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Our aim was to understand the involvement of Sonic hedgehog (Shh) morphogen in the oriented distribution of neural crest cells (NCCs) toward the optic vesicle and to look for potential disorders of this guiding mechanism after ethanol exposure. In vitro directional analysis showed the chemotactic response of NCCs up Shh gradients and to notochord co-cultures (Shh source) or to their conditioned medium, a response inhibited by anti-Shh antibody, receptor inhibitor cyclopamine and anti-Smo morpholino (MO). Expression of the Ptch-Smo receptor complex on in vitro NCCs was also shown. In whole embryos, the expression of Shh mRNA and protein was seen in the ocular region, and of Ptch, Smo and Gli/Sufu system on cephalic NCCs. Anti-Smo MO or Ptch-mutated plasmid (Ptch1(Δloop2)) impaired cephalic NCC migration/distribution, with fewer cells invading the optic region and with higher cell density at the homolateral mesencephalic level. Beads embedded with cyclopamine (Smo-blocking) or Shh (ectopic signal) supported the role of Shh as an in vivo guide molecule for cephalic NCCs. Ethanol exposure perturbed in vitro and in vivo NCC migration. Early stage embryos treated with ethanol, in a model reproducing Fetal Alcohol Syndrome, showed later disruptions of craniofacial development associated with abnormal in situ expression of Shh morphogen. The results show the Shh/Ptch/Smo-dependent migration of NCCs toward the optic vesicle, with the support of specific inactivation with genetic and pharmacological tools. They also help to understand mechanisms of accurate distribution of embryonic cells and of their perturbation by a commonly consumed teratogen, and demonstrate, in addition to its other known developmental functions, a new biological activity of cellular guidance for Shh., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Chemotactic cell migration is triggered by extracellular concentration gradients of molecules segregated by target fields. Neural crest cells (NCCs), paradigmatic as an accurately moving cell population, undergo wide dispersion along multiple pathways, invading with precision defined sites of the embryo to differentiate into many derivatives. This report addresses the involvement of NT-3 in early colonization by cephalic NCCs invading the optic vesicle region. The results of in vitro and in vivo approaches showed that NCCs migrate directionally up an NT-3 concentration gradient. We also demonstrated the expression of NT-3 in the ocular region as well as their functional TrkB, TrkC and p75 receptors on cephalic NCCs. On whole-mount embryo, a perturbed distribution of NCCs colonizing the optic vesicle target field was shown after morpholino cancelation of cephalic NT-3 or TrkC receptor on NCCs, as well as in situ blocking of TrkC receptor of mesencephalic NCCs by specific antibody released from inserted microbeads. The present results strongly suggest that, among other complementary cell guidance factor(s), the chemotactic response of NCCs toward the ocular region NT-3 gradient is essential for spatiotemporal cell orientation, amplifying the functional scope of this neurotrophic factor as a molecular guide for the embryo cells, besides its well-known canonical functions., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

