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SUMMARYThe orientation of epithelial cells in the plane of the tissue, known as planar cell polarity (PCP), is regulated by interactions of asymmetrically localized PCP protein complexes. In the Xenopus neural plate, Van Gogh-like2 (Vangl2) and Prickle3 (Pk3) proteins form a complex at the anterior cell boundaries, but how this complex is regulated in vivo remains largely unknown. Here we show that Vangl2-Pk3 association is inhibited by Frizzled3 (Fz3), a core PCP protein that is specifically expressed in the neuroectoderm and is essential for the establishment of PCP in this tissue. Proximity biotinylation and crosslinking studies revealed that the Vangl2-Pk3 interaction is suppressed by overexpressed Fz3, but enhanced in Fz3 morphants. In addition, Fz3 induced Vangl2 phosphorylation on T76 and T78, and this phosphorylation was required for Fz3-mediated inhibition of Vangl2-Pk3 complex formation. Consistent with this observation, the complex of Pk3 with nonphosphorylatable Vangl2 was not polarized in the neural plate. These findings provide evidence for in vivo regulation of the Vangl2-Pk3 complex formation and localization by a Frizzled receptor.

Coordinated polarization of cells in the tissue plane, known as planar cell polarity (PCP), is associated with a signaling pathway critical for the control of morphogenetic processes. Although the segregation of PCP components to opposite cell borders is believed to play a critical role in this pathway, whether PCP derives from egg polarity or preexistent long-range gradient, or forms in response to a localized cue remains a challenging question. Here we investigate the Xenopus neural plate, a tissue that has been previously shown to exhibit PCP. By imaging Vangl2 and Prickle3, we show that PCP is progressively acquired in the neural plate and requires a signal from the posterior region of the embryo. Tissue transplantations indicated that PCP is triggered in the neural plate by a planar cue from the dorsal blastopore lip. The PCP cue did not depend on the orientation of the graft and was distinct from neural inducers. These observations suggest that neuroectodermal PCP is not instructed by a preexisting molecular gradient, but induced by a signal from the dorsal blastopore lip.HighlightsThe Xenopus neural plate progressively acquires PCP in a posterior-to-anterior direction.The dorsal blastopore lip is likely the source of the PCP-instructing signal for the Xenopus neural plate.The PCP cue is distinct from neural inducers and has a planar mode of transmission.

Genetically diverse pluripotent stem cells (PSCs) display varied, heritable responses to differentiation cues in the culture environment. By harnessing these disparities through derivation of embryonic stem cells (ESCs) from the BXD mouse genetic reference panel, along with C57BL/6J (B6) and DBA/2J (D2) parental strains, we demonstrate genetically determined biases in lineage commitment and identify major regulators of the pluripotency epigenome. Upon transition to formative pluripotency using epiblast-like cells (EpiLCs), B6 quickly dissolves naïve networks adopting gene expression modules indicative of neuroectoderm lineages; whereas D2 retains aspects of naïve pluripotency with little bias in differentiation. Genetic mapping identifies 6 majortrans-acting loci co-regulating chromatin accessibility and gene expression in ESCs and EpiLCs, indicating a common regulatory system impacting cell state transition. These loci distally modulate occupancy of pluripotency factors, including TRIM28, P300, and POU5F1, at hundreds of regulatory elements. Onetrans-acting locus on Chr 12 primarily impacts chromatin accessibility in ESCs; while in EpiLCs the same locus subsequently influences gene expression, suggesting early chromatin priming. Consequently, the distal gene targets of this locus are enriched for neurogenesis genes and were more highly expressed when cells carried B6 haplotypes at this Chr 12 locus, supporting genetic regulation of biases in cell fate. Spontaneous formation of embryoid bodies validated this with B6 showing a propensity towards neuroectoderm differentiation and D2 towards definitive endoderm, confirming the fundamental importance of genetic variation influencing cell fate decisions.

Advances in the development of three-dimensional (3D) brain organoids maintained in vitro have provided excellent opportunities to study brain development and neurodegenerative disorders, including Alzheimer's disease (AD). However, there remains a need to generate AD organoids bearing patient-specific genomic backgrounds that can functionally recapitulate the key features observed in the AD patient's brain. To address this need, we described a strategy to generate self-organizing 3D cerebral organoids which develop a functional neuronal network connectivity. This was achieved by neuroectoderm induction of human pluripotent stem cell (hPSCs) aggregates and subsequent differentiation into desired neuroepithelia and mature neurons in a 3D Matrigel matrix. Using this approach, we successfully generated AD cerebral organoids from human pluripotent stem cells (hPSCs) derived from a familial AD patient with a common mutation in presenilin 2 (PSEN2N141I). An isogenic control with an identical genetic background but wild-type PSEN2 was generated using CRISPR/Cas9 technology. Both control and AD organoids were characterized by analyzing their morphology, the Aβ42/Aβ40 ratio, functional neuronal network activity, drug sensitivity, and the extent of neural apoptosis. The spontaneous activity of the network and its synchronization was measured in the organoids via calcium imaging. We found that compared with the mutation-corrected control organoids, AD organoids had a higher Aβ42/Aβ40 ratio, asynchronous calcium transients, and enhanced neuronal hyperactivity, successfully recapitulating an AD-like pathology at the molecular, cellular, and network level in a human genetic context. Moreover, two drugs which increase neuronal activity, 4-aminopyridine (4-AP) and bicuculline methochloride, induced high-frequency synchronized network bursting to a similar extent in both organoids. Therefore, our study presents a promising organoid-based biosystem for the study of the pathophysiology of AD and a platform for AD drug development.

