Telencephalic eversion in embryos and early larvae of four teleost species.

The telencephalon of ray-finned fishes undergoes eversion, which is very different to the evagination that occurs in most other vertebrates. Ventricle morphogenesis is key to build an everted telencephalon. Thus, here we use the apical marker zona occludens 1 to understand ventricle morphology, extension of the tela choroidea and the eversion process during early telencephalon development of four teleost species: giant danio (Devario aequipinnatus), blind cavefish (Astyanax mexicanus), medaka (Oryzias latipes), and paradise fish (Macroposus opercularis). In addition, by using immunohistochemistry against tubulin and calcium-binding proteins, we analyze the general morphology of the telencephalon, showing changes in the location and extension of the olfactory bulb and other telencephalic regions from 2 to 5 days of development. We also analyze the impact of abnormal eye and telencephalon morphogenesis on eversion, showing that cyclops mutants do undergo eversion despite very dramatic abnormal eye morphology. We discuss how the formation of the telencephalic ventricle in teleost fish, with its characteristic shape, is a crucial event during eversion.

Microtubules are not required to generate a nascent axon in embryonic spinal neurons in vivo.

Our understanding of the cell behaviours and cytoskeletal requirements of axon formation is largely derived from in vitro models but how these relate to axon formation in vivo is not clear. In vitro, neurons progress through a well-defined multineurite stage to form an axon and both actin and microtubules cooperate to drive the first steps in neurite and axon morphogenesis. However, these steps are not recapitulated in vivo, and it is not clear whether the underlying cell biological mechanisms may differ also. Here, we investigate the mechanisms that regulate axon formation in embryonic zebrafish spinal neurons in vivo. We find microtubule organising centres are located distant from the site of axon initiation, and microtubule plus-ends are not enriched in the axon during axon initiation. Focal F-actin accumulation precedes axon formation, and we find that nocodazole-treated neurons with no detectable microtubules are still able to form nascent axonal protrusions that are approximately 10-µm long, dilated and relatively long-lived. We suggest spinal axon formation in vivo is fundamentally different from axon formation in in vitro models.

Coordinated assembly and release of adhesions builds apical junctional belts during de novo polarisation of an epithelial tube.

Using the zebrafish neural tube as a model, we uncover the in vivo mechanisms allowing the generation of two opposing apical epithelial surfaces within the centre of an initially unpolarised, solid organ. We show that Mpp5a and Rab11a play a dual role in coordinating the generation of ipsilateral junctional belts whilst simultaneously releasing contralateral adhesions across the centre of the tissue. We show that Mpp5a- and Rab11a-mediated resolution of cell-cell adhesions are both necessary for midline lumen opening and contribute to later maintenance of epithelial organisation. We propose that these roles for both Mpp5a and Rab11a operate through the transmembrane protein Crumbs. In light of a recent conflicting publication, we also clarify that the junction-remodelling role of Mpp5a is not specific to dividing cells.

Mirror-symmetric microtubule assembly and cell interactions drive lumen formation in the zebrafish neural rod.

By analysing the cellular and subcellular events that occur in the centre of the developing zebrafish neural rod, we have uncovered a novel mechanism of cell polarisation during lumen formation. Cells from each side of the neural rod interdigitate across the tissue midline. This is necessary for localisation of apical junctional proteins to the region where cells intersect the tissue midline. Cells assemble a mirror-symmetric microtubule cytoskeleton around the tissue midline, which is necessary for the trafficking of proteins required for normal lumen formation, such as partitioning defective 3 and Rab11a to this point. This occurs in advance and is independent of the midline cell division that has been shown to have a powerful role in lumen organisation. To our knowledge, this is the first example of the initiation of apical polarisation part way along the length of a cell, rather than at a cell extremity. Although the midline division is not necessary for apical polarisation, it confers a morphogenetic advantage by efficiently eliminating cellular processes that would otherwise bridge the developing lumen.

Extracellular matrix couples the convergence movements of mesoderm and neural plate during the early stages of neurulation.

BACKGROUND: During the initial stages zebrafish neurulation, neural plate cells undergo highly coordinated movements before they assemble into a multicellular solid neural rod. We have previously identified that the underlying mesoderm is critical to ensure such coordination and generate correct neural tube organization. However, how intertissue coordination is achieved in vivo during zebrafish neural tube morphogenesis is unknown. RESULTS: In this work, we use quantitative live imaging to study the coordinated movements of neural ectoderm and mesoderm during dorsal tissue convergence. We show the extracellular matrix components laminin and fibronectin that lie between mesoderm and neural plate act to couple the movements of neural plate and mesoderm during early stages of neurulation and to maintain the close apposition of these two tissues. CONCLUSIONS: Our study highlights the importance of the extracellular matrix proteins laminin and libronectin in coupling the movements and spatial proximity of mesoderm and neuroectoderm during the morphogenetic movements of neurulation. Developmental Dynamics 245:580-589, 2016. © 2016 Wiley Periodicals, Inc.

