Embryonic stem cell factor FOXD3 (Genesis) defects in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms, believed to originate from the interstitial cells of Cajal (ICC), often caused by overexpression of tyrosine kinase receptors (TKR) KIT or PDGFRA. Here, we present evidence that the embryonic stem cell factor FOXD3, first identified as 'Genesis' and involved in both gastrointestinal and neural crest cell development, is implicated in GIST pathogenesis; its involvement is investigated both in vitro and in zebrafish and a mouse model of FOXD3 deficiency. Samples from a total of 58 patients with wild-type GISTs were used for molecular analyses, including Sanger sequencing, comparative genomic hybridization, and methylation analysis. Immunohistochemistry and western blot evaluation were used to assess FOXD3 expression. Additionally, we conducted in vitro functional studies in tissue samples and in transfected cells to confirm the pathogenicity of the identified genetic variants. Germline partially inactivating FOXD3 sequence variants (p.R54H and p.Ala88_Gly91del) were found in patients with isolated GISTs. Chromosome 1p loss was the most frequent chromosomal abnormality identified in tumors. In vitro experiments demonstrate the impairment of FOXD3 in the presence of those variants. Animal studies showed disruption of the GI neural network and changes in the number and distribution in the ICC. FOXD3 suppresses KIT expression in human cells; its inactivation led to an increase in ICC in zebrafish, as well as mice, providing evidence for a functional link between FOXD3 defects and KIT overexpression leading to GIST formation.

The Warburg effect is necessary to promote glycosylation in the blastema during zebrafish tail regeneration.

Throughout their lifetime, fish maintain a high capacity for regenerating complex tissues after injury. We utilized a larval tail regeneration assay in the zebrafish Danio rerio, which serves as an ideal model of appendage regeneration due to its easy manipulation, relatively simple mixture of cell types, and superior imaging properties. Regeneration of the embryonic zebrafish tail requires development of a blastema, a mass of dedifferentiated cells capable of replacing lost tissue, a crucial step in all known examples of appendage regeneration. Using this model, we show that tail amputation triggers an obligate metabolic shift to promote glucose metabolism during early regeneration similar to the Warburg effect observed in tumor forming cells. Inhibition of glucose metabolism did not affect the overall health of the embryo but completely blocked the tail from regenerating after amputation due to the failure to form a functional blastema. We performed a time series of single-cell RNA sequencing on regenerating tails with and without inhibition of glucose metabolism. We demonstrated that metabolic reprogramming is required for sustained TGF-ß signaling and blocking glucose metabolism largely mimicked inhibition of TGF-ß receptors, both resulting in an aberrant blastema. Finally, we showed using genetic ablation of three possible metabolic pathways for glucose, that metabolic reprogramming is required to provide glucose specifically to the hexosamine biosynthetic pathway while neither glycolysis nor the pentose phosphate pathway were necessary for regeneration.

Zebrafish Posterior Lateral Line primordium migration requires interactions between a superficial sheath of motile cells and the skin.

The Zebrafish Posterior Lateral Line primordium migrates in a channel between the skin and somites. Its migration depends on the coordinated movement of its mesenchymal-like leading cells and trailing cells, which form epithelial rosettes, or protoneuromasts. We describe a superficial population of flat primordium cells that wrap around deeper epithelialized cells and extend polarized lamellipodia to migrate apposed to the overlying skin. Polarization of lamellipodia extended by both superficial and deeper protoneuromast-forming cells depends on Fgf signaling. Removal of the overlying skin has similar effects on superficial and deep cells: lamellipodia are lost, blebs appear instead, and collective migration fails. When skinned embryos are embedded in Matrigel, basal and superficial lamellipodia are recovered; however, only the directionality of basal protrusions is recovered, and migration is not rescued. These observations support a key role played by superficial primordium cells and the skin in directed migration of the Posterior Lateral Line primordium.

Rapid image deconvolution and multiview fusion for optical microscopy.

