Extracellular hyaluronate pressure shaped by cellular tethers drives tissue morphogenesis.

How tissues acquire complex shapes is a fundamental question in biology and regenerative medicine. Zebrafish semicircular canals form from invaginations in the otic epithelium (buds) that extend and fuse to form the hubs of each canal. We find that conventional actomyosin-driven behaviors are not required. Instead, local secretion of hyaluronan, made by the enzymes uridine 5'-diphosphate dehydrogenase (ugdh) and hyaluronan synthase 3 (has3), drives canal morphogenesis. Charged hyaluronate polymers osmotically swell with water and generate isotropic extracellular pressure to deform the overlying epithelium into buds. The mechanical anisotropy needed to shape buds into tubes is conferred by a polarized distribution of actomyosin and E-cadherin-rich membrane tethers, which we term cytocinches. Most work on tissue morphogenesis ascribes actomyosin contractility as the driving force, while the extracellular matrix shapes tissues through differential stiffness. Our work inverts this expectation. Hyaluronate pressure shaped by anisotropic tissue stiffness may be a widespread mechanism for powering morphological change in organogenesis and tissue engineering.

Adhesion-Based Self-Organization in Tissue Patterning.

Since the proposal of the differential adhesion hypothesis, scientists have been fascinated by how cell adhesion mediates cellular self-organization to form spatial patterns during development. The search for molecular tool kits with homophilic binding specificity resulted in a diverse repertoire of adhesion molecules. Recent understanding of the dominant role of cortical tension over adhesion binding redirects the focus of differential adhesion studies to the signaling function of adhesion proteins to regulate actomyosin contractility. The broader framework of differential interfacial tension encompasses both adhesion and nonadhesion molecules, sharing the common function of modulating interfacial tension during cell sorting to generate diverse tissue patterns. Robust adhesion-based patterning requires close coordination between morphogen signaling, cell fate decisions, and changes in adhesion. Current advances in bridging theoretical and experimental approaches present exciting opportunities to understand molecular, cellular, and tissue dynamics during adhesion-based tissue patterning across multiple time and length scales.

Interplay of cell shape and division orientation promotes robust morphogenesis of developing epithelia.

Epithelial cells acquire functionally important shapes (e.g., squamous, cuboidal, columnar) during development. Here, we combine theory, quantitative imaging, and perturbations to analyze how tissue geometry, cell divisions, and mechanics interact to shape the presumptive enveloping layer (pre-EVL) on the zebrafish embryonic surface. We find that, under geometrical constraints, pre-EVL flattening is regulated by surface cell number changes following differentially oriented cell divisions. The division pattern is, in turn, determined by the cell shape distribution, which forms under geometrical constraints by cell-cell mechanical coupling. An integrated mathematical model of this shape-division feedback loop recapitulates empirical observations. Surprisingly, the model predicts that cell shape is robust to changes of tissue surface area, cell volume, and cell number, which we confirm in vivo. Further simulations and perturbations suggest the parameter linking cell shape and division orientation contributes to epithelial diversity. Together, our work identifies an evolvable design logic that enables robust cell-level regulation of tissue-level development.

A bioelectrical phase transition patterns the first vertebrate heartbeats.

A regular heartbeat is essential to vertebrate life. In the mature heart, this function is driven by an anatomically localized pacemaker. By contrast, pacemaking capability is broadly distributed in the early embryonic heart1-3, raising the question of how tissue-scale activity is first established and then maintained during embryonic development. The initial transition of the heart from silent to beating has never been characterized at the timescale of individual electrical events, and the structure in space and time of the early heartbeats remains poorly understood. Using all-optical electrophysiology, we captured the very first heartbeat of a zebrafish and analysed the development of cardiac excitability and conduction around this singular event. The first few beats appeared suddenly, had irregular interbeat intervals, propagated coherently across the primordial heart and emanated from loci that varied between animals and over time. The bioelectrical dynamics were well described by a noisy saddle-node on invariant circle bifurcation with action potential upstroke driven by CaV1.2. Our work shows how gradual and largely asynchronous development of single-cell bioelectrical properties produces a stereotyped and robust tissue-scale transition from quiescence to coordinated beating.

Specified neural progenitors sort to form sharp domains after noisy Shh signaling.

