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Development of the neural crest: achieving specificity in regulatory pathways.

Recent studies have revealed the signaling pathways and downstream effectors involved in the specification of the neural crest. Neural crest cells are generated from a zone at the neurectoderm border in response to Wnt and BMP signals. BMP signals are involved in establishing a competency zone at the border of the neurectoderm, while subsequent Wnt signals specify neural crest cells. Combinations of transcription factors, including pax and msx gene products, act downstream of these pathways to integrate signals and establish the neural crest. Mechanisms are emerging for how specificity is generated from reiterated signals and effectors.

Animals↗

Morphology and distribution of Müller cells in the retina of the toad Bufo marinus.

We have previously shown that an antibody against neuron-specific enolase (NSE) selectively labels Müller cells (MCs) in the anuran retina (Wilhelm et al. 1992). In the present study the light- and electron-microscopic morphology of MCs and their distribution were described in the retina of the toad, Bufo marinus, using the above antibody. The somata of MCs were located in the proximal part of the inner nuclear layer and were interconnected with each other by their processes. The MCs were uniformly distributed across the retina with an average density of 1500 cells/mm2. Processes of MCs encircled the somata of photoreceptor cells isolating them from each other by glial sheath, except for those of the double cones. Some of the photoreceptor pedicles remained free of glial sheath. Electron-microscopic observations confirmed that MC processes provide an extensive scaffolding across the neural retina. At the outer border of the ganglion cell layer these processes formed a non-continuous sheath. The MC processes traversed through the ganglion cell layer and spread beneath it between the neuronal somata and the underlying optic axons. These processes formed a continuous inner limiting membrane separating the optic fibre layer from the vitreous tissue. Neither astrocytic nor oligodendrocytic elements were found in the optic fibre layer. The significance of the uniform MC distribution and the functional implications of the observed pattern of MC scaffolding are discussed.

Animals↗

Separation of multiple functions in ventilatory control of pneumotaxic mechanisms.

Multiple functions have been ascribed to the pontile pneumotaxic center. We hypothesized that these functions might be separable among neurons in different regions. In decerebrate, vagotomized, paralyzed and ventilated cats, activities of the phrenic and triangularis sterni nerves were recorded. Microinjections of kainic acid were used to destroy neurons. Neurons in the rostrolateral tegmentum at the ponto-mesencephalic border controlled the duration of neural inspiration. Expiratory duration was controlled by neurons in the more caudal nucleus parabrachialis medialis and Kolliker-Fuse nucleus. Ventilatory responses to hypercapnia were depressed following injections of kainic acid into regions controlling either inspiratory or expiratory durations. The phases of expiration were regulated by two groups of neurons, located medial and lateral in the rostral pons. We conclude that rostral pontile and mesencephalic mechanisms control multiple aspects of the eupneic ventilatory cycle. There mechanisms are served by neurons in separable anatomical regions.

Animals↗

Characterization and retinoic acid responsiveness of the murine Hoxd4 transcription unit.

We have characterized the transcription unit of a murine Hox gene in the fourth paralogous group, Hoxd4. We have identified two Hoxd4 transcription start sites by S1 analysis. The upstream promoter (P2) is 5.2 kilobase pairs upstream from the coding region, while the downstream promoter (P1) is 1.1 kilobase pairs distant. Both promoters bear a cluster of start sites. Multiple transcripts were identified by Northern blot, originating from both promoters and multiple polyadenylation signals. Expression of P1 transcripts in the neural tube shows an anterior border at the rhombomere 6/7 boundary, corresponding to previous reports (Gaunt, S. J., Krumlauf, R., and Duboule, D. (1989) Development 107, 131-141; Morrison, A., Moroni, M. C., Ariza-McNaughton, L., Krumlauf, R., and Mavilio, F. (1996) Development 122, 1895-1907). A more posterior boundary in the central nervous system was observed for P2 transcripts. We observed strong expression up to somite 6 and weak expression in somite 5, correlating with the phenotype of Hoxd4 null mutant mice (Horan, G. S. B., Nagy Kovàcs, E., Behringer, R. R., and Featherstone, M. S. (1995) Dev. Biol. 169, 359-372). In response to retinoic acid, expression from P1 in the hindbrain was anteriorized after 4 or 24 h of treatment. P2 transcripts seemed to be less responsive and/or to have an indirect response to retinoic acid. The long 5'-untranslated region found in all Hoxd4 transcripts suggests that translation does not occur by a classical ribosome scanning mechanism.

