Early Neural Patterning Research Articles

Hif1α and Wnt are required for posterior gene expression during Xenopus tropicalis tail regeneration

Regeneration of complex tissues is initiated by an injury-induced stress response, eventually leading to activation of developmental signaling pathways such as Wnt signaling. How early injury cues are interpreted and coupled to activation of these developmental signals and their targets is not well understood. Here, we show that Hif1α, a stress induced transcription factor, is required for tail regeneration in Xenopus tropicalis. We find that Hif1α is required for regeneration of differentiated axial tissues, including axons and muscle. Using RNA-sequencing, we find that Hif1α and Wnt converge on a broad set of genes required for posterior specification and differentiation, including the posterior hox genes. We further show that Hif1α is required for transcription via a Wnt-responsive element, a function that is conserved in both regeneration and early neural patterning. Our findings indicate that Hif1α has regulatory roles in Wnt target gene expression across multiple tissue contexts.

Modeling Bainbridge-Ropers Syndrome in Xenopus laevis Embryos.

The Additional sex combs-like (ASXL1-3) genes are linked to human neurodevelopmental disorders. The de novo truncating variants in ASXL1-3 proteins serve as the genetic basis for severe neurodevelopmental diseases such as Bohring-Opitz, Shashi-Pena, and Bainbridge-Ropers syndromes, respectively. The phenotypes of these syndromes are similar but not identical, and include dramatic craniofacial defects, microcephaly, developmental delay, and severe intellectual disability, with a loss of speech and language. Bainbridge-Ropers syndrome resulting from ASXL3 gene mutations also includes features of autism spectrum disorder. Human genomic studies also identified missense ASXL3 variants associated with autism spectrum disorder, but lacking more severe Bainbridge-Ropers syndromic features. While these findings strongly implicate ASXL3 in mammalian brain development, its functions are not clearly understood. ASXL3 protein is a component of the polycomb deubiquitinase complex that removes mono-ubiquitin from Histone H2A. Dynamic chromatin modifications play important roles in the specification of cell fates during early neural patterning and development. In this study, we utilize the frog, Xenopus laevis as a simpler and more accessible vertebrate neurodevelopmental model system to understand the embryological cause of Bainbridge-Ropers syndrome. We have found that ASXL3 protein knockdown during early embryo development highly perturbs neural cell fate specification, potentially resembling the Bainbridge-Ropers syndrome phenotype in humans. Thus, the frog embryo is a powerful tool for understanding the etiology of Bainbridge-Ropers syndrome in humans.

Amphibian Zic Genes.

Studies in Xenopus laevis have greatly contributed to understanding the roles that the Zic family of zinc finger transcription factors play as essential drivers of early development. Explant systems that are not readily available in other organisms give Xenopus embryos a unique place in these studies, facilitated by the recent sequencing of the Xenopus laevis genome. A number of upstream regulators of zic gene expression have been identified, such as inhibition of BMP signaling, as well as calcium, FGF, and canonical Wnt signaling. Screens using induced ectodermal explants have identified genes that are direct targets of Zic proteins during early neural development and neural crest specification. These direct targets include Xfeb (also called glipr2; hindbrain development), aqp3b (dorsal marginal zone in gastrula embryos and neural folds), snail family members (premigratory neural crest), genes that play roles in retinoic acid signaling, noncanonical Wnt signaling, and mesoderm development, in addition to a variety of genes some with and many without known roles during neural or neural crest development. Functional experiments in Xenopus embryos demonstrated the involvement of Zic family members in left-right determination, early neural patterning, formation of the midbrain-hindbrain boundary, and neural crest specification. The role of zic genes in cell proliferation vs. differentiation remains unclear, and the activities of Zic factors as inhibitors or activators of canonical Wnt signaling may be dependent on developmental context. Overall, Xenopus has contributed much to our understanding of how Zic transcriptional activities shape the development of the embryo and contribute to disease.