Fibroblast growth factor (Fgf) and anti-bone morphogenetic protein (Bmp) signals are derived from the organiser of mesoderm origin and cooperate to promoteXenopusneural development from the gastrula ectoderm. Using antisense oligos to Fgf2 and Fgf8 and dominant-negative Ets transcription factors, we showed that the expression of Fgf2, Fgf8, and Ets in ectoderm cells is essential to initiate neural induction bothin vivoandin vitro. Our findings show that neural induction is initiated primarily by autonomous signalling in ectoderm cells, rather than by paracrine signalling from organiser cells. The signalling in ectoderm cells is transduced via the Fgf/Ras/Mapk/Ets pathway, independent of Bmp signal inhibition via the Fgf/Ras/Mapk/Smad1 route, as indicated by earlier studies. Through the same pathway, Fgfs activated position-specific neural genes dose-dependently along the anteroposterior axis in cultured ectoderm cells. The expression of these genes coincides with the establishment of the activated Ets gradient within the gastrula ectoderm. Organiser cells, being located posteriorly to the ectoderm, secrete Fgfs as gastrulation proceeds, which among several candidate molecules initially promote neural patterning of the induced neuroectoderm as morphogens.Summary statementFgf/Ets signalling in ectodermal cells is required to initiate the expression of both anterior and posterior neural genes from the late blastula to gastrula stages, independent of anti-Bmp signalling.

The internalization of the central nervous system, termed neurulation in vertebrates, is a critical step in embryogenesis. Open questions remain as to how force propels coordinated tissue movement during the process, and little is known as to how internalization happens in invertebrates. We show that inC. elegansmorphogenesis, apical constriction in the retracting pharynx drives involution of the adjacent neuroectoderm. Localized HMR-1/Cadherin mediates the inter-tissue attachment, as well as within the neuroectoderm to maintain intratissue cohesion. Our results demonstrate that localized HMR-1 is capable of mediating embryo wide reorganization driven by a centrally located force generator, and indicate a non-canonical use of Cadherin on the basal side of an epithelium that may apply to vertebrate neurulation. Additionally, we highlight shared morphology and gene expression in tissues driving involution, which suggests that neuroectoderm involution inC. elegansis potentially homologous with vertebrate neurulation and thus may help elucidate the evolutionary origin of the brain.

XBF-1 is an anterior neural plate-specific, winged helix transcription factor that affects neural development in a concentration-dependent manner. A high concentration of XBF-1 results in suppression of endogenous neuronal differentiation and an expansion of undifferentiated neuroectoderm. Here we investigate the mechanism by which this expansion is achieved. Our findings suggest that XBF-1 converts ectoderm to a neural fate and it does so independently of any effects on the mesoderm. In addition, we show that a high dose of XBF-1 promotes the proliferation of neuroectodermal cells while a low dose inhibits ectodermal proliferation. Thus, the neural expansion observed after high dose XBF-1 misexpression is due both to an increase in the number of ectodermal cells devoted to a neural fate and an increase in their proliferation. We show that the effect on cell proliferation is likely to be mediated by p27(XIC1), a cyclin-dependent kinase (cdk) inhibitor. We show that p27(XIC1) is expressed in a spatially restricted pattern in the embryo, including the anterior neural plate, and when misexpressed it is sufficient to block the cell cycle in vivo. We find that p27(XIC1)is transcriptionally regulated by XBF-1 in a dose-dependent manner such that it is suppressed or ectopically induced by a high or low dose of XBF-1, respectively. However, while a low dose of XBF-1 induces ectopic p27(XIC1)and ectopic neurons, misexpression of p27(XIC1)does not induce ectopic neurons, suggesting that the effects of XBF-1 on cell fate and cell proliferation are distinct. Finally, we show that p27(XIC1)is suppressed by XBF-1 in the absence of protein synthesis, suggesting that at least one component of p27(XIC1)regulation by XBF-1 may be direct. Thus, XBF-1 is a neural-specific transcription factor that can independently affect both the cell fate choice and the proliferative status of the cells in which it is expressed.

