Embryonic cell motility can be guided by physiological electric fields

Migratory embryonic quail somitic fibroblasts display a striking sensitivity to small, steady electric fields. There are three components to their response. They begin to orient their long axes perpendicular to the field lines within 5 min of current application at the optimal field strength of 600 mV/mm. The threshold field for significant orientation in 90 min is 150 mV/mm (only 3 mV/cell width). The cells migrate toward the cathode with a similar low threshold. At field strengths greater than 400 mV/mm, the cells also elongate beginning about 1 h after field application. The importance of this embryonic cell galvanotaxis and orientation by electric fields lies in the possible utilization of this behavior both by the embryo in the guidance of embryonic cell migration in vivo and by the investigator to control cell morphology and directionality of movement in vitro in order to study mechanisms of motility.     

Endogenous electrical currents and the resultant voltage gradients in the chick embryo

We have studied some of the electrophysiological properties of 2 1/2- to 4-day-old (stage 14-22) chick embryos. Using a recently developed two-dimensional vibrating probe, large currents were found to exit the posterior intestinal portal (p.i.p.) during the period of tail gut reduction. During this period, epithelial cells lining cloacal regions of the hindgut are dying, thus creating a low-resistance pathway for current flow out of the embryo. Currents entered the intact epithelium over other regions of the embryo. The outward currents at the p.i.p. were first detected at stage 15 and reached their average maximum current density of 112 +/- 10 microA/cm2 at stage 17. After stage 17, the magnitude of the currents decreased, dropping to 16 +/- 0.3 microA/cm2 by stage 22. The currents were reversibly reduced by about 50% when Na+ was replaced by choline in the bathing solution. The magnitude of the currents leaving the p.i.p. suggested the existence of a measurable intraembryonic voltage gradient. The transepithelial potential (TEP) of stage 14-21 embryos was measured lateral to the neural tube through the dorsal ectoderm. For all stages, the combined average TEP was 16 +/- 0.5 mV. Differences in the TEP between various regions of the embryo were used to calculate an intraembryonic voltage gradient. At stage 14, before outward current was found at the p.i.p., no significant intraembryonic voltage gradient was detected. At stage 17, when the outward current at the p.i.p. was maximum, a voltage gradient of 21 +/- 5 mV/mm (mean +/- SEM; N = 6) was measured in the caudal end of the embryo. This gradient in some cases was as steep as 33 mV/mm. This is well above the minimum level needed to affect the direction of embryonic cell migration in vitro. We hypothesize that this endogenous electrical field acts as a directional cue for neural crest cell movements in the developing chick embryo.     

Mapping of neural crest pathways in Xenopus laevis using inter- and intra-specific cell markers

This study examines the pathways of migration followed by neural crest cells in Xenopus embryos using two recently described cell marking techniques. The first is an interspecific chimera created by grafting Xenopus borealis cells into Xenopus laevis hosts. The cells of these closely related species can be distinguished by their nuclear dimorphism. The second type of marker is created by microinjection of lysinated dextrans into fertilized eggs which can then be used for intraspecific grafting. These recently developed fluorescent dyes are fixable and identifiable in both living and fixed embryos. After grafting labeled donor neural tubes into unlabeled host embryos, the distribution of neural crest cells at various stages after grafting was used to define the pathways of neural crest migration. To control for possible grafting artifacts, fluorescent lysinated dextran was injected into a single blastomere which gives rise to a large number of neural crest cells, thereby labeling the neural crest without grafting. By all three techniques, Xenopus neural crest cells were observed along two predominant pathways in the trunk. The majority of neural crest cells were observed along a "ventral" route, between the neural tube and somite, the notochord and somite, and along the dorsal mesentery. A second group of neural crest cells was observed "dorsally" where they populated the dorsal fin. A third minor "lateral" pathway was observed primarily in borealis/laevis chimerae and in blastomere-injected embryos; some neural crest cells were observed underneath the ectoderm lateral to the neural tube. Along the rostrocaudal axis, neural crest cells were not continuously distributed but were primarily located across from the caudal two-thirds of the somite. Fewer than 3% of the neural crest cells were observed across from the rostral third of each somite. When grafted to ventral locations, neural crest cells were not able to migrate dorsally but migrated laterally along the dorsal mesentery. Labeled neural crest cells gave rise to cells of the spinal, sympathetic, and enteric ganglia as well as to adrenal chromaffin cells, Schwann cells, pigment cells, mesenchymal cells of the dorsal fin, and some cells in the integuments and in the region of the pronephros. These results show that the neural crest migratory pathways in Xenopus differ from those in the avian embryo. In avians NC cells migrate as a closely associated sheet of cells while in Xenopus they migrate as individual cells. Both species exhibit a metamerism in the neural crest cell distribution pattern along the rostrocaudal axis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protein Tyrosine Phosphatase 4A3 (PTP4A3) Is Required for Xenopus laevis Cranial Neural Crest Migration In Vivo

