Exploiting spatiotemporal regulation of FZD5 during neural patterning for efficient ventral midbrain specification.

The Wnt/ß-catenin signaling governs anterior-posterior neural patterning during development. Current human pluripotent stem cell (hPSC) differentiation protocols use a GSK3 inhibitor to activate Wnt signaling to promote posterior neural fate specification. However, GSK3 is a pleiotropic kinase involved in multiple signaling pathways and, as GSK3 inhibition occurs downstream in the signaling cascade, it bypasses potential opportunities for achieving specificity or regulation at the receptor level. Additionally, the specific roles of individual FZD receptors in anterior-posterior patterning are poorly understood. Here, we have characterized the cell surface expression of FZD receptors in neural progenitor cells with different regional identity. Our data reveal unique upregulation of FZD5 expression in anterior neural progenitors, and this expression is downregulated as cells adopt a posterior fate. This spatial regulation of FZD expression constitutes a previously unreported regulatory mechanism that adjusts the levels of ß-catenin signaling along the anterior-posterior axis and possibly contributes to midbrain-hindbrain boundary formation. Stimulation of Wnt/ß-catenin signaling in hPSCs, using a tetravalent antibody that selectively triggers FZD5 and LRP6 clustering, leads to midbrain progenitor differentiation and gives rise to functional dopaminergic neurons in vitro and in vivo.

BMP controls dorsoventral and neural patterning in indirect-developing hemichordates providing insight into a possible origin of chordates.

A defining feature of chordates is the unique presence of a dorsal hollow neural tube that forms by internalization of the ectodermal neural plate specified via inhibition of BMP signaling during gastrulation. While BMP controls dorsoventral (DV) patterning across diverse bilaterians, the BMP-active side is ventral in chordates and dorsal in many other bilaterians. How this phylum-specific DV inversion occurs and whether it is coupled to the emergence of the dorsal neural plate are unknown. Here we explore these questions by investigating an indirect-developing enteropneust from the hemichordate phylum, which together with echinoderms form a sister group of the chordates. We found that in the hemichordate larva, BMP signaling is required for DV patterning and is sufficient to repress neurogenesis. We also found that transient overactivation of BMP signaling during gastrulation concomitantly blocked mouth formation and centralized the nervous system to the ventral ectoderm in both hemichordate and sea urchin larvae. Moreover, this mouthless, neurogenic ventral ectoderm displayed a medial-to-lateral organization similar to that of the chordate neural plate. Thus, indirect-developing deuterostomes use BMP signaling in DV and neural patterning, and an elevated BMP level during gastrulation drives pronounced morphological changes reminiscent of a DV inversion. These findings provide a mechanistic basis to support the hypothesis that an inverse chordate body plan emerged from an indirect-developing ancestor by tinkering with BMP signaling.

Transcriptional regulation of the Ciona Gsx gene in the neural plate.

The ascidian neural plate consists of a defined number of identifiable cells organized in a grid of rows and columns, representing a useful model to investigate the molecular mechanisms controlling neural patterning in chordates. Distinct anterior brain lineages are specified via unique combinatorial inputs of signalling pathways with Nodal and Delta-Notch signals patterning along the medial-lateral axis and FGF/MEK/ERK signals patterning along the anterior-posterior axis of the neural plate. The Ciona Gsx gene is specifically expressed in the a9.33 cells in the row III/column 2 position of anterior brain lineages, characterised by a combinatorial input of Nodal-OFF, Notch-ON and FGF-ON. Here, we identify the minimal cis-regulatory element (CRE) of 376 bp, which can recapitulate the early activation of Gsx. We show that this minimal CRE responds in the same way as the endogenous Gsx gene to manipulation of FGF- and Notch-signalling pathways and to overexpression of Snail, a mediator of Nodal signals, and Six3/6, which is required to demarcate the anterior boundary of Gsx expression at the late neurula stage. We reveal that sequences proximal to the transcription start site include a temporal regulatory element required for the precise transcriptional onset of gene expression. We conclude that sufficient spatial and temporal information for Gsx expression is integrated in 376 bp of non-coding cis-regulatory sequences.

Patterning of brain precursors in ascidian embryos.

