VolumePeeler: a novel FIJI plugin for geometric tissue peeling to improve visualization and quantification of 3D image stacks.

MOTIVATION: Quantitative descriptions of multi-cellular structures from optical microscopy imaging are prime to understand the variety of three-dimensional (3D) shapes in living organisms. Experimental models of vertebrates, invertebrates and plants, such as zebrafish, killifish, Drosophila or Marchantia, mainly comprise multilayer tissues, and even if microscopes can reach the needed depth, their geometry hinders the selection and subsequent analysis of the optical volumes of interest. Computational tools to "peel" tissues by removing specific layers and reducing 3D volume into planar images, can critically improve visualization and analysis. RESULTS: We developed VolumePeeler, a versatile FIJI plugin for virtual 3D "peeling" of image stacks. The plugin implements spherical and spline surface projections. We applied VolumePeeler to perform peeling in 3D images of spherical embryos, as well as non-spherical tissue layers. The produced images improve the 3D volume visualization and enable analysis and quantification of geometrically challenging microscopy datasets. AVAILABILITY: ImageJ/FIJI software, source code, examples, and tutorials are openly available in https://cimt.uchile.cl/mcerda.

Cell migration driven by substrate deformation gradients.

Identifying the cues followed by cells is key to understand processes as embryonic development, tissue homeostasis, or several pathological conditions. Based on a durotaxis model, it is shown that cells moving on predeformed thin elastic membrane follow the direction of increasing strain of the substrate. This mechanism, straintaxis, does not distinguish the origin of the strain, but the active stresses produce large strains on cells or tissues being used as substrates. Hence, straintaxis is the natural realization of duratoaxis in vivo. Considering a circular geometry for the substrate cells, it is shown that if the annular component of the active stress component increases with the radial distance, cells migrate toward the substrate cell borders. With appropriate estimation for the different parameters, the migration speeds are similar to those obtained in recent experiments (Reig et al 2017 Nat. Commun. 8 15431). In these, during the annual killifish epiboly, deep cells that move in contact with the epithelial enveloping cell layer (EVL), migrate toward the EVL cell borders with speeds of microns per minute.

Cell migration: from tissue culture to embryos.

Cell migration is a fundamental process that occurs during embryo development. Classic studies using in vitro culture systems have been instrumental in dissecting the principles of cell motility and highlighting how cells make use of topographical features of the substrate, cell-cell contacts, and chemical and physical environmental signals to direct their locomotion. Here, we review the guidance principles of in vitro cell locomotion and examine how they control directed cell migration in vivo during development. We focus on developmental examples in which individual guidance mechanisms have been clearly dissected, and for which the interactions among guidance cues have been explored. We also discuss how the migratory behaviours elicited by guidance mechanisms generate the stereotypical patterns of migration that shape tissues in the developing embryo.

Origin, form and function of extraembryonic structures in teleost fishes.

Teleost eggs have evolved a highly derived early developmental pattern within vertebrates as a result of the meroblastic cleavage pattern, giving rise to a polar stratified architecture containing a large acellular yolk and a small cellular blastoderm on top. Besides the acellular yolk, the teleost-specific yolk syncytial layer (YSL) and the superficial epithelial enveloping layer are recognized as extraembryonic structures that play critical roles throughout embryonic development. They provide enriched microenvironments in which molecular feedback loops, cellular interactions and mechanical signals emerge to sculpt, among other things, embryonic patterning along the dorsoventral and left-right axes, mesendodermal specification and the execution of morphogenetic movements in the early embryo and during organogenesis. An emerging concept points to a critical role of extraembryonic structures in reinforcing early genetic and morphogenetic programmes in reciprocal coordination with the embryonic blastoderm, providing the necessary boundary conditions for development to proceed. In addition, the role of the enveloping cell layer in providing mechanical, osmotic and immunological protection during early stages of development, and the autonomous nutritional support provided by the yolk and YSL, have probably been key aspects that have enabled the massive radiation of teleosts to colonize every ecological niche on the Earth. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

Geometrical characterization of active contraction pulses in epithelial cells using the two-dimensional vertex model.