Uveal melanoma is the most common intraocular malignancy in adults, representing between about 4% and 5% of all melanomas. High expression levels of Protein Tyrosine Phosphatase 4A3, a dual phosphatase, is highly predictive of metastasis development and PTP4A3 overexpression in uveal melanoma cells increases their in vitro migration and in vivo invasiveness. Melanocytes, including uveal melanocytes, are derived from the neural crest during embryonic development. We therefore suggested that PTP4A3 function in uveal melanoma metastasis may be related to an embryonic role during neural crest cell migration. We show that PTP4A3 plays a role in cephalic neural crest development in Xenopus laevis. PTP4A3 loss of function resulted in a reduction of neural crest territory, whilst gain of function experiments increased neural crest territory. Isochronic graft experiments demonstrated that PTP4A3-depleted neural crest explants are unable to migrate in host embryos. Pharmacological inhibition of PTP4A3 on dissected neural crest cells significantly reduced their migration velocity in vitro. Our results demonstrate that PTP4A3 is required for cephalic neural crest migration in vivo during embryonic development.     

An assay system to study migratory behavior of cranial neural crest cells in Xenopus

An increasing number of genes are known to show expression in the cranial neural crest area. So far it is very difficult to analyze their effect on neural crest cell migration because of the lack of transplantation techniques. This paper presents a simple method to study the migratory behavior of cranial neural crest cells by homo- and heterotopic transplantations: Green fluorescent protein (GFP) RNA was injected into one blastomere of Xenopus laevis embryos at the 2-cell stage. The cranial neural crest area of stage 14 embryos was transplanted into the head or trunk region of an uninjected host embryo, and the migration was monitored by GFP fluorescence. The transplants were further examined by double immunostaining and confocal microscopy to trace migratory routes inside the embryo, and to exclude contaminations of grafts with foreign tissues. Our results demonstrate that we developed a highly efficient and reproducible technique to study the migratory ability of cranial neural crest cells. It offers the possibility to analyze genes involved in neural crest cell migration by coinjecting their RNA with that of GFP. Received: 28 September 1999 / Accepted: 17 November 1999     

The distribution of tenascin coincides with pathways of neural crest cell migration

The distribution of the extracellular matrix (ECM) glycoprotein, tenascin, has been compared with that of fibronectin in neural crest migration pathways of Xenopus laevis, quail and rat embryos. In all species studied, the distribution of tenascin, examined by immunohistochemistry, was more closely correlated with pathways of migration than that of fibronectin, which is known to be important for neural crest migration. In Xenopus laevis embryos, anti-tenascin stained the dorsal fin matrix and ECM along the ventral route of migration, but not the ECM found laterally between the ectoderma and somites where neural crest cells do not migrate. In quail embryos, the appearance of tenascin in neural crest pathways was well correlated with the anterior-to-posterior wave of migration. The distribution of tenascin within somites was compared with that of the neural crest marker, HNK-1, in quail embryos. In the dorsal halves of quail somites which contained migrating neural crest cells, the predominant tenascin staining was in the anterior halves of the somites, codistributed with the migrating cells. In rat embryos, tenascin was detectable in the somites only in the anterior halves. Tenascin was not detectable in the matrix of cultured quail neural crest cells, but was in the matrix surrounding somite and notochord cells in vitro. Neural crest cells cultured on a substratum of tenascin did not spread and were rounded. We propose that tenascin is an important factor controlling neural crest morphogenesis, perhaps by modifying the interaction of neural crest cells with fibronectin.     