Alternative Splicing↗

Hallucinations following occipital lobe damage: the pathological activation of visual representations.

Neuropsychological investigation of hallucinations may provide insight into the nature of these subjective phenomena, as well as inform theories of perception and recall. We studied a man who described continuous visual hallucinations of object fragments (e.g., lines, corners, patterns) in the left visual field following a stroke in the right occipital cortex. The subject performed normally on standardized measures of visual perception and other cognitive abilities. He had no personality disturbance, and EEG during hallucinations was normal. Review of our records of 211 cases with focal lesions involving visual cortex revealed 5 patients with similar complaints. The hallucinatory experience of such patients probably reflects pathological activation of neural ensembles in the regions bordering an occipital lesion. These regions are presumed to contain records of visual feature fragments which are co-activated by feedback projections in the earliest visual association cortices, where they produce meaningful patterns during normal recall.

Aged↗

Analysis of the Hoxd-3 gene: structure and localization of its sense and natural antisense transcripts.

This study set out to investigate the structure and localized expression of the mouse homeobox-containing gene Hoxd-3. In addition to identifying a transcript of the type known from other Antennapedia (Antp)-like mammalian homeobox cDNAs, an antisense transcript was also detected. The antisense form of Hoxd-3 overlaps with 603 bp of the sense transcript including the homeobox. Active antisense transcription has been confirmed by RNA blot analysis with single-stranded probes and by the direction of splicing of an intron in the antisense transcript. The localized expression of sense and antisense transcripts was compared by in situ hybridization. Hoxd-3 expression was observed from 8.5 days p.c., in the neural tube with a sharp border in the hind brain at the level of rhombomeres 4-5. In contrast, the earliest antisense expression was detected at 10.5 days p.c. in cDNA libraries. At 12.5 days p.c., sense and antisense transcripts colocalized in the liver. The possible role of antisense homeobox transcripts during liver and the hematopoietic development is discussed.

Amino Acid Sequence↗

The coding of uniform colour figures in monkey visual cortex.

Psychophysical studies indicate that perception of the colour and brightness of a surface depends on neural signals evoked by the borders of the surface rather than its interior. The visual cortex emphasizes contrast borders, but it is unclear whether colour surface signals also exist, whether colour border signals are orientation selective or mainly non-oriented, and whether cortical processing tends to separate colour and form information. To address these questions we examined the representation of uniform colour figures by recording single neuron activity from areas V1 and V2 in alert macaque monkeys during behaviourally induced fixation. Three aspects of coding were quantified: colour, orientation and edge selectivity. The occurrence of colour selectivity was not correlated with orientation or edge selectivity. The fraction of colour-selective cells was the same (64 % in layers 2 and 3 of V1, 45 % in V2) for oriented and non-oriented cells, and for edge-selective and surface-responsive cells. Oriented cells were often highly selective in colour space, and about 40 % of them were selective for edge polarity or border ownership. Thus, contrary to the idea of feature maps, colour, orientation and edge polarity are multiplexed in cortical signals. The results from V2 were similar to those from upper-layer V1, indicating that cortical processing does not strive to separate form and colour information. Oriented cells were five times more frequent than non-oriented cells. Thus, the vast majority of colour-coded cells are orientation tuned. Based on response profiles across a 4 deg square figure, and the relative frequency of oriented and non-oriented cells, we estimate that the cortical colour signal is 5-6 times stronger for the edges than for the surface of the figure. The frequency of oriented colour cells and their ability to code edge polarity indicate that these cells play a major role in the representation of surface colour.

