Forebrain Specification Research Articles

Transcriptional Dysregulation and Impaired Neuronal Activity in FMR1 Knock-Out and Fragile X Patients' iPSC-Derived Models.

Fragile X syndrome (FXS) is caused by a repression of the FMR1 gene that codes the Fragile X mental retardation protein (FMRP), an RNA binding protein involved in processes that are crucial for proper brain development. To better understand the consequences of the absence of FMRP, we analyzed gene expression profiles and activities of cortical neural progenitor cells (NPCs) and neurons obtained from FXS patients' induced pluripotent stem cells (IPSCs) and IPSC-derived cells from FMR1 knock-out engineered using CRISPR-CAS9 technology. Multielectrode array recordings revealed in FMR1 KO and FXS patient cells, decreased mean firing rates; activities blocked by tetrodotoxin application. Increased expression of presynaptic mRNA and transcription factors involved in the forebrain specification and decreased levels of mRNA coding AMPA and NMDA subunits were observed using RNA sequencing on FMR1 KO neurons and validated using quantitative PCR in both models. Intriguingly, 40% of the differentially expressed genes were commonly deregulated between NPCs and differentiating neurons with significant enrichments in FMRP targets and autism-related genes found amongst downregulated genes. Our findings suggest that the absence of FMRP affects transcriptional profiles since the NPC stage, and leads to impaired activity and neuronal differentiation over time, which illustrates the critical role of FMRP protein in neuronal development.

The CUL4B‐based E3 ubiquitin ligase regulates mitosis and brain development by recruiting phospho‐specific DCAFs

The paralogs CUL4A and CUL4B assemble cullin‐RING E3 ubiquitin ligase (CRL) complexes regulating multiple chromatin‐associated cellular functions. Although they are structurally similar, we found that the unique N‐terminal extension of CUL4B is heavily phosphorylated during mitosis, and the phosphorylation pattern is perturbed in the CUL4B‐P50L mutation causing X‐linked intellectual disability (XLID). Phenotypic characterization and mutational analysis revealed that CUL4B phosphorylation is required for efficient progression through mitosis, controlling spindle positioning and cortical tension. While CUL4B phosphorylation triggers chromatin exclusion, it promotes binding to actin regulators and to two previously unrecognized CUL4B‐specific substrate receptors (DCAFs), LIS1 and WDR1. Indeed, co‐immunoprecipitation experiments and biochemical analysis revealed that LIS1 and WDR1 interact with DDB1, and their binding is enhanced by the phosphorylated N‐terminal domain of CUL4B. Finally, a human forebrain organoid model demonstrated that CUL4B is required to develop stable ventricular structures that correlate with onset of forebrain differentiation. Together, our study uncovers previously unrecognized DCAFs relevant for mitosis and brain development that specifically bind CUL4B, but not the CUL4B‐P50L patient mutant, by a phosphorylation‐dependent mechanism.

Generation of the Human Pluripotent Stem-Cell-Derived Astrocyte Model with Forebrain Identity.

Astrocytes form functionally and morphologically distinct populations of cells with brain-region-specific properties. Human pluripotent stem cells (hPSCs) offer possibilities to generate astroglia for studies investigating mechanisms governing the emergence of astrocytic diversity. We established a method to generate human astrocytes from hPSCs with forebrain patterning and final specification with ciliary neurotrophic factor (CNTF). Transcriptome profiling and gene enrichment analysis monitored the sequential expression of genes determining astrocyte differentiation and confirmed activation of forebrain differentiation pathways at Day 30 (D30) and D60 of differentiation in vitro. More than 90% of astrocytes aged D95 in vitro co-expressed the astrocytic markers glial fibrillary acidic protein (GFAP) and S100β. Intracellular calcium responses to ATP indicated differentiation of the functional astrocyte population with constitutive monocyte chemoattractant protein-1 (MCP-1/CCL2) and tissue inhibitor of metalloproteinases-2 (TIMP-2) expression. The method was reproducible across several hPSC lines, and the data demonstrated the usefulness of forebrain astrocyte modeling in research investigating forebrain pathology.