The Timing of Appearance and Pathway of Migration of Cranial Neural Crest-derived Precursors of Melanocytes in Chick Embryos

The timing of appearance and pathway of migration of precursors of melanocytes in cranial regions of chick embryos were examined by the monoclonal antibody MEBL-1, which can identify precursors of melanocytes soon after their emigration from the neural tube (7). Precursors of melanocytes were first detected on the dorsal side of the mesencephalic neural tube at stage 16, when other neural crest cells had already left the dorsal side of the neural tube. Then precursors of melanocytes at more rostral and caudal levels appeared. After the first appearance on the neural tube, precursors of melanocytes migrated along a dorsolateral pathway under the superficial ectoderm, which followed other neural crest cells. These results indicate that precursors of melanocytes migrate along spatially the same pathway as other neural crest cells, but temporally the different time as considered previously.     

Gene controlling a differentiation step in the quail melanocyte

Albino mutation in animals blocks pigmentation owing to a deficiency in tyrosinase, although it does not affect the differentiation of colorless melanocytes from the neural crest. In the albino Japanese quail (al, sex-linked), it was demonstrated that morphologically normal melanocytes differentiated from neural crest cells in culture and that these cells contained unmelanized melanosomes as expected for the mutant cells. The mutant melanocytes, however, were shown to exhibit tyrosinase activity in the Golgi-endoplasmic reticulum-lysosome region and in the Golgi vesicles. Our results seem to indicate that the mutation at the al locus affects the transport of tyrosinase from the Golgi area to melanosomes.     

The Delayed Entry of Thoracic Neural Crest Cells into the Dorsolateral Path Is a Consequence of the Late Emigration of Melanogenic Neural Crest Cells from the Neural Tube

Neural crest cells migrate along two pathways in the trunk: the ventral path, between the neural tube and somite, and the dorsolateral path, between the somite and overlying ectoderm. In avian embryos, ventral migration precedes dorsolateral migration by nearly 24 h, and the onset of dorsolateral migration coincides with the cessation of ventral migration. Neural crest cells in the ventral path differentiate predominantly as neurons and glial cells of the peripheral nervous system, whereas those in the dorsolateral path give rise to the melanocytes of the skin. Thus, early- and late-migrating neural crest cells exhibit unique morphogenetic behaviors and give rise to different subsets of neural crest derivatives. Here we present evidence that these differences reflect the appearance of specified melanocyte precursors, or melanoblasts, from late- but not early-migrating neural crest cells. We demonstrate that serum from Smyth line (SL) chickens specifically immunolabels melanocyte precursors, or melanoblasts. Using SL serum as a marker, we first detect melanoblasts immediately dorsal and lateral to the neural tube beginning at stage 18, which is prior to the onset of dorsolateral migration. At later stages every neural crest cell in the dorsolateral path is SL-positive, demonstrating that only melanoblasts migrate dorsolaterally. Thus, melanoblast specification precedes dorsolateral migration, and only melanoblasts migrate dorsolaterally at the thoracic level. Together with previous work (Erickson, C. A., and Goins, T. L.,Development121, 915–924, 1995), these data argue that specification as a melanoblast is a prerequisite for dorsolateral migration. This conclusion suggested that the delay in dorsolateral migration (relative to ventral migration) may reflect a delay in the emigration of melanogenic neural crest cells from the neural tube. Several experiments support this hypothesis. There are no melanoblasts in the ventral path, as revealed by the absence of SL-positive cells in the ventral path, and neural crest cells isolated from the ventral path do not give rise to melanocytes when explanted in culture, suggesting that early, ventrally migrating neural crest cells are limited in their ability to differentiate as melanocytes. Similarly, neural crest cells that emigrate from the neural tubein vitroduring the first 6 h fail to give rise to any melanocytes or SL-positive melanoblasts, whereas neural crest cells that emigrate at progressively later times show a dramatic increase in melanogenesis under identical culture conditions. Thus, the timing of dorsolateral migration at the thoracic level is ultimately controlled by the late emigration of melanogenic neural crest cells from the neural tube.     

