Organizing activities of axial mesoderm.

For almost a century, developmental biologists have appreciated that the ability of the embryonic organizer to induce and pattern the body plan is intertwined with its differentiation into axial mesoderm. Despite this, we still have a relatively poor understanding of the contribution of axial mesoderm to induction and patterning of different body regions, and the manner in which axial mesoderm-derived information is interpreted in tissues of changing competence. Here, with a particular focus on the nervous system, we review the evidence that axial mesoderm notochord and prechordal mesoderm/mesendoderm act as organizers, discuss how their influence extends through the different axes of the developing organism, and describe how the ability of axial mesoderm to direct morphogenesis impacts on its role as a local organizer.

A Methodology for the Enzymatic Isolation of Embryonic Hypothalamus Tissue and Its Acute or Post-Culture Analysis by Multiplex Hybridisation Chain Reaction.

The hypothalamus is an evolutionarily ancient part of the vertebrate ventral forebrain that integrates the dialogue between environment, peripheral body, and brain to centrally govern an array of physiologies and behaviours. Characterizing the mechanisms that control hypothalamic development illuminates both hypothalamic organization and function. Critical to the ability to unravel such mechanisms is the skill to isolate hypothalamic tissue, enabling both its acute analysis and its analysis after explant and culture. Tissue explants, in which cells develop in a manner analogous to their in vivo counterparts, are a highly effective tool to investigate the extrinsic signals and tissue-intrinsic self-organising features that drive hypothalamic development. The hypothalamus, however, is induced and patterned at neural tube stages of development, when the tissue is difficult to isolate, and its resident cells complex to define. No single molecular marker distinguishes early hypothalamic progenitor subsets from other cell types in the neural tube, and so their accurate dissection requires the simultaneous analysis of multiple proteins or mRNAs, techniques that were previously limited by antibody availability or were arduous to perform. Here, we overcome these challenges. We describe methodologies to precisely isolate early hypothalamic tissue from the embryonic chick at three distinct patterning stages and to culture hypothalamic explants in three-dimensional gels. We then describe optimised protocols for the analysis of embryos, isolated embryonic tissue, or cultured hypothalamic explants by multiplex hybridisation chain reaction. These methods can be applied to other vertebrates, including mouse, and to other tissue types. Key features • Detailed protocols for enzymatic isolation of embryonic chick hypothalamus at three patterning stages; methods can be extended to other vertebrates and tissues. • Brief methodologies for three-dimensional culture of hypothalamic tissue explants. • Optimised protocols for multiplex hybridisation chain reaction for analysis of embryos, isolated embryonic tissues, or explants.

Accurate staging of chick embryonic tissues via deep learning of salient features.

Recent work shows that the developmental potential of progenitor cells in the HH10 chick brain changes rapidly, accompanied by subtle changes in morphology. This demands increased temporal resolution for studies of the brain at this stage, necessitating precise and unbiased staging. Here, we investigated whether we could train a deep convolutional neural network to sub-stage HH10 chick brains using a small dataset of 151 expertly labelled images. By augmenting our images with biologically informed transformations and data-driven preprocessing steps, we successfully trained a classifier to sub-stage HH10 brains to 87.1% test accuracy. To determine whether our classifier could be generally applied, we re-trained it using images (269) of randomised control and experimental chick wings, and obtained similarly high test accuracy (86.1%). Saliency analyses revealed that biologically relevant features are used for classification. Our strategy enables training of image classifiers for various applications in developmental biology with limited microscopy data.

Fgf signalling triggers an intrinsic mesodermal timer that determines the duration of limb patterning.

Complex signalling between the apical ectodermal ridge (AER - a thickening of the distal epithelium) and the mesoderm controls limb patterning along the proximo-distal axis (humerus to digits). However, the essential in vivo requirement for AER-Fgf signalling makes it difficult to understand the exact roles that it fulfils. To overcome this barrier, we developed an amenable ex vivo chick wing tissue explant system that faithfully replicates in vivo parameters. Using inhibition experiments and RNA-sequencing, we identify a transient role for Fgfs in triggering the distal patterning phase. Fgfs are then dispensable for the maintenance of an intrinsic mesodermal transcriptome, which controls proliferation/differentiation timing and the duration of patterning. We also uncover additional roles for Fgf signalling in maintaining AER-related gene expression and in suppressing myogenesis. We describe a simple logic for limb patterning duration, which is potentially applicable to other systems, including the main body axis.