The extracellular environment through which neural crest cells (NCCs) translocate and differentiate plays a crucial role in the determination of cell migration and homing. In the trunk, NCC-derived melanocyte precursor cells (MPCs) take the dorsolateral pathway and colonize the skin, where they differentiate into pigment cells (PCs). Our hypothesis was that the skin, the MPCs' target tissue, may induce a directional response of NCCs toward diffusible factor(s). We show that the treatment of in vitro NCCs with skin extract (SE) or Stem Cell Factor (SCF) contributes to maintaining proliferative activity, accelerates melanocyte differentiation, and guides a subpopulation of NCCs by chemotaxis toward the gradient source of these factors, suggesting that they may represent the MPCs' subpopulation. Current data on stimulated directional persistence of NCCs supports the participation of diffusible molecules in the target colonization mechanism, guiding MPCs to migrate and invade the skin. Our results show similar effects of SE and SCF on NCC growth, proliferation and pigment cell differentiation. Also, the use of a proven real-time directionality-based objective assay shows the directional migration of NCCs toward SE and SCF, indicating that the epidermal SCF molecule may be involved in the chemotactic guidance mechanism of in vivo NCCs. Although SCF is the strongest candidate to account for these phenomena, the nature of other factor(s) affecting NCC-oriented migration remains to be investigated. This data amplifies the functional scope of trophic factors by involving them in new cell behaviors such as molecular guidance in the colonization mechanism of embryonic cells., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Neural crest cells (NCCs), a transient population that migrates from the developing neural tube, distributes through the embryo and differentiates into many derivatives, are clearly involved in the damage induced by prenatal exposure to ethanol. The aim of this work was to evaluate alterations of trophic parameters of in vivo (in ovo) and in vitro NCCs exposed to teratogenic ethanol doses, and their possible prevention by trophic factor treatment. Chick embryos of 24-30h of incubation were treated during 10h with 100mM ethanol, or 40 ng/ml Neurotrophin 3 (NT3), or 10 ng/ml Ciliary Neurotrophic Factor (CNTF), or ethanol plus NT3 or CNTF, or defined medium; then the topographic distribution of NCC apoptosis was assessed using a whole-mount acridine orange supravital method. Cultures of cephalic NCCs were exposed to the same ethanol or NT3, or CNTF treatments, or ethanol plus one of both trophic factors, or N2 medium. A viability assay was performed using the calcein-ethidium test, apoptosis was evaluated with the TUNEL test, and proliferative capacity after BrdU labeling. After direct exposure of embryos to 100mM ethanol for 10h, a high level of NCC apoptosis was coincident with the abnormal closure of the neural tube. These anomalies were prevented in embryos exposed to ethanol plus NT3 but not with CNTF. In NCC cultures, high cell mortality and a diminution of proliferative activity were observed after 3h of ethanol treatment. Incubation with ethanol plus NT3 (but not with CNTF) prevented NCC mortality as well as a fall in NCC proliferation. The consequences of direct exposure to ethanol expand data from our and other laboratories, supporting current opinion on the potential risk of alcohol ingestion (even at low doses and/or during a short time), in any period of pregnancy or lactation. Our in vivo/in vitro model encourages us to examine the pathogenic mechanism(s) of the ethanol-exposed embryo as well as the use of trophic factors for the treatment and/or prevention of anomalies induced by prenatal alcohol., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The effect of moderate exposure to ethanol during late gestation was studied in terms of its interaction with moderate exposure during nursing from an intoxicated dam. A further issue was whether behavioral effects of ethanol, especially the enhanced ethanol intake known to occur after moderate ethanol prenatally or during nursing, depend upon teratological effects that may include death of neurons in the main olfactory bulb (MOB). During gestational days 17-20 rats were given 0, 1 or 2g/kg ethanol doses intragastrically (i.g.). After parturition these dams were given a dose of 2.5g/kg ethanol i.g. each day and allowed to perform regular nursing activities. During postnatal days (PDs) 15 and 16, ethanol intake of pups was assessed along with aspects of their general activity. In a second experiment pups given the same prenatal treatment as above were tested for blood ethanol concentration (BEC) in response to an ethanol challenge on PD6. A third experiment (Experiment 2b) assessed stereologically the number of cells in the granular cell layer of the MOB on PD7, as a function of analogous pre- and postnatal ethanol exposures. Results revealed that ethanol intake during the third postnatal week was increased by prenatal as well as postnatal ethanol exposure, with a few interesting qualifications. For instance, pups given 1g/kg prenatally did not have increased ethanol intake unless they also had experienced ethanol during nursing. There were no effects of ethanol on either BECs or conventional teratology (cell number). This increases the viability of an explanation of the effects of prenatal and early postnatal ethanol on later ethanol intake in terms of learning and memory., (2010 Elsevier Inc. All rights reserved.)