The distribution of fibronectin and tenascin along migratory pathways of the neural crest in the trunk of amphibian embryos

It is generally assumed that in amphibian embryos neural crest cells migrate dorsally, where they form the mesenchyme of the dorsal fin, laterally (between somites and epidermis), where they give rise to pigment cells, and ventromedially (between somites and neural tube), where they form the elements of the peripheral nervous system. While there is agreement about the crest migratory routes in the axolotl (Ambystoma mexicanum), different opinions exist about the lateral pathway in Xenopus. We investigated neural crest cell migration in Xenopus (stages 23, 32, 35/36 and 41) using the X. laevis-X. borealis nuclear marker system and could not find evidence for cells migrating laterally. We have also used immunohistochemistry to study the distribution of the extracellular matrix (ECM) glycoproteins fibronectin (FN) and tenascin (TN), which have been implicated in directing neural crest cells during their migrations in avian and mammalian embryos, in the neural crest migratory pathways of Xenopus and the axolotl. In premigratory stages of the crest, both in Xenopus (stage 22) and the axolotl (stage 25), FN was found subepidermally and in extracellular spaces around the neural tube, notochord and somites. The staining was particularly intense in the dorsal part of the embryo, but it was also present along the visceral and parietal layers of the lateral plate mesoderm. TN, in contrast, was found only in the anterior trunk mesoderm in Xenopus; in the axolotl, it was absent. During neural crest cell migration in Xenopus (stages 25-33) and the axolotl (stages 28-35), anti-FN stained the ECM throughout the embryo, whereas anti-TN staining was limited to dorsal regions. There it was particularly intense medially, i.e. in the dorsal fin, around the neural tube, notochord, dorsal aorta and at the medial surface of the somites (stage 35 in both species). During postmigratory stages in Xenopus (stage 40), anti-FN staining was less intense than anti-TN staining. In culture, axolotl neural crest cells spread differently on FN- and TN-coated substrata. On TN, the onset of cellular outgrowth was delayed for about 1 day, but after 3 days the extent of outgrowth was indistinguishable from cultures grown on FN. However, neural crest cells in 3-day-old cultures were much more flattened on FN than on TN. We conclude that both FN and TN are present in the ECM that lines the neural crest migratory pathways of amphibian embryos at the time when the neural crest cells are actively migrating. FN is present in the embryonic ECM before the onset of neural crest migration.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cardiac neural crest is dispensable for outflow tract septation in Xenopus

In vertebrate embryos, cardiac precursor cells of the primary heart field are specified in the lateral mesoderm. These cells converge at the ventral midline to form the linear heart tube, and give rise to the atria and the left ventricle. The right ventricle and the outflow tract are derived from an adjacent population of precursors known as the second heart field. In addition, the cardiac neural crest contributes cells to the septum of the outflow tract to separate the systemic and the pulmonary circulations. The amphibian heart has a single ventricle and an outflow tract with an incomplete spiral septum; however, it is unknown whether the cardiac neural crest is also involved in outflow tract septation, as in amniotes. Using a combination of tissue transplantations and molecular analyses in Xenopus we show that the amphibian outflow tract is derived from a second heart field equivalent to that described in birds and mammals. However, in contrast to what we see in amniotes, it is the second heart field and not the cardiac neural crest that forms the septum of the amphibian outflow tract. In Xenopus, cardiac neural crest cells remain confined to the aortic sac and arch arteries and never populate the outflow tract cushions. This significant difference suggests that cardiac neural crest cell migration into the cardiac cushions is an amniote-specific characteristic, presumably acquired to increase the mass of the outflow tract septum with the evolutionary need for a fully divided circulation.     

