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A time-resolved single-cell roadmap of the logic driving anterior neural crest diversification from neural border to migration stages.

Neural crest cells exemplify cellular diversification from a multipotent progenitor population. However, the full sequence of early molecular choices orchestrating the emergence of neural crest heterogeneity from the embryonic ectoderm remains elusive. Gene-regulatory-networks (GRN) govern early development and cell specification toward definitive neural crest. Here, we combine ultradense single-cell transcriptomes with machine-learning and large-scale transcriptomic and epigenomic experimental validation of selected trajectories, to provide the general principles and highlight specific features of the GRN underlying neural crest fate diversification from induction to early migration stages using Xenopus frog embryos as a model. During gastrulation, a transient neural border zone state precedes the choice between neural crest and placodes which includes multiple converging gene programs. During neurulation, transcription factor connectome, and bifurcation analyses demonstrate the early emergence of neural crest fates at the neural plate stage, alongside an unbiased multipotent-like lineage persisting until epithelial-mesenchymal transition stage. We also decipher circuits driving cranial and vagal neural crest formation and provide a broadly applicable high-throughput validation strategy for investigating single-cell transcriptomes in vertebrate GRNs in development, evolution, and disease.

Animals

Distribution of surface coat material on fusing neural folds of mouse embryos during neurulation.

Fusing and non-fusing regions of neural folds from mouse embryos were examined during neurulation for the distribution of extracellular macromolecules (surface coats) prior to and at the time of closure. Ruthenium red staining of 10th day ICR/DUB mouse embryos was used to detect the distribution of surface coat material. Light microscopic examination of fusing and non-fusing regions in the midbrain, hindbrain, and spinal cord showed a consistent increase in ruthenium red positive material immediately prior to closure. Heavy deposits of positive staining material were present along apical neural fold borders and overlying ectoderm cells. This staining pattern was consistent in the three regions examined, but the pattern of initial contact between opposing neural folds differed. In mid- and hindbrain areas contact was initiated by overlying ectoderm, whereas in spinal cord regions contact was first established by neuroepithelial cells. Once contact between opposing neural folds was initiated a decrease in stainable material was observed.

Animals

Ultrastructure of hyaline, border, and vacuole cells in chick inner ear.

The sense organ for hearing in birds, the basilar papilla, is capable of replacing lost or damaged hair cells and supporting cells through regeneration. Potential candidates for precursor-cell populations include cells within the auditory receptor epithelium and nonsensory cells inferior to the sensory epithelium. Ultrastructural characteristics of hyaline cells, border cells, and vacuole cells, nonsensory cells which border or lie inferior to the receptor epithelium proper, were studied with transmission electron microscopy. Data were obtained from normal neonatal and adult chickens. Several rows of epithelial cells separate hyaline cells from inferiorly located organ supporting cells and hair cells. Ultrastructural characteristics and location of these epithelial cells differentiate them from organ supporting cells and hyaline cells; consequently, we have termed them "border cells." Synaptic specializations are observed between neural elements and border cells, and gap junctions are found between adjacent border cells, between border cells and neighboring organ supporting cells, and between juxtaposed border and hyaline cells. Hyaline cells, in contrast to border cells, are highly specialized. Dense bundles of filaments are present in hyaline cells from the basal one-half of the papilla, and an unusual structure, a rough tubular aggregate, is present in hyaline-cell cytoplasm. Pre- and postsynaptic specializations are observed between neural elements and hyaline cells, and gap-junctional complexes link neighboring hyaline cells. Vacuole cells lie inferior to the hyaline cells and rest on the inferior fibrocartilaginous plate. They are unspecialized morphologically. Their only remarkable morphological feature is the abundance of spherical vacuoles within their cytoplasmic matrix.

Animals

Identification of early developing axon projections from spinal interneurons in the chick embryo with a neuron specific beta-tubulin antibody: evidence for a new 'pioneer' pathway in the spinal cord.