Ybx1 fine-tunes PRC2 activities to control embryonic brain development

Chromatin modifiers affect spatiotemporal gene expression programs that underlie organismal development. The Polycomb repressive complex 2 (PRC2) is a crucial chromatin modifier in executing neurodevelopmental programs. Here, we find that PRC2 interacts with the nucleic acid–binding protein Ybx1. In the mouse embryo in vivo, Ybx1 is required for forebrain specification and restricting mid-hindbrain growth. In neural progenitor cells (NPCs), Ybx1 controls self-renewal and neuronal differentiation. Mechanistically, Ybx1 highly overlaps PRC2 binding genome-wide, controls PRC2 distribution, and inhibits H3K27me3 levels. These functions are consistent with Ybx1-mediated promotion of genes involved in forebrain specification, cell proliferation, or neuronal differentiation. In Ybx1-knockout NPCs, H3K27me3 reduction by PRC2 enzymatic inhibitor or genetic depletion partially rescues gene expression and NPC functions. Our findings suggest that Ybx1 fine-tunes PRC2 activities to regulate spatiotemporal gene expression in embryonic neural development and uncover a crucial epigenetic mechanism balancing forebrain–hindbrain lineages and self-renewal–differentiation choices in NPCs.

PAX 6D instructs neural retinal specification from human embryonic stem cell‐derived neuroectoderm

PAX6 is essential for neural retina (NR) and forebrain development but how PAX6 instructs NR versus forebrain specification remains unknown. We found that the paired-less PAX6, PAX6D, is expressed in NR cells during human eye development and along human embryonic stem cell (hESC) specification to retinal cells. hESCs deficient for PAX6D failed to enter NR specification. Induced expression of PAX6D but not PAX6A in a PAX6-null background restored the NR specification capacity. ChIP-Seq, confirmed by functional assays, revealed a set of retinal genes and non-retinal neural genes that are potential targets of PAX6D, including WNT8B. Inhibition of WNTs or knocking down of WNT8B restored the NR specification capacity of neuroepithelia with PAX6D knockout, whereas activation of WNTs blocked NR specification even when PAX6D was induced. Thus, PAX6D specifies neuroepithelia to NR cells via the regulation of WNT8B.

Distinct Vulnerability and Resilience of Human Neuroprogenitor Subtypes in Cerebral Organoid Model of Prenatal Hypoxic Injury.

Prenatal hypoxic injury (HI) is a leading cause of neurological disability. The immediate and long-term effects of hypoxia on progenitor homeostasis and developmental progression during early human brain development remain unclear. This gap is due to difficulty to access human fetal brain tissues and inadequate animal models to study human corticogenesis. Recent optimizations of cerebral organoid models derived from human embryonic stem (ES) cells present new opportunities to investigate pathophysiology of prenatal HI. Here, we implemented a transient HI model using human cerebral organoids with dorsal forebrain specification. We demonstrated that transient hypoxia resulted in immediate and prolonged apoptosis in cerebral organoids, with outer radial glia (oRG), a progenitor population more prominent in primates, and differentiating neuroblasts/immature neurons suffering larger losses. In contrast, neural stem cells in ventricular zone displayed relative resilience to HI and exhibited a shift of cleavage plane angle favoring symmetric division, thereby providing a mechanism to replenish the stem cell pool. Furthermore, we defined the vulnerable window and neurodifferentiation stages that are particularly sensitive to HI. Understanding cell type-specific and stage-dependent effects of prenatal HI on survival and mitotic behavior of human neuroprogenitor subtypes during early human corticogenesis helps elucidate the etiology of neurodevelopmental disorders, and provides a therapeutic starting point to protect the vulnerable populations at critical timeframes.

A concerted metabolic shift in early forebrain alters the CSF proteome and depends on MYC downregulation for mitochondrial maturation.