Glucocorticoid receptor regulates protein chaperone, circadian clock and affective disorder genes in the zebrafish brain.

Glucocorticoid resistance is commonly observed in depression, and has been linked to reduced expression and/or function of the glucocorticoid receptor (NR3C1 in human, hereafter referred to as GR). Previous studies have shown that GR-mutant zebrafish exhibit behavioural abnormalities that are indicative of an affective disorder, suggesting that GR plays a role in brain function. We compared the brain methylomes and brain transcriptomes of adult wild-type and GR-mutant zebrafish, and identified 249 differentially methylated regions (DMRs) that are regulated by GR. These include a cluster of CpG sites within the first intron of fkbp5, the gene encoding the glucocorticoid-inducible heat shock protein co-chaperone Fkbp5. RNA-sequencing analysis revealed that genes associated with chaperone-mediated protein folding, the regulation of circadian rhythm and the regulation of metabolism are particularly sensitive to loss of GR function. In addition, we identified subsets of genes exhibiting GR-regulated transcription that are known to regulate behaviour, and are linked to unipolar depression and anxiety. Taken together, our results identify key biological processes and novel molecular mechanisms through which the GR is likely to mediate responses to stress in the adult zebrafish brain, and they provide further support for the zebrafish GR mutant as a model for the study of affective disorders.

A neuroepithelial wave of BMP signalling drives anteroposterior specification of the tuberal hypothalamus.

The tuberal hypothalamus controls life-supporting homeostatic processes, but despite its fundamental role, the cells and signalling pathways that specify this unique region of the central nervous system in embryogenesis are poorly characterised. Here, we combine experimental and bioinformatic approaches in the embryonic chick to show that the tuberal hypothalamus is progressively generated from hypothalamic floor plate-like cells. Fate-mapping studies show that a stream of tuberal progenitors develops in the anterior-ventral neural tube as a wave of neuroepithelial-derived BMP signalling sweeps from anterior to posterior through the hypothalamic floor plate. As later-specified posterior tuberal progenitors are generated, early specified anterior tuberal progenitors become progressively more distant from these BMP signals and differentiate into tuberal neurogenic cells. Gain- and loss-of-function experiments in vivo and ex vivo show that BMP signalling initiates tuberal progenitor specification, but must be eliminated for these to progress to anterior neurogenic progenitors. scRNA-Seq profiling shows that tuberal progenitors that are specified after the major period of anterior tuberal specification begin to upregulate genes that characterise radial glial cells. This study provides an integrated account of the development of the tuberal hypothalamus.

Loss of Function of the Neural Cell Adhesion Molecule NrCAM Regulates Differentiation, Proliferation and Neurogenesis in Early Postnatal Hypothalamic Tanycytes.

Hypothalamic tanycytes are neural stem and progenitor cells, but little is known of how they are regulated. Here we provide evidence that the cell adhesion molecule, NrCAM, regulates tanycytes in the adult niche. NrCAM is strongly expressed in adult mouse tanycytes. Immunohistochemical and in situ hybridization analysis revealed that NrCAM loss of function leads to both a reduced number of tanycytes and reduced expression of tanycyte-specific cell markers, along with a small reduction in tyrosine hydroxylase-positive arcuate neurons. Similar analyses of NrCAM mutants at E16 identify few changes in gene expression or cell composition, indicating that NrCAM regulates tanycytes, rather than early embryonic hypothalamic development. Neurosphere and organotypic assays support the idea that NrCAM governs cellular homeostasis. Single-cell RNA sequencing (scRNA-Seq) shows that tanycyte-specific genes, including a number that are implicated in thyroid hormone metabolism, show reduced expression in the mutant mouse. However, the mild tanycyte depletion and loss of markers observed in NrCAM-deficient mice were associated with only a subtle metabolic phenotype.