In terms of their embryonic origins, the anterior and posterior parts of the ascidian central nervous system (CNS) are associated with distinct germ layers. The anterior part of the sensory vesicle, or brain, originates from ectoderm lineages following a neuro-epidermal binary fate decision. In contrast, a large part of the remaining posterior CNS is generated following neuro-mesodermal binary fate decisions. Here, we address the mechanisms that pattern the anterior brain precursors along the medial-lateral axis (future ventral-dorsal) at neural plate stages. Our functional studies show that Nodal signals are required for induction of lateral genes, including Delta-like, Snail, Msxb and Trp Delta-like/Notch signalling induces intermediate (Gsx) over medial (Meis) gene expression in intermediate cells, whereas the combinatorial action of Snail and Msxb prevents the expression of Gsx in lateral cells. We conclude that despite the distinct embryonic lineage origins within the larval CNS, the mechanisms that pattern neural precursors are remarkably similar.

Analysis of the wnt1 regulatory chromosomal landscape.

One of the earliest patterning events in the vertebrate neural plate is the specification of mes/r1, the territory comprising the prospective mesencephalon and the first hindbrain rhombomere. Within mes/r1, an interface of gene expression defines the midbrain-hindbrain boundary (MHB), a lineage restriction that separates the mesencephalon and rhombencephalon. wnt1 is critical to mes/r1 development and functions within the MHB as a component of the MHB gene regulatory network (GRN). Despite its importance to these critical and early steps of vertebrate neurogenesis, little is known about the factors responsible for wnt1 transcriptional regulation. In the zebrafish, wnt1 and its neighboring paralog, wnt10b, are expressed in largely overlapping patterns, suggesting co-regulation. To understand wnt1 and wnt10b transcriptional control, we used a comparative genomics approach to identify relevant enhancers. We show that the wnt1-wnt10b locus contains multiple cis-regulatory elements that likely interact to generate the wnt1 and wnt10b expression patterns. Two of 11 conserved enhancers tested show activity restricted to the midbrain and MHB, an activity that is conserved in the distantly related spotted gar orthologous elements. Three non-conserved elements also play a likely role in wnt1 regulation. The identified enhancers display dynamic modes of chromatin accessibility, suggesting controlled deployment during embryogenesis. Our results suggest that the control of wnt1 and wnt10b expression is under complex regulation involving the interaction of multiple enhancers.

Ventral neural patterning in the absence of a Shh activity gradient from the floorplate.

BACKGROUND: Vertebrate spinal cord development requires Sonic Hedgehog (Shh) signaling from the floorplate and notochord, where it is thought to act in concentration dependent manner to pattern distinct cell identities along the ventral-to-dorsal axis. While in vitro experiments demonstrate naïve neural tissues are sensitive to small changes in Shh levels, genetic studies illustrate that some degree of ventral patterning can occur despite significant perturbations in Shh signaling. Consequently, the mechanistic relationship between Shh morphogen levels and acquisition of distinct cell identities remains unclear. RESULTS: We addressed this using Hedgehog acetyltransferase (HhatCreface ) and Wiggable mouse mutants. Hhat encodes a palmitoylase required for the secretion of Hedgehog proteins and formation of the Shh gradient. In its absence, the spinal cord develops without floorplate cells and V3 interneurons. Wiggable is an allele of the Shh receptor Patched1 (Ptch1Wig ) that is unable to inhibit Shh signal transduction, resulting in expanded ventral progenitor domains. Surprisingly, HhatCreface/Creface ; Ptch1Wig/Wig double mutants displayed fully restored ventral patterning despite an absence of Shh secretion from the floorplate. CONCLUSIONS: The full range of neuronal progenitor types can be generated in the absence of a Shh gradient provided pathway repression is dampened, illustrating the complexity of morphogen dynamics in vertebrate patterning. Developmental Dynamics 247:170-184, 2018. © 2017 Wiley Periodicals, Inc.

Tbx2 regulates anterior neural specification by repressing FGF signaling pathway.