The early development of interneurons in the chick embryo spinal cord was studied using a monoclonal antibody against a neuron-specific beta-tubulin isoform. Early developing interneurons were divided into two cell groups on the basis of their location and the pattern of growth of their axons. One group is composed of cells that establish a primitive longitudinal pathway (PL-cells), whereas the other group contains cells constituting a circumferential pathway (C-cells). The onset of axonal development in both cell groups occurs at stage (st.) 15 (embryonic day, (E), 2) in the branchial segments, which is prior to axonogenesis of motoneurons. PL-cells develop in the region between the floor plate and the motoneuron nucleus. Their axons are the first neuronal processes ('pioneer axons') to arrive in the ventrolateral marginal zone and they project both rostrally and caudally to establish a primitive longitudinal association pathway at the ventrolateral surface of the neural tube. This pathway is formed before axons of C-cells arrive in the ventrolateral region. The first C-cells are initially located in the most dorsal portion of the neural tube, whereas later appearing C-cells are also located in both intermediate and ventral regions of the neural tube. The axons of C-cells project ventrally, without fasciculating, along the lateral border of the neural tube. Some of their axons enter the ipsilateral ventrolateral longitudinal pathway at st. 17. We often observed apparent contacts and interactions between preexisting axons of PL-cells and newly arriving axons of C-cells. The axons of commissural C-cells first enter the floor plate at st. 17 and cross the midline at st. 18. Axons of C cells begin to join the contralateral ventrolateral longitudinal pathway at st. 18+ to st. 19. In the floor plate region, contacts between growth cones and axons were often observed. However, axons in the floor plate at these stages were not fasciculated. These observations establish the timing and pattern of growth of axons from two specific populations of early developing interneurons in the chick spinal cord. Additionally, we have identified an early and apparently previously undescribed 'pioneer' pathway that constitutes the first longitudinal pathway in the chick spinal cord.

Animals

Border effects on brightness: a review of findings, models and issues.

This paper presents a summary of experimental findings, theoretical models and unresolved issues regarding border effects on brightness, of which the Cornsweet illusion (Cornsweet, 1970 Visual Perception. Academic Press: New York) is the best-known example. It is argued that no current theoretical model completely accounts for the wide variety of effects described. Contrast sensitivity function (CSF) models can explain many low-contrast, but not high-contrast, border effects. Lightness integration models based on Land and McCann's retinex theory (Land and McCann, 1971. J. Opt. Soc. Am. 61, pp. 1-11) have the advantage over CSF models in that they predict transitivity of border effects where they are found to occur. However, they fail to predict the appearance of a variety of Cornsweet-like figures, have never been tested with relatively high contrast versions of those figures, and have only been implemented by qualitative demonstration. It is argued that edge-detector models are potentially the most promising theoretical candidates but, as with lightness-integration models, they have invariably relied on qualitative demonstrations and have only dealt with low-contrast border effects. A computational edge-detector model which predicts the appearance of both high and low contrast Cornsweet figures is proposed and its advantages over other models, as well as its current limitations, are discussed. The final section discusses the neural locus for border effects in brightness.

Animals

Scanning and transmission electron microscope studies of interkinetic nuclear migration in the cerebral vesicles of the rat.

A scanning electron microscope (SEM) was used to examine the morphology and surface texture of neuroepithelial cells during interkinetic nuclear migration in the cerebral vesicles of the rat at 12, 13 and 14 days of gestation. Serial sections of embryonic material of the same age were also prepared for the transmission electron microscope (TEM). Particular attention was paid to the SEM and TEM appearance of mitotic neuroepithelial cells which occur exclusively along the ventricular border of the neural epithelium. Three distinctly-shaped classes of mitotic cells were recognised in scanning micrographs. (1) Pyriform cells. This type of mitotic cell was characterised by the presence of very long, fine processes radiating from the tip and shoulders of a short external (basal) process. These fine processes were termed "intramitotic filopodia." Microvilli were found on the surface of most pyriform cells. (2) Conical cells. These lacked an external process but there were large numbers of intramitotic filopodia at the basal pole of the cell body, and the perikaryal surface was rich in microvilli. (3) Globular cells. It was possible to subdivide this class of cell into large and small sizes, but usually a few short intramitotic filopdia were present at the basal pole. The perikaryal surfaces of the globular population were raised in coarse lumps and bubble-like protrusions. By pooling TEM and SEM information we were able to deduce that pyriform cells probably possess a prophase or prometaphase chromosome morphology, while conical cells exhibit a chromosome morphology somewhere between prometaphase and early anaphase. Large globular mitotic cells were found to be between metaphase and late anaphase and small globular cells were identified as early telophase cells. On the basis of these findings we have proposed that as a bipolar neuroepithelial cell rounds up for mitosis it passes first through a pyriform stage during which the external process is retracted or broken down, and then through a conical stage when the cell consolidates its position on the ventricular surface. Finally, the cell enters a large globular stage before dividing into two small globular telophase cells. It is not known what part, if any, the intramitotic filopodia play in this process of rounding up.