A mirror-symmetric cell division that orchestrates neuroepithelial morphogenesis.

The development of cell polarity is an essential prerequisite for tissue morphogenesis during embryogenesis, particularly in the development of epithelia. In addition, oriented cell division can have a powerful influence on tissue morphogenesis. Here we identify a novel mode of polarized cell division that generates pairs of neural progenitors with mirror-symmetric polarity in the developing zebrafish neural tube and has dramatic consequences for the organization of embryonic tissue. We show that during neural rod formation the polarity protein Pard3 is localized to the cleavage furrow of dividing progenitors, and then mirror-symmetrically inherited by the two daughter cells. This allows the daughter cells to integrate into opposite sides of the developing neural tube. Furthermore, these mirror-symmetric divisions have powerful morphogenetic influence: when forced to occur in ectopic locations during neurulation, they orchestrate the development of mirror-image pattern formation and the consequent generation of ectopic neural tubes.

Focal electroporation in zebrafish embryos and larvae.

A method is described that allows the introduction by electroporation of either small dyes or larger RNA, DNA, or morpholino constructs into single cells or small groups of cells in zebrafish embryos or larvae. The dye or construct is delivered to cells via a patch-like microelectrode that also delivers the electroporation stimulus train. This technique allows the experimenter to target cells of their choice at a particular time of development and at a particular location in the embryo, and is useful for fate mapping, analysing neuronal organisation, ectopic expression and gene knockdown experiments.

Publisher Correction: Cdh2 coordinates Myosin-II dependent internalisation of the zebrafish neural plate.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Cdh2 coordinates Myosin-II dependent internalisation of the zebrafish neural plate.

Tissue internalisation is a key morphogenetic mechanism by which embryonic tissues generate complex internal organs and a number of studies of epithelia have outlined a general view of tissue internalisation. Here we have used quantitative live imaging and mutant analysis to determine whether similar mechanisms are responsible for internalisation in a tissue that apparently does not have a typical epithelial organisation - the zebrafish neural plate. We found that although zebrafish embryos begin neurulation without a conventional epithelium, medially located neural plate cells adopt strategies typical of epithelia in order to constrict their dorsal surface membrane during cell internalisation. Furthermore, we show that Myosin-II activity is a significant driver of this transient cell remodeling which also depends on Cdh2 (N-cadherin). Abrogation of Cdh2 results in defective Myosin-II distribution, mislocalised internalisation events and defective neural plate morphogenesis. Our work suggests Cdh2 coordinates Myosin-II dependent internalisation of the zebrafish neural plate.

Basal Protrusions Mediate Spatiotemporal Patterns of Spinal Neuron Differentiation.

During early spinal cord development, neurons of particular subtypes differentiate with a sparse periodic pattern while later neurons differentiate in the intervening space to eventually produce continuous columns of similar neurons. The mechanisms that regulate this spatiotemporal pattern are unknown. In vivo imaging in zebrafish reveals that differentiating spinal neurons transiently extend two long protrusions along the basal surface of the spinal cord before axon initiation. These protrusions express Delta protein, consistent with the hypothesis they influence Notch signaling at a distance of several cell diameters. Experimental reduction of Laminin expression leads to smaller protrusions and shorter distances between differentiating neurons. The experimental data and a theoretical model support the proposal that neuronal differentiation pattern is regulated by transient basal protrusions that deliver temporally controlled lateral inhibition mediated at a distance. This work uncovers a stereotyped protrusive activity of newborn neurons that organize long-distance spatiotemporal patterning of differentiation.

Local tissue interactions across the dorsal midline of the forebrain establish CNS laterality.

The mechanisms that establish behavioral, cognitive, and neuroanatomical asymmetries are poorly understood. In this study, we analyze the events that regulate development of asymmetric nuclei in the dorsal forebrain. The unilateral parapineal organ has a bilateral origin, and some parapineal precursors migrate across the midline to form this left-sided nucleus. The parapineal subsequently innervates the left habenula, which derives from ventral epithalamic cells adjacent to the parapineal precursors. Ablation of cells in the left ventral epithalamus can reverse laterality in wild-type embryos and impose the direction of CNS asymmetry in embryos in which laterality is usually randomized. Unilateral modulation of Nodal activity by Lefty1 can also impose the direction of CNS laterality in embryos with bilateral expression of Nodal pathway genes. From these data, we propose that laterality is determined by a competitive interaction between the left and right epithalamus and that Nodal signaling biases the outcome of this competition.

Modelling size constraints on carbonate platform formation in groundwater upwelling zones.