Role of morphogens in neural crest cell determination

The neural crest is a transient, migratory cell population found in all vertebrate embryos that generate a diverse range of cell and tissue derivatives including, but not limited, to the neurons and glia of the peripheral nervous system, smooth muscle, connective tissue, melanocytes, craniofacial cartilage, and bone. Over the past few years, many studies have provided tremendous insights into understanding the mechanisms regulating the induction and migration of neural crest cell development. This review highlights the surprising and perhaps unexpected roles for morphogens in these distinct processes. A comparison of studies performed in several different vertebrates emphasizes the requirement for coordination between multiple signaling pathways in the induction and migration of neural crest cells in the developing embryo.     

Migration and proliferation of cultured neural crest cells in W mutant neural crest chimeras.

Chimeric mice, generated by aggregating preimplantation embryos, have been instrumental in the study of the development of coat color patterns in mammals. This approach, however, does not allow for direct experimental manipulation of the neural crest cells, which are the precursors of melanoblasts. We have devised a system that allows assessment of the developmental potential and migration of neural crest cells in vivo following their experimental manipulation in vitro. Cultured C57Bl/6 neural crest cells were microinjected in utero into neurulating Balb/c or W embryos and shown to contribute efficiently to pigmentation in the host animal. The resulting neural crest chimeras showed, however, different coat pigmentation patterns depending on the genotype of the host embryo. Whereas Balb/c neural crest chimeras showed very limited donor cell pigment contribution, restricted largely to the head, W mutant chimeras displayed extensive pigmentation throughout, often exceeding 50% of the coat. In contrast to Balb/c chimeras, where the donor melanoblasts appeared to have migrated primarily in the characteristic dorsoventral direction, in W mutants the injected cells appeared to migrate in the longitudinal as well as the dorsoventral direction, as if the cells were spreading through an empty space. This is consistent with the absence of a functional endogenous melanoblast population in W mutants, in contrast to Balb/c mice, which contain a full complement of melanocytes. Our results suggest that the W mutation disturbs migration and/or proliferation of endogenous melanoblasts. In order to obtain information on clonal size and extent of intermingling of donor cells, two genetically marked neural crest cell populations were mixed and coinjected into W embryos. In half of the tricolored chimeras, no co-localization of donor crest cells was observed, while, in the other half, a fine intermingling of donor-derived colors had occurred. These results are consistent with the hypothesis that pigmented areas in the chimeras can be derived from extensive proliferation of a few donor clones, which were able to colonize large territories in the host embryo. We have also analyzed the development of pigmentation in neural crest cultures in vitro, and found that neural tubes explanted from embryos carrying wt or weak W alleles produced pigmented melanocytes while more severe W genotypes were associated with deficient pigment formation in vitro.     

Myosin-X is critical for migratory ability of Xenopus cranial neural crest cells

The neural crest is a highly migratory cell population, unique to vertebrates, that forms much of the craniofacial skeleton and peripheral nervous system. In exploring the cell biological basis underlying this behavior, we have identified an unconventional myosin, myosin-X (Myo10) that is required for neural crest migration. Myo10 is highly expressed in both premigratory and migrating cranial neural crest (CNC) cells in Xenopus embryos. Disrupting Myo10 expression using antisense morpholino oligonucleotides leads to impaired neural crest migration and subsequent cartilage formation, but only a slight delay in induction. In vivo grafting experiments reveal that Myo10-depleted CNC cells migrate a shorter distance and fail to segregate into distinct migratory streams. Finally, in vitro cultures and cell dissociation-reaggregation assays suggest that Myo10 may be critical for cell protrusion and cell-cell adhesion. These results demonstrate an essential role for Myo10 in normal cranial neural crest migration and suggest a link to cell-cell interactions and formation of processes.