Massive, coordinated cellular changes accompany the transition of central nervous system (CNS) progenitors from forebrain neurectodermal cells to specified neuroepithelial cells. We have previously found that MYC regulates the changing ribosomal and proteostatic landscapes in mouse forebrain precursors at embryonic days E8.5 and E10.5 (before and after neural tube closure; NTC) (Chau et al., 2018). Here, we demonstrate parallel coordinated transcriptional changes in metabolic machinery during this same stage of forebrain specification. Progenitors showed striking mitochondrial structural changes transitioning from glycolytic cristae at E8.5, to more traditional mitochondria at E10.5. Accordingly, glucose use shifted in progenitors such that E8.5 progenitors relied on glycolysis, and after NTC increasingly used oxidative phosphorylation. This metabolic shift was matched by changes in surrounding amniotic and cerebrospinal fluid proteomes. Importantly, these mitochondrial morphological shifts depend on MYC downregulation. Together, our findings demonstrate that metabolic shifting accompanies dynamic organelle and proteostatic remodeling of progenitor cells during the earliest stages of forebrain development.

Activation of Wnt/ß-catenin signaling in ESC promotes rostral forebrain differentiation in vitro.

Wnt/ß-catenin signaling is crucial for maintenance of pluripotent state of embryonic stem cell (ESC). However, it is unclear how Wnt/ß-catenin signaling affects the differentiation ability of ESC, especially with regard to rostral forebrain cells. Here, using Rax, rostral forebrain marker, and Wnt/ß-catenin reporter lines, we report ratio of Rax+ and Wnt responding tissue (Wnt+) patterns, which were affected by seeding number of ESC in three-dimensional culture system. Surprisingly, we found ß-catenin level and localization are heterogeneous in ESC colony by immunostaining and time-laps imaging of ß-catenin-mEGFP signals. Moreover, activation of Wnt signaling in ESC promoted expression level and nuclear localization of ß-catenin, and mRNA levels of Wnt antagonists, axin2 and dkk1, leading to upregulating Wnt/ß-catenin reporter in ESC state and Rax expression at differentiation culture day 7. Together, our results suggest that activation of Wnt signaling in ESC promotes the differentiation efficacy of rostral forebrain cells. Wnt-priming culture method may provide a useful tool for applications in the areas of basic science and molecular therapeutics for regenerative medicine.Electronic supplementary materialThe online version of this article (doi:10.1007/s11626-015-9975-y) contains supplementary material, which is available to authorized users.

Differentiation of forebrain and hippocampal dopamine 1-class receptors, D1R and D5R, in spatial learning and memory.

Activation of prefrontal cortical (PFC), striatal, and hippocampal dopamine 1-class receptors (D1R and D5R) is necessary for normal spatial information processing. Yet the precise role of the D1R versus the D5R in the aforementioned structures, and their specific contribution to the water-maze spatial learning task remains unknown. D1R- and D5R-specific in situ hybridization probes showed that forebrain restricted D1R and D5R KO mice (F-D1R/D5R KO) displayed D1R mRNA deletion in the medial (m)PFC, dorsal and ventral striatum, and the dentate gyrus (DG) of the hippocampus. D5R mRNA deletion was limited to the mPFC, the CA1 and DG hippocampal subregions. F-D1R/D5R KO mice were given water-maze training and displayed subtle spatial latency differences between genotypes and spatial memory deficits during both regular and reversal training. To differentiate forebrain D1R from D5R activation, forebrain restricted D1R KO (F-D1R KO) and D5R KO (F-D5R KO) mice were trained on the water-maze task. F-D1R KO animals exhibited escape latency deficits throughout regular and reversal training as well as spatial memory deficits during reversal training. F-D1R KO mice also showed perseverative behavior during the reversal spatial memory probe test. In contrast, F-D5R KO animals did not present observable deficits on the water-maze task. Because F-D1R KO mice showed water-maze deficits we tested the necessity of hippocampal D1R activation for spatial learning and memory. We trained DG restricted D1R KO (DG-D1R KO) mice on the water-maze task. DG-D1R KO mice did not present detectable spatial memory deficit, but did show subtle deficits during specific days of training. Our data provides evidence that forebrain D5R activation plays a unique role in spatial learning and memory in conjunction with D1R activation. Moreover, these data suggest that mPFC and striatal, but not DG D1R activation are essential for spatial learning and memory.