Carbonate depositional systems related to groundwater upwelling are ubiquitous around the world and form ecologically and culturally important features of many landscapes. Spring carbonate deposits record past climatic and hydrological conditions. The reconstruction of past processes using spring carbonate proxies requires fundamental understanding of the factors that control their geometry. In this work, we show that the spatial extent of spring carbonate platforms is amenable to quantitative prediction by simulating the early growth stage of their formation for the iconic mound springs in the central Australian outback. We exploit their well-defined, circular geometry to demonstrate the existence of two size-limiting regimes: one controlled by the spring flow rate and the other by the concentration of lattice ions. Deviations between modelled and observed size metrics are attributable to diminishing spring flow rates since formation, enabling assessment of the relative vulnerability of springs to further hydrological change.

Expression of pcp4a in subpopulations of CNS neurons in zebrafish.

The molecular organization of the zebrafish brain and its relation to neuroanatomical divisions are still largely unknown. In this study we have analyzed the expression of a small transcript encoding for the IQ containing polypeptide Pcp4a in developing and juvenile zebrafish. The transcript is exclusively expressed in neural structures with a pattern that is highly specific for restricted domains and cell populations throughout development, and it allows us to follow the development of these structures at different times. The expression of pcp4a characterizes the dorsocaudal telencephalon, dorsal habenula, pretectal nuclei, preglomerular complex, mammillary bodies, and deep layers of the optic tectum and is a hallmark of a subpopulation of reticulospinal neurons. In the telencephalon, comparison of the expression of pcp4a with other pallial markers showed a rostrocaudal gradient in the expression of these genes, which suggests that the dorsal telencephalon of zebrafish may be organized in distinct areas with different molecular natures. Pcp4 has been involved in modulating calcium signals and in binding to calmodulin, but its precise role in neuronal functions is not known. The analysis of pcp4a expression and localization in the zebrafish brain suggests that pcp4a may be a useful marker for sensory and some motor neuronal circuitries and for telencephalic areas processing sensory inputs.

Reversible Optogenetic Control of Subcellular Protein Localization in a Live Vertebrate Embryo.

We demonstrate the utility of the phytochrome system to rapidly and reversibly recruit proteins to specific subcellular regions within specific cells in a living vertebrate embryo. Light-induced heterodimerization using the phytochrome system has previously been used as a powerful tool to dissect signaling pathways for single cells in culture but has not previously been used to reversibly manipulate the precise subcellular location of proteins in multicellular organisms. Here we report the experimental conditions necessary to use this system to manipulate proteins in vivo. As proof of principle, we demonstrate that we can manipulate the localization of the apical polarity protein Pard3 with high temporal and spatial precision in both the neural tube and the embryo's enveloping layer epithelium. Our optimizations of optogenetic component expression and chromophore purification and delivery should significantly lower the barrier for establishing this powerful optogenetic system in other multicellular organisms.

Paradoxical sensation of nasal airflow in patients with common cold. Are we measuring the correct modality?

CONCLUSIONS: A paradoxical relationship between objective and subjective measures of nasal obstruction exists in participants not exposed to any treatment. The sensation of nasal obstruction may be due to the amalgamation of many different nasal sensations. Improved methods for measuring nasal sensations are required to further investigate the relationship between objective and subjective measures of nasal obstruction. OBJECTIVES: In a recent study it was shown that the subjective sensation of nasal patency increased as the nasal passages became objectively more obstructed in patients who received a placebo compared to those who received an oral decongestant. This paradoxical response may be explained as a placebo effect, i.e. patients who received a placebo may have expected to feel less obstructed. The aim of the present study was to investigate this interesting paradox by determining objective and subjective measures of nasal obstruction over time in participants not exposed to any treatment. MATERIAL AND METHODS: A total of 60 healthy participants with common cold were recruited. Objective and subjective measures of nasal obstruction were recorded at baseline and at 1 and 2 h using posterior rhinomanometry and a visual analogue scale. RESULTS: Objective measures demonstrated an increase in nasal obstruction over time for both nasal passages considered together and for individual nasal passages. Subjective measures demonstrated a sensation of decreased nasal obstruction over time for both nasal passages considered together and for individual nasal passages.

Post-transplant lymphoproliferative disorder presenting as epistaxis.

An unusual case of epistaxis resulting from post-transplant lymphoproliferative disorder is described. A 30-year-old woman who had undergone renal transplantation 12 years previously presented with profuse, posterior, unilateral epistaxis. The initial findings, workup and treatment are presented. A post-nasal space (PNS) mass was detected and biopsy showed this to be an Epstein-Barr virus-positive polymorphous B-cell post-transplant lymphoproliferative disorder. Computed tomography findings showed a polypoid lesion protruding from the sphenoethmoidal recess and filling the left PNS. Post-transplant lymphoproliferative disorder is well known to involve tonsil tissue. Commonly, this is the first presentation of the disease in children. However, until now post-transplant lymphoproliferative disorder has not been described in the PNS or nasal cavity presenting as epistaxis. We conclude that all transplant patients presenting with epistaxis should be followed up for an accurate examination of the PNS and nasal cavity after the acute episode.