Background: Due to the fact that different biological parameters play a key role in colonic malignant behavior, with nuclear morphometry being a prognostic marker in many malignancies, then predictive approaches in colorectal cancer (CRC) carried out on histologically well-defined groups may prevent interpretative errors. Subsequently, in the present study, CRC patients were screened according to the morphometric features of tumor cell nuclei, using an accurate histotechnical approach, to analyze their clinical evolution according to Dukes' stratification., Materials and Methods: A total of 66 cases were grouped according to Dukes' classification (5 y of follow-up). The perimeter, nuclear area, and shape factor of 50 interphase carcinoma nuclei were recorded through microphotographs obtained from each subject. Nuclei boundaries were drawn by an electronic pencil and examined by a computerized system. Data were submitted to a variance analysis, and a multi-regression model compared results., Results: The sample was made up of 44 males (66.67%) and 22 females (33.33%) aged 59.7 +/- 6 y old. Forty-nine patients (74.24%) were classified as stage B, and 17 (25.76%) as stage C. Nuclear homogeneity was confirmed by analysis of variance. The nuclear parameters were (mean +/- SD): area (3.17 +/- 1.74), perimeter (6.72 +/- 1.83), and shape factor (0.82 +/- 0.03). A multiple logistic regression model showed that stage C subjects had a higher risk of developing a worse clinical evolution than those at stage B (P < 0.02), independent of sex and age., Conclusions: Dukes' classification remains the best predictor of evolution. Although nucleomorphometric suitability is still controversial, this technique has become an additional tool to establish CRC prognosis.

Attraction of spermatozoa by way of chemotaxis to substances secreted from the egg or its surrounding cells has been demonstrated in marine species, amphibians, and mammals. This process is species- or family-specific in marine invertebrates: a chemoattractant for one marine species is usually not recognized by another species or by a member of another family. It is not known whether this selectivity is also the rule in other phyla. Furthermore, it is not at all obvious that such selectivity would be advantageous to species with internal fertilization. Here, using a directionality-based assay for chemotaxis, we studied in vitro the chemotactic response of human and rabbit spermatozoa to human, rabbit, and bovine egg-related factors. We found that spermatozoa from each of the two sources responded similarly well to egg-related factors obtained from any of the three species examined. These results indicate lack of chemotaxis-related, species specificity between these species, suggesting that their sperm chemoattractants are common or very similar. The findings further suggest that mammals do not rely on species specificity of sperm chemotaxis for avoidance of interspecies fertilization.

Precontact communication between gametes is established by chemotaxis. Sperm chemotaxis toward factor(s) in follicular fluid (FF) has been demonstrated in humans and mice. In humans, the chemotactic responsiveness is restricted to capacitated spermatozoa. Here, we investigated whether sperm chemotaxis to factor(s) present in FF also occurs in rabbits and, if so, whether only capacitated spermatozoa are chemotactically responsive. Chemotaxis assays were performed by videomicroscopy in a Zigmond chamber. We measured chemotactic responsiveness as a function of FF dilution by means of a novel directionality-based method that considers the ratio between the distances traveled by the spermatozoa both parallel to the chemoattractant gradient and perpendicular to it. A peak of maximal response was observed at 10(-4) dilution of FF, resulting in a typical chemotactic concentration-dependent curve in which 23% of the spermatozoa were chemotactically responsive. In contrast, the percentage of cells exhibiting FF-dependent enhanced speed of swimming increased with the FF concentration, whereas the percentage of cells maintaining linear motility decreased with the FF concentration. The percentages of chemotactically responsive cells were very similar to those of capacitated spermatozoa. Depletion of the latter by stimulation of the acrosome reaction resulted in a total loss of the chemotactic response, whereas the reappearance of capacitated cells resulted in a recovery of chemotactic responsiveness. We conclude that rabbit spermatozoa, like human spermatozoa, are chemotactically responsive to FF factor(s) and acquire this responsiveness as part of the capacitation process.

Background: Fetal alcohol syndrome (FAS) is an embryopathology related to maternal alcohol drinking. The information concerning the factors involved in the prenatal mechanisms of ethanol action at the cellular and molecular levels is scarce. Because several abnormal changes in FAS involve regions colonized by cell lineages derived from neural crest cells (NCCs), it is reasonable to propose that epigenetic alteration of this cell population can represent an important component of the etiopathogeny. The aim of this work was to evaluate the direct effect of ethanol on a chick embryo model, as well as on in vitro NCC morphology and dynamic behavior., Methods: After ethanol treatment, in ovo or cultured chick embryos were used to determine the anatomical development of and to quantify the migratory parameters and apoptosis of NCCs. Scanning electron microscopy was performed on ethanol-perfused (and control) cultures of cephalic and trunk NCCs; the actin cytoskeleton was evaluated, and morphometric and dynamic parameters were determined after time-lapse videorecording. Recovery capacity after ethanol treatment was also determined., Results: Chick embryos submitted to conditions sufficient to induce FAS in mammals displayed developmental disruptions frequently accompanied by cephalic/facial anomalies. In vitro studies also indicated that cephalic and trunk NCCs exposed to ethanol exhibited significant and permanent changes regarding cell shape, surface morphology, apoptotic cell death, cytoskeleton, and distance and velocity traveled, as well as an abnormal pattern of migration., Conclusions: Taking into account that even a limited period of abnormal behavior may imply serious consequences in the final cues of an embryonic cell population, our results indicate that the biological effects of ethanol on early development-even during a short time-could induce permanent ontogenetic perturbations of NCCs, with potentially dramatic effects on embryonic morphogenesis. These results support an important participation of NCCs in the etiopathogeny of FAS.