MicroRNAs (miRNAs), a class of small, non-coding RNA molecules represent important regulators of gene expression. Recent reports have implicated their role in the cell specification process acting as “fine-tuners” to ensure the precise gene expression at the specific stage of cell differentiation. Here we used retinal organoids differentiated from human pluripotent stem cells (hPSCs) as a model to closely investigate the role of a sensory organ-specific and evolutionary conserved miR-183/96/182 cluster. Using a miRNA tough decoy approach, we inhibited the miR-183/96/182 cluster in hPSCs. Inhibition of the miRNA cluster resulted in an increased expansion of neuroepithelium leading to abnormal “bulged” neural retina in organoids, associated with upregulation of neural-specific and retinal-specific genes. Importantly, we identifiedPAX6, a well-known essential gene in neuroectoderm specification, as a target of the miR-183/96/182 cluster members. Taken together, the miR-183/96/182 cluster not only represents an important regulator ofPAX6expression, but it also plays a crucial role in retinal tissue morphogenesis.

Summary Induced pluripotency provides a tool to explore mechanisms underlying establishment, maintenance, and differentiation of naive pluripotent stem cells (nPSCs). Here, we report that self-renewal of nPSCs requires minimal Sox2 expression (Sox2-low). Sox2-low nPSCs do not show impaired neuroectoderm specification and differentiate efficiently in vitro into all embryonic germ lineages. Strikingly, upon the removal of self-renewing cues Sox2-low nPSCs differentiate into both embryonic and extraembryonic cell fates in vitro and in vivo. This differs from previous studies which only identified conditions that allowed cells to differentiate to one fate or the other. At the single-cell level self-renewing Sox2-low nPSCs exhibit a naive molecular signature. However, they display a nearer trophoblast identity than controls and decreased ability of Oct4 to bind naïve-associated regulatory sequences. In sum, this work defines wild-type levels of Sox2 as a restrictor of developmental potential and suggests perturbation of naive network as a mechanism to increase cell plasticity., Graphical abstract, Highlights • Low Sox2 expression is sufficient for naive pluripotent stem cell self-renewal • Low Sox2 expression does not impair neurectoderm differentiation in vitro • Low Sox2 expression impairs Oct4 genomic occupancy • Low Sox2 increases naive pluripotent stem cell plasticity in vitro and in vivo, Biological Sciences; Cell Biology; Stem Cells Research

ADNP (Activity Dependent Neuroprotective Protein) is a neuroprotective protein whose aberrant expression has been frequently linked to neural developmental disorders, including the Helsmoortel-Van der Aa syndrome (also called the ADNP syndrome). However, its role in neural development and pathology remains unclear. Here, we show that ADNP is required for neural induction and differentiation by enhancing Wnt signaling. Mechanistically, ADNP functions to stabilize β-Catenin through binding to its armadillo domain which prevents its association with key components of the degradation complex: Axin and APC. Loss of ADNP promotes the formation of the degradation complex and β-Catenin degradation via ubiquitin-proteasome pathway, resulting in down-regulation of key neuroectoderm developmental genes. In addition, adnp gene disruption in zebrafish leads to defective neurogenesis and reduced Wnt signaling. Our work provides important insights into the role of ADNP in neural development and the pathology of the Helsmoortel-Van der Aa syndrome caused by ADNP gene mutation., ADNP has been connected to neural developmental disorders. Here, the authors uncover a role for ADNP in neural induction and differentiation via β-Catenin stabilization, with ADNP disruption in zebrafish leading to defective neurogenesis and decreased Wnt signaling.

The human brain has changed dramatically since humans diverged from our closest living relatives, chimpanzees and the other great apes1–5. However, the genetic and developmental programs underlying this divergence are not fully understood6–8. Here, we have analyzed stem cell-derived cerebral organoids using single-cell transcriptomics (scRNA-seq) and accessible chromatin profiling (scATAC-seq) to explore gene regulatory changes that are specific to humans. We first analyze cell composition and reconstruct differentiation trajectories over the entire course of human cerebral organoid development from pluripotency, through neuroectoderm and neuroepithelial stages, followed by divergence into neuronal fates within the dorsal and ventral forebrain, midbrain and hindbrain regions. We find that brain region composition varies in organoids from different iPSC lines, yet regional gene expression patterns are largely reproducible across individuals. We then analyze chimpanzee and macaque cerebral organoids and find that human neuronal development proceeds at a delayed pace relative to the other two primates. Through pseudotemporal alignment of differentiation paths, we identify human-specific gene expression resolved to distinct cell states along progenitor to neuron lineages in the cortex. We find that chromatin accessibility is dynamic during cortex development, and identify instances of accessibility divergence between human and chimpanzee that correlate with human-specific gene expression and genetic change. Finally, we map human-specific expression in adult prefrontal cortex using single-nucleus RNA-seq and find developmental differences that persist into adulthood, as well as cell state-specific changes that occur exclusively in the adult brain. Our data provide a temporal cell atlas of great ape forebrain development, and illuminate dynamic gene regulatory features that are unique to humans.