Sharply delineated domains of cell types arise in developing tissues under instruction of inductive signal (morphogen) gradients, which specify distinct cell fates at different signal levels. The translation of a morphogen gradient into discrete spatial domains relies on precise signal responses at stable cell positions. However, cells in developing tissues undergoing morphogenesis and proliferation often experience complex movements, which may affect their morphogen exposure, specification, and positioning. How is a clear pattern achieved with cells moving around? Using in toto imaging of the zebrafish neural tube, we analyzed specification patterns and movement trajectories of neural progenitors. We found that specified progenitors of different fates are spatially mixed following heterogeneous Sonic Hedgehog signaling responses. Cell sorting then rearranges them into sharply bordered domains. Ectopically induced motor neuron progenitors also robustly sort to correct locations. Our results reveal that cell sorting acts to correct imprecision of spatial patterning by noisy inductive signals.

Tetrahedral serial multiview microscopy and image fusion for improved resolution and extent in stained zebrafish embryos.

BACKGROUND: Spatial mapping on the single-cell level over the whole organism can uncover roles of molecular players involved in vertebrate development. Custom microscopes have been developed that use multiple objectives to view a sample from multiple views at the same time. Such multiview imaging approaches can improve resolution and uniformity of image quality as well as allow whole embryos to be imaged (Swoger et al., Opt Express, 2007;15(13):8029). However, multiview imaging is highly restricted to specialized equipment requiring multiple objectives or sample rotation with automated hardware. RESULTS: Our approach uses a standard single-objective confocal microscope to perform serial multiview imaging. Multiple views are imaged sequentially by mounting the fixed sample in an agarose tetrahedron that is manually rotated in between imaging each face. Computational image fusion allows for a joint 3D image to be created from multiple tiled Z-stacks acquired from different angles. The resulting fused image has improved resolution and imaging extent. CONCLUSION: With this technique, multiview imaging can be performed on a variety of common single-objective microscopes to allow for whole-embryo, high-resolution imaging.

Hydrostatic pressure as a driver of cell and tissue morphogenesis.

Morphogenesis, the process by which tissues develop into functional shapes, requires coordinated mechanical forces. Most current literature ascribes contractile forces derived from actomyosin networks as the major driver of tissue morphogenesis. Recent works from diverse species have shown that pressure derived from fluids can generate deformations necessary for tissue morphogenesis. In this review, we discuss how hydrostatic pressure is generated at the cellular and tissue level and how the pressure can cause deformations. We highlight and review findings demonstrating the mechanical roles of pressures from fluid-filled lumens and viscous gel-like components of the extracellular matrix. We also emphasise the interactions and mechanochemical feedbacks between extracellular pressures and tissue behaviour in driving tissue remodelling. Lastly, we offer perspectives on the open questions in the field that will further our understanding to uncover new principles of tissue organisation during development.

Feedback between tissue packing and neurogenesis in the zebrafish neural tube.

Balancing the rate of differentiation and proliferation in developing tissues is essential to produce organs of robust size and composition. Although many molecular regulators have been established, how these connect to physical and geometrical aspects of tissue architecture is poorly understood. Here, using high-resolution timelapse imaging, we find that changes to cell geometry associated with dense tissue packing play a significant role in regulating differentiation rate in the zebrafish neural tube. Specifically, progenitors that are displaced away from the apical surface due to crowding, tend to differentiate in a Notch-dependent manner. Using simulations we show that interplay between progenitor density, cell shape and changes in differentiation rate could naturally result in negative-feedback control on progenitor cell number. Given these results, we suggest a model whereby differentiation rate is regulated by density dependent effects on cell geometry to: (1) correct variability in cell number; and (2) balance the rates of proliferation and differentiation over development to 'fill' the available space.

Size-reduced embryos reveal a gradient scaling-based mechanism for zebrafish somite formation.

Little is known about how the sizes of animal tissues are controlled. A prominent example is somite size, which varies widely both within an individual and across species. Despite intense study of the segmentation clock governing the timing of somite generation, how it relates to somite size is poorly understood. Here, we examine somite scaling and find that somite size at specification scales with the length of the presomitic mesoderm (PSM) despite considerable variation in PSM length across developmental stages and in surgically size-reduced embryos. Measurement of clock period, axis elongation speed and clock gene expression patterns demonstrate that existing models fail to explain scaling. We posit a 'clock and scaled gradient' model, in which somite boundaries are set by a dynamically scaling signaling gradient across the PSM. Our model not only explains existing data, but also makes a unique prediction that we confirm experimentally - the formation of periodic 'echoes' in somite size following perturbation of the size of one somite. Our findings demonstrate that gradient scaling plays a central role in both progression and size control of somitogenesis.

Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA.

