Inhibition of the 3-hydroxy-3-methyl-glutaryl-CoA reductase diminishes the survival and size of chondrocytes during orofacial morphogenesis in zebrafish.

OBJECTIVES: The 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) is the enzyme controlling the rate-limiting step in the synthesis of cholesterol, sterols, and isoprenoids in the mevalonate biosynthetic pathway. Impaired function of HMGCR in zebrafish produces craniofacial malformations and orofacial cleft, mainly affecting the post-migratory neural crest cells with little earlier effect. Here we investigate morphogenetic and cellular mechanisms underlying the generation of these malformations. METHODS: The morphology of chondrocytes in the lower jaw and the proliferation/apoptosis in the ethmoid plate were analysed in hmgcr1b mutants and in embryos treated with atorvastatin. In the ceratohyal of treated embryos, we measured the number and dimensions of chondrocytes. In the ethmoid plate, we performed proliferation and apoptosis assays to quantify the number of cells undergoing each process in both hmgcr1b mutants and pharmacologically treated embryos. All embryos were imaged using confocal microscopy and processed to obtain maximum intensity z-projection. RESULTS: The shortening of the ceratohyal is produced by a moderate reduction in the number of cells combined with isometric shrinkage of the chondrocytes. At the same time, the shortening of the ethmoid plate is due to a combination of a slightly diminished proliferation with massive abnormal apoptosis at the proliferation front. CONCLUSION: HMGCR function is necessary for the normal survival and morphology of chondrocytes during condensation and chondrogenesis in the developing palate and jaw. Further studies are required to establish the pathways through which HMGCR acts on apoptosis, proliferation, and cell size during normal craniofacial development.

Ontogenesis of the asymmetric parapineal organ in the zebrafish epithalamus.

The parapineal organ is a midline-derived epithalamic structure that in zebrafish adopts a left-sided position at embryonic stages to promote the development of left-right asymmetries in the habenular nuclei. Despite extensive knowledge about its embryonic and larval development, it is still unknown whether the parapineal organ and its profuse larval connectivity with the left habenula are present in the adult brain or whether, as assumed from historical conceptions, this organ degenerates during ontogeny. This paper addresses this question by performing an ontogenetic analysis using an integrative morphological, ultrastructural and neurochemical approach. We find that the parapineal organ is lost as a morphological entity during ontogeny, while parapineal cells are incorporated into the posterior wall of the adult left dorsal habenular nucleus as small clusters or as single cells. Despite this integration, parapineal cells retain their structural, neurochemical and connective features, establishing a reciprocal synaptic connection with the more dorsal habenular neuropil. Furthermore, we describe the ultrastructure of parapineal cells using transmission electron microscopy and report immunoreactivity in parapineal cells with antibodies against substance P, tachykinin, serotonin and the photoreceptor markers arrestin3a and rod opsin. Our findings suggest that parapineal cells form an integral part of a neural circuit associated with the left habenula, possibly acting as local modulators of the circuit. We argue that the incorporation of parapineal cells into the habenula may be part of an evolutionarily relevant developmental mechanism underlying the presence/absence of the parapineal organ in teleosts, and perhaps in a broader sense in vertebrates.

Developmental Biology in Chile: historical perspectives and future challenges.

Developmental Biology is a growing discipline in Chile. It started in the 1950s when Luis Izquierdo challenged the traditional descriptive perspective of embryology and comparative anatomy to explore the mechanisms underlying the origin of form. After this initial drive, Claudio Barros, beginning in the late 1960s and Juan Fernández, in the late 1970s, contributed with unique and complementary facets to the early growth of the discipline. In the 1980s, the community of developmental biologists created its first forms of association together with the reproduction biology community, and in 1993 the first international course of developmental biology was organised. During the 1990s and 2000s, a group of young investigators arrived in Chile after postdocs in Europe and the US to build the first research centres of developmental biology, fostering the discipline to an unprecedented level. In the 2010s, as these centres consolidated, a stream of young developmental biologists established new labs at several institutions, expanding the community size and broadening its scope. The recent organisation of developmental biology meetings fostered the sense of community and nurtured the need of formal organisation, setting the bases for the foundation of the Chilean Society for Developmental Biology. Today, the community of developmental biologists is a mix of young and experienced investigators working in a variety of geographical locations, institutions, topics and model organisms. These characteristics are a strength of an active community that is pushing the discipline to the next level, aiming to make it a relevant actor in national and international settings.

Heterochrony and Morphological Variation of Epithalamic Asymmetry.

