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The patterning of the CNS relies on the interaction of multiple signaling molecules such as Sonic Hedgehog, Wnts, and BMPs and their antagonists Chordin and Noggin. The identification of the secreted molecule Tiarin (Tsuda et al., 2002, this issue of Neuron), produced by the nonneural ectoderm at border of the anterior and lateral neural plate, now introduces a novel signaling pathway participating in CNS development.
The relation between the role of the organizer at the gastrula stage and the activity of earlier signals in the specification, maintenance, and regionalization of the developing brain anlage is still controversial. Mouse embryos homozygous for null mutation in the cripto gene die at about 9.0 days postcoitum (d.p.c.) and fail to gastrulate and to form the node (the primary organizer). Here, we study the presence and the distribution of anterior neural plate molecular domains in cripto null mutants. We demonstrate that, in cripto(-/-) embryos, the main prosencephalic and mesencephalic regions are present and that they assume the correct topological organization. The identity of the anterior neural domains is maintained in mutant embryos at 8.5 d.p.c., as well as in mutant explants dissected at 8.5 d.p.c. and cultured in vitro for 24 h. Our data imply the existence of a stable neural regionalization of anterior character inside the cripto(-/-) embryos, despite the failure in both the gastrulation process and node formation. These results suggest that, in mouse embryos, the specification of the anterior neural identities can be maintained without an absolute requirement for the embryonic mesoderm and the node.
Neuronal differentiation is regulated by both positive and negative regulatory factors; however, precisely how these factors interact to regulate retinogenesis is still unclear. We have examined the ability of the Notch pathway to modulate the function of the basic helix-loop-helix factor Xath5. Overexpression of Xath5 by RNA injection into cleavage-stage blastomeres promotes ectopic neurogenesis at neural plate stages and ganglion cell differentiation in the developing retina. We found that these activities of Xath5 could be inhibited by coexpression of activated Notch. Notch inhibition of Xath5 function was reversed by coexpression with the zinc finger protein X-MyT1. The Notch effector enhancer-of-split related 1 (ESR1) also blocked Xath5 activity but efficient inhibition by ESR1 required the DNA binding basic domain and the conserved WRPW motif. In addition, ESR1 inhibited the ability of Xath5 to directly activate the expression of XBrn3d, a transcription factor involved in retinal ganglion cell development. Xath5 could upregulate expression of X-Delta-1, ESR1, and ESR3, suggesting that Xath5 participates in a regulatory loop with the Notch pathway.
Amphioxus, as the closest living invertebrate relative of the vertebrates, can give insights into the evolutionary origin of the vertebrate body plan. Therefore, to investigate the evolution of genetic mechanisms for establishing and patterning the neuroectoderm, we cloned and determined the embryonic expression of two amphioxus transcription factors, AmphiSox1/2/3 and AmphiNeurogenin. These genes are the earliest known markers for presumptive neuroectoderm in amphioxus. By the early neurula stage, AmphiNeurogenin expression becomes restricted to two bilateral columns of segmentally arranged neural plate cells, which probably include precursors of motor neurons. This is the earliest indication of segmentation in the amphioxus nerve cord. Later, expression extends to dorsal cells in the nerve cord, which may include precursors of sensory neurons. By the midneurula, AmphiSox1/2/3 expression becomes limited to the dorsal part of the forming neural tube. These patterns resemble those of their vertebrate and Drosophila homologs. Taken together with the evolutionarily conserved expression of the dorsoventral patterning genes, BMP2/4 and chordin, in nonneural and neural ectoderm, respectively, of chordates and Drosophila, our results are consistent with the evolution of the chordate dorsal nerve cord and the insect ventral nerve cord from a longitudinal nerve cord in a common bilaterian ancestor. However, AmphiSox1/2/3 differs from its vertebrate homologs in not being expressed outside the CNS, suggesting that additional roles for this gene have evolved in connection with gene duplication in the vertebrate lineage. In contrast, expression in the midgut of AmphiNeurogenin together with the gene encoding the insulin-like peptide suggests that amphioxus may have homologs of vertebrate pancreatic islet cells, which express neurogenin3. In addition, AmphiNeurogenin, like its vertebrate and Drosophila homologs, is expressed in apparent precursors of epidermal chemosensory and possibly mechanosensory cells, suggesting a common origin for protostome and deuterostome epidermal sensory cells in the ancestral bilaterian.
