Developmental potential of trunk neural crest cells in the mouse

The availability of naturally occurring and engineered mutations in mice which affect the neural crest makes the mouse embryo an important experimental system for studying neural crest cell differentiation. Here, we determine the normal developmental potential of neural crest cells by performing in situ cell lineage analysis in the mouse by microinjecting lysinated rhodamine dextran (LRD) into individual dorsal neural tube cells in the trunk. Labeled progeny derived from single cells were found in the neural tube, dorsal root ganglia, sympathoadrenal derivatives, presumptive Schwann cells and/or pigment cells. Most embryos contained labeled cells both in the neural tube and at least one neural crest derivative, and numerous clones contributed to multiple neural crest derivatives. The time of injection influenced the derivatives populated by the labeled cells. Injections at early stages of migration yielded labeled progeny in both dorsal and ventral neural crest derivatives, whereas those performed at later stages had labeled cells only in more dorsal neural crest derivatives, such as dorsal root ganglion and presumptive pigment cells. The results suggest that in the mouse embryo: (1) there is a common precursor for neural crest and neural tube cells; (2) some neural crest cells are multipotent; and (3) the timing of emigration influences the range of possible neural crest derivatives

Pathways of trunk neural crest cell migration in the mouse embryo as revealed by vital dye labelling

Analysis of neural crest cell migration in the mouse has been difficult due to the lack of reliable cell markers. Recently, we found that injection of DiI into the chick neural tube marks premigratory neural crest cells whose endfeet are in contact with the lumen of the neural tube (Serbedzija et al. Development 106, 809–819 (1989)). In the present study, this technique was applied to study neural crest cell migratory pathways in the trunk of the mouse embryo. Embryos were removed from the mother between the 8th and the 10th days of development and DiI was injected into the lumen of the neural tube. The embryos were then cultured for 12 to 24 h, and analyzed at the level of the forelimb. We observed two predominant pathways of neural crest cell migration: (1) a ventral pathway through the rostral portion of the somite and (2) a dorsolateral pathway between the dermamyotome and the epidermis. Neural crest cells were observed along the dorsolateral pathway throughout the period of migration. The distribution of labelled cells along the ventral pathway suggested that there were two overlapping phases of migration. An early ventrolateral phase began before E9 and ended by E9.5; this pathway consisted of a stream of cells within the rostral sclerotome, adjacent to the dermamyotome, that extended ventrally to the region of the sympathetic ganglia and the dorsal aorta

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

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

Vital dye labelling demonstrates a sacral neural crest contribution to the enteric nervous system of chick and mouse embryos

We have used the vital dye, DiI, to analyze the contribution of sacral neural crest cells to the enteric nervous system in chick and mouse embryos. In order to label premigratory sacral neural crest cells selectively, DiI was injected into the lumen of the neural tube at the level of the hindlimb. In chick embryos, DiI injections made prior to stage 19 resulted in labelled cells in the gut, which had emerged from the neural tube adjacent to somites 29–37. In mouse embryos, neural crest cells emigrated from the sacral neural tube between E9 and E9.5. In both chick and mouse embryos, DiI-labelled cells were observed in the rostral half of the somitic sclerotome, around the dorsal aorta, in the mesentery surrounding the gut, as well as within the epithelium of the gut. Mouse embryos, however, contained consistently fewer labelled cells than chick embryos. DiI-labelled cells first were observed in the rostral and dorsal portion of the gut. Paralleling the maturation of the embryo, there was a rostral-to-caudal sequence in which neural crest cells populated the gut at the sacral level. In addition, neural crest cells appeared within the gut in a dorsal-to-ventral sequence, suggesting that the cells entered the gut dorsally and moved progressively ventrally. The present results resolve a long-standing discrepancy in the literature by demonstrating that sacral neural crest cells in both the chick and mouse contribute to the enteric nervous system in the postumbilical gut

Multidisciplinary approaches to understanding collective cell migration in developmental biology

Mathematical models are becoming increasingly integrated with experimental efforts in the study of biological systems. Collective cell migration in developmental biology provides a particularly fruitful application area for the development and application of theoretical models to predict the behaviour of complex multicellular systems with many interacting parts. By doing so, mathematical models provide a tool to assess the consistency of experimental observations with testable mechanistic hypotheses. In this review article we showcase examples from recent years of multidisciplinary investigations of neural crest cell migration. The neural crest model system has been used to study how collective migration of cell populations is shaped by cell-cell interactions, cell-environmental interactions, and heterogeneity between cells. The wide range of emergent behaviours exhibited by neural crest cells in different embryonal locations and in different organisms helps us chart out the spectrum of collective cell migration. At the same time, this diversity in migratory characteristics highlights the need to reconcile or unify the array of currently hypothesised mechanisms through the next generation of experimental data and generalised theoretical descriptions