Heterochrony is one proposed mechanism to explain how morphological variation and novelty arise during evolution. To experimentally approach heterochrony in a comprehensive manner, we must consider all three aspects of developmental time (sequence, timing, duration). This task is only possible in developmental models that allow the acquisition of high-quality temporal data in the context of normalized developmental time. Here we propose that epithalamic asymmetry of teleosts is one such model. Comparative studies among related teleost species have revealed heterochronic shifts in the timing of ontogenic events leading to the development of epithalamic asymmetry. Such temporal changes involve neural structures critical for tissue-tissue interactions underlying the generation of asymmetry and are concurrent with the appearance of morphological differences in the pattern of asymmetry between species. Based on these findings, we hypothesize that interspecies variation of epithalamic asymmetry results from changes in the timing of tissue-tissue interactions critical for the establishment of asymmetry during ontogeny. Importantly, this hypothesis can be tested by systematic comparative approaches among teleosts species based on normalized developmental time, combined with experimental manipulation of epithalamic asymmetry development.

Nodal signalling and asymmetry of the nervous system.

The role of Nodal signalling in nervous system asymmetry is still poorly understood. Here, we review and discuss how asymmetric Nodal signalling controls the ontogeny of nervous system asymmetry using a comparative developmental perspective. A detailed analysis of asymmetry in ascidians and fishes reveals a critical context-dependency of Nodal function and emphasizes that bilaterally paired and midline-unpaired structures/organs behave as different entities. We propose a conceptual framework to dissect the developmental function of Nodal as asymmetry inducer and laterality modulator in the nervous system, which can be used to study other types of body and visceral organ asymmetries. Using insights from developmental biology, we also present novel evolutionary hypotheses on how Nodal led the evolution of directional asymmetry in the brain, with a particular focus on the epithalamus. We intend this paper to provide a synthesis on how Nodal signalling controls left-right asymmetry of the nervous system.This article is part of the themed issue 'Provocative questions in left-right asymmetry'.

Inhibition of the 3-hydroxy-3-methyl-glutaryl-CoA reductase induces orofacial defects in zebrafish.

BACKGROUND: Orofacial clefts (OFCs) are common birth defects, which include a range of disorders with a complex etiology affecting formation of craniofacial structures. Some forms of syndromic OFCs are produced by defects in the cholesterol pathway. The principal enzyme of the cholesterol pathway is the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). Our aim is to study whether defects of HMGCR function would produce orofacial malformation similar to those found in disorders of cholesterol synthesis. METHODS: We used zebrafish hmgcrb mutants and HMGCR inhibition assay using atorvastatin during early and late stages of orofacial morphogenesis in zebrafish. To describe craniofacial phenotypes, we stained cartilage and bone and performed in situ hybridization using known craniofacial markers. Also, we visualized neural crest cell migration in a transgenic fish. RESULTS: Our results showed that mutants displayed loss of cartilage and diminished orofacial outgrowth, and in some cases palatal cleft. Late treatments with statin show a similar phenotype. Affected-siblings displayed a moderate phenotype, whereas early-treated embryos had a minor cleft. We found reduced expression of the downstream component of Sonic Hedgehog-signaling gli1 in ventral brain, oral ectoderm, and pharyngeal endoderm in mutants and in late atorvastatin-treated embryos. CONCLUSION: Our results suggest that HMGCR loss-of-function primarily affects postmigratory cranial neural crest cells through abnormal Sonic Hedgehog signaling, probably induced by reduction in metabolites of the cholesterol pathway. Malformation severity correlates with the grade of HMGCR inhibition, developmental stage of its disruption, and probably with availability of maternal lipids. Together, our results might help to understand the spectrum of orofacial phenotypes found in cholesterol synthesis disorders. Birth Defects Research (Part A) 106:814-830, 2016. © 2016 Wiley Periodicals, Inc.

Genetics: a common origin for neuronal asymmetries?

A new study reveals an unexpected genetic link between two distinct types of neuronal asymmetries in the nematode Caenorhabditis elegans. This finding suggests a common origin of genetically determined asymmetries and raises intriguing questions about their evolution.

Morfógenos durante el desarrollo embrionario de vertebrados / Morphogens during embryonic development of vertebrates

Durante el desarrollo embrionario, las células de muchos tejidos se diferencian de acuerdo con la información de posición que se establece por las gradientes de concentración de morfógenos. Estas son moléculas de señalización secretadas en una región restringida de un tejido y se difunden lejos de su fuente para formar una gradiente de concentración. La molécula de un mismo morfógeno actúa generalmente en distintas etapas de desarrollo de un organismo y puede provocar reacciones muy diferentes en las células en función de su historia de diferenciación. Los morfógenos más conocidos son miembros del factor de crecimiento beta (TGF-b), Hedgehog (Hh), familias Wnt y los microRNAs.

During embryonic development, cells in many tissues differ according to the positional information that is set by the concentration of morphogen gradients. These are signaling molecules that are secreted in a restricted region of a tissue and diffuse away from their source forming a concentration gradient. Morphogens generally act at different development stages in an organism and cause different reactions in cells depending on their history of differentiation. The best known example of morphogens are members of growth factor beta (TGF-beta), Hedgehog(Hh), and Wnt families or microRNAs.