Animals

The onset of dopaminergic innervation during ontogeny decreases melanotrope proliferation in the intermediate lobe of the rat pituitary.

The onset of dopaminergic innervation and its effects on melanotrope proliferation were investigated in the rat pituitary intermediate lobe. Dopamine, and its synthetic rate-limiting enzyme tyrosine hydroxylase, were first detected immunohistochemically on late post-natal day 3 or early postnatal day 4. Axon density was highest at the neural lobe/intermediate lobe border, and decreased toward the pituitary cleft. By postnatal day 10, the adult pattern of tyrosine hydroxylase immunoreactivity was established and remained through post-natal day 14. Neurointermediate lobe dopamine levels, measured by HPLC, correlated well with the increased axon density observed in the immunohistochemical studies. Dopamine could not be measured by our assay (100 fg limit) until post-natal day 3 (439.32 fg/NIL). Dopamine concentration increased to 2.09 +/- 0.425 ng at PN 4, 86.31 +/- 20.42 ng at PN 7, 168.72 +/- 18.37 ng at PN 10. Melanotrope proliferation was determined by [3H]thymidine incorporation before and after innervation. Concomitant with the onset of innervation, the proliferation index dropped from 13.4 +/- 0.01% to 6.5 +/- 0.002% at PN 4, and continued to decrease until a level of 3 +/- 0.003% was established by PN 10. To confirm the inhibitory action of dopaminergic innervation on melanotrope proliferation, rat neonates were injected intracisternally with 150 mg 6-hydroxydopamine to destroy dopaminergic axons within the intermediate lobe. Measurement of dopamine concentrations in neurointermediate lobes of injected animals showed a decrease in dopamine levels as compared to controls. From PN 4 (0.88 +/- 0.165 ng), DA levels gradually increased during development: at PN 5, [DA] = 0.689 +/- 0.104 ng; PN 6 [DA] = 11.60 +/- 2.24 ng; PN 7 [DA] = 20.93 +/- 3.80 ng; and PN 10 [DA] = 27.95 +/- 3.46 ng. Melanotrope proliferation also increased in 6-hydroxydopamine-treated animals. At PN 4, the onset of innervation reduced the pre-innervation proliferation index to 8.75 +/- 0.002%, only a 30% reduction in contrast to the greater than 50% decrease observed in control animals. A stable proliferation level of approximately 7.5% persisted in all subsequent stages with 6-OHDA administration. Our results demonstrated the time of dopamine innervation onset and a characteristic developmental pattern for axons within the rat intermediate lobe. The onset of innervation and increased dopamine concentration suggests increased dopaminergic control of the melanotropes, illustrated specifically by a decrease in their level of proliferation. This is the first presentation of evidence showing that dopaminergic innervation within the intermediate lobe of the rat pituitary regulates melanotrope proliferation.

Animals

Functional characteristics of cochlear nucleus in behaving cat examined by acoustic masking of electrical stimuli.