Algorithms↗

Differential effects of dorsomedial medulla lesion size on ingestive behavior in rats.

Previous studies indicate that rats with lesions centered on the area postrema (AP) drink more saline and consume abnormally large amounts of water after treatment with subcutaneous isoproterenol (Iso) angiotensin II. In addition, lesioned rats lose a significant amount of body weight immediately after surgery. Nonetheless, there are disparate reports on the effects of lesions of the AP on fluid intake and body weight. These reports suggest that the adjacent nucleus of the solitary tract (NTS) may play a role in the effects observed subsequent to the lesion. In this study we evaluated the effects of varying lesion size on body weight, fluid intake, and the baroreflex. As the lesion included more of the NTS, the effect on body weight was reduced. Moreover, water intake induced by Iso increased as more NTS was involved in the lesion. Conversely, 3-h ad libitum saline intake and saline intake after sodium depletion decreased with more involvement of the NTS in the lesion. These data suggest that the neural population in the NTS bordering the AP may play a critical role in the control of water and saline intake as well as the regulation of body weight.

Animals↗

Autoradiographic localization of quinuclidinyl benzilate binding to rat pituitary gland.

The location of muscarinic receptors in the rat pituitary gland was examined with an autoradiographic technique. Slides containing 10 or 20 micron horizontal sections of tissue were incubated in a solution of 1 n M[3H]quinuclidinyl benzilate (QNB) in phosphate-buffered saline to label muscarinic receptors. Autoradiograms were produced by placing the slides into X-ray cassettes with tritium-sensitive film and processing the film 8-10 weeks later. Minimal binding occurred when sections were incubated in 1 nM[3H]QNB plus 1 microM atropine. Highest specific binding of QNB was found in the anterior and intermediate lobes along their border with the pituitary cleft. Patches of high binding were also seen penetrating the intermediate lobe and at the border between the intermediate and neural lobes. The remainder of the intermediate lobe had background density of autoradiographic grains. Moderate density of specific binding was found throughout the anterior lobe. Low specific binding occurred in the neural lobe.

Animals↗

The effects of cerebrosides on model membrane shape.

Model membranes composed of phosphatidylcholine, various cerebrosides, and cholesterol have been studied by electron microscopy, deuterium NMR, and phosphorus-31 NMR. Large variations in phosphatidylcholine membrane morphology are observed when cerebrosides are present in phosphatidylcholine bilayers. Large spherical liposomes, small spherical liposomes, and long tubular liposomes are formed, depending on the relative acyl chain lengths of the phosphatidylcholine and cerebroside. In some cases, cholesterol can reverse the morphological effects of cerebrosides. These results suggest that cerebrosides, which are found in high concentrations in certain highly specialized membranes, can affect membrane shape and may be involved in the mechanism of formation of membranes with unusual morphology, e.g. neural myelin and the brush border membrane of the intestinal epithelium.

1,2-Dipalmitoylphosphatidylcholine↗

The Xenopus laevis homeobox gene Xgbx-2 is an early marker of anteroposterior patterning in the ectoderm.

In a search for homeobox genes expressed during early Xenopus development, we have isolated a gene which appears to be the Xenopus cognate of the mouse Gbx-2 gene. Expression of Xgbx-2 is first detectable by in situ hybridization at the midgastrula stage when it is predominantly expressed in the dorsolateral ectoderm, with a gap in expression at the dorsal midline. By the end of gastrulation and during neurulation, Xgbx-2 is expressed dorsolaterally in the neural ectoderm and laterally and ventrally in the epidermis with sharp anterior expression borders in both tissues. The anteriormost expression in the neural ectoderm persists throughout the early stages of development, and was mapped to the region of rhombomere 1, with an anterior expression border in the region of the midbrain-hindbrain boundary. Thus Xgbx-2 is expressed anterior to the Hox genes. Xgbx-2 expression is induced by retinoic acid (RA) in animal caps, and RA treatment of whole embryos expands and enhances Xgbx-2 expression in the ectoderm. We suggest a role for Xgbx-2 in establishing the midbrain-hindbrain boundary, which appears to separate early neurectodermal regions expressing genes that are positively and negatively regulated by RA.