Proper development of metazoan embryos requires cell to cell communications. In many instances, these communications involve diffusible molecules, particularly members of the Transforming Growth Factor beta superfamily. In an effort to identify new members of this superfamily involved in the control of early zebrafish embryogenesis, we have isolated a gene, Radar, which appears to be conserved throughout vertebrate evolution and defines a new subfamily within the superfamily. Its pattern of expression suggests that Radar plays a role in the dorso-ventral polarity of the neural plate, blood islands formation, blood cells differentiation, the establishment of retinal dorso-ventral polarity and/or proper axonal retinotectal projections. Radar expression in ntl homozygous mutants indicates that notochord and hypochord development are intimately linked.
Cysteine-rich repeats (CRs) of the type described in Chordin constitute conserved domains present in an expanding family of secreted molecules. These motifs were shown to mediate directly the antagonism of BMP signaling by Chordin and play a major role during development. Here we report the cloning and expression pattern of neuralin-1, a new member of the chordin family. The mouse cDNA was cloned by homology with a human genomic sequence encoding putative CRs. In the human genome, neuralin-1 transcripts are encoded by 8 exons that span a region of at least 80 kilobases located on chromosome Xq22.1-23. Neuralin-1 is a 333 amino acid protein containing three CRs, two of them highly similar to the Chordin CRs that bind BMP. Like chordin, neuralin-1 is able to induce secondary axes after mRNA injection in Xenopus embryos. Interestingly, during late gastrulation, neuralin-1 and chordin present distinct and complementary expression patterns in the mouse: neuralin-1 expression starts in the neural plate at mid-gastrulation, whereas chordin expression at that stage is restricted to the node and midline mesendoderm. Later on, neuralin-1 expression becomes restricted to discrete regions of the central nervous system and to derivatives of the neural crest cells. During organogenesis, neuralin-1 presents a broad expression pattern in many tissues such as dorsal root ganglia, gut, condensing cartilages of the skeleton and developing hair follicles.
Glucose is an important cellular nutrient, and in the early embryo, which is dependent mostly on anaerobic glycolysis, it is even more essential. Based on tissue culture cells in which glucose utilization has become membrane-limited, a concept has been developed that the tip of the microvilli is the entrance compartment for glucose and that the shaft sets up a diffusion barrier. An increase in length of the microvillus is associated with decreased entry of phosphorylated hexose into the cells. Our previous findings of lengthening of the microvilli of the neural plate cells after 40 min exposure to glucose at room temperature have been extended to a 17 hr whole embryo culture system. In cultures where the final concentration of glucose was 20 and 24 mg/dl there was embryonic death. In those cultures ending with 29-137 mg/dl of glucose the embryos developed normally. Those grown in dialyzed serum supplemented with B vitamins and glucose grew equally as well as those in whole rat serum. Somite numbers attained did not change with increasing glucose concentration but a modest increase in micromoles of glucose used per embryo was found, suggesting the presence of another source of energy at lower glucose concentrations. The average glucose utilization per gram of protein per hour was 844 mumol in these day 9.5-10 embryos and this compares to 733 mumol previously found using uniformly labeled 14C glucose on day 10.3. Lactate production averaged 85% of the glucose utilized. Pyruvate did not support growth in the absence of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)
The generation of anterior-posterior polarity in the vertebrate brain requires the establishment of regional domains of gene expression at early somite stages. Wnt-1 encodes a signal that is expressed in the developing midbrain and is essential for midbrain and anterior hindbrain development. Previous work identified a 5.5 kilobase region located downstream of the Wnt-1 coding sequence which is necessary and sufficient for Wnt-1 expression in vivo. Using a transgenic mouse reporter assay, we have now identified a 110 base pair regulatory sequence within the 5.5 kilobase enhancer, which is sufficient for expression of a lacZ reporter in the approximate Wnt-1 pattern at neural plate stages. Multimers of this element driving Wnt-1 expression can partially rescue the midbrain-hindbrain phenotype of Wnt-1(-/-) embryos. The possibility that this region represents an evolutionarily conserved regulatory module is suggested by the identification of a highly homologous region located downstream of the wnt-1 gene in the pufferfish (Fugu rubripes). These sequences are capable of appropriate temporal and spatial activation of a reporter gene in the embryonic mouse midbrain; although, later aspects of the Wnt-1 expression pattern are absent. Genetic evidence has implicated Pax transcription factors in the regulation of Wnt-1. Although Pax-2 binds to the 110 base pair murine regulatory element in vitro, the location of the binding sites could not be precisely established and mutation of two putative low affinity sites did not abolish activation of a Wnt-1 reporter transgene in vivo. Thus, it is unlikely that Pax proteins regulate Wnt-1 by direct interactions with this cis-acting regulatory region. Our analysis of the 110 base pair minimal regulatory element suggests that Wnt-1 regulation is complex, involving different regulatory interactions for activation and the later maintenance of transgene expression in the dorsal midbrain and ventral diencephalon, and at the midbrain-hindbrain junction.