BACKGROUND: Directed DNA methylation on N6-adenine (6mA), N4-cytosine (4mC), and C5-cytosine (5mC) can potentially increase DNA coding capacity and regulate a variety of biological functions. These modifications are relatively abundant in bacteria, occurring in about a percent of all bases of most bacteria. Until recently, 5mC and its oxidized derivatives were thought to be the only directed DNA methylation events in metazoa. New and more sensitive detection techniques (ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-ms/ms) and single molecule real-time sequencing (SMRTseq)) have suggested that 6mA and 4mC modifications could be present in a variety of metazoa. RESULTS: Here, we find that both of these techniques are prone to inaccuracies, which overestimate DNA methylation concentrations in metazoan genomic DNA. Artifacts can arise from methylated bacterial DNA contamination of enzyme preparations used to digest DNA and contaminating bacterial DNA in eukaryotic DNA preparations. Moreover, DNA sonication introduces a novel modified base from 5mC that has a retention time near 4mC that can be confused with 4mC. Our analyses also suggest that SMRTseq systematically overestimates 4mC in prokaryotic and eukaryotic DNA and 6mA in DNA samples in which it is rare. Using UHPLC-ms/ms designed to minimize and subtract artifacts, we find low to undetectable levels of 4mC and 6mA in genomes of representative worms, insects, amphibians, birds, rodents and primates under normal growth conditions. We also find that mammalian cells incorporate exogenous methylated nucleosides into their genome, suggesting that a portion of 6mA modifications could derive from incorporation of nucleosides from bacteria in food or microbiota. However, gDNA samples from gnotobiotic mouse tissues found rare (0.9-3.7 ppm) 6mA modifications above background. CONCLUSIONS: Altogether these data demonstrate that 6mA and 4mC are rarer in metazoa than previously reported, and highlight the importance of careful sample preparation and measurement, and need for more accurate sequencing techniques.

A versatile gene trap to visualize and interrogate the function of the vertebrate proteome.

We report a multifunctional gene-trapping approach, which generates full-length Citrine fusions with endogenous proteins and conditional mutants from a single integration event of the FlipTrap vector. We identified 170 FlipTrap zebrafish lines with diverse tissue-specific expression patterns and distinct subcellular localizations of fusion proteins generated by the integration of an internal citrine exon. Cre-mediated conditional mutagenesis is enabled by heterotypic lox sites that delete Citrine and "flip" in its place mCherry with a polyadenylation signal, resulting in a truncated fusion protein. Inducing recombination with Cerulean-Cre results in fusion proteins that often mislocalize, exhibit mutant phenotypes, and dramatically knock down wild-type transcript levels. FRT sites in the vector enable targeted genetic manipulation of the trapped loci in the presence of Flp recombinase. Thus, the FlipTrap captures the functional proteome, enabling the visualization of full-length fluorescent fusion proteins and interrogation of function by conditional mutagenesis and targeted genetic manipulation.

Mathematically guided approaches to distinguish models of periodic patterning.

How periodic patterns are generated is an open question. A number of mechanisms have been proposed--most famously, Turing's reaction-diffusion model. However, many theoretical and experimental studies focus on the Turing mechanism while ignoring other possible mechanisms. Here, we use a general model of periodic patterning to show that different types of mechanism (molecular, cellular, mechanical) can generate qualitatively similar final patterns. Observation of final patterns is therefore not sufficient to favour one mechanism over others. However, we propose that a mathematical approach can help to guide the design of experiments that can distinguish between different mechanisms, and illustrate the potential value of this approach with specific biological examples.

Otolith tethering in the zebrafish otic vesicle requires Otogelin and α-Tectorin.

Otoliths are biomineralised structures important for balance and hearing in fish. Their counterparts in the mammalian inner ear, otoconia, have a primarily vestibular function. Otoliths and otoconia form over sensory maculae and are attached to the otolithic membrane, a gelatinous extracellular matrix that provides a physical coupling between the otolith and the underlying sensory epithelium. In this study, we have identified two proteins required for otolith tethering in the zebrafish ear, and propose that there are at least two stages to this process: seeding and maintenance. The initial seeding step, in which otolith precursor particles tether directly to the tips of hair cell kinocilia, fails to occur in the einstein (eis) mutant. The gene disrupted in eis is otogelin (otog); mutations in the human OTOG gene have recently been identified as causative for deafness and vestibular dysfunction (DFNB18B). At later larval stages, maintenance of otolith tethering to the saccular macula is dependent on tectorin alpha (tecta) function, which is disrupted in the rolling stones (rst) mutant. α-Tectorin (Tecta) is a major constituent of the tectorial membrane in the mammalian cochlea. Mutations in the human TECTA gene can cause either dominant (DFNA8/12) or recessive (DFNB21) forms of deafness. Our findings indicate that the composition of extracellular otic membranes is highly conserved between mammals and fish, reinforcing the view that the zebrafish is an excellent model system for the study of deafness and vestibular disease.