Single-cell analysis of early chick hypothalamic development reveals that hypothalamic cells are induced from prethalamic-like progenitors.

The hypothalamus regulates many innate behaviors, but its development remains poorly understood. Here, we used single-cell RNA sequencing (RNA-seq) and hybridization chain reaction (HCR) to profile multiple stages of early hypothalamic development in the chick. Hypothalamic neuroepithelial cells are initially induced from prethalamic-like cells. Two distinct hypothalamic progenitor populations then emerge and give rise to tuberal and mammillary/paraventricular hypothalamic cells. At later stages, the regional organization of the chick and mouse hypothalamus is highly similar. We identify selective markers for major subdivisions of the developing chick hypothalamus and many previously uncharacterized candidate regulators of hypothalamic induction, regionalization, and neurogenesis. As proof of concept for the power of the dataset, we demonstrate that prethalamus-derived follistatin inhibits hypothalamic induction. This study clarifies the organization of the nascent hypothalamus and identifies molecular mechanisms that may control its induction and subsequent development.

Zebrafish as a model to investigate the CRH axis and interactions with DISC1.

Release of corticotropin-releasing hormone (CRH) from CRH neurons activates the hypothalamo-pituitary-adrenal (HPA) axis, one of the main physiological stress response systems. Complex feedback loops operate in the HPA axis and understanding the neurobiological mechanisms regulating CRH neurons is of great importance in the context of stress disorders. In this article, we review how in vivo studies in zebrafish have advanced knowledge of the neurobiology of CRH neurons. Disrupted-in-schizophrenia 1 (DISC1) mutant zebrafish have blunted stress responses and can be used to model human stress disorders. We propose that DISC1 influences the development and functioning of CRH neurons as a mechanism linking DISC1 to psychiatric disorders.

PINK1 deficiency impairs adult neurogenesis of dopaminergic neurons.

Recent evidence suggests neurogenesis is on-going throughout life but the relevance of these findings for neurodegenerative disorders such as Parkinson's disease (PD) is poorly understood. Biallelic PINK1 mutations cause early onset, Mendelian inherited PD. We studied the effect of PINK1 deficiency on adult neurogenesis of dopaminergic (DA) neurons in two complementary model systems. Zebrafish are a widely-used model to study neurogenesis in development and through adulthood. Using EdU analyses and lineage-tracing studies, we first demonstrate that a subset of ascending DA neurons and adjacent local-projecting DA neurons are each generated into adulthood in wild type zebrafish at a rate that decreases with age. Pink1-deficiency impedes DA neurogenesis in these populations, most significantly in early adult life. Pink1 already exerts an early effect on Th1+ progenitor cells rather than on differentiated DA neurons only. In addition, we investigate the effect of PINK1 deficiency in a human isogenic organoid model. Global neuronal differentiation in PINK1-deficient organoids and isogenic controls is similar, but PINK1-deficient organoids display impeded DA neurogenesis. The observation of impaired adult dopaminergic neurogenesis in Pink1 deficiency in two complementing model systems may have significant consequences for future therapeutic approaches in human PD patients with biallelic PINK1 mutations.

Defining the signalling determinants of a posterior ventral spinal cord identity in human neuromesodermal progenitor derivatives.

The anteroposterior axial identity of motor neurons (MNs) determines their functionality and vulnerability to neurodegeneration. Thus, it is a crucial parameter in the design of strategies aiming to produce MNs from human pluripotent stem cells (hPSCs) for regenerative medicine/disease modelling applications. However, the in vitro generation of posterior MNs corresponding to the thoracic/lumbosacral spinal cord has been challenging. Although the induction of cells resembling neuromesodermal progenitors (NMPs), the bona fide precursors of the spinal cord, offers a promising solution, the progressive specification of posterior MNs from these cells is not well defined. Here, we determine the signals guiding the transition of human NMP-like cells toward thoracic ventral spinal cord neurectoderm. We show that combined WNT-FGF activities drive a posterior dorsal pre-/early neural state, whereas suppression of TGFß-BMP signalling pathways promotes a ventral identity and neural commitment. Based on these results, we define an optimised protocol for the generation of thoracic MNs that can efficiently integrate within the neural tube of chick embryos. We expect that our findings will facilitate the comparison of hPSC-derived spinal cord cells of distinct axial identities.