Methylmercury exposure during early Xenopus laevis development affects cell proliferation and death but not neural progenitor specification

Methylmercury (MeHg) is a widespread environmental toxin that preferentially and adversely affects developing organisms. To investigate the impact of MeHg toxicity on the formation of the vertebrate nervous system at physiologically relevant concentrations, we designed a graded phenotype scale for evaluating Xenopus laevis embryos exposed to MeHg in solution. Embryos displayed a range of abnormalities in response to MeHg, particularly in brain development, which is influenced by both MeHg concentration and the number of embryos per ml of exposure solution. A TC50 of ~50μg/l and LC50 of ~100μg/l were found when maintaining embryos at a density of one per ml, and both increased with increasing embryo density. In situ hybridization and microarray analysis showed no significant change in expression of early neural patterning genes including sox2, en2, or delta; however a noticeable decrease was observed in the terminal neural differentiation genes GAD and xGAT, but not xVGlut. PCNA, a marker for proliferating cells, was negatively correlated with MeHg dose, with a significant reduction in cell number in the forebrain and spinal cord of exposed embryos by tadpole stages. Conversely, the number of apoptotic cells in neural regions detected by a TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay was significantly increased. These results provide evidence that disruption of embryonic neural development by MeHg may not be directly due to a loss of neural progenitor specification and gene transcription, but to a more general decrease in cell proliferation and increase in cell death throughout the developing nervous system.

Evolutionary Development of Neural Systems in Vertebrates and Beyond

The emerging field of “neuro-evo-devo” is beginning to reveal how the molecular and neural substrates that underlie brain function are based on variations in evolutionarily ancient and conserved neurochemical and neural circuit themes. Comparative work across bilaterians is reviewed to highlight how early neural patterning specifies modularity of the embryonic brain, which lays a foundation on which manipulation of neurogenesis creates adjustments in brain size. Small variation within these developmental mechanisms contributes to the evolution of brain diversity. Comparing the specification and spatial distribution of neural phenotypes across bilaterians has also suggested some major brain evolution trends, although much more work on profiling neural connections with neurochemical specificity across a wide diversity of organisms is needed. These comparative approaches investigating the evolution of brain form and function hold great promise for facilitating a mechanistic understanding of how variation in brain morphology, neural phenotypes, and neural networks influences brain function and behavioral diversity across organisms.

Neurologic and ocular phenotype in Pitt–Hopkins syndrome and a zebrafish model

In this study, we performed an in-depth analysis of the neurologic and ophthalmologic phenotype in a patient with Pitt-Hopkins syndrome (PTHS), a disorder characterized by severe mental and motor retardation, carrying a uniallelic TCF4 deletion, and studied a zebrafish model. The PTHS-patient was characterized by high-resolution magnetic resonance imaging (MRI) with diffusion tensor imaging to analyze the brain structurally, spectral-domain optical coherence tomography to visualize the retinal layers, and electroretinography to evaluate retinal function. A zebrafish model was generated by knockdown of tcf4-function by injection of morpholino antisense oligos into zebrafish embryos and the morphant phenotype was characterized for expression of neural differentiation genes neurog1, ascl1b, pax6a, zic1, atoh1a, atoh2b. Data from PTHS-patient and zebrafish morphants were compared. While a cerebral MRI-scan showed markedly delayed myelination and ventriculomegaly in the 1-year-old PTHS-patient, no structural cerebral anomalies including no white matter tract alterations were detected at 9years of age. Structural ocular examinations showed highly myopic eyes and an increase in ocular length, while retinal layers were normal. Knockdown of tcf4-function in zebrafish embryos resulted in a developmental delay or defects in terminal differentiation of brain and eyes, small eyes with a relative increase in ocular length and an enlargement of the hindbrain ventricle. In summary, tcf4-knockdown in zebrafish embryos does not seem to affect early neural patterning and regionalization of the forebrain, but may be involved in later aspects of neurogenesis and differentiation. We provide evidence for a role of TCF4/E2-2 in ocular growth control in PTHS-patients and the zebrafish model.