Amino Acid Sequence↗

Neural plate patterning by secreted signals.

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.

Animals↗

Correlation of a chicken stage 4 neural plate fate map with early gene expression patterns.

A number of gene markers are currently claimed to allow positive or negative visualization of the early chick neural plate at stages 3d/4, when its fate becomes determined. Some markers labeled by various authors as either "neural" or "non-neural" indeed show ectodermal expression patterns roughly correlative with widespread yet vague ideas on the shape and size of the early neural plate, based on previous fate maps. However, for technical reasons, it is not clear how precisely these expression patterns correlate with any experimentally determined fate boundaries. An eventual mismatch between fate and marker interpretation might bear importantly on ideas about gene functions and causal hypotheses in issues such as the establishment of the neural/non-neural border or the earliest mechanisms of neural regionalization. In this review, we correlated a set of epiblastic and mesendodermal gene expression patterns with the novel neuroectoderm proportions suggested by our recent fate map of the chick neural plate at stages HH 3d/4 [P. Fernández-Garre, L. Rodriguez-Gallardo, V. Gallego-Diaz, I.S. Alvarez, L. Puelles, Fate map of the chicken neural plate at stage 4, Development 129 (2002) 2807-2822.]. This analysis suggests the existence of various nested subregions of the epiblast with boundaries codefined by given sets of gene patterns. No gene expression studied reproduces exactly or even approximately the entire neural plate shape, leading to a combinatorial hypothesis on its specification. This kind of analysis (fate and molecular maps), jointly with competence maps, provides the basis for understanding gene functions and the mechanisms of neural induction, specification and regionalization. Several gene patterns observed are consistent with precocious incipient regionalization of the neural plate along the dorsoventral and anteroposterior axes.

Animals↗

Vitamin A deficiency results in the dose-dependent acquisition of anterior character and shortening of the caudal hindbrain of the rat embryo.

The developing nervous system is particularly vulnerable to vitamin A deficiency. Retinoid has been proposed to be a posteriorizing factor during hindbrain development, although direct evidence in the mammalian embryo is lacking. In the present study, pregnant vitamin A-deficient (VAD) rats were fed purified diets containing varying levels of all-trans-retinoic acid (atRA; 0, 0.5, 1.5, 6, 12, 25, 50, 125, or 250 microg/g diet) or were supplemented with retinol. Hindbrain development was studied from embryonic day 10 to 12.5 ( approximately 6 to 40 somites). Normal morphogenesis was observed in all embryos from groups fed 250 microg atRA/g diet or retinol. The most caudal region of the hindbrain was the most sensitive to retinoid insufficiency, as evidenced by a loss of the hypoglossal nerve (cranial nerve XII) in embryos from the 125 microg atRA/g diet group. Further reduction of atRA to 50 microg/g diet led to the loss of cranial nerves IX, X, XI, and XII and associated sensory ganglia IX and X in all embryos as well as the loss of hindbrain segmentation caudal to the rhombomere (r) 3/4 border in a subset of embryos. Dysmorphic orthotopic otic vesicles or immature otic-like vesicles in both orthotopic and caudally ectopic locations were also observed. As the level of atRA was reduced, a loss of caudal hindbrain segmentation was observed in all embryos and the incidence of otic vesicle abnormalities increased. Perturbations in hindbrain segmentation, cranial nerve formation, and otic vesicle development were associated with abnormal patterning of the posterior hindbrain. Embryos from VAD dams fed between 0.5 and 50 microg atRA/g diet exhibited Hoxb-1 protein expression along the entire neural tube caudal to the r3/r4 border at a time when it should be restricted to r4. Krox-20 protein expression was expanded in r3 but absent or reduced in presumptive r5. Hoxd-4 mRNA expression was absent in the posterior hindbrain, and the rostral limit of Hoxb-5 protein expression in the neural tube was anteriorized, suggesting that the most posterior hindbrain region (r7/r8) had been deleted and/or improperly patterned. Thus, when limiting amounts of atRA are provided to VAD dams, the caudal portion of the hindbrain is shortened and possesses r4/r5-like characteristics, with this region finally exhibiting r4-like gene expression when retinoid is restricted even more severely. Thus, regions of the anterior hindbrain (i.e., r3 and r4) appear to be greatly expanded, whereas the posterior hindbrain (r5-r8) is reduced or absent. This work shows that retinoid plays a critical role in patterning, segmentation, and neurogenesis of the caudal hindbrain and serves as an essential posteriorizing signal for this region of the central nervous system in the mammal.