1. Cats were trained, using an operant procedure, to detect and respond to electrical stimulation delivered in the vicinity of the cochlear nucleus. The electrical stimuli were presented both in silence and in synchrony with repeated noise bursts to determine whether detection thresholds for the electrical stimuli were elevated by the acoustic masking noise. 2. For stimulation sites centered within auditory structures (cochlear nucleus or acoustic nerve root), the acoustic maskers caused a consistent elevation of the electrical detection thresholds. For stimulation sites that were in or bordered on nonacoustic neural structures (e.g., vestibular), the acoustic maskers caused little or no elevation of electrical detection thresholds. 3. The magnitude of the acoustic masking effect was monotonically related to the intensity of the acoustic masker across the range of intensities tested. 4. The magnitude of the masking effect was strongly dependent on the relative timing of the stimulus pulse and the masker noise burst. Maximum masking occurred when the pulse just followed the onset of the neural activity in cochlear nucleus evoked by the masker burst. Less masking occurred when the electrical pulse occurred at the middle or end of the masker burst, and still less when the pulse occurred just prior to the onset (backward masking) or just after the offset (forward masking) of the masker burst. 5. The magnitude of the masking effect also depended on the frequency of the acoustic masker. For tone bursts, masking was maximal for each electrode at a particular frequency and declined monotonically for masker frequencies above or below the optimal frequency. 6. It is concluded that the masking of an electrical stimulus by an acoustic stimulus depends on a direct interaction between the neural responses evoked by the two stimuli, and that similar central, neural interactions may contribute to acoustic masking of acoustic stimuli. It is also concluded that the technique of masking an electrical stimulus by an acoustical stimulus is a precise and useful tool for the study of sensory-neural organization in intact behaving animals.

Animals

Effects of retinoic acid on the distribution of glycoconjugates during mouse tail bud development.

Retinoic acid (RA), a potent teratogen of caudal axial development in rodents, has been shown to alter glycoconjugates in a variety of embryonic tissues and teratocarcinomas. In this study, we examined its effects on the expression of cell surface and extracellular matrix glycoconjugates during tail bud development in mouse embryos by using lectin histochemistry. The lectins WGA, sWGA, and PNA showed striking differences in binding between RA-exposed and control embryos. Computer-assisted densitometry revealed a significant increase in binding of all three lectins to the extracellular material of the luminal and abluminal borders of the secondary neural tube and surrounding the notochord in RA-exposed embryos. RA-treated embryos also showed an increased binding affinity for the lectins sWGA and PNA to the cells of the notochord, while WGA showed increased binding to the neuroepithelial cells of the secondary neural tube. The results suggest that RA affects the expression of lectin binding sites during the early development of RA-induced caudal axial defects.

Animals

Origin of infrasound sensitive neurones in the papilla basilaris of the pigeon: an HRP study.

Infrasound sensitive afferent fibres recorded in the pigeon cochlear ganglion were marked by intracellular injections of horseradish peroxidase (HRP). All stained fibres were found to innervate hair cells in the basilar membrane between 90 and 950 microns from its apical end. No fibres to the macula lagenae were found. Nine of the 10 completely stained fibres contacted hair cells located abneurally on the free basilar membrane, the tenth ended over the neural limbus near its abneural border. All fibres innervated between two and nine hair cells. This is in contrast to common auditory fibres in the bird that were reported to innervate only one hair cell located neurally over the neural limbus. This paper, therefore, demonstrates, for the first time, physiologically defined fibres that do not end on 'inner' hair cells.

Animals

The morphogenesis of hindbrain crowding associated with lumbosacral myeloschisis.

The teratogenicity of ethylenethiourea (ETU) was investigated in Sprague-Dawley rats and Landrace pigs. Pregnant rats each received a single intragastric dose of ETU on a given day from day 8 to day 19 of gestation, and pregnant pigs were given a single dose of ETU from day 15 to day 19. Control animals received an ETU-free vehicle. The newborn pigs were all normal, whereas there were high incidences of specific types of congenital malformation of the central nervous and other systems in the rats. A high incidence of lumbosacral myeloschisis associated with hindbrain crowding was observed in rat fetuses exposed to ETU on day 11 of gestation. This abnormality is considered comparable to Chiari type II malformation associated with spinal dysraphism (Arnold-Chiari malformation) in humans. Morphogenetic fetuses were examined by light and scanning electron microscopy and interactive image analysis. At gestational days 12 and 13, the volume of the caudal end of the neural tube from the anterior border of the hindlimb bud to the most caudal portion of the fetus was significantly greater in ETU-exposed than in control rat embryos. Experimental rat embryos also exhibited disturbance in closure of the posterior neuropore, with extrusion of neural tissue through the opening. An apparently unrelated abnormality in ETU-exposed rat fetuses was underdevelopment of the cranium, leading to hindbrain crowding.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

Central neural pathways for angiotensin-induced thirst.