The contrast and resolution of images obtained with optical microscopes can be improved by deconvolution and computational fusion of multiple views of the same sample, but these methods are computationally expensive for large datasets. Here we describe theoretical and practical advances in algorithm and software design that result in image processing times that are tenfold to several thousand fold faster than with previous methods. First, we show that an 'unmatched back projector' accelerates deconvolution relative to the classic Richardson-Lucy algorithm by at least tenfold. Second, three-dimensional image-based registration with a graphics processing unit enhances processing speed 10- to 100-fold over CPU processing. Third, deep learning can provide further acceleration, particularly for deconvolution with spatially varying point spread functions. We illustrate our methods from the subcellular to millimeter spatial scale on diverse samples, including single cells, embryos and cleared tissue. Finally, we show performance enhancement on recently developed microscopes that have improved spatial resolution, including dual-view cleared-tissue light-sheet microscopes and reflective lattice light-sheet microscopes.

NetLogo agent-based models as tools for understanding the self-organization of cell fate, morphogenesis and collective migration of the zebrafish posterior Lateral Line primordium.

Interactions between primordium cells and their environment determines the self-organization of the zebrafish posterior Lateral Line primordium as it migrates under the skin from the ear to the tip of the tail forming and depositing neuromasts to spearhead formation of the posterior Lateral Line sensory system. In this review we describe how the NetLogo agent-based programming environment has been used in our lab to visualize and explore how self-generated chemokine gradients determine collective migration, how the dynamics of Wnt signaling can be used to predict patterns of neuromast deposition, and how previously defined interactions between Wnt and Fgf signaling systems have the potential to determine the periodic formation of center-biased Fgf signaling centers in the wake of a shrinking Wnt system. We also describe how NetLogo was used as a database for storing and visualizing the results of in toto lineage analysis of all cells in the migrating primordium. Together, the models illustrate how this programming environment can be used in diverse ways to integrate what has been learnt from biological experiments about the nature of interactions between cells and their environment, and explore how these interactions could potentially determine emergent patterns of cell fate specification, morphogenesis and collective migration of the zebrafish posterior Lateral Line primordium.

Time-lapse imaging beyond the diffraction limit.

The zebrafish, with its rapid external development, optical transparency, and the relative ease with which transgenic lines can be created, is rapidly becoming the model of choice for examining developmental processes via time-lapse microscopy. The recent proliferation of techniques for super-resolution imaging now allows for an unprecedented view of embryonic development at high spatial and temporal resolution in live tissues. This review examines both the theoretical basis and practical application of a number of established and emerging super-resolution microscopy techniques, focusing on their application in time-lapse imaging of live zebrafish embryos.

Cxcl12a induces snail1b expression to initiate collective migration and sequential Fgf-dependent neuromast formation in the zebrafish posterior lateral line primordium.

The zebrafish posterior lateral line primordium migrates along a path defined by the chemokine Cxcl12a, periodically depositing neuromasts, to pioneer formation of the zebrafish posterior lateral line system. snail1b, known for its role in promoting cell migration, is expressed in leading cells of the primordium in response to Cxcl12a, whereas its expression in trailing cells is inhibited by Fgf signaling. snail1b knockdown delays initiation of primordium migration. This delay is associated with aberrant expansion of epithelial cell adhesion molecule (epcam) and reduction of cadherin 2 expression in the leading part of the primordium. Co-injection of snail1b morpholino with snail1b mRNA prevents the initial delay in migration and restores normal expression of epcam and cadherin 2 The delay in initiating primordium migration in snail1b morphants is accompanied by a delay in sequential formation of trailing Fgf signaling centers and associated protoneuromasts. This delay is not specifically associated with knockdown of snail1b but also with other manipulations that delay migration of the primordium. These observations reveal an unexpected link between the initiation of collective migration and sequential formation of protoneuromasts in the primordium.

Self-organizing spots get under your skin.