Neural crest cells (NCC) segregate from neural folds, swim through extracellular matrix, and differentiate in several cell types. During in vivo and in vitro dispersion, NCC display a substantial proliferative rate. Here, we have evaluated this biological behavior under the influence of different substrates. NCC exhibit the highest proliferative activity on fibronectin substrate, with shortening of the G1 period. This fact is reverted by specific antibody pre-treatment of the substrate. Taking into account that NCC migrate along narrow and restricted pathways rich in essential fibronectin, with high proliferative rates, these results indicate that the fibronectin pathway contributes to increase the cell number, leading to a high population pressure that could participate in the early dispersion of NCC.

The dynamic parameters of mouse sperm cells exposed to follicular and oviductal fluids were assessed. Spermatozoa were tracked on a chemotactic Zigmond chamber and recorded using a videomicroscopy system. The results were evaluated with computer-supported image analysis. Follicular fluid at a dilution of 10(-4) markedly increased the proportion of spermatozoa with high velocity, and stimulated chemotactic behaviour. The highest velocities were observed in sperm cells exposed to oviductal fluid, and a greater proportion of these cells had high velocity compared with those exposed to follicular fluid. Chemotaxis was induced in spermatozoa exposed to oviductal fluid at dilutions of 10(-3) and 10(-5). These results suggest the presence of temporal subpopulations of responsive spermatozoa, considering the distance travelled towards both follicular and oviductal fluids and the proportion of sperm cells migrating towards the gradient in the highest distance ranges. This is the first report on the effect of isolated follicular and oviductal fluids on dynamic parameters and chemotaxis of mouse spermatozoa. The findings support previous work showing that the motility and directionality of mouse sperm cells is increased by factors in the microenvironment of the egg. Although the significance of these factors in vivo is unknown, it is possible that there is a relay mechanism involving sequential activity of both oviductal and follicular fluids to direct the male gametes towards the egg.

The aim of this work was to evaluate the period of time required for the induction of changes in motility of mouse spermatozoa in response to factors from the microenvironment of oocytes. To determine the effects of the latency time, the period of preincubation before contact with the oocyte product(s), sperm samples were incubated for 15 or 90 min and then exposed to either Dulbecco's Modified Eagle's Medium (DMEM) or to a crude extract of superovulated oocytes (CE). The assays were performed in a Zigmond chamber by filling one compartment with either DMEM or CE, and the other compartment with the sperm suspension. A videomicroscopy system was used for tracking the spermatozoa. Sperm motility analysis was assessed using a semiautomatic objective method, and the following parameters were determined; (1) dynamic parameters: curvilinear velocity, linear velocity and linearity; (2) progressive motility: percentage of spermatozoa showing either circular or linear patterns of movement; and (3) directional motility: percentage of spermatozoa that moved towards either the DMEM or the CE. The results of this work showed that when the spermatozoa contacted soluble factors in CE after only 15 min of previous incubation, there was a significant increase in their dynamic parameters, change in their progressive motility, and induction of directional movement of the spermatozoa towards the CE components, while a longer period of preincubation did not significantly modify these effects. On the other hand, in the presence of culture medium (with or without addition of bovine serum albumin), the spermatozoa needed a more extended period of incubation to significantly increase their dynamic parameters and to modify their progressive motility, while maintaining a random direction of movement.