Recovery of shape and size in a developing organ pair.

BACKGROUND: Paired organs in animals are largely bilaterally symmetric despite inherent noise in most biological processes. How is precise organ shape and size achieved during development despite this noise? Examining paired organ development is a challenge because it requires repeated quantification of two structures in parallel within living embryos. Here we combine bilateral quantification of morphology through time with asymmetric perturbations to study regulation of organ shape, size, and symmetry in developing organ pairs. RESULTS: We present quantitative live imaging tools to measure the shape and size of the developing inner ears on both the left and right side simultaneously over time. By quantifying variation between the left and right inner ear (intrinsic noise) and between different individuals (extrinsic noise), we find that initial variability decreases over time in normal development to achieve symmetry. Early asymmetry is increased by environmental stress, but symmetry is still recovered over subsequent developmental time. Using multiple unilateral perturbations including Fgf signaling and ultraviolet light, we find that growth can be adjusted to compensate for a range of initial size and shape differences. CONCLUSIONS: We propose that symmetry in developmental systems does not emerge through precise deterministic bilateral development, but rather through feedback mechanisms that adjust morphogenesis rates to account for variation. Developmental Dynamics 246:451-465, 2016. © 2017 Wiley Periodicals, Inc.

Iterative use of nuclear receptor Nr5a2 regulates multiple stages of liver and pancreas development.

The stepwise progression of common endoderm progenitors into differentiated liver and pancreas organs is regulated by a dynamic array of signals that are not well understood. The nuclear receptor subfamily 5, group A, member 2 gene nr5a2, also known as Liver receptor homolog-1 (Lrh-1) is expressed in several tissues including the developing liver and pancreas. Here, we interrogate the role of Nr5a2 at multiple developmental stages using genetic and chemical approaches and uncover novel pleiotropic requirements during zebrafish liver and pancreas development. Zygotic loss of nr5a2 in a targeted genetic null mutant disrupted the development of the exocrine pancreas and liver, while leaving the endocrine pancreas intact. Loss of nr5a2 abrogated exocrine pancreas markers such as trypsin, while pancreas progenitors marked by ptf1a or pdx1 remained unaffected, suggesting a role for Nr5a2 in regulating pancreatic acinar cell differentiation. In the developing liver, Nr5a2 regulates hepatic progenitor outgrowth and differentiation, as nr5a2 mutants exhibited reduced hepatoblast markers hnf4α and prox1 as well as differentiated hepatocyte marker fabp10a. Through the first in vivo use of Nr5a2 chemical antagonist Cpd3, the iterative requirement for Nr5a2 for exocrine pancreas and liver differentiation was temporally elucidated: chemical inhibition of Nr5a2 function during hepatopancreas progenitor specification was sufficient to disrupt exocrine pancreas formation and enhance the size of the embryonic liver, suggesting that Nr5a2 regulates hepatic vs. pancreatic progenitor fate choice. Chemical inhibition of Nr5a2 at a later time during pancreas and liver differentiation was sufficient to block the formation of mature acinar cells and hepatocytes. These findings define critical iterative and pleiotropic roles for Nr5a2 at distinct stages of pancreas and liver organogenesis, and provide novel perspectives for interpreting the role of Nr5a2 in disease.

RNA-seq-based mapping and candidate identification of mutations from forward genetic screens.

Forward genetic screens have elucidated molecular pathways required for innumerable aspects of life; however, identifying the causal mutations from such screens has long been the bottleneck in the process, particularly in vertebrates. We have developed an RNA-seq-based approach that identifies both the region of the genome linked to a mutation and candidate lesions that may be causal for the phenotype of interest. We show that our method successfully identifies zebrafish mutations that cause nonsense or missense changes to codons, alter transcript splicing, or alter gene expression levels. Furthermore, we develop an easily accessible bioinformatics pipeline allowing for implementation of all steps of the method. Overall, we show that RNA-seq is a fast, reliable, and cost-effective method to map and identify mutations that will greatly facilitate the power of forward genetics in vertebrate models.

POC1A truncation mutation causes a ciliopathy in humans characterized by primordial dwarfism.