During early embryogenesis, FGF signals regulate the antero-posterior (AP) patterning of the neural plate by promoting posterior cell fates. In particular, BMP signal-mediated attenuation of FGF pathway plays a critical role in the determination of the anterior neural region. Here we show that Tbx2, a T-box transcriptional repressor regulates anterior neural specification by suppressing FGF8 signaling pathway in Xenopus embryo. Tbx2 is expressed in the anterior edge of the neural plate in early neurulae. Overexpression and knockdown of Tbx2 induce expansion and reduction in the expression of anterior neural markers, respectively. It also suppresses FGF8-induced ERK phosphorylation and neural caudalization. Tbx2, which is a target gene of BMP signal, down-regulates FGF8 signaling by inhibiting the expression of Flrt3, a positive regulator of this pathway. We found that Tbx2 binds directly to the T-box element located in the promoter region of Flrt3 gene, thereby interfering with the activity of the promoter. Consistently, Tbx2 augmentation of anterior neural formation is inhibited by co-expression of Flrt3. Furthermore, disruption of the anterior-most structures such as eyes in Tbx2-depleted embryos can be rescued by inhibition of Flrt3 function or FGF signaling. Taken together, our results suggest that Tbx2 mediates BMP signal to down-regulate FGF signaling pathway by repressing Flrt3 expression for anterior tissue formation.

Spop regulates Gli3 activity and Shh signaling in dorsoventral patterning of the mouse spinal cord.

Sonic Hedgehog (Shh) signaling regulates the patterning of ventral spinal cord through the effector Gli family of transcription factors. Previous in vitro studies showed that an E3 ubiquitin ligase containing Speckle-type POZ protein (Spop) targets Gli2 and Gli3 for ubiquitination and degradation, but the role of Spop in Shh signaling and mammalian spinal cord patterning remains unknown. Here, we show that loss of Spop does not alter spinal cord patterning, but it suppresses the loss of floor plate and V3 interneuron phenotype of Gli2 mutants, suggesting a negative role of Spop in Gli3 activator activity, Shh signaling and the specification of ventral cell fates in the spinal cord. This correlates with a moderate but significant increase in the level of Gli3 protein in the Spop mutant spinal cords. Furthermore, loss of Spop restores the maximal Shh pathway activation and ventral cell fate specification in the Gli1;Sufu double mutant spinal cord. Finally, we show that loss of Spop-like does not change the spinal cord patterning in either wild type or Spop mutants, suggesting that it does not compensate for the loss of Spop in Shh signaling and spinal cord patterning. Therefore, our results demonstrate a negative role of Spop in the level and activity of Gli3, Shh signaling and ventral spinal cord patterning.

sall1 and sall4 repress pou5f3 family expression to allow neural patterning, differentiation, and morphogenesis in Xenopus laevis.

The embryonic precursor of the vertebrate central nervous system, the neural plate, is patterned along the anterior-posterior axis and shaped by morphogenetic movements early in development. We previously identified the genes sall1 and sall4, known regulators of pluripotency in other contexts, as transcriptional targets of developmental signaling pathways that regulate neural development. Here, we demonstrate that these two genes are required for induction of posterior neural fates, the cell shape changes that contribute to neural tube closure, and later neurogenesis. Upon sall1 or sall4 knockdown, defects are associated with the failure of the neural plate to differentiate. Consistent with this, sall-deficient neural tissue exhibits an aberrant upregulation of pou5f3 family genes, the Xenopus homologs of the mammalian stem cell maintenance factor Pou5f1 (Oct4). Furthermore, overexpression of pou5f3 genes in Xenopus causes defects in neural patterning, morphogenesis, and differentiation that phenocopy those observed in sall1 and sall4 morphants. In all, this work shows that both sall1 and sall4 act to repress pou5f3 family gene expression in the neural plate, thereby allowing vertebrate neural development to proceed.

Spalt-like 4 promotes posterior neural fates via repression of pou5f3 family members in Xenopus.

Amphibian neural development occurs as a two-step process: (1) induction specifies a neural fate in undifferentiated ectoderm; and (2) transformation induces posterior spinal cord and hindbrain. Signaling through the Fgf, retinoic acid (RA) and Wnt/ß-catenin pathways is necessary and sufficient to induce posterior fates in the neural plate, yet a mechanistic understanding of the process is lacking. Here, we screened for factors enriched in posterior neural tissue and identify spalt-like 4 (sall4), which is induced by Fgf. Knockdown of Sall4 results in loss of spinal cord marker expression and increased expression of pou5f3.2 (oct25), pou5f3.3 (oct60) and pou5f3.1 (oct91) (collectively, pou5f3 genes), the closest Xenopus homologs of mammalian stem cell factor Pou5f1 (Oct4). Overexpression of the pou5f3 genes results in the loss of spinal cord identity and knockdown of pou5f3 function restores spinal cord marker expression in Sall4 morphants. Finally, knockdown of Sall4 blocks the posteriorizing effects of Fgf and RA signaling in the neurectoderm. These results suggest that Sall4, activated by posteriorizing signals, represses the pou5f3 genes to provide a permissive environment allowing for additional Wnt/Fgf/RA signals to posteriorize the neural plate.

