Fluorescence-Activated Cell Sorting and NanoString Profiling of Single Neural Crest Cells and Pigment Cells

Jaws and branchial arches together are a basic, segmented feature of the vertebrate head. Seven arches develop in the zebrafish embryo (Danio rerio), derived largely from neural crest cells that form the cartilaginous skeleton. In this and the following paper we describe the phenotypes of 109 arch mutants, focusing here on three classes that affect the posterior pharyngeal arches, including the hyoid and five gill-bearing arches. In lockjaw, the hyoid arch is strongly reduced and subsets of branchial arches do not develop. Mutants of a large second class, designated the flathead group, lack several adjacent branchial arches and their associated cartilages. Five alleles at the flathead locus all lead to larvae that lack arches 4–6. Among 34 other flathead group members complementation tests are incomplete, but at least six unique phenotypes can be distinguished. These all delete continuous stretches of adjacent branchial arches and unpaired cartilages in the ventral midline. Many show cell death in the midbrain, from which some neural crest precursors of the arches originate. lockjaw and a few mutants in the flathead group, including pistachio, affect both jaw cartilage and pigmentation, reflecting essential functions of these genes in at least two neural crest lineages. Mutants of a third class, including boxer, dackel and pincher, affect pectoral fins and axonal trajectories in the brain, as well as the arches. Their skeletal phenotypes suggest that they disrupt cartilage morphogenesis in all arches. Our results suggest that there are sets of genes that: (1) specify neural crest cells in groups of adjacent head segments, and (2) function in common genetic pathways in a variety of tissues including the brain, pectoral fins and pigment cells as well as pharyngeal arches.

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An experimental study on neural crest migration in Barbus conchonius (Cyprinidae, Teleostei), with special reference to the origin of the enteroendocrine cells

Development ◽

10.1242/dev.62.1.309 ◽

1981 ◽

Vol 62 (1) ◽

pp. 309-323

Author(s):

C. H. J. Lamers ◽

J. W. H. M. Rombout ◽

L. P. M. Timmermans

Keyword(s):

Neural Crest ◽

Neural Tube ◽

Ganglion Cells ◽

Neural Crest Cells ◽

Enteroendocrine Cells ◽

Pigment Cells ◽

Transplantation Technique ◽

Spinal Ganglion Cells ◽

Neural Crest Origin ◽

Neural Crest Migration

A neural crest transplantation technique is described for fish. As in other classes ofvertebrates, two pathways of neural crest migration can be distinguished: a lateroventral pathway between somites and ectoderm, and a medioventral pathway between somites and neural tube/notochord. In this paper evidence is presented for a neural crest origin of spinal ganglion cells and pigment cells, and indication for such an origin is obtained for sympathetic and enteric ganglion cells and for cells that are probably homologues to adrenomedullary and paraganglion cells in the future kidney area. The destiny of neural crest cells near the developing lateral-line sense organs is discussed. When grafted into the yolk, neural crest cells or neural tube cells appear to differentiate into ‘periblast cells’; this suggests a highly activating influence of the yolk. Many neural crest cells are found around the urinary ducts and, when grafted below the notochord, even within the urinary duct epithelium. These neural crest cells do not invade the gut epithelium, even when grafted adjacent to the developing gut. Consequently enteroendocrine cells in fish are not likely to have a trunkor rhombencephalic neural crest origin. Another possible origin of these cells will be proposed.

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A vital dye analysis of the timing and pathways of avian trunk neural crest cell migration

Development ◽

10.1242/dev.106.4.809 ◽

1989 ◽

Vol 106 (4) ◽

pp. 809-816 ◽

Cited By ~ 33

Author(s):

G.N. Serbedzija ◽

M. Bronner-Fraser ◽

S.E. Fraser

Keyword(s):

Neural Crest ◽

Neural Tube ◽

Neural Crest Cell ◽

Neural Crest Cells ◽

Sympathetic Ganglia ◽

Pigment Cells ◽

Root Cells ◽

Vital Dye ◽

Rostral Portion ◽

Crest Cell

To permit a more detailed analysis of neural crest cell migratory pathways in the chick embryo, neural crest cells were labelled with a nondeleterious membrane intercalating vital dye, DiI. All neural tube cells with endfeet in contact with the lumen, including premigratory neural crest cells, were labelled by pressure injecting a solution of DiI into the lumen of the neural tube. When assayed one to three days later, migrating neural crest cells, motor axons, and ventral root cells were the only cells types external to the neural tube labelled with DiI. During the neural crest cell migratory phase, distinctly labelled cells were found along: (1) a dorsolateral pathway, under the epidermis, as well adjacent to and intercalating through the dermamyotome; and (2) a ventral pathway, through the rostral portion of each sclerotome and around the dorsal aorta as described previously. In contrast to those cells migrating through the sclerotome, labelled cells on the dorsolateral pathway were not segmentally arranged along the rostrocaudal axis. DiI-labelled cells were observed in all truncal neural crest derivatives, including subepidermal presumptive pigment cells, dorsal root ganglia, and sympathetic ganglia. By varying the stage at which the injection was performed, neural crest cell emigration at the level of the wing bud was shown to occur from stage 13 through stage 22. In addition, neural crest cells were found to populate their derivatives in a ventral-to-dorsal order, with the latest emigrating cells migrating exclusively along the dorsolateral pathway.