Conserved roles of Rax/rx3 genes in hypothalamus and pituitary development.

Rax (Rx) genes encode paired-type homeodomain-containing transcription factors present in virtually all metazoan groups. In vertebrates, studies in fish, amphibian, chick and mouse models have revealed that these genes play important roles in the development of structures located at the anterior portion of the central nervous system, in particular the eyes, the hypothalamus and the pituitary gland. In addition, human patients with eye and brain defects carry mutations in the two human Rax paralogues, RAX and RAX2. Here, we review work done in the last years on Rax genes, focusing especially on the function that mouse Rax and its zebrafish homologue, rx3, play in hypothalamic and pituitary development. Work on both of these model organisms indicate that Rax genes are necessary for the patterning, growth and differentiation of the hypothalamus, in particular the ventro-tuberal and dorso-anterior hypothalamus, where they effect their action by controlling expression of the secreted signalling protein, Sonic hedgehog (Shh). In addition, Rax/rx3 mutations disturb the development of the pituitary gland, mimicking phenotypes observed in human subjects carrying mutations in the RAX gene. Thus, along with their crucial role in eye morphogenesis, Rax genes play a conserved role in the development of the hypothalamus and adjacent structures in the vertebrate clade.

A multiorganism pipeline for antiseizure drug discovery: Identification of chlorothymol as a novel γ-aminobutyric acidergic anticonvulsant.

OBJECTIVE: Current medicines are ineffective in approximately one-third of people with epilepsy. Therefore, new antiseizure drugs are urgently needed to address this problem of pharmacoresistance. However, traditional rodent seizure and epilepsy models are poorly suited to high-throughput compound screening. Furthermore, testing in a single species increases the chance that therapeutic compounds act on molecular targets that may not be conserved in humans. To address these issues, we developed a pipeline approach using four different organisms. METHODS: We sequentially employed compound library screening in the zebrafish, Danio rerio, chemical genetics in the worm, Caenorhabditis elegans, electrophysiological analysis in mouse and human brain slices, and preclinical validation in mouse seizure models to identify novel antiseizure drugs and their molecular mechanism of action. RESULTS: Initially, a library of 1690 compounds was screened in an acute pentylenetetrazol seizure model using D rerio. From this screen, the compound chlorothymol was identified as an effective anticonvulsant not only in fish, but also in worms. A subsequent genetic screen in C elegans revealed the molecular target of chlorothymol to be LGC-37, a worm γ-aminobutyric acid type A (GABAA ) receptor subunit. This GABAergic effect was confirmed using in vitro brain slice preparations from both mice and humans, as chlorothymol was shown to enhance tonic and phasic inhibition and this action was reversed by the GABAA receptor antagonist, bicuculline. Finally, chlorothymol exhibited in vivo anticonvulsant efficacy in several mouse seizure assays, including the 6-Hz 44-mA model of pharmacoresistant seizures. SIGNIFICANCE: These findings establish a multiorganism approach that can identify compounds with evolutionarily conserved molecular targets and translational potential, and so may be useful in drug discovery for epilepsy and possibly other conditions.

Of mitogens and morphogens: modelling Sonic Hedgehog mechanisms in vertebrate development.

Sonic Hedgehog (Shh) Is a critical protein in vertebrate development, orchestrating patterning and growth in many developing systems. First described as a classic morphogen that patterns tissues through a spatial concentration gradient, subsequent studies have revealed a more complex mechanism, in which Shh can also regulate proliferation and differentiation. While the mechanism of action of Shh as a morphogen is well understood, it remains less clear how Shh might integrate patterning, proliferation and differentiation in a given tissue, to ultimately direct its morphogenesis. In tandem with experimental studies, mathematical modelling can help gain mechanistic insights into these processes and bridge the gap between Shh-regulated patterning and growth, by integrating these processes into a common theoretical framework. Here, we briefly review the roles of Shh in vertebrate development, focusing on its functions as a morphogen, mitogen and regulator of differentiation. We then discuss the contributions that modelling has made to our understanding of the action of Shh and highlight current challenges in using mathematical models in a quantitative and predictive way. This article is part of a discussion meeting issue 'Contemporary morphogenesis'.

