivo, time-lapse imaging we show that as centrally-projecting pioneer axons from dorsal root ganglia (DRG) enter the spinal cord, they initiate expression of the cytokine TNFalpha. This induction coincides with ensheathment of these axons by associated glia via a TNF receptor 2 (TNFR2)-mediated process. This work identifies a signaling cascade that mediates peripheral glial-axon interactions and it functions to ensure that DRG afferent projections are ensheathed after pioneer axons complete their navigation, which promotes efficient somatosensory neural function.

In contrast to mammals, adult zebrafish have a high capacity to regenerate damaged or lost myocardium through proliferation of cardiomyocytes spared from damage. The epicardial sheet covering the heart is activated by injury and aids muscle regeneration through paracrine effects and as a multipotent cell source, and has received recent attention as a target in cardiac repair strategies. Although it is recognized that epicardium is required for muscle regeneration and itself has high regenerative potential, the extent of cellular heterogeneity within epicardial tissue is largely unexplored. Here, we performed transcriptome analysis on dozens of epicardial lineage cells purified from zebrafish harboring a transgenic reporter for the pan-epicardial gene tcf21. Hierarchical clustering analysis suggested the presence of at least three epicardial cell subsets defined by expression signatures. We validated many new pan-epicardial and epicardial markers by alternative expression assays. Additionally, we explored the function of the scaffolding protein and main component of caveolae, caveolin 1 (cav1), which was present in each epicardial subset. In BAC transgenic zebrafish, cav1 regulatory sequences drove strong expression in ostensibly all epicardial cells and in coronary vascular endothelial cells. Moreover, cav1 mutant zebrafish generated by genome editing showed grossly normal heart development and adult cardiac anatomy, but displayed profound defects in injury-induced cardiomyocyte proliferation and heart regeneration. Our study defines a new platform for the discovery of epicardial lineage markers, genetic tools, and mechanisms of heart regeneration., (© 2016. Published by The Company of Biologists Ltd.)

Inflammatory bowel diseases (IBD), which include Crohn's disease and ulcerative colitis, contribute to significant morbidity and mortality globally. Despite an increase in incidence, IBD onset is still poorly understood. Mouse models of IBD recapitulate several aspects of human disease, but limited accessibility for live imaging and the lack of forward genetics highlight the need for new model systems for disease onset characterization. Zebrafish represent a powerful platform to model IBD using forward and reverse genetics, live imaging of transgenic lines and physiological assays. In this review, we address current models of IBD in zebrafish and newly developed reagents available for future studies.

Over several decades genetic studies have unraveled many molecular mechanisms that underlie the signaling networks guiding morphogenesis, but the mechanical forces at work remain much less well understood. Accumulation of fluid within a luminal space can generate outward hydrostatic pressure capable of shaping morphogenesis at several scales, ranging from individual organs to the entire vertebrate body-plan. Here, we focus on recent work that uncovered mechanical roles for fluid secretion during morphogenesis. Identifying the roles and regulation of fluid secretion will be instrumental for understanding the mechanics of morphogenesis as well as many human diseases of complex genetic and environmental origin including secretory diarrheas and scoliosis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The development and function of many internal organs requires precisely regulated fluid secretion. A key regulator of vertebrate fluid secretion is an anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Loss of CFTR function leads to defects in fluid transport and cystic fibrosis (CF), a complex disease characterized by a loss of fluid secretion and mucus buildup in many organs including the lungs, liver, and pancreas. Several animal models including mouse, ferret and pig have been generated to investigate the pathophysiology of CF. However, these models have limited accessibility to early processes in the development of CF and are not amenable for forward genetic or chemical screens. Here, we show that Cftr is expressed and localized to the apical membrane of the zebrafish pancreatic duct and that loss of cftr function leads to destruction of the exocrine pancreas and a cystic fibrosis phenotype that mirrors human disease. Our analyses reveal that the cftr mutant pancreas initially develops normally, then rapidly loses pancreatic tissue during larval life, reflecting pancreatic disease in CF. Altogether, we demonstrate that the cftr mutant zebrafish is a powerful new model for pancreatitis and pancreatic destruction in CF. This accessible model will allow more detailed investigation into the mechanisms that drive CF of the pancreas and facilitate development of new therapies to treat the disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The intestinal epithelium forms a barrier protecting the organism from microbes and other proinflammatory stimuli. The integrity of this barrier and the proper response to infection requires precise regulation of powerful immune homing signals such as tumor necrosis factor (TNF). Dysregulation of TNF leads to inflammatory bowel diseases (IBD), but the mechanism controlling the expression of this potent cytokine and the events that trigger the onset of chronic inflammation are unknown. Here, we show that loss of function of the epigenetic regulator ubiquitin-like protein containing PHD and RING finger domains 1 (uhrf1) in zebrafish leads to a reduction in tnfa promoter methylation and the induction of tnfa expression in intestinal epithelial cells (IECs). The increase in IEC tnfa levels is microbe-dependent and results in IEC shedding and apoptosis, immune cell recruitment, and barrier dysfunction, consistent with chronic inflammation. Importantly, tnfa knockdown in uhrf1 mutants restores IEC morphology, reduces cell shedding, and improves barrier function. We propose that loss of epigenetic repression and TNF induction in the intestinal epithelium can lead to IBD onset.