PTK7 modulates Wnt signaling activity via LRP6.

Protein tyrosine kinase 7 (PTK7) is a transmembrane protein expressed in the developing Xenopus neural plate. PTK7 regulates vertebrate planar cell polarity (PCP), controlling mesodermal and neural convergent-extension (CE) cell movements, neural crest migration and neural tube closure in vertebrate embryos. Besides CE phenotypes, we now show that PTK7 protein knockdown also inhibits Wnt/ß-catenin activity. Canonical Wnt signaling caudalizes the neural plate via direct transcriptional activation of the meis3 TALE-class homeobox gene, which subsequently induces neural CE. PTK7 controls meis3 gene expression to specify posterior tissue and downstream PCP activity. Furthermore, PTK7 morphants phenocopy embryos depleted for Wnt3a, LRP6 and Meis3 proteins. PTK7 protein depletion inhibits embryonic Wnt/ß-catenin signaling by strongly reducing LRP6 protein levels. LRP6 protein positively modulates Wnt/ß-catenin, but negatively modulates Wnt/PCP activities. The maintenance of high LRP6 protein levels by PTK7 triggers PCP inhibition. PTK7 and LRP6 proteins physically interact, suggesting that PTK7 stabilization of LRP6 protein reciprocally regulates both canonical and noncanonical Wnt activities in the embryo. We suggest a novel role for PTK7 protein as a modulator of LRP6 that negatively regulates Wnt/PCP activity.

Snail mediates medial-lateral patterning of the ascidian neural plate.

The ascidian neural plate exhibits a regular, grid-like arrangement of cells. Patterning of the neural plate across the medial-lateral axis is initiated by bilateral sources of Nodal signalling, such that Nodal signalling induces expression of lateral neural plate genes and represses expression of medial neural plate genes. One of the earliest lateral neural plate genes induced by Nodal signals encodes the transcription factor Snail. Here, we show that Snail is a critical downstream factor mediating this Nodal-dependent patterning. Using gain and loss of function approaches, we show that Snail is required to repress medial neural plate gene expression at neural plate stages and to maintain the lateral neural tube genetic programme at later stages. A comparison of these results to those obtained following Nodal gain and loss of function indicates that Snail mediates a subset of Nodal functions. Consistently, overexpression of Snail can partially rescue a Nodal inhibition phenotype. We conclude that Snail is an early component of the gene regulatory network, initiated by Nodal signals, that patterns the ascidian neural plate.

Diverse ETS transcription factors mediate FGF signaling in the Ciona anterior neural plate.

The ascidian Ciona intestinalis is a marine invertebrate belonging to the sister group of the vertebrates, the tunicates. Its compact genome and simple, experimentally tractable embryos make Ciona well-suited for the study of cell-fate specification in chordates. Tunicate larvae possess a characteristic chordate body plan, and many developmental pathways are conserved between tunicates and vertebrates. Previous studies have shown that FGF signals are essential for neural induction and patterning at sequential steps of Ciona embryogenesis. Here we show that two different ETS family transcription factors, Ets1/2 and Elk1/3/4, have partially redundant activities in the anterior neural plate of gastrulating embryos. Whereas Ets1/2 promotes pigment cell formation in lateral lineages, both Ets1/2 and Elk1/3/4 are involved in the activation of Myt1L in medial lineages and the restriction of Six3/6 expression to the anterior-most regions of the neural tube. We also provide evidence that photoreceptor cells arise from posterior regions of the presumptive sensory vesicle, and do not depend on FGF signaling. Cells previously identified as photoreceptor progenitors instead form ependymal cells and neurons of the larval brain. Our results extend recent findings on FGF-dependent patterning of anterior-posterior compartments in the Ciona central nervous system.

Distinct structural requirements for CDON and BOC in the promotion of Hedgehog signaling.