Pharmacological characterisation of ligand- and voltage-gated ion channels expressed in human iPSC-derived forebrain neurons

Genetic causes, or predisposition, are increasingly accepted to be part of the ethiopathogenesis of many neuropsychiatric diseases. While genes can be studied in any type of cells, their physiological function in human brain cells is difficult to evaluate, particularly in living subjects. As a first step towards the characterisation of human inducible pluripotent stem cell (iPSC)-derived neurons from autism spectrum disorder (ASD) patients, we used gene expression and functional studies to define the regional identity of the typical forebrain differentiation, demonstrate expression patterns of genes of interest in ASD and understand the properties of 'control' iPSC-derived neurons (iCell-Neurons™), with a focus on receptors and ion channels that play a central role in synaptic physio-pathology. The gene expression profile of the iCell-Neurons™ closely resembled that observed in neonatal prefrontal cortex tissues. Functional studies, performed mainly using calcium flux assays, demonstrated the presence of ionotropic glutamate (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate) and gamma-aminobutyric acid type A receptors. Voltage-gated sodium and calcium channels were also identified using similar techniques. Overall, the results reported here suggest that iCell-Neurons™ are a good cellular model of a relatively immature forebrain human neuron population that can be used both as a control in comparison to patients cells, and as host cells in which mutations, insertions and deletions can be used in order to study the molecular mechanisms of ASD and other neurological disorders in an isogenic cellular background.

Multiple roles of Activin/Nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures

Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/β-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/β-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification.

Characterization of forebrain neurons derived from late-onset Huntington's disease human embryonic stem cell lines

Huntington's disease (HD) is an incurable neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the Huntingtin (HTT) gene. Recently, induced pluripotent stem cell (iPSC) lines carrying atypical and aggressive (CAG60+) HD variants have been generated and exhibit disparate molecular pathologies. Here we investigate two human embryonic stem cell (hESC) lines carrying CAG37 and CAG51 typical late-onset repeat expansions in comparison to wildtype control lines during undifferentiated states and throughout forebrain neuronal differentiation. Pluripotent HD lines demonstrate growth, viability, pluripotent gene expression, mitochondrial activity and forebrain specification that is indistinguishable from control lines. Expression profiles of crucial genes known to be dysregulated in HD remain unperturbed in the presence of mutant protein and throughout differentiation; however, elevated glutamate-evoked responses were observed in HD CAG51 neurons. These findings suggest typical late-onset HD mutations do not alter pluripotent parameters or the capacity to generate forebrain neurons, but that such progeny may recapitulate hallmarks observed in established HD model systems. Such HD models will help further our understanding of the cascade of pathological events leading to disease onset and progression, while simultaneously facilitating the identification of candidate HD therapeutics.

Developmental origins of brain disorders: roles for dopamine

Neurotransmitters and neuromodulators, such as dopamine, participate in a wide range of behavioral and cognitive functions in the adult brain, including movement, cognition, and reward. Dopamine-mediated signaling plays a fundamental neurodevelopmental role in forebrain differentiation and circuit formation. These developmental effects, such as modulation of neuronal migration and dendritic growth, occur before synaptogenesis and demonstrate novel roles for dopaminergic signaling beyond neuromodulation at the synapse. Pharmacologic and genetic disruptions demonstrate that these effects are brain region- and receptor subtype-specific. For example, the striatum and frontal cortex exhibit abnormal neuronal structure and function following prenatal disruption of dopamine receptor signaling. Alterations in these processes are implicated in the pathophysiology of neuropsychiatric disorders, and emerging studies of neurodevelopmental disruptions may shed light on the pathophysiology of abnormal neuronal circuitry in neuropsychiatric disorders.

Hmx4 regulates Sonic hedgehog signaling through control of retinoic acid synthesis during forebrain patterning

Mutations in H6-homeobox ( HMX) genes are linked to neural mispatterning and neural tube closure defects in humans. We demonstrate that zebrafish Hmx4 regulates the signaling of two morphogens critical for neural development, retinoic acid (RA) and Sonic hedgehog (Shh). Hmx4-depleted embryos have a strongly narrowed eye field and reduced forebrain Shh target gene expression. hmx4 morphants fail to properly transcribe the Shh signal transducer gli3, and have reduced ventral forebrain specification. Hmx4-depleted embryos also have neural tube patterning defects that phenocopy RA-deficiency. We show that Hmx4 is required for the initiation and maintenance of aldh1a2, the principal RA-synthesizing gene. Loss of RA is the primary defect in Hmx4-depleted embryos, as RA treatment rescues a number of the neural patterning defects. Surprisingly, RA treatment also rescues forebrain morphology, gli3 transcription, and Shh signaling. We propose that Hmx4 is a critical regulator of retinoic acid synthesis in a developing embryo, and that this regulation is essential for controlling Shh signaling and forebrain development.