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Fluorescence-Activated Cell Sorting of EGFP-Labeled Neural Crest Cells From Murine Embryonic Craniofacial Tissue

Journal of Biomedicine and Biotechnology ◽

10.1155/jbb.2005.232 ◽

2005 ◽

Vol 2005 (3) ◽

pp. 232-237 ◽

Cited By ~ 12

Author(s):

Saurabh Singh ◽

Vasker Bhattacherjee ◽

Partha Mukhopadhyay ◽

Christopher A. Worth ◽

Samuel R. Wellhausen ◽

...

Keyword(s):

Neural Crest ◽

Cell Sorting ◽

Present Report ◽

Neural Crest Cells ◽

Egfp Expression ◽

Marker Genes ◽

Flow Cytometric Method ◽

Flow Cytometric ◽

Polymerase Chain ◽

Craniofacial Tissue

During the early stages of embryogenesis, pluripotent neural crest cells (NCC) are known to migrate from the neural folds to populate multiple target sites in the embryo where they differentiate into various derivatives, including cartilage, bone, connective tissue, melanocytes, glia, and neurons of the peripheral nervous system. The ability to obtain pure NCC populations is essential to enable molecular analyses of neural crest induction, migration, and/or differentiation. CrossingWnt1-CreandZ/EGtransgenic mouse lines resulted in offspring in which theWnt1-Cretransgene activated permanent EGFP expression only in NCC. The present report demonstrates a flow cytometric method to sort and isolate populations of EGFP-labeled NCC. The identity of the sorted neural crest cells was confirmed by assaying expression of known marker genes by TaqMan Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR). The molecular strategy described in this report provides a means to extract intact RNA from a pure population of NCC thus enabling analysis of gene expression in a defined population of embryonic precursor cells critical to development.

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

Development ◽

10.1242/dev.103.4.743 ◽

1988 ◽

Vol 103 (4) ◽

pp. 743-756 ◽

Cited By ~ 2

Author(s):

H.H. Epperlein ◽

W. Halfter ◽

R.P. Tucker

Keyword(s):

Cell Migration ◽

Neural Crest ◽

Neural Tube ◽

Neural Crest Cell ◽

Neural Crest Cells ◽

Pigment Cells ◽

Dorsal Fin ◽

Amphibian Embryos ◽

Neural Crest Cell Migration ◽

Crest Cell

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)

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Vital dye labelling of Xenopus laevis trunk neural crest reveals multipotency and novel pathways of migration

Development ◽

10.1242/dev.118.2.363 ◽

1993 ◽

Vol 118 (2) ◽

pp. 363-376 ◽

Cited By ~ 16

Author(s):

A. Collazo ◽

M. Bronner-Fraser ◽

S.E. Fraser

Keyword(s):

Xenopus Laevis ◽

Neural Crest ◽

Neural Tube ◽

Single Cells ◽

Neural Crest Cell ◽

Neural Crest Cells ◽

Pigment Cells ◽

Spinal Ganglia ◽

Posterior Portion ◽

Precise Position

Although the Xenopus embryo has served as an important model system for both molecular and cellular studies of vertebrate development, comparatively little is known about its neural crest. Here, we take advantage of the ease of manipulation and relative transparency of Xenopus laevis embryos to follow neural crest cell migration and differentiation in living embryos. We use two techniques to study the lineage and migratory patterns of frog neural crest cells: (1) injections of DiI or lysinated rhodamine dextran (LRD) into small populations of neural crest cells to follow movement and (2) injections of LRD into single cells to follow cell lineage. By using non-invasive approaches that allow observations in living embryos and control of the time and position of labelling, we have been able to expand upon the results of previous grafting experiments. Migration and differentiation of the labelled cells were observed over time in individual living embryos, and later in sections to determine precise position and morphology. Derivatives populated by the neural crest are the fins, pigment stripes, spinal ganglia, adrenal medulla, pronephric duct, enteric nuclei and the posterior portion of the dorsal aorta. In the rostral to mid-trunk levels, most neural crest cells migrate along two paths: a dorsal pathway into the fin, followed by presumptive fin cells, and a ventral pathway along the neural tube and notochord, followed by presumptive pigment, sensory ganglion, sympathetic ganglion and adrenal medullary cells. In the caudal trunk, two additional paths were noted. One group of cells moves circumferentially within the fin, in an arc from dorsal to ventral; another progresses ventrally to the anus and subsequently populates the ventral fin. By labelling individual precursor cells, we find that neural tube and neural crest cells often share a common precursor. The majority of clones contain labelled progeny cells in the dorsal fin. The remainder have progeny in multiple derivatives including spinal ganglion cells, pigment cells, enteric cells, fin cells and/or neural tube cells in all combinations, suggesting that many premigratory Xenopus neural crest precursors are multipotent.