Animals↗

Mechanisms of axonal guidance used by interneurons in the chick embryo spinal cord.

Axonal guidance mechanisms involved in pathway formation by avian spinal interneurons have been studied using a variety of different approaches. The earliest developing spinal interneurons can be divided into two groups on the basis of the initial direction of axonal outgrowth: circumferential cells (C-cells) and primitive longitudinal cells. Perturbation studies suggest that a chemotropic-like mechanism plays a role in the guidance of the C-cell axons toward the floor plate. The shape of floor plate cells is polarized mediolaterally (direction of axon growth) and only in this region do growth cones express integrin and contact the basement membrane. Pathway formation following dorsal to ventral spinal cord rotation indicates that the ventrolateral region of the neural tube may provide a permissive substrate for longitudinally advancing growth cones. In addition, following rostrocaudal rotation of three segments of the neural tube, the axons of dorsolateral border cells in the rotated segments modified their directionality according to the general rostrocaudal polarity of the embryo. Changes in cell adhesion molecule expression by the C-cell growth cones and axons is region and age specific. These observations suggest that a variety of mechanisms and cues are working in the guidance of spinal interneurons in each region of the pathway.

Animals↗

Neural crest cell migratory pathways in the trunk of the chick embryo.

Neural crest cells migrate during embryogenesis to give rise to segmented structures of the vertebrate peripheral nervous system: namely, the dorsal root ganglia and the sympathetic chain. However, neural crest cell arise from the dorsal neural tube where they are apparently unsegmented. It is generally agreed that the somites impose segmentation on migrating crest cells, but there is a disagreement about two basic questions: exactly pathways do neural crest cells use to move through or around somites, and do neural crest cells actively migrate or are they passively dispersed by the movement of somite cells? The answers to both questions are critically important to any further understanding of the mechanisms underlying the precise distribution of the neural crest cells that develop into ganglia. We have done an exhaustive study of the locations of neural crest cells in chick embryos during early stages of their movement, using antibodies to neural crest cells (HNK-1), to neural filament-associated protein in growing nerve processes (E/C8), and to the extracellular matrix molecule laminin. Our results show that Some neural crest cells invade the extracellular space between adjacent somites, but the apparent majority move into the somites themselves along the border between the dermatome/myotome (DM) and the sclerotome. Neural crest cells remain closely associated with the anterior half of the DM of developing somites as they travel, suggesting that the basal lamina of the DM may be used as a migratory substratum. Supporting this idea is our observation that the development of the DM basal lamina coincides in time and location with the onset of crest migration through the somite. The leading front of neural crest cells advance through the somite while the length of the DM pathway remains constant, suggesting active locomotion, at least in this early phase of development. Neural crest cells leave the DM at a later stage of development to associate with the dorsal aorta, where sympathetic ganglia form, and to associate with newly emerging fibers of the ventral root nerve, where they presumably give rise to neuronal supportive cells. Thus we propose that the establishment of the segmental pattern of the peripheral ganglia and nerves depends on the timely development of appropriate substrata to guide and distribute migrating neural crest cells during the early stages of embryogenesis.