Several models have been proposed to describe the dynamics of epithelial tissues undergoing morphogenetic changes driven by apical constriction pulses, which differ in where the constriction is applied, either at the perimeter or in the medial regions. To help discriminate between these models, we analyse the impact of where constriction is applied on the final geometry of the active contracted cell, using the two-dimensional vertex model. We find that medial activity, characterized by a reduction in the reference area, generates anisotropic cell shapes, whereas isotropic cell shapes are produced when the reference perimeter is reduced. When plasticity is included, sufficiently slow processes of medial contractile activity, compared with the characteristic times of elasticity and plasticity, cells can achieve less elongated shapes. Similarly, for perimeter activity, the highest level of contraction is achieved. Finally, we apply the model to describe the apical contractile pulses observed within the epithelial enveloping cell layer during the pre-epiboly of the annual killifish Austrolebias nigripinnis. The analysis of the cell shape changes allowed a global fit of all parameters of the vertex model, with the pulses being quantitatively captured using perimeter activity and area plasticity.

Author Correction: The Reprimo gene family member, reprimo-like (rprml), is required for blood development in embryonic zebrafish.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Reprimo gene family member, reprimo-like (rprml), is required for blood development in embryonic zebrafish.

The Reprimo gene family comprises a group of single-exon genes for which their physiological function remains poorly understood. Heretofore, mammalian Reprimo (RPRM) has been described as a putative p53-dependent tumor suppressor gene that functions at the G2/M cell cycle checkpoint. Another family member, Reprimo-like (RPRML), has not yet an established role in physiology or pathology. Importantly, RPRML expression pattern is conserved between zebrafish and human species. Here, using CRISPR-Cas9 and antisense morpholino oligonucleotides, we disrupt the expression of rprml in zebrafish and demonstrate that its loss leads to impaired definitive hematopoiesis. The formation of hemangioblasts and the primitive wave of hematopoiesis occur normally in absence of rprml. Later in development there is a significant reduction in erythroid-myeloid precursors (EMP) at the posterior blood island (PBI) and a significant decline of definitive hematopoietic stem/progenitor cells (HSPCs). Furthermore, loss of rprml also increases the activity of caspase-3 in endothelial cells within the caudal hematopoietic tissue (CHT), the first perivascular niche where HSPCs reside during zebrafish embryonic development. Herein, we report an essential role for rprml during hematovascular development in zebrafish embryos, specifically during the definitive waves of hematopoiesis, indicating for the first time a physiological role for the rprml gene.

Heparin activates Wnt signaling for neuronal morphogenesis.

Wnt factors are secreted ligands that affect different aspects of the nervous system behavior like neurodevelopment, synaptogenesis and neurodegeneration. In different model systems, Wnt signaling has been demonstrated to be regulated by heparan sulfate proteoglycans (HSPGs). Whether HSPGs modulate Wnt signaling in the context of neuronal behavior is currently unknown. Here we demonstrate that activation of Wnt signaling with the endogenous ligand Wnt-7a results in an increased of neurite outgrowth in the neuroblastoma N2a cell line. Interestingly, heparin induces glycogen synthase kinase-3beta (GSK-3beta) inhibition, beta-catenin stabilization and morphological differentiation in both N2a cells and in rat primary hippocampal neuronal cultures. We also show that heparin modulates Wnt-3a-induced stabilization of beta-catenin. Several extracellular matrix and membrane-attached HSPGs were found to be expressed in both in vitro neuronal models. Changes in the expression of specific HSPGs were observed upon differentiation of N2a cells. Taken together, our findings suggest that HSPGs may modulate canonical Wnt signaling for neuronal morphogenesis.

Extra-embryonic tissue spreading directs early embryo morphogenesis in killifish.

The spreading of mesenchymal-like cell layers is critical for embryo morphogenesis and tissue repair, yet we know little of this process in vivo. Here we take advantage of unique developmental features of the non-conventional annual killifish embryo to study the principles underlying tissue spreading in a simple cellular environment, devoid of patterning signals and major morphogenetic cell movements. Using in vivo experimentation and physical modelling we reveal that the extra-embryonic epithelial enveloping cell layer, thought mainly to provide protection to the embryo, directs cell migration and the spreading of embryonic tissue during early development. This function relies on the ability of embryonic cells to couple their autonomous random motility to non-autonomous signals arising from the expansion of the extra-embryonic epithelium, mediated by cell membrane adhesion and tension. Thus, we present a mechanism of extra-embryonic control of embryo morphogenesis that couples the mechanical properties of adjacent tissues in the early killifish embryo.