Zebrafish model of holoprosencephaly demonstrates a key role for TGIF in regulating retinoic acid metabolism

Holoprosencephaly (HPE) is the most common human congenital forebrain defect, affecting specification of forebrain tissue and subsequent division of the cerebral hemispheres. The causes of HPE are multivariate and heterogeneous, and include exposure to teratogens, such as retinoic acid (RA), and mutations in forebrain patterning genes. Many of the defects in HPE patients resemble animal models with aberrant RA levels, which also show severe forebrain abnormalities. RA plays an important role in early neural patterning of the vertebrate embryo: expression of RA-synthesizing enzymes initiates high RA levels in the trunk, which are required for proper anterior-posterior patterning of the hindbrain and spinal cord. In the forebrain and midbrain, RA-degrading enzymes are expressed, protecting these regions from the effects of RA. However, the mechanisms that regulate RA-synthesizing and RA-degrading enzymes are poorly understood. Mutations in the gene TGIF are associated with incidence of HPE. We demonstrate in zebrafish that Tgif plays a key role in regulating RA signaling, and is essential to properly pattern the forebrain. Tgif is necessary for normal initiation of genes that control RA synthesis and degradation, resulting in defects in RA-dependent central nervous system patterning in Tgif-depleted embryos. The loss of the forebrain-specific RA-degrading enzyme cyp26a1 causes a forebrain phenotype that mimics tgif morphants. We propose a model in which Tgif controls forebrain patterning by regulating RA degradation. The consequences of abnormal RA levels for forebrain patterning are profound, and imply that in human patients with TGIF deficiencies, increased forebrain RA levels contribute to the development of HPE.

Integration of multiple signal transducing pathways on Fgf response elements of the Xenopus caudal homologue Xcad3.

Early neural patterning along the anteroposterior (AP) axis appears to involve a number of signal transducing pathways, but the precise role of each of these pathways for AP patterning and how they are integrated with signals that govern neural induction step is not well understood. We investigate the nature of Fgf response element (FRE) in a posterior neural gene, Xcad3 (Xenopus caudal homologue) that plays a crucial role of posterior neural development. We provide evidence that FREs of Xcad3 are widely dispersed in its intronic sequence and that these multiple FREs comprise Ets-binding and Tcf/Lef-binding motifs that lie in juxtaposition. Functional and physical analyses indicate that signaling pathways of Fgf, Bmp and Wnt are integrated on these FREs to regulate the expression of Xcad3 in the posterior neural tube through positively acting Ets and Sox family transcription factors and negatively acting Tcf family transcription factor(s).

Distinct roles for Fgf, Wnt and retinoic acid in posteriorizing the neural ectoderm.

Early neural patterning in vertebrates involves signals that inhibit anterior (A) and promote posterior (P) positional values within the nascent neural plate. In this study, we have investigated the contributions of, and interactions between, retinoic acid (RA), Fgf and Wnt signals in the promotion of posterior fates in the ectoderm. We analyze expression and function of cyp26/P450RAI, a gene that encodes retinoic acid 4-hydroxylase, as a tool for investigating these events. Cyp26 is first expressed in the presumptive anterior neural ectoderm and the blastoderm margin at the late blastula. When the posterior neural gene hoxb1b is expressed during gastrulation, it shows a strikingly complementary pattern to cyp26. Using these two genes, as well as otx2 and meis3 as anterior and posterior markers, we show that Fgf and Wnt signals suppress expression of anterior genes, including cyp26. Overexpression of cyp26 suppresses posterior genes, suggesting that the anterior expression of cyp26 is important for restricting the expression of posterior genes. Consistent with this, knock-down of cyp26 by morpholino oligonucleotides leads to the anterior expansion of posterior genes. We further show that Fgf- and Wnt-dependent activation of posterior genes is mediated by RA, whereas suppression of anterior genes does not depend on RA signaling. Fgf and Wnt signals suppress cyp26 expression, while Cyp26 suppresses the RA signal. Thus, cyp26 has an important role in linking the Fgf, Wnt and RA signals to regulate AP patterning of the neural ectoderm in the late blastula to gastrula embryo in zebrafish.