We have investigated the early development expressional of the heat shock protein genes (hsps) and HSP synthesis and their role during neuroectoderm induction, differentiation and early CNS formation. The expression and kinetics of 90, 73/71, 47 and 27 HSPs on neuroectoderm differentiation was compared under normal and stressed conditions. The role of HSPs on neuroctoderm cell fate including thermotolerance and apoptosis using a whole in vitro embryo culture system was studied. Hsp expression appears closely linked in early mammalian development to critical differentiation and proliferation stages in early brain and heart formation. The hsps are developmentally activated around blastula stage and HSPs are constitutively expressed at high levels during neural tube closure and are heat shock responsive. Using both Northern analysis, confocal microscopy and whole mount in situ hybridisation we have identified the mRNA hsp transcripts and HSPs during organogenesis. HSPs were detected during neuroectoderm cell induction and differentiation with the hsp mRNA being tightly regulated during the cell cycle of neuroectoderm especially at early fore-, mid-, hindbrain and heart formation. The 'chaperone' functions of the HSPs are well known, recently during gastrulation the HSP47 and 27 have been shown to specifically bind and fold to nascent collagen and actin molecules respectively. This role is essential for the formation of the basement membrane, extra cellular matrix and neural crest migration during neural plate development. HSP function was observed by using anti-sense strategy, short '5 anti-sense cDNA' hsp oligonucleotides inhibited hsp expression during gastrulation in the whole embryo cultures. The developmental activation of the heat shock element (HSE) is essential to our understanding of the HSPs role in neuronal cell fate. Using specific polyclonal antibodies to HSF1 and 2 (Dr Nakai, Kyoto University) the expression of heat shock factors (HSFs) during neuroectoderm differentiation was examined. Using Western analysis, confocal microscopy and flow cytometry HSF1 and 2 were identified and studied under both normal and heat shocked conditions. During gastrulation higher levels of HSF1 and 2 were identified in the neuroectoderm layer especially in regions of the fore-, mid- and hindbrain. The heat shock response and activation of the HSPs 90, 70, 47 and 27 families have been correlated with HSF1 and 2. The HSF1 appears to be present in all early embryonic cells but appears not to bind to the HSE until early head fold stage at gastrulation when the presence of HSF2 is observed. During neuroectoderm differentiation the activation of HSF1 and 2 appears to correlate with high constitutive expression of many of the hsps specifically hsp90, 73, 71, 47 and 27 being tightly regulated by the cell cycle at neurulation.
Targeted disruption of effectors molecules of the apoptotic pathway have demonstrated the occurrence and magnitude of early programmed cell death (EPCD), a form of apoptosis that affects proliferating and newly differentiated cells in vertebrates, and most dramatically cells of the central nervous system (CNS). Little is known about the molecular pathways controlling apoptosis at these early developmental stages, as the roles of EPCD during patterning of the developing nervous system. We describe a new function, in Xenopus neurodevelopment, for a highly conserved homeodomain protein Barhl2. Barhl2 promotes apoptosis in the Xenopus neuroectoderm and mesoderm, acting as a transcriptional repressor, through a mechanism that cannot be attributed to an unspecific cellular stress response. We show that the pro-apoptotic activity of Barhl2 is essential during normal neural plate formation as it limits the number of chordin- and Xshh-expressing cells in the prospective notochord and floorplate, which act as organizing centers. Our findings show that Barhl2 is part of a pathway regulating EPCD. They also provide evidence that apoptosis plays an important role in regulating the size of organizing centers.
During gastrulation, optimal adhesion and receptivity to signalling cues are essential for cells to acquire new positions and identities via coordinated cell movements. T-box transcription factors and the Wnt signalling pathways are known to play important roles in these processes. Zebrafish tbx2b, a member of the TBX2 family, has previously been shown to be required for the specification of midline mesoderm. We show here that tbx2b transcripts are present during mid-gastrula before its expression is detected by whole-mount in situ hybridization. Isolated ectodermal cells deficient in Tbx2b have altered cell surface properties and the level of cadherins in these cells is lower. In chimaeric embryos generated by cell transplantation and single blastomere injections, Tbx2b-deficient cells are defective in cell movement in a cell-autonomous manner, resulting in their exclusion from the developing neural plate. Using this ;exclusion' phenotype as a screen, we show that Tbx2b acts within the context of Fz7 signalling. The exclusion of cells lacking T-box proteins in chimeras during development was demonstrated with other T-box genes and may indicate a general functional mechanism for T-box proteins.