Proper levels of Hedgehog (HH) signaling are essential during embryonic development and adult tissue homeostasis. A central mechanism to control HH pathway activity is through the regulation of secreted HH ligands at the plasma membrane. Recent studies have revealed a collective requirement for the cell surface co-receptors GAS1, CDON and BOC in HH signal transduction. Despite their requirement in HH pathway function, the mechanisms by which these proteins act to promote HH signaling remain poorly understood. Here we focus on the function of the two structurally related co-receptors, CDON and BOC. We utilized an in vivo gain-of-function approach in the developing chicken spinal cord to dissect the structural requirements for CDON and BOC function in HH signal transduction. Notably, we find that although CDON and BOC display functional redundancy during HH-dependent ventral neural patterning, these molecules utilize distinct molecular mechanisms to execute their HH-promoting effects. Specifically, we define distinct membrane attachment requirements for CDON and BOC function in HH signal transduction. Further, we identify novel and separate extracellular motifs in CDON and BOC that are required to promote HH signaling. Together, these data suggest that HH co-receptors employ distinct mechanisms to mediate HH pathway activity.

Xenopus pax6 mutants affect eye development and other organ systems, and have phenotypic similarities to human aniridia patients.

Mutations in the Pax6 gene cause ocular defects in both vertebrate and invertebrate animal species, and the disease aniridia in humans. Despite extensive experimentation on this gene in multiple species, including humans, we still do not understand the earliest effects on development mediated by this gene. This prompted us to develop pax6 mutant lines in Xenopus tropicalis taking advantage of the utility of the Xenopus system for examining early development and in addition to establish a model for studying the human disease aniridia in an accessible lower vertebrate. We have generated mutants in pax6 by using Transcription Activator-Like Effector Nuclease (TALEN) constructs for gene editing in X. tropicalis. Embryos with putative null mutations show severe eye abnormalities and changes in brain development, as assessed by changes in morphology and gene expression. One gene that we found is downregulated very early in development in these pax6 mutants is myc, a gene involved in pluripotency and progenitor cell maintenance and likely a mediator of some key pax6 functions in the embryo. Changes in gene expression in the developing brain and pancreas reflect other important functions of pax6 during development. In mutations with partial loss of pax6 function eye development is initially relatively normal but froglets show an underdeveloped iris, similar to the classic phenotype (aniridia) seen in human patients with PAX6 mutations. Other eye abnormalities observed in these froglets, including cataracts and corneal defects, are also common in human aniridia. The frog model thus allows us to examine the earliest deficits in eye formation as a result of pax6 lesions, and provides a useful model for understanding the developmental basis for the aniridia phenotype seen in humans.

Gene regulatory network for neurogenesis in a sea star embryo connects broad neural specification and localized patterning.

A great challenge in development biology is to understand how interacting networks of regulatory genes can direct the often highly complex patterning of cells in a 3D embryo. Here, we detail the gene regulatory network that describes the distribution of ciliary band-associated neurons in the bipinnaria larva of the sea star. This larva, typically for the ancestral deuterostome dipleurula larval type that it represents, forms two loops of ciliary bands that extend across much of the anterior-posterior and dorsal-ventral ectoderm. We show that the sea star first likely uses maternally inherited factors and the Wnt and Delta pathways to distinguish neurogenic ectoderm from endomesoderm. The broad neurogenic potential of the ectoderm persists throughout much of gastrulation. Nodal, bone morphogenetic protein 2/4 (Bmp2/4), and Six3-dependent pathways then sculpt a complex ciliary band territory that is defined by the expression of the forkhead transcription factor, foxg. Foxg is needed to define two molecularly distinct ectodermal domains, and for the formation of differentiated neurons along the edge of these two territories. Thus, significantly, Bmp2/4 signaling in sea stars does not distinguish differentiated neurons from nonneuronal ectoderm as it does in many other animals, but instead contributes to the patterning of an ectodermal territory, which then, in turn, provides cues to permit the final steps of neuronal differentiation. The modularity between specification and patterning likely reflects the evolutionary history of this gene regulatory network, in which an ancient module for specification of a broad neurogenic potential ectoderm was subsequently overlaid with a module for patterning.

Hedgehog Signaling in Cortical Development.