Floor plate develops upon depletion of tiggy-winkle and sonic hedgehog.

Hedgehog signaling is thought to play an important role in the differentiation of the vertebrate neural tube (Tanabe and Jessell, 1996). By injecting a morpholino (MO) directed against tiggy-winkle hedgehog (twhh) (Ekker et al., 1995) into zebrafish sonic-you (syu) embryos mutant for the sonic hedgehog (shh) gene (Schauerte et al., 1998), we examined Hedgehog-dependent and independent aspects of early neural patterning. The floor plate (FP) is a specialized row of cells located in the ventral midline of the vertebrate neural tube that influences axon guidance and neuronal differentiation (Placzek et al., 2000). Experiments performed on chick embryos indicated that the FP is specified by Shh signaling from the notochord to the overlying neural plate (Tanabe and Jessell, 1996). This model of a classic cellular induction was supported by studies showing that notochord-derived signals induce FP formation (Yamada et al., 1991), and that notochord removal results in loss of FP (Placzek et al., 1990). Analyses of chick-quail chimeric embryos and of zebrafish mutants with midline defects suggest that FP specification commences before notochord formation (Le Douarin and Halpern, 2000). When Hensen’s node of chick embryos is replaced by the quail counterpart, for example, the regressing node produces FP, notochord, and dorsal endoderm of quail origin (Charrier et al., 1999). Together with the finding that FP has a different origin than the rest of the neural plate (Catala et al., 1996), the data suggest that notochord and FP originate from a population of multipotential midline precursor cells that reside in Hensen’s node in the chick (Le Douarin and Halpern, 2000). Analogous cells are predicted to be found in the dorsal shield or early organizer region of the zebrafish gastrula (Halpern et al., 1997; Appel et al., 1999). Zebrafish floating head (flh) and no tail (ntl) mutants develop FP even though they lack an Shh-expressing notochord (Halpern et al., 1993, 1995; Talbot et al., 1995). In fact, ntl mutants develop a wider FP than wild-type (WT) embryos (Odenthal et al., 1996; Strahle et al., 1996; Halpern et al., 1997), suggesting that ntl regulates allocation of notochord and FP from the proposed common precursor population (Halpern et al., 1997). The Notch-Delta signaling pathway has been similarly implicated in mediating cell fate choices in the zebrafish gastrula midline (Appel et al., 1999). The role of Shh in FP formation has also come under question from the study of zebrafish syu/shh mutants. Homozygous null syu mutants develop the medial FP row, indicating that Shh signaling is not essential for induction of these cells (Schauerte et al., 1998; Odenthal et al., 2000). One explanation is that the shh paralogue twhh is also expressed in the dorsal shield and in the gastrula midline, where it could function redundantly with shh in FP specification (Fig. 1a–e). Later, during segmentation, shh is expressed in the developing notochord and FP (Krauss et al., 1993), whereas twhh expression is confined to the FP (Ekker et al., 1995). Expression of a third zebrafish gene, echidna hedgehog (ehh), is first detected at midgastrulation, following shh and twhh expression in the midline (Currie and Ingham, 1996), and presumably too late to be involved in the initial steps of FP specification. Recent experiments using the ehh-MO support this view (Lewis and Eisen, 2001). An argument in favor of the early specification of FP comes from the analysis of FP-specific gene expression, which is evident shortly after cells emerge from the dorsal shield during extension of the embryonic axis. We find that twhh and shh expression segregates into discrete cell layers during gastrulation (Fig. 1c–e), with shh transcripts colocalized to the Ntl immunoreactive cells of the presumptive notochord and twhh transcripts confined to overlying cells (Fig. 1c, d). These cells are presumed to develop as FP on the basis of their position and continuous and specific expression of twhh. Al-

Initial patterning of the central nervous system: how many organizers?