Crumbs2 mediates ventricular layer remodelling to form the spinal cord central canal.

In the spinal cord, the central canal forms through a poorly understood process termed dorsal collapse that involves attrition and remodelling of pseudostratified ventricular layer (VL) cells. Here, we use mouse and chick models to show that dorsal ventricular layer (dVL) cells adjacent to dorsal midline Nestin(+) radial glia (dmNes+RG) down-regulate apical polarity proteins, including Crumbs2 (CRB2) and delaminate in a stepwise manner; live imaging shows that as one cell delaminates, the next cell ratchets up, the dmNes+RG endfoot ratchets down, and the process repeats. We show that dmNes+RG secrete a factor that promotes loss of cell polarity and delamination. This activity is mimicked by a secreted variant of Crumbs2 (CRB2S) which is specifically expressed by dmNes+RG. In cultured MDCK cells, CRB2S associates with apical membranes and decreases cell cohesion. Analysis of Crb2F/F/Nestin-Cre+/- mice, and targeted reduction of Crb2/CRB2S in slice cultures reveal essential roles for transmembrane CRB2 (CRB2TM) and CRB2S on VL cells and dmNes+RG, respectively. We propose a model in which a CRB2S-CRB2TM interaction promotes the progressive attrition of the dVL without loss of overall VL integrity. This novel mechanism may operate more widely to promote orderly progenitor delamination.

Development of the basal hypothalamus through anisotropic growth.

The adult hypothalamus is subdivided into distinct domains: pre-optic, anterior, tuberal and mammillary. Each domain harbours an array of neurones that act together to regulate homeostasis. The embryonic origins and the development of hypothalamic neurones, however, remain enigmatic. Here, we summarise recent studies in model organisms that challenge current views of hypothalamic development, which traditionally have attempted to map adult domains to correspondingly located embryonic domains. Instead, new studies indicate that hypothalamic neurones arise from progenitor cells that undergo anisotropic growth, expanding to a greater extent than other progenitors, and grow in different dimensions. We describe in particular how a multipotent Shh/ Fgf10-expressing progenitor population gives rise to progenitors throughout the basal hypothalamus that grow anisotropically and sequentially: first, a subset displaced rostrally give rise to anterior-ventral/tuberal neuronal progenitors; then a subset displaced caudally give rise to mammillary neuronal progenitors; and, finally, a subset(s) displaced ventrally give rise to tuberal infundibular glial progenitors. As this occurs, stable populations of Shh+ive and Fgf10+ive progenitors form. We describe current understanding of the mechanisms that induce Shh+ive /Fgf10+ive progenitors and begin to direct their differentiation to anterior-ventral/tuberal neuronal progenitors, mammillary neuronal progenitors and tuberal infundibular progenitors. Taken together, these studies suggest a new model for hypothalamic development that we term the "anisotropic growth model". We discuss the implications of the model for understanding the origins of adult hypothalamic neurones.

Human axial progenitors generate trunk neural crest cells in vitro.

The neural crest (NC) is a multipotent embryonic cell population that generates distinct cell types in an axial position-dependent manner. The production of NC cells from human pluripotent stem cells (hPSCs) is a valuable approach to study human NC biology. However, the origin of human trunk NC remains undefined and current in vitro differentiation strategies induce only a modest yield of trunk NC cells. Here we show that hPSC-derived axial progenitors, the posteriorly-located drivers of embryonic axis elongation, give rise to trunk NC cells and their derivatives. Moreover, we define the molecular signatures associated with the emergence of human NC cells of distinct axial identities in vitro. Collectively, our findings indicate that there are two routes toward a human post-cranial NC state: the birth of cardiac and vagal NC is facilitated by retinoic acid-induced posteriorisation of an anterior precursor whereas trunk NC arises within a pool of posterior axial progenitors.