Sixty-five years after Turing first revealed the potential of systems with local activation and long-range inhibition to generate pattern, we have only recently begun to identify the biological elements that operate at many scales to generate periodic patterns in nature. In this Primer, we first review the theoretical framework provided by Turing, Meinhardt, and others that suggests how periodic patterns could self-organize in developing animals. This Primer was developed to provide context for recent studies that reveal how diverse molecular, cellular, and physical mechanisms contribute to the establishment of the periodic pattern of hair or feather buds in the developing skin. From an initial emphasis on trying to disambiguate which specific mechanism plays a primary role in hair or feather bud development, we are beginning to discover that multiple mechanisms may, in at least some contexts, operate together. While the emergence of the diverse mechanisms underlying pattern formation in specific biological contexts probably reflects the contingencies of evolutionary history, an intriguing possibility is that these mechanisms interact and reinforce each other, producing emergent systems that are more robust.

Adaptive optics improves multiphoton super-resolution imaging.

We improve multiphoton structured illumination microscopy using a nonlinear guide star to determine optical aberrations and a deformable mirror to correct them. We demonstrate our method on bead phantoms, cells in collagen gels, nematode larvae and embryos, Drosophila brain, and zebrafish embryos. Peak intensity is increased (up to 40-fold) and resolution recovered (up to 176 ± 10 nm laterally, 729 ± 39 nm axially) at depths ∼250 µm from the coverslip surface.

A framework for understanding morphogenesis and migration of the zebrafish posterior Lateral Line primordium.

A description of zebrafish posterior Lateral Line (pLL) primordium development at single cell resolution together with the dynamics of Wnt, FGF, Notch and chemokine signaling in this system has allowed us to develop a framework to understand the self-organization of cell fate, morphogenesis and migration during its early development. The pLL primordium migrates under the skin, from near the ear to the tip of the tail, periodically depositing neuromasts. Nascent neuromasts, or protoneuromasts, form sequentially within the migrating primordium, mature, and are deposited from its trailing end. Initially broad Wnt signaling inhibits protoneuromast formation. However, protoneuromasts form sequentially in response to FGF signaling, starting from the trailing end, in the wake of a progressively shrinking Wnt system. While proliferation adds to the number of cells, the migrating primordium progressively shrinks as its trailing cells stop moving and are deposited. As it shrinks, the length of the migrating primordium correlates with the length of the leading Wnt system. Based on these observations we show how measuring the rate at which the Wnt system shrinks, the proliferation rate, the initial size of the primordium, its speed, and a few additional parameters allows us to predict the pattern of neuromast formation and deposition by the migrating primordium in both wild-type and mutant contexts. While the mechanism that links the length of the leading Wnt system to that of the primordium remains unclear, we discuss how it might be determined by access to factors produced in the leading Wnt active zone that are required for collective migration of trailing cells. We conclude by reviewing how FGFs, produced in response to Wnt signaling in leading cells, help determine collective migration of trailing cells, while a polarized response to a self-generated chemokine gradient serves as an efficient mechanism to steer primordium migration along its relatively long journey.

Polarization and migration in the zebrafish posterior lateral line system.

Collective cell migration plays an important role in development. Here, we study the posterior lateral line primordium (PLLP) a group of about 100 cells, destined to form sensory structures, that migrates from head to tail in the zebrafish embryo. We model mutually inhibitory FGF-Wnt signalling network in the PLLP and link tissue subdivision (Wnt receptor and FGF receptor activity domains) to receptor-ligand parameters. We then use a 3D cell-based simulation with realistic cell-cell adhesion, interaction forces, and chemotaxis. Our model is able to reproduce experimentally observed motility with leading cells migrating up a gradient of CXCL12a, and trailing (FGF receptor active) cells moving actively by chemotaxis towards FGF ligand secreted by the leading cells. The 3D simulation framework, combined with experiments, allows an investigation of the role of cell division, chemotaxis, adhesion, and other parameters on the shape and speed of the PLLP. The 3D model demonstrates reasonable behaviour of control as well as mutant phenotypes.

In toto imaging of the migrating Zebrafish lateral line primordium at single cell resolution.