Daam1a mediates asymmetric habenular morphogenesis by regulating dendritic and axonal outgrowth.

Although progress has been made in resolving the genetic pathways that specify neuronal asymmetries in the brain, little is known about genes that mediate the development of structural asymmetries between neurons on left and right. In this study, we identify daam1a as an asymmetric component of the signalling pathways leading to asymmetric morphogenesis of the habenulae in zebrafish. Daam1a is a member of the Formin family of actin-binding proteins and the extent of Daam1a expression in habenular neuron dendrites mirrors the asymmetric growth of habenular neuropil between left and right. Local loss and gain of Daam1a function affects neither cell number nor subtype organisation but leads to a decrease or increase of neuropil, respectively. Daam1a therefore plays a key role in the asymmetric growth of habenular neuropil downstream of the pathways that specify asymmetric cellular domains in the habenulae. In addition, Daam1a mediates the development of habenular efferent connectivity as local loss and gain of Daam1a function impairs or enhances, respectively, the growth of habenular neuron terminals in the interpeduncular nucleus. Abrogation of Daam1a disrupts the growth of both dendritic and axonal processes and results in disorganised filamentous actin and α-tubulin. Our results indicate that Daam1a plays a key role in asymmetric habenular morphogenesis mediating the growth of dendritic and axonal processes in dorsal habenular neurons.

Insights into the organization of dorsal spinal cord pathways from an evolutionarily conserved raldh2 intronic enhancer.

Comparative studies of the tetrapod raldh2 (aldh1a2) gene, which encodes a retinoic acid (RA) synthesis enzyme, have led to the identification of a dorsal spinal cord enhancer. Enhancer activity is directed dorsally to the roof plate and dorsal-most (dI1) interneurons through predicted Tcf- and Cdx-homeodomain binding sites and is repressed ventrally via predicted Tgif homeobox and ventral Lim-homeodomain binding sites. Raldh2 and Math1/Cath1 expression in mouse and chicken highlights a novel, transient, endogenous Raldh2 expression domain in dI1 interneurons, which give rise to ascending circuits and intraspinal commissural interneurons, suggesting roles for RA in the ontogeny of spinocerebellar and intraspinal proprioceptive circuits. Consistent with expression of raldh2 in the dorsal interneurons of tetrapods, we also found that raldh2 is expressed in dorsal interneurons throughout the agnathan spinal cord, suggesting ancestral roles for RA signaling in the ontogenesis of intraspinal proprioception.

Mechanisms of directional asymmetry in the zebrafish epithalamus.

The epithalamus of zebrafish presents the best-studied case of directional asymmetry in the vertebrate brain. Epithalamic asymmetries are coupled to visceral asymmetry and include left-sided migration of a single midline structure (the parapineal organ) and asymmetric differentiation of paired bilateral nuclei (habenulae). The mechanisms underlying the establishment of epithalamic asymmetry involve the interplay between anti-symmetry and laterality signals to guide asymmetric parapineal migration. This event triggers the amplification of habenular asymmetries and the subsequent organisation of lateralised circuits in the interpeduncular nucleus. This review will summarise our current understanding on these processes and propose a sequential modular organisation of the events controlling the development of asymmetry along the parapineal-habenular-interpeduncular axis.

Zebrafish and medaka: model organisms for a comparative developmental approach of brain asymmetry.

Comparison between related species is a successful approach to uncover conserved and divergent principles of development. Here, we studied the pattern of epithalamic asymmetry in zebrafish (Danio rerio) and medaka (Oryzias latipes), two related teleost species with 115-200 Myr of independent evolution. We found that these species share a strikingly conserved overall pattern of asymmetry in the parapineal-habenular-interpeduncular system. Nodal signalling exhibits comparable spatial and temporal asymmetric expressions in the presumptive epithalamus preceding the development of morphological asymmetries. Neuroanatomical asymmetries consist of left-sided asymmetric positioning and connectivity of the parapineal organ, enlargement of neuropil in the left habenula compared with the right habenula and segregation of left-right habenular efferents along the dorsoventral axis of the interpeduncular nucleus. Despite the overall conservation of asymmetry, we observed heterotopic changes in the topology of parapineal efferent connectivity, heterochronic shifts in the timing of developmental events underlying the establishment of asymmetry and divergent degrees of canalization of embryo laterality. We offer new tools for developmental time comparison among species and propose, for each of these transformations, novel hypotheses of ontogenic mechanisms that explain interspecies variations that can be tested experimentally. Together, these findings highlight the usefulness of zebrafish and medaka as comparative models to study the developmental mechanisms of epithalamic asymmetry in vertebrates.