Midbrain and forebrain patterning delivers immunocytochemically and functionally similar populations of neuropeptide Y containing GABAergic neurons

Neurons differentiated in vitro from embryonic stem cells (ESCs) have the potential to serve both as models of disease states and in drug discovery programs. In this study, we use sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF-8) to enrich for forebrain and midbrain phenotypes from mouse ESCs. We then investigate, using Ca 2+ imaging and [ 3H]-GABA release studies, whether the GABAergic neurons produced exhibit distinct functional phenotypes. At day 24 of differentiation, reverse transcriptase-PCR showed the presence of both forebrain (Bf-1, Hesx1, Pgc-1α, Six3) and midbrain (GATA2, GATA3) selective mRNA markers in developing forebrain-enriched cultures. All markers were present in midbrain cultures except for Bf-1 and Pgc-1α. Irrespective of culture conditions all GABA immunoreactive neurons were also immunoreactive to neuropeptide Y (NPY) antibodies. Forebrain and midbrain GABAergic neurons responded to ATP (1 mM), l-glutamate (30 μM), noradrenaline (30 μM), acetylcholine (30 μM) and dopamine (30 μM), with similar elevations of intracellular Ca 2+([Ca 2+] i ). The presence of GABA A and GABA B antagonists, bicuculline (30 μM) and CGP55845 (1 μM), increased the elevation of [Ca 2+] i in response to dopamine (30 μM) in midbrain, but not forebrain GABAergic neurons. All agonists, except dopamine, elicited similar [ 3H]-GABA release from forebrain and midbrain cultures. Dopamine (30 μM) did not stimulate significant [ 3H]-GABA release in midbrain cultures, although it was effective in forebrain cultures. This study shows that differentiating neurons toward a midbrain fate restricts the expression of forebrain markers. Forebrain differentiation results in the expression of forebrain and midbrain markers. All GABA + neurons contain NPY, and show similar agonist-induced elevations of [Ca 2+] i and [ 3H]-GABA release. This study indicates that the pharmacological phenotype of these particular neurons may be independent of the addition of the patterning factors that direct neurons toward forebrain and midbrain fates.

Development of head organizer of the mouse embryo depends on a high level of mitochondrial metabolism

Mouse genetic studies have defined a set of signaling molecules and transcription factors that are necessary to induce the forebrain. Here we describe an ENU-induced mouse mutation, nearly headless (nehe), that was identified based on the specific absence of most of the forebrain at midgestation. Positional cloning and genetic analysis show that, unlike other mouse mutants that disrupt specification of the forebrain, the nehe mutation disrupts mitochondrial metabolism. nehe is a hypomorphic allele of Lipoic acid Synthetase (Lias), the enzyme that catalyzes the synthesis of lipoic acid, an essential cofactor for several mitochondrial multienzyme complexes required for oxidative metabolism. The defect in forebrain development in nehe mutants is apparent as soon as the forebrain is specified, without a concomitant increase in apoptosis. Two tissues required for forebrain specification, the anterior visceral endoderm and the anterior definitive endoderm, develop normally in nehe mutants. However, a third head organizer tissue, the prechordal plate, fails to express markers of cell type determination and shows abnormal morphology in the mutants. We find that the level of phosphorylated (active) AMPK, a cellular energy sensor that affects cell polarity, is up-regulated in nehe mutants at the time when the prechordal plate is normally specified. The results suggest that the nehe phenotype arises because high levels of energy production are required for the specialized morphogenetic movements that generate the prechordal plate, which is required for normal development of the mammalian forebrain. We suggest that a requirement for high levels of ATP for early forebrain patterning may contribute to certain human microcephaly syndromes.