During embryogenesis, the distinct morphogenetic cell behavior programs that shape tissues are influenced both by the fate of cells and their position with respect to the embryonic axes, making embryonic patterning a prerequisite for morphogenesis. These two essential processes must therefore be coordinated in space and time to ensure proper development, but mechanisms by which patterning information is translated to the cellular machinery that drives morphogenesis remain poorly understood. Here, we address the role of Nodal morphogen signaling at the intersection of cell fate specification, patterning, and anteroposterior (AP) axis extension in zebrafish gastrulae and embryonic explants. AP axis extension is impaired in Nodal-deficient embryos, but it is unclear whether this defect is strictly secondary to their severe mesendoderm deficiencies or also results from loss of Nodal signalingper se. We find that convergence & extension (C&E) gastrulation movements and underlying mediolateral (ML) cell polarization are reduced in the neuroectoderm of Nodal-deficient mutants and exacerbated by simultaneous disruption of Planar Cell Polarity (PCP) signaling, demonstrating at least partially parallel functions of Nodal and PCP. ML polarity of mutant neuroectoderm cells is not fully restored upon transplantation into wild-type gastrulae, demonstrating a cell autonomous, mesoderm-independent role for Nodal in neural cell polarization. This is further demonstrated by the ability of Nodal ligands to promote neuroectoderm-driven C&E of naïve blastoderm explants in a tissue-autonomous fashion. Finally, temporal manipulation of signaling reveals that Nodal contributes to neural C&E in explants after mesoderm is specified and promotes C&E even in the absence of mesoderm. Together these results reveal a mesoderm-independent, cell-autonomous role for Nodal signaling in neural C&E that may cooperate with previously-described mesoderm-dependent mechanisms to drive AP embryonic axis extension.

Unlike the Hox genes in arthropods and vertebrates, those in molluscs show diverse expression patterns and, with some exceptions, have generally been described as lacking the canonical staggered pattern along the anterior-posterior (AP) axis. This difference is unexpected given that almost all molluscs share highly conserved early development. Here, we show that molluscan Hox expression can undergo dynamic changes, which may explain why previous research observed different expression patterns. Moreover, we reveal that a key character of molluscan Hox expression is that the dorsal and ventral expression is dissociated. We then deduce a generalized molluscan Hox expression model, including conserved staggered Hox expression in the neuroectoderm on the ventral side and lineage-specific dorsal expression that strongly correlates with shell formation. This generalized model clarifies a long-standing debate over whether molluscs possess staggered Hox expression and it can be used to explain the diversification of molluscs. In this scenario, the dorsoventral dissociation of Hox expression allows lineage-specific dorsal and ventral patterning in different clades, which may have permitted the evolution of diverse body plans in different molluscan clades.

Coordination of differentiation and cell cycle progression represents an essential process for embryonic development and adult tissue homeostasis. These mechanisms ultimately determine the quantities of specific cell types that are generated. Despite their importance, the precise molecular interplays between cell cycle machinery and master regulators of cell fate choice remain to be fully uncovered. Here, we demonstrate that cell cycle regulators Cyclin D1–3 control cell fate decisions in human pluripotent stem cells by recruiting transcriptional corepressors and coactivator complexes onto neuroectoderm, mesoderm, and endoderm genes. This activity results in blocking the core transcriptional network necessary for endoderm specification while promoting neuroectoderm factors. The genomic location of Cyclin Ds is determined by their interactions with the transcription factors SP1 and E2Fs, which result in the assembly of cell cycle-controlled transcriptional complexes. These results reveal how the cell cycle orchestrates transcriptional networks and epigenetic modifiers to instruct cell fate decisions.

The vertebrate body forms by continuous generation of new tissue from progenitors at the posterior end of the embryo. In mice, these axial progenitors initially reside in the epiblast, from where they form the trunk; and later relocate to the chordo-neural hinge of the tail bud to form the tail. Among them, a small group of bipotent neuromesodermal progenitors (NMPs) are thought to generate the spinal cord and paraxial mesoderm to the end of axis elongation. The study of these progenitors, however, has proven challengingin vivodue to their small numbers and dynamic nature, and the lack of a unique molecular marker to identify them. Here, we report the generation of the Nkx1.2CreERT2transgenic mouse line in which the endogenousNkx1.2promoter drives tamoxifen-inducible CreERT2recombinase. We show that Nkx1.2CreERT2targets axial progenitors, including NMPs and early neural and mesodermal progenitors. Using a YFP reporter, we demonstrate thatNkx1.2-expressing epiblast cells contribute to all three germ layers, mostly neuroectoderm and mesoderm excluding notochord; and continue contributing neural and paraxial mesoderm tissues from the tail bud. This study identifies theNkx1.2-expressing cell population as the source of most trunk and tail tissues in the mouse; and provides a key tool to genetically label and manipulate this progenitor populationin vivo.