Background: In vertebrate embryos, migration of trunk neural crest cells (NCC) proceeds mainly in two streams: a dorsoventral path between the neural tube and somites, and a dorsolateral one between somites and ectoderm. This last pathway is taken by melanocyte precursor cells (MPC) homing the skin, while pigment cells seeding internal organs and the peritoneal wall follow the dorsoventral pathway. Early routes taken by subpopulations of NCC have been well documented using the quail-chick chimaera system and monoclonal antibodies to NCC. However, very little is known about the advanced migratory behavior of MPC, which determines their late distribution patterns at different embryonic axial levels., Methods: Histological sections of neck, thorax, and abdomen of 6.5 to 9 day quail embryos submitted to DOPA reaction (tyrosinase activity) were used. In four concentric areas--dorsal and ventrally subdivided--the relative density of MPC was determined by morphometric methods., Results: The relative regional density of MPC from their individualization as DOPA-positive putative pigment cells until their definitive seeding in the epidermis showed a progressively higher cell density from deeper to peripheral zones in all three levels studied, with peaks of cell density suggesting a centrifugal pattern occurring in at least two waves of migratory cells., Conclusions: The spatial distribution of the MPC varies according to both the axial level and the developmental stage of the embryo. Furthermore, the general pattern of centrifugal distribution observed might be attributed to a different timing of cell differentiation closely related to their migratory behavior.

A method is presented for softening the hard tissues of stem and strobile of Equisetum giganteum allowing the preparation of representative histological slides suitable for teaching and research. Segments of aerial portions of the Equisetum giganteum shoot system were fixed with formaldehyde:ethanol:acetic acid (5:10:5) for 24 hr at room temperature, washed in distilled water and immersed in a mixture of 5% hydrofluoric acid and 0.5% sulfuric acid for 1 hr at room temperature. Hydrofluoric acid has a higher affinity for silica components, and the sulfuric acid acts as a catalyst favoring the separation of calcium silcates. This simple, inexpensive and rapid method allows paraffin sections to be prepared while preserving the topographic microanatomy by decreasing technical artifacts produced by conventional softeners, and preserving PAS-positive polysaccharides.

We have previously demonstrated that directional migration of neural crest cells (NCC) is associated with a high cell density, resulting from an active cell proliferation. It is also known that treatment with retinoic acid (RA) causes a dose-dependent inhibition of proliferation of some cell types, and that administration of RA during the early stages of embryonic development, induces cranio-facial abnormal patterns corresponding to NCC derivatives. In view of these findings, it was of interest to determine if exogenous RA is a potential modulator of the mitotic rate of NCC, and to explore the hypothesis of an inhibitory effect exerted by RA on the proliferative behaviour of NCC in vivo and in vitro. Homogenates of RA-treated chick embryos showed a low [3H]dT incorporation, indicating a generalized diminution of DNA synthesis. The labelling index (LI = number of labelled cells/total number of cells) revealed that NCC from RA-treated and control embryos had higher values of [3H]dT incorporation than neural tube cells (P < 0.0001). Autoradiographs of RA-treated chick embryos showed a significantly lower [3H]dT incorporation in NCC at the prosencephalic and mesencephalic levels, as well as in the neural tube cells at the prosencephalic, mesencephalic and rhombencephalic levels, than in control chick embryos (P < 0.0001). NCC cultures treated with 1 or 10 microM RA had a significantly lower LI than in cultures treated with 0.1 microM RA or control cultures (P < 0.04). In chick embryos, the mitotic index of NCC was 0.026 for RA-treated and 0.033 for controls, while the duration of the cell cycle was significantly longer in the NCC of RA-treated embryos (approximately 40 h) than in controls (approximately 25 h). The length of the cell cycle phases of NCC was similar in both experimental conditions, except for G1 phase, which was significantly longer in the RA-treated group than in controls. These results show that RA blocks DNA synthesis and lengthens the proliferative behaviour of NCC both in early chick embryos and in vitro, effects that could modify the morphogenetic patterns of NCC distribution through a decreased cell population.