Evidence is reviewed implicating the preoptic region in angiotensin-induced thirst. The most responsive area according to results obtained with behavioral, electrophysiological, and autoradiographic mapping techniques is at the caudal border of the medial preoptic region and rostral border of the anterior hypothalamus. The neural pathway from this preoptic site for angiotensin-induced thirst extends along the medial forebrain bundle through the midlateral hypothalamus to the paramedial midbrain tegmentum and to an area ventrolateral to the central gray. Lesions of this pathway in the midlateral hypothalamus and rostral midbrain significantly attenuated drinking induced by microinjections of angiotensin II into the preoptic area but did not disrupt water intake induced by microinjections of angiotensin II into the subfornical organ or cerebral ventricles. Although the efferent pathways from angiotensin-receptive sites in the subfornical organ and cerebral ventricles are unknown, it appears from these observations that the medial forebrain bundle is not involved. Lesions of the medial forebrain bundle-lateral hypothalamus also do not disrupt drinking induced by microinjections of hypertonic saline into the preoptic region although lesions placed 1 mm further lateral do. Since fat lateral hypothalamic lesions are without effect on drinking induced by centrally administered angiotensin II, this suggests that intracellular and extracellular thirst signals are subserved by separate neural pathways in the hypothalamus.

Action Potentials

Ultrastructure of the neurohemal hypothalamic floor of the frog, Rana catesbeiana.

The fine structure of the hypothalamic floor was studied in the frog, Rana catesbeiana. The regions slightly anterior and posterior to thw swollen hypothalamic floor part, which has been classically designated as the median eminence, contained neurosecretory axon terminals abutting on the capillary walls. The region relatively far anterior to the swollen part did not show neurosecretory axons terminating on capillary walls. The neural stalk contiguous to the rostral border of the pars intermedia had few neurosecretory axon terminals which end on the terminal portions of the ependymal processes covering the capillary wall. The functional significance of the regional differentiation of the frog neurohypophysis is discussed in connection with the regional differentiation of various secretory cells in the adenohypophysis.

Animals

Horner's syndrome: an electron microscopic study of a human iris.

Electron microscopy was performed on the irides of a man with a history of a long standing Horner's syndrome which resulted in iris heterochromia. Comparison of his normal brown iris with the depigmented blue iris showed depletion of anterior border cells and absence of sympathetic nerve fibres. Stromal melanocyte numbers were also diminished but melanosome numbers within the residual cells were not significantly different. Postnatal maintenance of stromal and anterior border zone pigmentation, derived from the neural crest, would appear to be dependent on an intact sympathetic nerve supply in contrast to the iris pigment epithelium which remains normally unaffected in Horner's syndrome.

Adrenergic Fibers

Catecholamine-synthesizing enzymes in the rat pituitary. An immunohistochemical study.

The catecholamine-containing nerve fibers of the rat pituitary were studied by immunohistochemical demonstration of the catecholamine-synthesizing enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Immunohistochemical demonstration of TH confirms earlier catecholamine fluorescence histochemical studies showing a fine network of varicose fibers in both the intermediate and the neural lobe, with the most dense aggregation of fibers at the border between the lobes. DBH-immunoreactive fibers were much less in number, and confined to the neural lobe, where both vascular and parenchymal fibers were seen. With the antibody to PNMT bright staining was seen in all the glandular cells of the intermediate lobe, while the neural lobe was negative. No immunoreactive structures were observed in the anterior lobe. Functionally the study confirms the presence of an extensive dopaminergic innervation of the neurointermediate lobe, giving an anatomical basis for the tonic inhibitory action of dopamine on the intermediate lobe cells and for recent observations attributing dopamine a local regulatory function also in the neural lobe. In addition to vascular noradrenaline-containing fibers as described earlier the study shows parenchymal DBH-immunoreactive fibers in the neural lobe, suggesting a local role for noradrenaline in this lobe. The nature of the cellular PNMT-immunoreactivity in the intermediate lobe remains to be established. The cellular localization of the PNMT-immunoreactivity was distinctly different than that of the alpha-MSH-immunoreactivity within the intermediate lobe cells and reserpine treatment did not affect the PNMT-immunoreactivity although it induced a heterogeneous depletion of alpha-MSH and related peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

[The use of multivariate mathematical methods in medical diagnostic systems--a model for the evaluation of cytological smears].