Epithelial organs develop through tightly coordinated events of cell proliferation and differentiation in which endocytosis plays a major role. Despite recent advances, how endocytosis regulates the development of vertebrate organs is still unknown. Here we describe a mechanism that facilitates the apical availability of endosomal SNARE receptors for epithelial morphogenesis through the developmental upregulation of plasmolipin (pllp) in a highly endocytic segment of the zebrafish posterior midgut. The protein PLLP (Pllp in fish) recruits the clathrin adaptor EpsinR to sort the SNARE machinery of the endolysosomal pathway into the subapical compartment, which is a switch for polarized endocytosis. Furthermore, PLLP expression induces apical Crumbs internalization and the activation of the Notch signalling pathway, both crucial steps in the acquisition of cell polarity and differentiation of epithelial cells. We thus postulate that differential apical endosomal SNARE sorting is a mechanism that regulates epithelial patterning.

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. Using light sheet microscopy and genetic approaches we have investigated single lumen formation in the zebrafish gut. Here we show that during gut development multiple lumens open and enlarge to generate a distinct intermediate, which consists of two adjacent unfused lumens separated by basolateral contacts. We observed that these lumens arise independently from each other along the length of the gut and do not share a continuous apical surface. Resolution of this intermediate into a single, continuous lumen requires the remodeling of contacts between adjacent lumens and subsequent lumen fusion. 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. Furthermore, we show that smo mutants exhibit perturbations in the Rab11 trafficking pathway and demonstrate that Rab11-mediated trafficking is necessary for single lumen formation. Thus, lumen resolution is a distinct genetically controlled process crucial for single, continuous lumen formation in the zebrafish gut.

Congenital vertebral malformations (CVM) occur in 1 in 1000 live births and in many cases can cause spinal deformities, such as scoliosis, and result in disability and distress of affected individuals. Many severe forms of the disease, such as spondylocostal dystostosis, are recessive monogenic traits affecting somitogenesis, however the etiologies of the majority of CVM cases remain undetermined. Here we demonstrate that morphological defects of the notochord in zebrafish can generate congenital-type spine defects. We characterize three recessive zebrafish leviathan/col8a1a mutant alleles ((m531, vu41, vu105)) that disrupt collagen type VIII alpha1a (col8a1a), and cause folding of the embryonic notochord and consequently adult vertebral column malformations. Furthermore, we provide evidence that a transient loss of col8a1a function or inhibition of Lysyl oxidases with drugs during embryogenesis was sufficient to generate vertebral fusions and scoliosis in the adult spine. Using periodic imaging of individual zebrafish, we correlate focal notochord defects of the embryo with vertebral malformations (VM) in the adult. Finally, we show that bends and kinks in the notochord can lead to aberrant apposition of osteoblasts normally confined to well-segmented areas of the developing vertebral bodies. Our results afford a novel mechanism for the formation of VM, independent of defects of somitogenesis, resulting from aberrant bone deposition at regions of misshapen notochord tissue., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Forward genetic approaches in zebrafish have provided invaluable information about developmental processes. However, the relative difficulty of mapping and isolating mutations has limited the number of new genetic screens. Recent improvements in the annotation of the zebrafish genome coupled to a reduction in sequencing costs prompted the development of whole genome and RNA sequencing approaches for gene discovery. Here we describe a whole exome sequencing (WES) approach that allows rapid and cost-effective identification of mutations. We used our WES methodology to isolate four mutations that cause kidney cysts; we identified novel alleles in two ciliary genes as well as two novel mutants. The WES approach described here does not require specialized infrastructure or training and is therefore widely accessible. This methodology should thus help facilitate genetic screens and expedite the identification of mutants that can inform basic biological processes and the causality of genetic disorders in humans.

Regulated fluid secretion is crucial for the function of most organs. In vertebrates, the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) is a master regulator of fluid secretion. Although the biophysical properties of CFTR have been well characterized in vitro, little is known about its in vivo role during development. Here, we investigated the function of Cftr during zebrafish development by generating several cftr mutant alleles using TAL effector nucleases. We found that loss of cftr function leads to organ laterality defects. In zebrafish, left-right (LR) asymmetry requires cilia-driven fluid flow within the lumen of Kupffer's vesicle (KV). Using live imaging we found that KV morphogenesis is disrupted in cftr mutants. Loss of Cftr-mediated fluid secretion impairs KV lumen expansion leading to defects in organ laterality. Using bacterial artificial chromosome recombineering, we generated transgenic fish expressing functional Cftr fusion proteins with fluorescent tags under the control of the cftr promoter. The transgenes completely rescued the cftr mutant phenotype. Live imaging of these transgenic lines showed that Cftr is localized to the apical membrane of the epithelial cells in KV during lumen formation. Pharmacological stimulation of Cftr-dependent fluid secretion led to an expansion of the KV lumen. Conversely, inhibition of ion gradient formation impaired KV lumen inflation. Interestingly, cilia formation and motility in KV were not affected, suggesting that fluid secretion and flow are independently controlled in KV. These findings uncover a new role for cftr in KV morphogenesis and function during zebrafish development.