The full spectrum of holoprosencephaly-associated mutations within theZIC2gene in humans predicts loss-of-function as the predominant disease mechanism

Mutations of the ZIC2 transcription factor gene are among the most common heterozygous variations detected in holoprosencephaly (HPE) patients, a patient group who lack critical midline forebrain specification due to defective embryonic signaling during development. Recent studies indicate that complete deficiency of the related murine Zic2 transcription factor can also be a contributing factor to variable midline deficiencies, presenting during mid-gastrulation, that could explain similar forebrain anomalies in this model system. Here we collect and summarize all available mutations in the human ZIC2 gene detected in HPE patients (21 published and 62 novel). Our analysis corroborates this mechanism proposed in mice by predicting loss-of-function as the likely pathogenetic mechanism common to most, if not all, of these mutations in HPE.

Eye field requires the function of Sfrp1 as a Wnt antagonist

Wnts have been shown to provide a posteriorizing signal that has to be repressed in the specification of vertebrate forebrain region. Previous studies have shown that Wnt activation by LiCl treatment causes an expansion of optic stalk and mid-hindbrain boundary, whereas eye and ventral diencephalon in the forebrain region were reduced. However, the molecular mechanism, by which inhibits Wnt activity in the forebrain remains poorly defined. To investigate relationship between forebrain specification and Wnt signaling, the zebrafish homologue of secreted frizzled related protein1 (sfrp1) has been characterized. The transcripts of sfrp1 are detected in the presumptive forebrain at gastrula and in the ventral telencephalon, ventral diencephalon, midbrain and optic vesicles at 24h after postfertilization (hpf). Overexpression of sfrp1 causes an anteriorization of embryo, with enlarged head and reduced posterior structure as in the embryo overexpressing dominant-negative form of Frizzled8a or Dkk1. Its overexpression restored the eye defects in the Wnt8b-overexpressing embryos, but not in the LiCl-treated embryos. These results suggest that Sfrp1 expressed in the forebrain and eye field plays a critical role in the extracellular events of antagonizing Wnt activity for the forebrain specification.

Roles of organizer factors and BMP antagonism in mammalian forebrain establishment

A critical question in mammalian development is how the forebrain is established. In amphibians, bone morphogenetic protein (BMP) antagonism emanating from the gastrula organizer is key. Roles of BMP antagonism and the organizer in mammals remain unclear. Anterior visceral endoderm (AVE) promotes early mouse head development, but its function is controversial. Here, we explore the timing and regulation of forebrain establishment in the mouse. Forebrain specification requires tissue interaction through the late streak stage of gastrulation. Foxa2 −/− embryos lack both the organizer and its BMP antagonists, yet about 25% show weak forebrain gene expression. A similar percentage shows ectopic AVE gene expression distally. The distal VE may thus be a source of forebrain promoting signals in these embryos. In wild-type ectoderm explants, AVE promoted forebrain specification, while anterior mesendoderm provided maintenance signals. Embryological and molecular data suggest that the AVE is a source of active BMP antagonism in vivo. In prespecification ectoderm explants, exogenous BMP antagonists triggered forebrain gene expression and inhibited posterior gene expression. Conversely, BMP inhibited forebrain gene expression, an effect that could be antagonized by anterior mesendoderm, and promoted expression of some posterior genes. These results lead to a model in which BMP antagonism supplied by exogenous tissues promotes forebrain establishment and maintenance in the murine ectoderm.

Twisted gastrulation is required for forebrain specification and cooperates with Chordin to inhibit BMP signaling during X. tropicalis gastrulation

In the developing vertebrate embryo, proper dorsal–ventral patterning relies on BMP antagonists secreted by the organizer during gastrulation. The BMP antagonist chordin has a complex interaction with BMPs that is governed in part by its interaction with the secreted protein twisted gastrulation (tsg). In different contexts, tsg has activity as either a BMP agonist or as a BMP antagonist. Using morpholino oligonucleotides in Xenopus tropicalis, we show that reducing tsg gene product results in a ventralized embryo, and that tsg morphants specifically lack a forebrain. We provide new evidence that tsg acts as a BMP antagonist during X. tropicalis gastrulation since the tsg depletion phenotype can be rescued in two ways: by chordin overexpression and by BMP depletion. We conclude that tsg acts as a BMP antagonist in the context of the frog gastrula, and that it acts cooperatively with chordin to establish dorsal structures and particularly forebrain tissue during development.