Sonic hedgehog in vertebrate neural tube development.

The formation and wiring of the vertebrate nervous system involves the spatially and temporally ordered production of diverse neuronal and glial subtypes that are molecularly and functionally distinct. The chick embryo has been the experimental model of choice for many of the studies that have led to our current understanding of this process, and has presaged and informed a wide range of complementary genetic studies, in particular in the mouse. The versatility and tractability of chick embryos means that it remains an important model system for many investigators in the field. Here we will focus on the role of Sonic hedgehog (Shh) signaling in coordinating the diversification, patterning, growth and differentiation of the vertebrate nervous system. We highlight how studies in chick led to the identification of the role Shh plays in the developing neural tube and how subsequent work, including studies in the chick and the mouse revealed details of the cell intrinsic programs controlling cell fate determination. We compare these mechanisms at different rostral-caudal positions along the neuraxis and discuss the particular experimental attributes of the chick that facilitated this work.

Fgf10+ progenitors give rise to the chick hypothalamus by rostral and caudal growth and differentiation.

Classical descriptions of the hypothalamus divide it into three rostro-caudal domains but little is known about their embryonic origins. To investigate this, we performed targeted fate-mapping, molecular characterisation and cell cycle analyses in the embryonic chick. Presumptive hypothalamic cells derive from the rostral diencephalic ventral midline, lie above the prechordal mesendoderm and express Fgf10Fgf10+ progenitors undergo anisotropic growth: those displaced rostrally differentiate into anterior cells, then those displaced caudally differentiate into mammillary cells. A stable population of Fgf10+ progenitors is retained within the tuberal domain; a subset of these gives rise to the tuberal infundibulum - the precursor of the posterior pituitary. Pharmacological approaches reveal that Shh signalling promotes the growth and differentiation of anterior progenitors, and also orchestrates the development of the infundibulum and Rathke's pouch - the precursor of the anterior pituitary. Together, our studies identify a hypothalamic progenitor population defined by Fgf10 and highlight a role for Shh signalling in the integrated development of the hypothalamus and pituitary.

Disrupted-in-Schizophrenia-1 is essential for normal hypothalamic-pituitary-interrenal (HPI) axis function.

Psychiatric disorders arise due to an interplay of genetic and environmental factors, including stress. Studies in rodents have shown that mutants for Disrupted-In-Schizophrenia-1 (DISC1), a well-accepted genetic risk factor for mental illness, display abnormal behaviours in response to stress, but the mechanisms through which DISC1 affects stress responses remain poorly understood. Using two lines of zebrafish homozygous mutant for disc1, we investigated behaviour and functioning of the hypothalamic-pituitary-interrenal (HPI) axis, the fish equivalent of the hypothalamic-pituitary-adrenal (HPA) axis. Here, we show that the role of DISC1 in stress responses is evolutionarily conserved and that DISC1 is essential for normal functioning of the HPI axis. Adult zebrafish homozygous mutant for disc1 show aberrant behavioural responses to stress. Our studies reveal that in the embryo, disc1 is expressed in neural progenitor cells of the hypothalamus, a conserved region of the vertebrate brain that centrally controls responses to environmental stressors. In disc1 mutant embryos, proliferating rx3+ hypothalamic progenitors are not maintained normally and neuronal differentiation is compromised: rx3-derived ff1b+ neurons, implicated in anxiety-related behaviours, and corticotrophin releasing hormone (crh) neurons, key regulators of the stress axis, develop abnormally, and rx3-derived pomc+ neurons are disorganised. Abnormal hypothalamic development is associated with dysfunctional behavioural and neuroendocrine stress responses. In contrast to wild type siblings, disc1 mutant larvae show altered crh levels, fail to upregulate cortisol levels when under stress and do not modulate shoal cohesion, indicative of abnormal social behaviour. These data indicate that disc1 is essential for normal development of the hypothalamus and for the correct functioning of the HPA/HPI axis.