The zebrafish Posterior Lateral Line primordium (PLLp) has emerged as an important model system for studying many aspects of development, including cell migration, cell type specification and tissue morphogenesis. Despite this, basic aspects of PLLp biology remain incompletely understood. The PLLp is a group of approximately 140 cells which pioneers the formation of the Posterior Lateral Line (LL) system by migrating along the length of the embryo, periodically depositing clusters of epithelial cells, which will go on to form the mature sense organs of the lateral line, called neuromasts. The neuromasts are formed within the migrating PLLp as protoneuromasts: the first protoneuromast is formed close to the trailing end and additional protoneuromasts are formed sequentially, progressively closer to the leading edge of the migrating collective. We imaged the migration of PLL primordia and tracked every cell in the lateral line system over the course of migration. From this data set we unambiguously determined the lineage and fate of every cell deposited by the migrating PLLp. We show that, on average, proliferation across the entire PLLp is weakly patterned, with leading cells tending to divide more slowly than trailing cells. Neuromasts are formed sequentially by local expansion of existing cells along the length of the PLLp, not by self-renewing stem cell-like divisions of a restricted leading population that is highly proliferative. The fate of deposited cells, either within neuromasts or as interneuromast cells (in between deposited neuromasts) is not determined by any obvious stereotyped lineages. Instead, it is determined somewhat stochasitcailly, as a function of a cells distance from the center of a maturing protoneuromast. Together, our data provide a rigorous baseline for the behavior of the PLLp, which can be used to inform further study of this important model system.

Epb41l5 competes with Delta as a substrate for Mib1 to coordinate specification and differentiation of neurons.

We identified Erythrocyte membrane protein band 4.1-like 5 (Epb41l5) as a substrate for the E3 ubiquitin ligase Mind bomb 1 (Mib1), which is essential for activation of Notch signaling. Although loss of Epb41l5 does not significantly alter the pattern of neural progenitor cells (NPCs) specified as neurons at the neural plate stage, it delays their delamination and differentiation after neurulation when NPCs normally acquire organized apical junctional complexes (AJCs) in the zebrafish hindbrain. Delays in differentiation are reduced by knocking down N-cadherin, a manipulation expected to help destabilize adherens junctions (AJs). This suggested that delays in neuronal differentiation in epb41l5-deficient embryos are related to a previously described role for Epb41l5 in facilitating disassembly of cadherin-dependent AJCs. Mib1 ubiquitylates Epb41l5 to promote its degradation. DeltaD can compete with Epb41l5 to reduce Mib1-dependent Epb41l5 degradation. In this context, increasing the number of NPCs specified to become neurons, i.e. cells expressing high levels of DeltaD, stabilizes Epb41l5 in the embryo. Together, these observations suggest that relatively high levels of Delta stabilize Epb41l5 in NPCs specified as neurons. This, we suggest, helps coordinate NPC specification with Epb41l5-dependent delamination and differentiation as neurons.

The Notch meeting: an odyssey from structure to function.

The Notch signaling pathway plays fundamental roles in diverse developmental processes. Studies of the basic biology of Notch function have provided insights into how its dysfunction contributes to multi-systemic diseases and cancer. In addition, our understanding of Notch signaling in maintaining stem/progenitor cell populations is revealing new avenues for rekindling regeneration. The Notch IX meeting, which was held in Athens, Greece in October 2015, brought together scientists working on different model systems and studying Notch signaling in various contexts. Here, we provide a summary of the key points that were presented at the meeting. Although we focus on the molecular mechanisms that determine Notch signaling and its role in development, we also cover talks describing roles for Notch in adulthood. Together, the talks revealed how interactions between adjacent cells mediated by Notch regulate development and physiology at multiple levels.

Case-based discussion: Lymphocytic interstitial pneumonia a rare presentation in an immunocompetent adult male.

Lymphocytic interstitial pneumonia (LIP) is a rare form of interstitial lung disease usually associated with other systemic diseases; however, idiopathic cases are being reported. As per recent ATS/ERS 2013 guidelines, diagnostic criteria of clinical, radiological and histopathological for LIP is same as 2002 except some cystic changes on HRCT chest. Many cases diagnosed in the past as LIP now turn out to be NSIP; therefore as per new ATS/ERS classification whenever anybody report a case of LIP, NSIP should always be kept in mind as differential diagnosis. Here we present a case of LIP in an immunocompetent adult male presented with history of persistent dry cough and breathlessness on exertion, confirmed on HRCT chest and histopathologically, treated successfully with steroids.