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. Although 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 in spine morphogenesis. Thus, the vertebrate notochord plays important structural roles beyond early development.

The intrahepatic biliary ducts transport bile produced by the hepatocytes out of the liver. Defects in biliary cell differentiation and biliary duct remodeling cause a variety of congenital diseases including Alagille Syndrome and polycystic liver disease. While the molecular pathways regulating biliary cell differentiation have received increasing attention (Lemaigre, 2010), less is known about the cellular behavior underlying biliary duct remodeling. Here, we have identified a novel gene, claudin 15-like b (cldn15lb), which exhibits a unique and dynamic expression pattern in the hepatocytes and biliary epithelial cells in zebrafish. Claudins are tight junction proteins that have been implicated in maintaining epithelial polarity, regulating paracellular transport, and providing barrier function. In zebrafish cldn15lb mutant livers, tight junctions are observed between hepatocytes, but these cells show polarization defects as well as canalicular malformations. Furthermore, cldn15lb mutants show abnormalities in biliary duct morphogenesis whereby biliary epithelial cells remain clustered together and form a disorganized network. Our data suggest that Cldn15lb plays an important role in the remodeling process during biliary duct morphogenesis. Thus, cldn15lb mutants provide a novel in vivo model to study the role of tight junction proteins in the remodeling of the biliary network and hereditary cholestasis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background & Aims: The nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) transcription factor pathway is activated in response to diverse microbial stimuli to regulate expression of genes involved in immune responses and tissue homeostasis. However, the temporal and spatial activation of NF-κB in response to microbial signals have not been determined in whole living organisms, and the molecular and cellular details of these responses are not well understood. We used in vivo imaging and molecular approaches to analyze NF-κB activation in response to the commensal microbiota in transparent gnotobiotic zebrafish., Methods: We used DNA microarrays, in situ hybridization, and quantitative reverse transcription polymerase chain reaction analyses to study the effects of the commensal microbiota on gene expression in gnotobiotic zebrafish. Zebrafish PAC2 and ZFL cells were used to study the NF-κB signaling pathway in response to bacterial stimuli. We generated transgenic zebrafish that express enhanced green fluorescent protein under transcriptional control of NF-κB, and used them to study patterns of NF-κB activation during development and microbial colonization., Results: Bacterial stimulation induced canonical activation of the NF-κB pathway in zebrafish cells. Colonization of germ-free transgenic zebrafish with a commensal microbiota activated NF-κB and led to up-regulation of its target genes in intestinal and extraintestinal tissues of the digestive tract. Colonization with the bacterium Pseudomonas aeruginosa was sufficient to activate NF-κB, and this activation required a functional flagellar apparatus., Conclusions: In zebrafish, transcriptional activity of NF-κB is spatially and temporally regulated by specific microbial factors. The observed patterns of NF-κB-dependent responses to microbial colonization indicate that cells in the gastrointestinal tract respond robustly to the microbial environment., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Transport of chloride through the cystic fibrosis transmembrane conductance regulator (CFTR) channel is a key step in regulating fluid secretion in vertebrates [1, 2]. Loss of CFTR function leads to cystic fibrosis [1, 3, 4], a disease that affects the lungs, pancreas, liver, intestine, and vas deferens. Conversely, uncontrolled activation of the channel leads to increased fluid secretion and plays a major role in several diseases and conditions including cholera [5, 6] and other secretory diarrheas [7] as well as polycystic kidney disease [8-10]. Understanding how CFTR activity is regulated in vivo has been limited by the lack of a genetic model. Here, we used a forward genetic approach in zebrafish to uncover CFTR regulators. We report the identification, isolation, and characterization of a mutation in the zebrafish cse1l gene that leads to the sudden and dramatic expansion of the gut tube. We show that this phenotype results from a rapid accumulation of fluid due to the uncontrolled activation of the CFTR channel. Analyses in zebrafish larvae and mammalian cells indicate that Cse1l is a negative regulator of CFTR-dependent fluid secretion. This work demonstrates the importance of fluid homeostasis in development and establishes the zebrafish as a much-needed model system to study CFTR regulation in vivo., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Most organs consist of networks of interconnected tubes that serve as conduits to transport fluid and cells and act as physiological barriers between compartments. Biological tubes are assembled through very diverse developmental processes that generate structures of different shapes and sizes. Nevertheless, all biological tubes invariably possess one single lumen. The mechanisms responsible for single lumen specification are not known. Here we show that zebrafish mutants for the MODY5 and familial GCKD gene tcf2 (also known as vhnf1) fail to specify a single lumen in their gut tube and instead develop multiple lumens. We show that Tcf2 controls single lumen formation by regulating claudin15 and Na+/K+-ATPase expression. Our in vivo and in vitro results indicate that Claudin15 functions in paracellular ion transport to specify single lumen formation. This work shows that single lumen formation is genetically controlled and appears to be driven by the accumulation of fluid.