Animals↗

Cell death in the avian sclerotome.

In this study the occurrence of apoptotic cells in chick embryo trunk somites, between 2.5 and 4 days of development, has been examined using an in situ nick-end-labeling method (TUNEL) to identify nuclei in which DNA is undergoing fragmentation. At 2.5 days of development, apoptotic cells were found in the sclerotome with a distribution that depended on the rostrocaudal level in the trunk. At the most rostral levels (somites 1-18), dying cells were present primarily in the rostral half of the ventral sclerotome; at midlevels (somites 19-26), they were present throughout the ventral sclerotome; and at caudal levels (somites 27-32), no dying cells were present. By 4 days of development, the number of dying cells in the sclerotome was sharply reduced, and those present were primarily distributed to the caudal side of the intrasclerotomal fissure. Double labeling of cells for both TUNEL and the HNK-1 epitope, at 2.5 days, indicated that the majority of the dying cells were not neural crest cells. Further, dying cells in the rostral somite half were present largely in regions of the sclerotome that labeled poorly for HNK-1. It was confirmed that apoptotic neural crest cells retain the HNK-1 epitope and therefore would have been observed if present. Neural crest cells only appeared to be apoptotic in relatively small numbers and only at the ventral border of the sclerotome. Examination of DiI-labeled neural crest cells confirmed that the dying cells in the body of the somite were not primarily neural crest cells. Two hypotheses regarding the TUNEL-positive cells in the sclerotome were experimentally tested. First, that they originate from the somitocoel compartment of the somite, because their distribution patterns at 4 days were similar to those of somitocoel cells. To test this, somitocoel cells were labeled with carboxyfluorescein and grafted into host embryos in ovo. Results showed that these cells did not become apoptotic and that the dying cells were therefore not derived from the somitocoel. Second, the hypothesis was tested that the distribution patterns of the dying cells in the sclerotome are determined by factors outside the somite itself. Somites and segmental plates were transplanted into hosts in ovo with reversed orientation, after which the patterns of dying cells were examined using nile blue sulfate staining. The results indicated that the patterns were unchanged after a further 2 days incubation, suggesting that the patterns of cell death in the sclerotome are not determined solely from within the somite. The distribution of the cell death-associated gene products, bcl-2, bax, and interleukin-1 beta converting enzyme, indicates that although these proteins are segmentally distributed in the dermomyotome and in the rostrodorsal quadrant of the sclerotome, their patterns are not directly correlated with the distribution of dying cells.

Animals↗

Expression of snail2, a second member of the zebrafish snail family, in cephalic mesendoderm and presumptive neural crest of wild-type and spadetail mutant embryos.

Transcripts of a newly discovered gene called snail2, encoding a zinc finger protein of the Snail family, first appear in rows of cephalic mesendodermal cells in gastrulating zebrafish embryos. At the end of gastrulation, snail2 RNA accumulates in a domain of ectodermal cells that mark the border between the epidermal epithelium and the neural plate and includes precursors of the neural crest. During somitogenesis, snail2 expression becomes restricted to neural crest. snail2 is thus one of the earliest genes yet known to be specifically expressed in neural crest in zebrafish embryos. Since snail2 is expressed in mesendoderm, a tissue layer whose convergence in the trunk is known to be altered in embryos homozygous for the spadetail mutation, we examined snail2 expression in spadetail embryos. In these mutants, the number of cephalic mesendodermal cells expressing snail2 is strongly reduced and the distribution of cells containing snail2 and no tail transcripts in the axial mesoderm is much broader than normal Moreover, the embryos are shorter than normal at the end of gastrulation. This shows that, in addition to the failure of paraxial mesoderm to converge normally in the trunk during gastrulation, spadetail also affects the elongation of the embryo and the convergence of axial and lateral mesendoderm in both trunk and head.

Amino Acid Sequence↗