Connecting physical cues and tissue patterning.

Several signaling pathways work together, via a protein called Amotl2a, to establish the size and shape of a zebrafish sense organ primordium.

Identification of the Mind Bomb1 Interaction Domain in Zebrafish DeltaD.

Ubiquitylation promotes endocytosis of the Notch ligands like Delta and Serrate and is essential for them to effectively activate Notch in a neighboring cell. The RING E3 ligase Mind bomb1 (Mib1) ubiquitylates DeltaD to facilitate Notch signaling in zebrafish. We have identified a domain in the intracellular part of the zebrafish Notch ligand DeltaD that is essential for effective interactions with Mib1. We show that elimination of the Mind bomb1 Interaction Domain (MID) or mutation of specific conserved motifs in this domain prevents effective Mib1-mediated ubiquitylation and internalization of DeltaD. Lateral inhibition mediated by Notch signaling regulates early neurogenesis in zebrafish. In this context, Notch activation suppresses neurogenesis, while loss of Notch-mediated lateral inhibition results in a neurogenic phenotype, where too many cells are allowed to become neurons. While Mib1-mediated endocytosis of DeltaD is essential for effective activation of Notch in a neighboring cell (in trans) it is not required for DeltaD to inhibit function of Notch receptors in the same cell (in cis). As a result, forms of DeltaD that have the MID can activate Notch in trans and suppress early neurogenesis when mRNA encoding it is ectopically expressed in zebrafish embryos. On the other hand, when the MID is eliminated/mutated in DeltaD, its ability to activate Notch in trans fails but ability to inhibit in cis is retained. As a result, ectopic expression of DeltaD lacking an effective MID results in a failure of Notch-mediated lateral inhibition and a neurogenic phenotype.

Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples.

Fluorescence imaging methods that achieve spatial resolution beyond the diffraction limit (super-resolution) are of great interest in biology. We describe a super-resolution method that combines two-photon excitation with structured illumination microscopy (SIM), enabling three-dimensional interrogation of live organisms with ~150 nm lateral and ~400 nm axial resolution, at frame rates of ~1 Hz. By performing optical rather than digital processing operations to improve resolution, our microscope permits super-resolution imaging with no additional cost in acquisition time or phototoxicity relative to the point-scanning two-photon microscope upon which it is based. Our method provides better depth penetration and inherent optical sectioning than all previously reported super-resolution SIM implementations, enabling super-resolution imaging at depths exceeding 100 µm from the coverslip surface. The capability of our system for interrogating thick live specimens at high resolution is demonstrated by imaging whole nematode embryos and larvae, and tissues and organs inside zebrafish embryos.

Leading and trailing cells cooperate in collective migration of the zebrafish posterior lateral line primordium.

Collective migration of cells in the zebrafish posterior lateral line primordium (PLLp) along a path defined by Cxcl12a expression depends on Cxcr4b receptors in leading cells and on Cxcr7b in trailing cells. Cxcr7b-mediated degradation of Cxcl12a by trailing cells generates a local gradient of Cxcl12a that guides PLLp migration. Agent-based computer models were built to explore how a polarized response to Cxcl12a, mediated by Cxcr4b in leading cells and prevented by Cxcr7b in trailing cells, determines unidirectional migration of the PLLp. These chemokine signaling-based models effectively recapitulate many behaviors of the PLLp and provide potential explanations for the characteristic behaviors that emerge when the PLLp is severed by laser to generate leading and trailing fragments. As predicted by our models, the bilateral stretching of the leading fragment is lost when chemokine signaling is blocked in the PLLp. However, movement of the trailing fragment toward the leading cells, which was also thought to be chemokine dependent, persists. This suggested that a chemokine-independent mechanism, not accounted for in our models, is responsible for this behavior. Further investigation of trailing cell behavior shows that their movement toward leading cells depends on FGF signaling and it can be re-oriented by exogenous FGF sources. Together, our observations reveal the simple yet elegant manner in which leading and trailing cells coordinate migration; while leading cells steer PLLp migration by following chemokine cues, cells further back play follow-the-leader as they migrate toward FGFs produced by leading cells.