Primordial dwarfism (PD) is a phenotype characterized by profound growth retardation that is prenatal in onset. Significant strides have been made in the last few years toward improved understanding of the molecular underpinning of the limited growth that characterizes the embryonic and postnatal development of PD individuals. These include impaired mitotic mechanics, abnormal IGF2 expression, perturbed DNA-damage response, defective spliceosomal machinery, and abnormal replication licensing. In three families affected by a distinct form of PD, we identified a founder truncating mutation in POC1A. This gene is one of two vertebrate paralogs of POC1, which encodes one of the most abundant proteins in the Chlamydomonas centriole proteome. Cells derived from the index individual have abnormal mitotic mechanics with multipolar spindles, in addition to clearly impaired ciliogenesis. siRNA knockdown of POC1A in fibroblast cells recapitulates this ciliogenesis defect. Our findings highlight a human ciliopathy syndrome caused by deficiency of a major centriolar protein.

Rapid positional cloning of zebrafish mutations by linkage and homozygosity mapping using whole-genome sequencing.

Forward genetic screens in zebrafish have identified >9000 mutants, many of which are potential disease models. Most mutants remain molecularly uncharacterized because of the high cost, time and labor investment required for positional cloning. These costs limit the benefit of previous genetic screens and discourage future screens. Drastic improvements in DNA sequencing technology could dramatically improve the efficiency of positional cloning in zebrafish and other model organisms, but the best strategy for cloning by sequencing has yet to be established. Using four zebrafish inner ear mutants, we developed and compared two approaches for 'cloning by sequencing': one based on bulk segregant linkage (BSFseq) and one based on homozygosity mapping (HMFseq). Using BSFseq we discovered that mutations in lmx1b and jagged1b cause abnormal ear morphogenesis. With HMFseq we validated that the disruption of cdh23 abolishes the ear's sensory functions and identified a candidate lesion in lhfpl5a predicted to cause nonsyndromic deafness. The success of HMFseq shows that the high intrastrain polymorphism rate in zebrafish eliminates the need for time-consuming map crosses. Additionally, we analyzed diversity in zebrafish laboratory strains to find areas of elevated diversity and areas of fixed homozygosity, reinforcing recent findings that genome diversity is clustered. We present a database of >15 million sequence variants that provides much of this approach's power. In our four test cases, only a single candidate single nucleotide polymorphism (SNP) remained after subtracting all database SNPs from a mutant's critical region. The saturation of the common SNP database and our open source analysis pipeline MegaMapper will improve the pace at which the zebrafish community makes unique discoveries relevant to human health.

Attenuation of Notch and Hedgehog signaling is required for fate specification in the spinal cord.

During the development of the spinal cord, proliferative neural progenitors differentiate into postmitotic neurons with distinct fates. How cells switch from progenitor states to differentiated fates is poorly understood. To address this question, we studied the differentiation of progenitors in the zebrafish spinal cord, focusing on the differentiation of Kolmer-Agduhr″ (KA″) interneurons from lateral floor plate (LFP) progenitors. In vivo cell tracking demonstrates that KA″ cells are generated from LFP progenitors by both symmetric and asymmetric cell divisions. A photoconvertible reporter of signaling history (PHRESH) reveals distinct temporal profiles of Hh response: LFP progenitors continuously respond to Hh, while KA″ cells lose Hh response upon differentiation. Hh signaling is required in LFP progenitors for KA″ fate specification, but prolonged Hh signaling interferes with KA″ differentiation. Notch signaling acts permissively to maintain LFP progenitor cells: activation of Notch signaling prevents differentiation, whereas inhibition of Notch signaling results in differentiation of ectopic KA″ cells. These results indicate that neural progenitors depend on Notch signaling to maintain Hh responsiveness and rely on Hh signaling to induce fate identity, whereas proper differentiation depends on the attenuation of both Notch and Hh signaling.

IQGAP2 regulates blood-brain barrier immune dynamics.

Brain endothelial cells (BECs) play an important role in maintaining central nervous system (CNS) homeostasis through blood-brain barrier (BBB) functions. BECs express low baseline levels of adhesion receptors, which limits entry of leukocytes. However, the molecular mediators governing this phenotype remain mostly unclear. Here, we explored how infiltration of immune cells across the BBB is influenced by the scaffold protein IQ motif containing GTPase activating protein 2 (IQGAP2). In mice and zebrafish, we demonstrate that loss of Iqgap2 increases infiltration of peripheral leukocytes into the CNS under homeostatic and inflammatory conditions. Using single-cell RNA sequencing and immunohistology, we further show that BECs from mice lacking Iqgap2 exhibit a profound inflammatory signature, including extensive upregulation of adhesion receptors and antigen-processing machinery. Human tissue analyses also reveal that Alzheimer's disease is associated with reduced hippocampal IQGAP2. Overall, our results implicate IQGAP2 as an essential regulator of BBB immune privilege and immune cell entry into the CNS.