The methods of the multivariate mathematics have been applied in several studies to increase the diagnostic reliability of medical decision support system. In the recent years some new algorithms for decision support (fuzzy logic) and for pattern recognition (neural nets), both specified by nonlinearity, were developed. This paper provides results for the application of this methods in the area of quantitative cytology and the comparison with the traditional classifiers. 21 normal, 15 dysplastic, 23 malignant, Feulgen stained gastric imprint smears were analysed on a Leitz Miamed DNA equipment. The determination of mean DNA content, the 2c deviation index (2cDI), 5c Exceeding rate (RcER), G1,S,G2 phase fraction ratios, cell nucleus area, form factor was performed. The discriminant analysis classified correctly the 95.6% of malignant cases, 86.7% of dysplasias, and 80.7% normal cases. Our diagnostic system using fuzzy logic made the diagnostic borders fine tuneable, and reliable. The back propagation neural net could classify all three diagnostic groups above 95% correctly. The application of nonlinear computational methods made the diagnostic system more reliable. The application of these algorithms are encouraged.

Algorithms

Immunochemical localization of aminopeptidase M in the alimentary tract of the guinea pig and rat.

Aminopeptidase M (APM) was localized in the kidney and alimentary tract of guinea pigs and rats by indirect immunohistochemistry. APM was detected in the brush border of the epithelium of the proximal convoluted tubule of the kidney and of the small intestine, and it was localized to cells scattered throughout lymphoid tissue in the small intestine and colon. The gastric mucosa was unstained. APM was localized to numerous fibers supplying the myenteric plexus of the stomach, small intestine, and colon. The submucosal plexus was sparsely supplied by immunoreactive fibers. Occasional cell bodies were stained in the myenteric plexus. Staining was abolished by preabsorption of the primary antibody with APM. APM was characterized in membranes prepared from the muscle and mucosa of the guinea pig and rat stomach, small intestine, and colon by Western blotting. The major immunoreactive protein identified in membranes prepared from all tissues had an apparent molecular weight of 140, corresponding to the monomer of APM. In the brush border APM has a digestive function, whereas in neural tissue it may degrade and inactivate neuropeptides.

Aminopeptidases

Absence of laminin deposition in breast cancer and metastases except to the brain.

Laminin, a major glycoprotein of basement membrane has been found to play significant roles during invasion and metastases. In this study, we have examined the distribution of laminin in several human brain carcinoma metastases, human breast cancers, skin and lymph node metastases of breast cancer as well as in an in vitro and an in vivo model of invasion. A laminin accumulation was demonstrated a) at the border between human metastatic carcinoma cells and surrounding neural tissue; b) at the invasive edge between MO4 cells (a highly malignant cell line which synthesizes large amounts of laminin) and host tissues of syngenic mice; c) at the front of invasion between MO4 cells and precultured heart fragments in an in vitro model of invasion. Laminin, but not type IV collagen, promoted attachment of MO4 cells. This attachment was inhibited by preincubation of laminin matrix support with (+)-catechin, a flavonoid which also prevented invasion of the precultured heart fragment in vitro. Our data demonstrate that laminin accumulates between malignant cells and host tissue in human brain metastases and in an in vitro and an in vivo model of invasion. In these later models, accumulation of laminin is the consequence, at least in part, of its biosynthesis by MO4 cells. Since laminin promotes attachment of malignant cells in vitro, increases invasiveness and metastatic activities of murine malignant cells, it is tempting to speculate that laminin synthesized by invasive cells and accumulated at the front of invasion plays a significant role in the first step of invasion.

Adenocarcinoma