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The role of cell-extracellular matrix interactions in neural crest cell migration

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156195 ◽

1989 ◽

Vol 47 ◽

pp. 842-843

Author(s):

Marianne Bronner-Fraser

Keyword(s):

Extracellular Matrix ◽

Cell Migration ◽

Neural Crest ◽

Cell Movement ◽

Neural Crest Cell ◽

Neural Crest Cells ◽

Pigment Cells ◽

Surrounding Matrix ◽

Adrenal Chromaffin ◽

Crest Cell

The formation of the embryo involves intricate cell movements, cell proliferation, and differentiation. The neural crest has long served as a model for the study of these processes because these cells: 1. migrate extensively along characteristic pathways during embryogenesis. 2. give rise to diverse and numerous derivatives, including pigment cells, adrenal chromaffin cells, and the ganglia of the peripheral nervous system; and 3. are accessible to surgical, immunological, and biochemical manipulations during both initial and certain later stages in their development. We are in the process of identifying factors that influence cell migration and differentiation in the neural crest system.Neural crest cells follow two primary migratory pathways in the trunk: a dorsolateral route underneath the skin, and a ventral route through the somite. Within the somites, neural crest cells preferentially migrate through the rostral half of each sclerotome but are absent from the caudal sclerotome. The regions through which neural crest cells migrate are lined with extracellular matrix (ECM) molecules. Because of the intimate relationship between neural crest cells and the surrounding matrix, it has been proposed that the ECM plays an important role in the initiation, guidance, and cessation of neural crest cell movement.

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Avian neural crest cells can migrate in the dorsolateral path only if they are specified as melanocytes

Development ◽

10.1242/dev.121.3.915 ◽

1995 ◽

Vol 121 (3) ◽

pp. 915-924 ◽

Cited By ~ 19

Author(s):

C.A. Erickson ◽

T.L. Goins

Keyword(s):

Neural Crest ◽

Normal Development ◽

Neural Crest Cells ◽

Pigment Cells ◽

Environmental Cues ◽

Avian Embryo ◽

Thoracic Level ◽

Branchial Arches ◽

Trunk Neural Crest ◽

Migratory Pathway

Neural crest cells are conventionally believed to migrate arbitrarily into various pathways and to differentiate according to the environmental cues that they encounter. We present data consistent with the notion that melanocytes are directed, by virtue of their phenotype, into the dorsolateral path, whereas other neural crest derivatives are excluded. In the avian embryo, trunk neural crest cells that migrate ventrally differentiate largely into neurons and glial cells of the peripheral nervous system. Neural crest cells that migrate into the dorsolateral path become melanocytes, the pigment cells of the skin. Neural crest cells destined for the dorsolateral path are delayed in their migration until at least 24 hours after migration commences ventrally. Previous studies have suggested that invasion into the dorsolateral path is dependent upon a change in the migratory environment. A complementary possibility is that as neural crest cells differentiate into melanocytes they acquire the ability to take this pathway. When quail neural crest cells that have been grown in culture for 12 hours are labeled with Fluoro-gold and then grafted into the early migratory pathway at the thoracic level, they migrate only ventrally and are coincident with the host neural crest. When fully differentiated melanocytes (96 hours old) are back-grafted under identical conditions, however, they enter the dorsolateral path and invade the ectoderm at least one day prior to the host neural crest. Likewise, neural crest cells that have been cultured for at least 20 hours and are enriched in melanoblasts immediately migrate in the dorsolateral path, in addition to the ventral path, when back-grafted into the thoracic level. A population of neural crest cells depleted of melanoblasts--crest cells derived from the branchial arches--are not able to invade the dorsolateral path, suggesting that only pigment cells or their precursors are able to take this migratory route. These results suggest that as neural crest cells differentiate into melanocytes they can exploit the dorsolateral path immediately. Even when 12-hour crest cells are grafted into stage 19–21 embryos at an axial level where host crest are invading the dorsolateral path, these young neural crest cells do not migrate dorsolaterally. Conversely, melanoblasts or melanocytes grafted under the same circumstances are found in the ectoderm. These latter results suggest that during normal development neural crest cells must be specified, if not already beginning to differentiate, as melanocytes in order to take this path.(ABSTRACT TRUNCATED AT 400 WORDS)

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