Recently emerging methodology for generating human tissues in vitro has the potential to revolutionize drug discovery and disease research. Currently, three-dimensional cell culture models either rely on the pronounced ability of mammalian cells to self organize in vitro1-6, or use bioengineered constructs to arrange cells in an organ-like configuration7,8. While self-organizing organoids can recapitulate developmental events at a remarkable level of detail, bioengineered constructs excel at reproducibly generating tissue of a desired architecture. Here, we combine these two approaches to reproducibly generate micropatterned human forebrain tissue while maintaining its self-organizing capacity. We utilize poly(lactide-co-glycolide) copolymer (PLGA) fiber microfilaments as a scaffold to generate elongated embryoid bodies and demonstrate that this influences tissue identity. Micropatterned engineered cerebral organoids (enCORs) display enhanced neuroectoderm formation and improved cortical development. Furthermore, we reconstitute the basement membrane at later stages leading to characteristic cortical tissue architecture including formation of a polarized cortical plate and radial units. enCORs provide the first in vitro system for modelling the distinctive radial organization of the cerebral cortex and allow for the study of neuronal migration. We demonstrate their utility by modelling teratogenic effects of ethanol and show that defects in leading process formation may be responsible for the neuronal migration deficits in fetal alcohol syndrome. Our data demonstrate that combining 3D cell culture with bioengineering can significantly enhance tissue identity and architecture, and establish organoid models for teratogenic compounds.

Coordination of differentiation and cell cycle progression represents an essential process for embryonic development and adult tissue homeostasis. These mechanisms ultimately determine the quantities of specific cell types that are generated. Despite their importance, the precise molecular interplays between cell cycle machinery and master regulators of cell fate choice remain to be fully uncovered. Here, we demonstrate that cell cycle regulators Cyclin D1-3 control cell fate decisions in human pluripotent stem cells by recruiting transcriptional corepressors and coactivator complexes onto neuroectoderm, mesoderm, and endoderm genes. This activity results in blocking the core transcriptional network necessary for endoderm specification while promoting neuroectoderm factors. The genomic location of Cyclin Ds is determined by their interactions with the transcription factors SP1 and E2Fs, which result in the assembly of cell cycle-controlled transcriptional complexes. These results reveal how the cell cycle orchestrates transcriptional networks and epigenetic modifiers to instruct cell fate decisions.

Experiments in Xenopus have illustrated the importance of extracellular morphogens for embryonic gene regulation in vertebrates. Much less is known about how induction leads to the correct positioning of boundaries; for example, between germ layers. Here we report that the neuroectoderm/mesoderm boundary is controlled by the chromatin remodeling ATPase CHD4/Mi-2β. Gain and loss of CHD4 function experiments shifted this boundary along the animal–vegetal axis at gastrulation, leading to excess mesoderm formation at the expense of neuroectoderm, or vice versa. This phenotype results from specific alterations in gene transcription, notably of the neural-promoting gene Sip1 and the mesodermal regulatory gene Xbra. We show that CHD4 suppresses Sip1 transcription by direct binding to the 5′ end of the Sip1 gene body. Furthermore, we demonstrate that CHD4 and Sip1 expression levels determine the “ON” threshold for Nodal-dependent but not for eFGF-dependent induction of Xbra transcription. The CHD4/Sip1 epistasis thus constitutes a regulatory module, which balances mesoderm and neuroectoderm formation.

In both chick and mouse, the otic placode, the rudiment of the inner ear, is induced by at least two signals, one from the cephalic paraxial mesoderm and the other from the neural ectoderm. In chick, the mesodermal signal, FGF19, induces neural ectoderm to express additional signals, including WNT8c and FGF3, resulting in induction of the otic placode. In mouse, mesodermalFgf10acting redundantly with neuralFgf3is required for induction of the placode. To determine how the mesodermal inducers of the otic placode are localized, we took advantage of the unique strengths of the two model organisms. We show that endoderm is necessary for otic induction in the chick and thatFgf8, expressed in the chick endoderm subjacent toFgf19, is both sufficient and necessary for the expression ofFgf19in the mesoderm. In the mouse,Fgf8is also expressed in endoderm as well as in other germ layers in the periotic placode region. We show that otic induction fails in embryos null forFgf3and hypomorphic forFgf8and expression of mesodermalFgf10is reduced. Thus,Fgf8plays a critical upstream role in an FGF signaling cascade required for otic induction in chick and mouse.