A method is presented for in situ treatment of whole chick embryos with drugs and immunocytochemical and fixative reagents that resembles conditions "in ovo." The chick embryo is placed in a "shell-less" culture system where it is contained by an agar ring allowing for treatment in vivo. The conceptus (embryo+membranes) is then mounted on a microporous membrane and inserted into a filter device connected to a three-way stopcock that permits fluids to be changed using syringes. The embryo is then processed in toto or after embedding and sectioning for light or electron microscopy. The proposed handling system decreases technical artifacts and changes in the topographic microanatomy produced by conventional manipulation of chick embryos. This method is useful also for directly observing and recording changes in the embryo during drug treatments and allows processing with dangerous reagents without their direct contact with the operator. It is simple, inexpensive and requires only minimal technical training.

The purpose of this work was to study the dispersion of early migratory neural crest cell (NCC) of chick embryos treated with ethanol concentration known to induce the Fetal Alcohol Syndrome (FAS). After a direct treatment with ethanol (250 mg/dl), there was a higher number of abnormal embryos than in the control group, showing neural and cardiac anomalies. After NC-1 immunostaining, ethanol-treated embryos showed smaller number of NCC at all neuraxis levels and presumptive NCC were frequently seen flowing towards the lumen of the neural tube. Present data support the view that ethanol impairment of migratory behaviour of NCC may explain certain anomalies of FAS such as those found at the cephalic end of the body, which is known to be largely derived from NCC.

Study of sperm motility is associated to the development of precise and economical systems of evaluation. The purpose of this work was to develop an Objective Semi-Automated Method (MOSA) to evaluate the sperm motility. Human semen samples were registered by video-microscopy. The same videofilms were analysed with the MOSA, the subjective method and the automated CellSoft method. The percentages of motile and immotile sperms were equivalent with the three methods. The percentages of rapidly and slowly motile sperms were similar both with the MOSA and the subjective method. The curvilinear and linear velocities as well as the linearity values obtained with the MOSA were different to those obtained with the CellSoft, although such differences would be biologically acceptable. MOSA is an inexpensive, objective and precise method that does not require trained technicians and allows evaluation of several parameters of sperm motility.

The chemotactic response of neoplastic cells (NC) induced by soluble platelet factors was investigated. NC suspensions isolated from murine mammary gland adenocarcinomas having different metastatic capabilities were incubated in Boyden's chambers and challenged with (1) 'Early Platelet Factors' (EP), obtained from the soluble fraction of recently collagen-activated human platelets, and (2) 'Late Platelet Factors' (LP), isolated after 24 hours incubation of the platelet aggregates. Chemotaxis was expressed as the distance travelled by NC through nitrocellulose filters. NC isolated from M3, the tumour line having the stronger metastatic potential, showed a significant chemotactic response towards LP factors, whereas NC from the M2 line exhibiting the lower metastatic behaviour, showed a chemotactic response towards EP factors. Both tumour cell lines lacked motion capability towards the well known chemoattractant peptide N-f-Met-Leu-Phe-Phe as well as to serum, plasma, collagen type I or culture medium. The different chemotactic response of both tumour lines when they were challenged by concentration gradients of factors released by early or late collagen-activated human platelets, confirm a relationship between platelet activity and metastatic capabilities and suggests that platelet chemoattractants might play a role in the metastatic dissemination of these mammary gland adenocarcinomas.

Fibronectins (FN) are adhesive glycoproteins whose role in platelet aggregation is unclear. Addition of 3, 6 and 12 micrograms/ml of human plasma FN in vitro to isolated human platelets, which had been freed from plasma FN by gel filtration and subsequently stimulated with collagen, inhibited the last stage of platelet aggregation. With 3 and 6 micrograms/ml of FN a shortening of the lag-time was also observed. These data showed that FN may play a role in platelet-collagen interaction as well as in platelet-platelet interaction.

Histochemical characterization of glycoconjugates elaborated by palatal pleomorphic adenomas showed that glycoproteins, glycosaminoglycans, and perhaps glycolipids were present in both the "epithelial" and "connective tissue" components of these tumors, as well as in the distinct glycocalyx present in the luminal surface of tubular and cystic areas.