Can early second-look tympanoplasty reduce the rate of conversion to modified radical mastoidectomy?

CONCLUSIONS: Combined approach tympanoplasty (CAT) allows for successful treatment of cholesteatoma with rates of recurrent and residual disease comparable to open mastoid surgery. Early timing of second-look procedures allows easier removal of any recurrent or residual disease, which reduces the conversion rate to open mastoidectomy. OBJECTIVES: The aims of the study were to report the rates of recurrent and residual cholesteatoma following primary CAT surgery and to report the rate of conversion to a modified radical mastoidectomy. METHODS: This was a retrospective review of a single surgeon series between 2006 and 2012. RESULTS: In total 132 second-look operations were undertaken, with a mean interval between primary surgery and second-look procedures of 6 months. The rate of cholesteatoma at second-look surgery was 19.7%, which was split into residual disease (10.6%) and recurrent disease (9.09%). New tympanic membrane defects with cholesteatoma were considered as recurrent disease. Residual disease was defined as cholesteatoma present behind an intact tympanic membrane. The majority of recurrent and residual disease was easily removed at second look (73.1%). Only four cases were converted to a modified radical mastoidectomy (3%) and three cases required a third-look procedure.

Developmental time rather than local environment regulates the schedule of epithelial polarization in the zebrafish neural rod.

BACKGROUND: Morphogenesis requires developmental processes to occur both at the right time and in the right place. During neural tube formation in the zebrafish embryo, the generation of the apical specializations of the lumen must occur in the center of the neural rod after the neural cells have undergone convergence, invagination and interdigitation across the midline. How this coordination is achieved is uncertain. One possibility is that environmental signaling at the midline of the neural rod controls the schedule of apical polarization. Alternatively, polarization could be regulated by a timing mechanism and then independent morphogenetic processes ensure the cells are in the correct spatial location. RESULTS: Ectopic transplantation demonstrates the local environment of the neural midline is not required for neural cell polarization. Neural cells can self-organize into epithelial cysts in ectopic locations in the embryo and also in three-dimensional gel cultures. Heterochronic transplants demonstrate that the schedule of polarization and the specialized cell divisions characteristic of the neural rod are more strongly regulated by time than local environmental signals. The cells' schedule for polarization is set prior to gastrulation, is stable through several rounds of cell division and appears independent of the morphogenetic movements of gastrulation and neurulation. CONCLUSIONS: Time rather than local environment regulates the schedule of epithelial polarization in zebrafish neural rod.

Morphogenesis underlying the development of the everted teleost telencephalon.

BACKGROUND: Although the mechanisms underlying brain patterning and regionalization are very much conserved, the morphology of different brain regions is extraordinarily variable across vertebrate phylogeny. This is especially manifest in the telencephalon, where the most dramatic variation is seen between ray-finned fish, which have an everted telencephalon, and all other vertebrates, which have an evaginated telencephalon. The mechanisms that generate these distinct morphologies are not well understood. RESULTS: Here we study the morphogenesis of the zebrafish telencephalon from 12 hours post fertilization (hpf) to 5 days post fertilization (dpf) by analyzing forebrain ventricle formation, evolving patterns of gene and transgene expression, neuronal organization, and fate mapping. Our results highlight two key events in telencephalon morphogenesis. First, the formation of a deep ventricular recess between telencephalon and diencephalon, the anterior intraencephalic sulcus (AIS), effectively creates a posterior ventricular wall to the telencephalic lobes. This process displaces the most posterior neuroepithelial territory of the telencephalon laterally. Second, as telencephalic growth and neurogenesis proceed between days 2 and 5 of development, the pallial region of the posterior ventricular wall of the telencephalon bulges into the dorsal aspect of the AIS. This brings the ventricular zone (VZ) into close apposition with the roof of the AIS to generate a narrow ventricular space and the thin tela choroidea (tc). As the pallial VZ expands, the tc also expands over the upper surface of the telencephalon. During this period, the major axis of growth and extension of the pallial VZ is along the anteroposterior axis. This second step effectively generates an everted telencephalon by 5 dpf. CONCLUSION: Our description of telencephalic morphogenesis challenges the conventional model that eversion is simply due to a laterally directed outfolding of the telencephalic neuroepithelium. This may have significant bearing on understanding the eventual organization of the adult fish telencephalon.