Nieuwkoop's two-signal model proposed that induced neural tissue is inherently anterior (forebrain) in character and that a graded transforming (or posteriorizing) signal specifies posterior identity to the anterior neuroectoderm (Nieuwkoop 1952). It has been suggested that during vertebrate head development, the level of Wnt activity may specify posterior-to-anterior fates within the neural plate (Niehrs 1999; Heisenberg et al. 2001; Kiecker and Niehrs 2001). Wnt signaling must be inhibited to allow the development of the rostral telencephalon, or the prospective forebrain will acquire a caudal diencephalic identity (Niehrs 1999; Heisenberg et al. 2001; Kiecker and Niehrs 2001). This anterior Wnt-signaling-free zone is maintained by Wnt antagonists secreted by the anterior neuroectoderm and adjacent anterior mesendoderm (Niehrs 1999; Houart et al. 2002). Head truncations occur when genes that are required for the development of the anterior visceral endoderm (AVE; i.e., Hex, Lim1, and Otx2) are mutated (Thomas and Beddington 1996; Shawlot et al. 1999; Martinez-Barbera and Beddington 2001; Perea-Gomez et al. 2001). The lack of anterior head structures also occurs in mice that are double-homozygous for chordin and noggin, which encode secreted bone morphogenetic protein antagonists (Bachiller et al. 2000). In addition, mouse embryos lacking Dickkopf1 (Dkk1), a secreted protein that acts as an inhibitor of the Wnt coreceptor LRP-6, lack head structures anterior to the midbrain; Dkk1 activity is required in the axial mesendoderm (Mukhopadhyay et al. 2001). Variable forebrain truncations are also observed in mice with inactivating mutations in the homeobox gene Hesx1, whose activity is required in the anterior neural ectoderm (Martinez-Barbera and Beddington 2001). We have previously shown that in mice, Six3 is expressed in the most anterior part of the developing neural plate (Oliver et al. 1995). To determine the role of Six3 during vertebrate development, we inactivated the mouse Six3 locus. We find that Six3 is required for development of the mammalian rostral forebrain. The absence of Six3 results in forebrain truncations and posteriorization of the remaining mutant head. We demonstrate that Six3 binds to the Wnt1 promoter region in vivo and represses Wnt1 expression in the most anterior neuroectoderm. Work recently performed in zebrafish embryos has suggested that telencephalic induction, as well as the subsequent patterning of the forebrain into telencephalic, eye, and diencephalic regions, is the result of the graded expression of Wnt signaling in the anterior neural plate (Houart et al. 2002). Thus, during vertebrate head regional specification, the maintenance and refinement of anterior neural fates requires that Wnt signaling is transcriptionally repressed in the anterior neuroectoderm, and Six3 is a key player during this process. We also show that Six3 is sufficient to suppress the loss of forebrain resulting from excess Wnt1 signaling in headless (Tlc3) zebrafish mutants. Taken together, these results not only identified Six3 as a key player in vertebrate head development, but also demonstrated the existence of another regulatory step in the complex Wnt signaling pathway, the direct repression of Wnt1 expression by a transcription factor in the mammalian anterior neural plate at the late headfold–early somite stage, a step that is probably required for the maintenance of the anterior neural fates.

Animal development involves a complex progression of tissue induction and morphogenesis, expansion of precursor cell populations, and the death or terminal differentiation of these cells into specific functional types. Bone morphogenetic protein (BMP) signaling is repeatedly used in this dynamic process for both vertebrates and invertebrates. In gastrulating Xenopus and Drosophila, BMPs and the BMP2/4 homolog Decapentaplegic (Dpp) contribute to specifying the embryonic dorsoventral axis. High activity of BMP/Dpp induces blood/amnioserosa, whereas maximal inhibition of BMP/Dpp leads to neural ectoderm induction (Dale 2000; Nakayama et al. 2000). Subsequently, BMP/Dpp signaling contributes to the specification and/or expansion of many tissues such as vertebrate limb and Drosophila wing (Vogt and Duboule 1999; Day and Lawrence 2000) and neural ectoderm (Cornell and Ohlen 2000). Vertebrate neural ectoderm undergoes a period of rapid proliferation and morphogenetic movements to form a neural tube, as signals from adjacent tissues induce dorsal–ventral and anterior-posterior identities on precursors (Altmann and Brivanlou 2001). BMPs are expressed at high levels in nonneural ectoderm and then in the roof plate of the neural tube (Liem et al. 1995; Furuta et al. 1997). A BMP activity gradient induces dorsal identity markers indicative of neural crest or dorsal interneuron precursors (Liem et al. 1997; Nguyen et al. 1998, 2000; Barth et al. 1999). However, BMPs also cause apoptosis in early central nervous system (CNS) precursor cells (Graham et al. 1996; Furuta et al. 1997), neuronal differentiation in mid-gestation CNS precursors (Li et al. 1998; Mehler et al. 2000), and glial differentiation in late embryonic or adult CNS precursors (Gross et al. 1996). It is unclear how BMPs mediate such a wide variety of responses, but it may be a result of diversity in the components of signal transduction. Some BMP-related ligands are known to be involved in the generation of specific cell types (Shah et al. 1996; Lee et al. 1998), but there is evidence that many act in a redundant manner (Dudley and Robertson 1997; Solloway et al. 1998; Zhao et al. 1999). BMPs exert their effects by activating a complex of type I and type II receptors. The type II receptor is the primary ligand-binding component; both BMPRII and ActRIIB are functional type II receptors for BMPs. Type I receptors also have BMP-binding properties but are primarily responsible for transducing the signal into the cell: BMPR-IA (Alk3), BMPR-IB (Alk6), and ActR-I (Alk2) are all known to transduce BMP signals (Kawabata et al. 1998). The activated type I receptors in turn phosphorylate the DNA-binding proteins Smad1, Smad5, and Smad8, each of which can then heteromerize with Smad4 and translocate to the nucleus to regulate transcription of downstream target genes (Itoh et al. 2000). The expression patterns of Bmpr-1a and Bmpr-1b in the CNS (Dewulf et al. 1995; Zhang et al. 1998) suggest that they have distinct roles in the transduction of BMP signals. In an effort to understand their role in neural development, we used multipotent CNS stem cells as a defined system to understand signals that control proliferation and fate choice (Johe et al. 1996; Studer et al. 1998). We have also developed a vector using POU-regulated genomic elements that control expression of the intermediate filament gene nestin in CNS precursor cells (Josephson et al. 1998). This vector, called pNERV, expresses mutant BMP receptors selectively in CNS stem cells in vitro and in transgenic mice. Using these tools, we show that most of the known actions of BMPs during neural precursor development can be attributed to the distinct actions of BMPR-IA and BMPR-IB. Furthermore, the activities of these receptors are linked sequentially, with BMPR-IA acting first and inducing the expression of Bmpr-1b. These results identify a feed-forward mechanism by which BMPs can control both the production and fate of precursor cells.