For three-quarters of a century, developmental biologists have been asking how the nervous system is specified as distinct from the rest of the ectoderm during early development, and how it becomes subdivided initially into distinct regions such as forebrain, midbrain, hindbrain and spinal cord. The two events of 'neural induction' and 'early neural patterning' seem to be intertwined, and many models have been put forward to explain how these processes work at a molecular level. Here I consider early neural patterning and discuss the evidence for and against the two most popular models proposed for its explanation: the idea that multiple signalling centres (organizers) are responsible for inducing different regions of the nervous system, and a model first articulated by Nieuwkoop that invokes two steps (activation/transformation) necessary for neural patterning. As recent evidence from several systems challenges both models, I propose a modification of Nieuwkoop's model that most easily accommodates both classical and more recent data, and end by outlining some possible directions for future research.

FGF is required for posterior neural patterning but not for neural induction.

Fibroblast growth factor (FGF) has been implicated in a variety of developmental processes including posterior mesoderm and neural patterning. Previous work has led to contradictory roles for FGF in neural induction and anteroposterior neural patterning. Launay et al. (Development 122, 869-880, 1996) suggested a requirement for FGF in anterior neural induction. In contrast, Kroll and Amaya (Development 122, 3173-3183, 1996) and Bang et al. (Development 124, 2075-2085, 1997) proposed that FGF is not required for early neural patterning. Here we use a loss-of-function assay to examine whether FGF is required for neural patterning in three experimental situations: (i) in Xenopus early embryos, (ii) in embryonic explants consisting of presumptive dorsal mesoderm and neurectoderm (Keller explants), and (iii) in explants of dorsal ectoderm and posterior mesoderm in which FGF signaling is specifically blocked in the ectoderm. When cultured until tailbud stages, Keller explants develop neural tissue with normal anteroposterior pattern. Overexpression of the dominant-negative FGF receptor (XFD) in Keller explants inhibited the posterior neural markers En-2, Krox-20, and HoxB9, but not the panneural marker nrp-1 and the anterior neurectodermal markers XAG-1 and Xotx-2. Similar results were seen in whole embryos, but only when XFD RNA was targeted to both the dorsal and lateral regions. In contrast, addition of FGF to Keller explants resulted in a shift of the midbrain-hindbrain boundary marker En-2 to a more anterior position normally fated to become cement gland. We also determined whether FGF is required specifically by the neurectoderm for anteroposterior neural patterning. Recombinants of dorsal ectoderm and posterior mesoderm were made in which FGF was specifically blocked in the ectoderm. Spinal cord and hindbrain markers were inhibited in these recombinants, whereas anterior markers and cement gland development were enhanced. Our results demonstrate that FGF is important for posterior development in both mesoderm and neurectoderm and that neural induction and posteriorization represent separable developmental events.

Guidance of circumferentially growing axons by the floor plate in the vertebrate central nervous system.

We describe the recent progress of studies on the mechanisms of early neural patterning, focusing on the guidance of circumferentially migrating axons in the vertebrate brain. We also refer to the underlying molecular mechanisms revealed by recent studies.

From gastrulation to neurulation: Transition in retinoic acid sensitivity identifies distinct stages of neural patterning in the rat

Early neural development is a multistep process with morphologically distinct stages; however, the molecular events that underlie morphologic development are poorly understood. Retinoic acid (RA) was chosen as a teratogen to perturb development because this endogenous molecule is thought to play an integral role in normal neuraxis formation in many vertebrate species. We have examined the effects of RA on early neural patterning in the rat at three morphologically distinct stages: late streak, foregut pocket, and early somite. In this model exogenous RA exposure during mid-gastrulation (late streak stage) leads to severe disruption of anterior neural development as determined by morphologic and molecular (Engrailed [En] gene expression) markers. This disruption in anterior neural development is associated with excessive cell death in the hindbrain posterior to the En expression domain. In contrast, at the time the neural folds begin to elevate (foregut pocket stage) there is a dramatic reduction in the sensitivity of anterior neural development to exogenous RA as reflected by En expression and cell death patterns. These results suggest that we have identified a major transition in the development of the anterior neuraxis that is reflected in a transition in sensitivity to RA. This transition in sensitivity demonstrates that the fundamental patterning mechanisms that separate fore- and midbrain from hindbrain occurs very early in neurogenesis.