The Hedgehog (Hh) pathway plays a crucial role in embryonic development, acting both as a morphogenic signal that organizes tissue formation and a potent mitogenic signal driving cell proliferation. Dysregulated Hh signaling leads to various developmental defects in the brain. This article aims to review the roles of Hh signaling in the development of the neocortex in the mammalian brain, focusing on its regulation of neural progenitor proliferation and neuronal production. The review will summarize studies on genetic mouse models that have targeted different components of the Hh pathway, such as the ligand Shh, the receptor Ptch1, the GPCR-like transducer Smo, the intracellular transducer Sufu, and the three Gli transcription factors. As key insights into the Hh signaling transduction mechanism were obtained from mouse models displaying neural tube defects, this review will also cover some studies on Hh signaling in neural tube development. The results from these genetic mouse models suggest an intriguing hypothesis that elevated Hh signaling may play a role in the gyrification of the brain in certain species. Additionally, the distinctive production of GABAergic interneurons in the dorsal cortex in the human brain may also be linked to the extension of Hh signaling from the ventral to the dorsal brain region. Overall, these results suggest key roles of Hh signaling as both a morphogenic and mitogenic signal during the forebrain development and imply the potential involvement of Hh signaling in the evolutionary expansion of the neocortex.

Retinoic Acid and POU Genes in Developing Amphioxus: A Focus on Neural Development.

POU genes are a family of evolutionarily conserved transcription factors with key functions in cell type specification and neurogenesis. In vitro experiments have indicated that the expression of some POU genes is controlled by the intercellular signaling molecule retinoic acid (RA). In this work, we aimed to characterize the roles of RA signaling in the regulation of POU genes in vivo. To do so, we studied POU genes during the development of the cephalochordate amphioxus, an animal model crucial for understanding the evolutionary origins of vertebrates. The expression patterns of amphioxus POU genes were assessed at different developmental stages by chromogenic in situ hybridization and hybridization chain reaction. Expression was further assessed in embryos subjected to pharmacological manipulation of endogenous RA signaling activity. In addition to a detailed description of the effects of these treatments on amphioxus POU gene expression, our survey included the first description of Pou2 and Pou6 expression in amphioxus embryos. We found that Pit-1, Pou2, Pou3l, and Pou6 expression are not affected by alterations of endogenous RA signaling levels. In contrast, our experiments indicated that Brn1/2/4 and Pou4 expression are regulated by RA signaling in the endoderm and the nerve cord, respectively. The effects of the treatments on Pou4 expression in the nerve cord revealed that, in developing amphioxus, RA signaling plays a dual role by (1) providing anteroposterior patterning information to neural cells and (2) specifying neural cell types. This finding is coherent with a terminal selector function of Pou4 for GABAergic neurons in amphioxus and represents the first description of RA-induced changes in POU gene expression in vivo.

Establishing the Molecular and Functional Diversity of Spinal Motoneurons.

Spinal motoneurons are a remarkably diverse class of neurons responsible for facilitating a broad range of motor behaviors and autonomic functions. Studies of motoneuron differentiation have provided fundamental insights into the developmental mechanisms of neuronal diversification, and have illuminated principles of neural fate specification that operate throughout the central nervous system. Because of their relative anatomical simplicity and accessibility, motoneurons have provided a tractable model system to address multiple facets of neural development, including early patterning, neuronal migration, axon guidance, and synaptic specificity. Beyond their roles in providing direct communication between central circuits and muscle, recent studies have revealed that motoneuron subtype-specific programs also play important roles in determining the central connectivity and function of motor circuits. Cross-species comparative analyses have provided novel insights into how evolutionary changes in subtype specification programs may have contributed to adaptive changes in locomotor behaviors. This chapter focusses on the gene regulatory networks governing spinal motoneuron specification, and how studies of spinal motoneurons have informed our understanding of the basic mechanisms of neuronal specification and spinal circuit assembly.

Development of a larval nervous system in the sea urchin.

This review reports recent findings on the specification and patterning of neurons that establish the larval nervous system of the sea urchin embryo. Neurons originate in three regions of the embryo. Perturbation analyses enabled construction of gene regulatory networks controlling the several neural cell types. Many of the mechanisms described reflect shared features of all metazoans and others are conserved among deuterostomes. This nervous system with a very small number of neurons supports the feeding and swimming behaviors of the larva until metamorphosis when an adult nervous system replaces that system.