Notochord signals to the endoderm are required for development of the chick dorsal pancreas. Sonic hedgehog (SHH) is normally absent from pancreatic endoderm, and we provide evidence that notochord, in contrast to its effects on adjacent neuroectoderm where SHH expression is induced, represses SHH expression in adjacent nascent pancreatic endoderm. We identify activin-βB and FGF2 as notochord factors that can repress endodermal SHH and thereby permit expression of pancreas genes including Pdx1 and insulin. Endoderm treatment with antibodies that block hedgehog activity also results in pancreatic gene expression. Prevention of SHH expression in prepancreatic dorsal endoderm by intercellular signals, like activin and FGF, may be critical for permitting early steps of chick pancreatic development.

Embryonic patterning in vertebrates is dependent upon the balance of inductive signals and their specific antagonists. We show thatNoggin, which encodes a bone morphogenetic protein (BMP) antagonist expressed in the node, notochord, and dorsal somite, is required for normal mouse development. Although Noggin has been implicated in neural induction, examination of null mutants in the mouse indicates that Noggin is not essential for this process. However, Noggin is required for subsequent growth and patterning of the neural tube. Early BMP-dependent dorsal cell fates, the roof plate and neural crest, form in the absence of Noggin. However, there is a progressive loss of early, Sonic hedgehog(Shh)-dependent ventral cell fates despite the normal expression of Shh in the notochord. Further, somite differentiation is deficient in both muscle and sclerotomal precursors. Addition of BMP2 or BMP4 to paraxial mesoderm explants blocks Shh-mediated induction of Pax-1, a sclerotomal marker, whereas addition of Noggin is sufficient to induce Pax-1. Noggin and Shh induce Pax-1 synergistically. Use of protein kinase A stimulators blocks Shh-mediated induction of Pax-1, but not induction by Noggin, suggesting that induction is mediated by different pathways. Together these data demonstrate that inhibition of BMP signaling by axially secreted Noggin is an important requirement for normal patterning of the vertebrate neural tube and somite.

Bone morphogenetic proteins (BMPs), which have been implicated in the patterning of mesoderm, are members of the transforming growth factor-beta (TGF-beta) superfamily. We have investigated the roles of Xenopus BMP-7 (XBMP-7) and BMP-4 (XBMP-4), and activin (another TGF-beta-related molecule) in early development by generating dominant-negative versions of these growth factors. Mutations were generated by altering the cleavage sites that are required for maturation of the active dimeric forms of XBMP-7, XBMP-4, and activin. These mutant constructs, designated Cm-XBMP-7, Cm-XBMP-4, and Cm-activin, result in polypeptides that allow for dimerization of the subunits, but are incapable of maturation. Expression of Cm-XBMP-7 and Cm-XBMP-4, but not Cm-activin, in the ventral marginal zone of the Xenopus embryo results in the development of a secondary axis, similar to that seen by ectopic expression of the truncated BMP receptor. These results suggest that the cleavage mutants interfere with BMP signaling during mesodermal patterning. We also found that expression of Cm-XBMP-7 or Cm-XBMP-4 in animal cap ectoderm directly induces neuroectoderm. The neural induction was specific for Cm-XBMP-7 and Cm-XBMP-4 because ectopic expression of Cm-activin or Vg-1 did not mimic the same phenotype. Molecular study of neural patterning by Cm-XBMP-7 and Cm-XBMP-4 revealed that only anterior neuroectodermal markers are expressed in response to these Cm-XBMPs. These results suggest that the BMPs are involved in the specification of ectoderm in Xenopus development, and that neural induction requires the removal of BMP signals in the ectoderm. We propose that neural induction occurs by a default mechanism, whereby the inhibition of BMP signaling is required for the conversion of ectoderm to neuroectoderm in the developing Xenopus embryo.