Regionalization of the embryonic brain is achieved through multi-step processes that operate sequentially and/or simultaneously. Localized sources of various signaling molecules act as organizing centers that pattern neighboring fields to create molecularly distinct domains. We investigated the mechanisms underlying the regionally distinct competence for two such organizing signals, Fibroblast growth factor 8 (Fgf8) and Sonic hedgehog (Shh), using chick embryos. First, we demonstrated that FGF receptor 1 (Fgfr1) and Fgfr3, expressed differentially in the developing brain, possess an equivalent potential to induce the regionally distinct Fgf8-responsive genes, depending on the anterior-posterior dimension of the brain. Next we found that homeodomain transcription factors Six3 and Irx3 can alter the regional responses to both Fgf8 and Shh in the forebrain. Six3 confers the ability to express Bf1, a gene essential for the telencephalon and eye development, and Nkx2.1, which is required for development of the hypothalamus. In contrast, Irx3 confers the ability to express En2 and Nkx6.1 in response to Fgf8 and Shh, respectively. Furthermore, an alteration in the region-specific response to Fgf8 upon misexpression of Irx3 resulted in transformation of diencephalic and possibly telencephalic tissues into the optic tectum. Finally, we demonstrated that Six3 and Irx3 can mutually repress their expression, which may contribute to the establishment of their complementary expression domains in the neural plate. These repressive interactions are specific, as Six3 did not repress Gbx2, and Irx3 did not disturb Otx2 expression. These findings provide evidence that the early embryonic forebrain is demarcated into two domains with distinct genetic programs, which argues against the authentic telendiencephalic subdivision.
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Approximately 50% of untreated presomite rat embryos in culture have demonstrated inversions of cardiac looping (laeval instead of dextral) or tail flexure (left-sided instead of right-sided), or both. This spontaneous situs inversus (SI) was not accompanied by growth inhibition or any other observable defects. The incidence of SI was directly related to the stage at dissection, and all heart defects and most flexure defects were eliminated by delaying explantation to the early somite stage. The incidence of SI was not lowered significantly either by removal of endogenous catecholamines from the culture serum by dialysis or by inclusion of alpha- or beta-adrenergic antagonists in the medium. However, the alpha-adrenergic agonist L-phenylephrine (50 micrograms/ml) increased the incidence of SI to 73%. These findings appear to rule out adrenergic mechanisms as a cause of spontaneous SI in cultured, neural plate-staged rat embryos but suggest a mechanism, yet unknown, that is additive with SI induced by alpha-adrenergic agonists. The low incidence of non-SI-related defects suggests that the high incidence of SI is not an artifact of suboptimal culture conditions. The virtual absence of SI in embryos cultured in bovine serum, a medium in which overall embryonic growth and development were retarded, provides further evidence against nonspecific artifacts.
The neural crest is a unique cell population induced at the lateral border of the neural plate. Neural crest is not produced at the anterior border of the neural plate, which is fated to become forebrain. Here, the roles of BMPs, FGFs, Wnts, and retinoic acid signaling in neural crest induction were analyzed by using an assay developed for investigating the posteriorization of the neural plate. Using specific markers for the anterior neural plate border and the neural crest, the posterior end of early neurula embryos was shown to be able to transform the anterior neural plate border into neural crest cells. In addition, tissue expressing anterior neural plate markers, induced by an intermediate level of BMP activity, was transformed into neural crest by posteriorizing signals. This transformation was mimicked by bFGF, Wnt-8, or retinoic acid treatment and was also inhibited by expression of the dominant negative forms of the FGF receptor, the retinoic acid receptor, and Wnt signaling molecules. The transformation of the anterior neural plate border into neural crest cells was also achieved in whole embryos, by retinoic acid treatment or by use of a constitutively active form of the retinoic acid receptor. By analyzing the expression of mesodermal markers and various graft experiments, the expression of the mutant retinoic acid receptor was shown to directly affect the ectoderm. We thereby propose a two-step model for neural crest induction. Initially, BMP levels intermediate to those required for neural plate and epidermal specification induce neural folds with an anterior character along the entire neural plate border. Subsequently, the most posterior region of this anterior neural plate border is transformed into the neural crest by the posteriorizing activity of FGFs, Wnts, and retinoic acid signals. We discuss a unifying model where lateralizing and posteriorizing signals are presented as two stages of the same inductive process required for neural crest induction.
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