The fat globules of the cream fractions of human and rat milk were stained with ruthenium red. Under the electron microscope, discrete granules and an amorphous coat of lesser density are seen at the surface of the milk fat globules. Since ruthenium red binds anionic groups selectively, it is probable that the granules contain the greatest concentration of these groups. The cream fraction of rat milk contains hexoses, hexosamines, methylpentoses and sialic acid. Methylpentoses and hexosamines are significantly enriched in the cream fraction. It is concluded that the finding of a surface coat in milk fat globules is in keeping with the Bargmann-Knoop model and suggests a distinct mechanism for carrying certain complex carbohydrates in milk. The role of the negative charges at the outer surface of the membrane coat is maintaining fat globules in suspension and in binding certain cations such as calcium is suggested.

Cells of the neural crest participate in a major class of cell migratory events during embryonic development. From indirect evidence, it has been suggested that fibronectin (FN) might be involved in these events. We have directly tested the role of FN in neural crest cell adhesion and migration using several in vitro model systems. Avian trunk neural crest cells adhered readily to purified plasma FN substrates and to extracellular matrices containing cellular FN. Their adhesion was inhibited by antibodies to a cell-binding fragment of FN. In contrast, these cells did not adhere to glass, type I collagen, or to bovine serum albumin in the absence of FN. Neural crest cell adhesion to laminin (LN) was significantly less than to FN; however, culturing of crest cells under conditions producing an epithelioid phenotype resulted in cells that could bind equally as well to LN as to FN. The migration of neural crest cells appeared to depend on both the substrate and the extent of cell interactions. Cells migrated substantially more rapidly on FN than on LN or type I collagen substrates; if provided a choice between stripes of FN and glass or LN, cells migrated preferentially on the FN. Migration was inhibited by antibodies against the cell-binding region of FN, and the inhibition could be reversed by a subsequent addition of exogenous FN. However, the migration on FN was random and displayed little persistence of direction unless cells were at high densities that permitted frequent contacts. The in vitro rate of migration of cells on FN-containing matrices was 50 microns/h, similar to their migration rates along the narrow regions of FN-containing extracellular matrix in migratory pathways in vivo. These results indicate that FN is important for neural crest cell adhesion and migration and that the high cell densities of neural crest cells in the transient, narrow migratory pathways found in the embryo are necessary for effective directional migration.

Carrageenans are widely used as food additives. Thus, it seemed of interest to test their possible teratogenic action. For this purpose, 530 chick eggs were injected in the yolk sac with 0.1 ml of a solution of 0.1% lambda-carrageenan in 0.9% sodium chloride. As controls, 286 eggs were injected with 0.1 ml of 9.0% sodium chloride. In addition, 284 eggs received no treatment. After incubation for 48--50 hours at 39 degrees C, embryos were fixed, cleared, and observed with a stereoscopic microscope. The frequency of abnormal embryos in the group receiving lambda-carrageenan was higher than in the controls (p less than 0.04). Partial duplication of the body, abnormal flexures of the trunk, anencephaly, a severely malformed brain, thickening of the neural tube wall, an irregular neural tube lumen with segmentary occlusion and a reduction in crown-rump length and number of somites were distinctly seen in the lambda-carrageenan-injected group. Moreover, the average number of anomalies per embryo in the lambda-carrageenan-injected group was nearly twice that in the controls. Present data indicate that lambda-carrageenan has teratogenic effects on early stages of the development of the chick embryo.

Chick embryos injected with lambda-carrageenan prior to incubation and studied with light microscopy at 48 hours of development presented various anomalies. Lack of closure at different levels of the neural tube was one of the lesions most frequently seen. Rachischisis and craniorachischisis were commonly associated with various degrees of hyperplasia of the neural tube wall. Diverse patterns of neural hyperplasia ranged from total occlusion of the neural tube to localized thickening of lateral walls leading to a typical "hourglass" appearance of the ependymal lumen. Multiple septa and cavitations of the neural tube lumen were also recorded. Cephalic and/or trunk duplication of the body as well as disorganization of notochord and somites were frequently associated with rachischisis and areas of necrosis or pyknosis of neural tissue. Neural crest cells, which are normally present in paraneural areas at the stages studied, were not encountered in the carrageenan-injected embryos. These areas were frequently occupied by hyperplastic ectodermic folds. Similarity of present findings on carrageenan-treated embryos with the anomalies induced by selective destruction or impairment of migration of neural crest cells suggests that involvement of this cell population may have a role in the causation of the anomalies observed.