We have isolated genomic and cDNA clones of Brca1, a mouse homolog of the recently cloned breast cancer-associated gene, BRCA1. Brca1 encodes an 1812-amino-acid protein with a conserved zinc finger domain and significant homology to the human protein. Brca1 maps to Chromosome 11 within a region of conserved synteny with human chromosome 17, consistent with the mapping of the human gene to 17q21. Brca1 transcripts are expressed in a variety of cultured cells but reveal a specific and dynamic expression pattern during embryonic development. For example, expression is observed first in the otic vesicle of embryonic day 9.5 (E9.5) embryos. This expression diminishes and is replaced by expression in the neuroectoderm at E10.5. By E11-12.5, higher levels are observed in differentiating keratinocytes and in whisker pad primordia. Transcripts also become evident in epithelial cells of the E14-17 kidney. Brca1 expression occurs in differentiating epithelial cells of several adult organs as well, suggesting a general role in the functional maturation of these tissues. Consistent with this, Brca1 transcripts are expressed in both alveolar and ductal epithelial cells of the mammary gland. During pregnancy, there is a large increase in Brca1 mRNA in mammary epithelial cells, an increase that parallels their functional differentiation. Because high rates of breast cancer are associated with loss of BRCA1 in humans, it is possible that this gene provides an important growth regulatory function in mammary epithelial cells. In addition, increased transcription of mammary Brca1 during pregnancy might contribute, in part, to the reduced cancer risk associated with exposure to pregnancy and lactation.

The dorsal (dl) nuclear gradient initiates the differentiation of the mesoderm, neuroectoderm, and dorsal ectoderm by activating and repressing gene expression in the early Drosophila embryo. This gradient is organized by a Toll signaling pathway that shares many common features with the mammalian IL-1 cytokine pathway. Here we present evidence that a second signaling pathway, controlled by the torso (tor) receptor tyrosine kinase, also modulates dl activity. Evidence is presented that the tor pathway selectively masks the ability of dl to repress gene expression but has only a slight effect on activation. Intracellular kinases that are thought to function downstream of tor, such as D-raf and the rolled MAP kinase, mediate this selective block in repression. Normally, the Toll and tor pathways are both active only at the embryonic poles, and consequently, target genes (zen and dpp) that are repressed in middle body regions are expressed at these sites. Constitutive activation of the tor pathway causes severe embryonic defects, including disruptions in gastrulation and mesoderm differentiation, as a result of misregulation of dl target genes. These results suggest that RTK signaling pathways can control gene expression by antirepression, and that multiple pathways can fine-tune the activities of a single transcription factor.

The first step in the differentiation of the Drosophila mesoderm is the activation of two regulatory genes, twist (twi) and snail (sna), in ventral regions of early embryos. sna is a transcriptional repressor that is uniformly expressed throughout the presumptive mesoderm. Its sharp lateral limits help to establish the boundary between the mesoderm and neuroectoderm. Genetic studies suggest that sna is a target of the dorsal (dl) morphogen, and this interaction provides a model for determining how a morphogen gradient establishes a sharp, on/off threshold response. We present evidence that dl and twi directly activate sna expression. Site-directed mutagenesis of dl- and twi-binding sites within defined regions of the sna promoter suggest that the two proteins (containing the Rel and helix-loop-helix domains, respectively) function multiplicatively to ensure strong, uniform expression of sna, particularly in ventral-lateral regions where there are diminishing amounts of dl. These results are consistent with the possibility that the sharp sna borders are formed by multiplying the shallow dl gradient and the steeper twi gradient.

Three embryonic tissue sources—the neural ectoderm, the surface ectoderm, and the periocular mesenchyme—contribute to the formation of the mammalian eye. For this reason, the developing eye has presented an invaluable system for studying the interactions among cells and, more recently, genes, in specifying cell fate. This article describes how the eye primordium is specified in the anterior neural plate by four eye field transcription factors and how the optic vesicle becomes regionalized into three distinct tissue types. Specific attention is given to how cross talk between the optic vesicle and surface ectoderm contributes to lens and optic cup formation. This article also describes how signaling networks and cell movements set up axes in the optic cup and establish the multiple cell fates important for vision. How multipotent retinal progenitor cells give rise to the six neuronal and one glial cell type in the mature retina is also explained. Finally, the history and progress of cellular therapeutics for the treatment of degenerative eye disease is outlined. Throughout this article, special attention is given to how disruption of gene function causes ocular malformation in humans. Indeed, the accessibility of the eye has contributed much to our understanding of the basic processes involved in mammalian development.

A gradient of Dorsal activity patterns the dorsoventral (DV) axis of the early Drosophila melanogaster embryo by controlling the expression of genes that delineate presumptive mesoderm, neuroectoderm, and dorsal ectoderm. The availability of the Drosophila melanogaster genome sequence has accelerated the study of embryonic DV patterning, enabling the use of systems-level approaches. As a result, our understanding of Dorsal-dependent gene regulation has expanded to encompass a collection of more than 50 genes and 30 cis-regulatory sequences. This information, which has been integrated into a spatiotemporal atlas of gene regulatory interactions, comprises one of the best-understood networks controlling any developmental process to date. In this article, we focus on how Dorsal controls differential gene expression and how recent studies have expanded our understanding of Drosophila embryonic development from the cis-regulatory level to that controlling morphogenesis of the embryo.