Zebrafish BarH-like genes define discrete neural domains in the early embryo.

BarH (Barhl) genes encode for highly conserved homeodomain-containing transcription factors involved in critical functions during development, including cell fate specification, migration and survival. Here, we report the dynamic and restricted expression of three zebrafish barhl within the developing central nervous system. barhl2 becomes expressed in the late gastrula as a transverse diencephalic domain located immediately caudal to the prospective eyes. At early somitogenesis, barhl1.1 and barhl1.2 are expressed in the diencephalon in domains that partially overlap with the ventral and dorsal aspects of barhl2 expression, respectively. At later stages, expression of all zebrafish barhl shows large extent of overlap in the pretectum, tectum and dorsal hindbrain. The presence of a unique territory of barhl2 expression in the dorsal telencephalon and the high levels of expression in the retina are both consistent with expression reports of other Barhl2 orthologues, and support the subdivision of vertebrate Barhl into two paralogue groups based on the phylogenetic analysis of nucleotide and amino acid sequences.

Impronta Genómica y Desarrollo Embrionario / Genomic Imprinting and Embryonic Development

En los organismos diploides, cada gen autosómico está representado por dos copias, o alelos, heredados de cada progenitor al momento de la fecundación. Para la gran mayoría de los genes la expresión ocurre desde ambos alelos de manera simultánea. Sin embargo, un número reducido de genes (menos del 1%) es afectado por un proceso de impronta genómica. Este proceso determina que la expresión del gen sea dependiente del origen parental, es decir, se comporte de manera distinta si su origen es materno o paterno. La metilación del ADN es una de las modificaciones epigenéticas mejor estudiadas y su participación resulta esencial durante el establecimiento de la impronta genómica. Si bien los patrones de metilación a nivel genómico son estables y heredables, existen al menos dos períodos del desarrollo embrionario de mamíferos durante los cuales los patrones de metilación globales son borrados y re-establecidos. Estos dos períodos del desarrollo coinciden con el borrado y establecimiento de la impronta genómica específica de cada individuo. Desde el punto de vista funcional, la mayoría de los genes sometidos a impronta cumplen roles en el control del crecimiento y desarrollo embrionario y placentario. Alteraciones en el patrón de expresión de ellos han sido relacionados a patologías tales como el Síndrome de Algelman y el Síndrome de Prader-Willi, entre otros.

In diploid organisms, autosomal genes are composed of two copies, or alleles, inherited from both parents at fertilization. For the vast majority of autosomal genes, expression occurs from both alleles simultaneously. However, a small proportion (<1%) of genes are imprinted, meaning that their expression depends on the parental origin . DNA methylation is one of the most known epigenetic modifications and its function is critical for the establishment of imprinting. The global pattern of genomic methylation is stable and inheritable, however, it is erased and re-established in a sex-depended manner at two critical periods of embryonic development. Functionally, the majority of imprinted genes play roles in the control of embryonic and placental growth and development. Alterations in imprinted genes have been correlated with several pathologies including the Angelman and Prader-Willi syndromes.

Functions of BarH transcription factors during embryonic development.

This paper reviews the developmental role of a group of homeobox-containing genes firstly described in the early nineties as critical factors regulating eye development in Drosophila. These genes received the name of BarH due to the Drosophila "Bar" mutant phenotype and, since then, vertebrate homologues (named BarH-like or Barhl) have been described in a number of species of fish, amphibians and mammals. During embryonic development, BarH/Barhl are expressed primarily in the central nervous system where they play essential roles in decisions of cell fate, migration and survival. Transcriptional regulation mediated by these proteins involves either repression or activation mechanisms. In Drosophila, BarH is involved in morphogenesis and fate determination of the eye and external sensory organs, in regional prepatterning of the notum, and in formation and specification of distal leg segments. Vertebrate Barhl shares some functional properties with the fly counterparts, such as the ability to interact with basic helix-loop-helix (bHLH) proneural proteins, and plays crucial roles during cell type specification within the retina, acquisition of commissural neuron identity in the spinal cord, migration of cerebellar cells, and in cell survival within the neural plate, cochlea and cerebellum.