Vital dye analysis of cranial neural crest cell migration in the mouse embryo

The spatial and temporal aspects of cranial neural crest cell migration in the mouse are poorly understood because of technical limitations. No reliable cell markers are available and vital staining of embryos in culture has had limited success because they develop normally for only 24 hours. Here, we circumvent these problems by combining vital dye labelling with exo utero embryological techniques. To define better the nature of cranial neural crest cell migration in the mouse embryo, premigratory cranial neural crest cells were labelled by injecting DiI into the amniotic cavity on embryonic day 8. Embryos, allowed to develop an additional 1 to 5 days exo utero in the mother before analysis, showed distinct and characteristic patterns of cranial neural crest cell migration at the different axial levels. Neural crest cells arising at the level of the forebrain migrated ventrally in a contiguous stream through the mesenchyme between the eye and the diencephalon. In the region of the midbrain, the cells migrated ventrolaterally as dispersed cells through the mesenchyme bordered by the lateral surface of the mesencephalon and the ectoderm. At the level of the hindbrain, neural crest cells migrated ventrolaterally in three subectodermal streams that were segmentally distributed. Each stream extended from the dorsal portion of the neural tube into the distal portion of the adjacent branchial arch. The order in which cranial neural crest cells populate their derivatives was determined by labelling embryos at different stages of development. Cranial neural crest cells populated their derivatives in a ventral-to-dorsal order, similar to the pattern observed at trunk levels. In order to confirm and extend the findings obtained with exo utero embryos, DiI (1,1-dioctadecyl-3,3,3′,3′-tetramethylindo-carbocyanine perchlorate) was applied focally to the neural folds of embryos, which were then cultured for 24 hours. Because the culture technique permitted increased control of the timing and location of the DiI injection, it was possible to determine the duration of cranial neural crest cell emigration from the neural tube. Cranial neural crest cell emigration from the neural folds was completed by the 11-somite stage in the region of the rostral hindbrain, the 14-somite stage in the regions of the midbrain and caudal hindbrain and not until the 16-somite stage in the region of the forebrain. At each level, the time between the earliest and latest neural crest cells to emigrate from the neural tube appeared to be 9 hours

Ancient evolutionary origin of vertebrate enteric neurons from trunk-derived neural crest

The enteric nervous system of jawed vertebrates arises primarily from vagal neural crest cells that migrate to the foregut and subsequently colonize and innervate the entire gastrointestinal tract. Here we examine development of the enteric nervous system in the basal jawless vertebrate the sea lamprey (Petromyzon marinus) to gain insight into its evolutionary origin. Surprisingly, we find no evidence for the existence of a vagally derived enteric neural crest population in the lamprey. Rather, labelling with the lipophilic dye DiI shows that late-migrating cells, originating from the trunk neural tube and associated with nerve fibres, differentiate into neurons within the gut wall and typhlosole. We propose that these trunk-derived neural crest cells may be homologous to Schwann cell precursors, recently shown in mammalian embryos to populate post-embryonic parasympathetic ganglia, including enteric ganglia. Our results suggest that neural-crest-derived Schwann cell precursors made an important contribution to the ancient enteric nervous system of early jawless vertebrates, a role that was largely subsumed by vagal neural crest cells in early gnathostomes

Advanced optical imaging in living embryos

Developmental biology investigations have evolved from static studies of embryo anatomy and into dynamic studies of the genetic and cellular mechanisms responsible for shaping the embryo anatomy. With the advancement of fluorescent protein fusions, the ability to visualize and comprehend how thousands to millions of cells interact with one another to form tissues and organs in three dimensions (xyz) over time (t) is just beginning to be realized and exploited. In this review, we explore recent advances utilizing confocal and multi-photon time-lapse microscopy to capture gene expression, cell behavior, and embryo development. From choosing the appropriate fluorophore, to labeling strategy, to experimental set-up, and data pipeline handling, this review covers the various aspects related to acquiring and analyzing multi-dimensional data sets. These innovative techniques in multi-dimensional imaging and analysis can be applied across a number of fields in time and space including protein dynamics to cell biology to morphogenesis

Hedgehog signaling via a calcitonin receptor-like receptor can induce arterial differentiation independently of VEGF signaling in zebrafish

Multiple signaling pathways control the specification of endothelial cells (ECs) to become arteries or veins during vertebrate embryogenesis. Current models propose that a cascade of Hedgehog (Hh), vascular endothelial growth factor (VEGF), and Notch signaling acts instructively on ECs to control the choice between arterial or venous fate. Differences in the phenotypes induced by Hh, VEGF, or Notch inhibition suggest that not all of the effects of Hh on arteriovenous specification are mediated by VEGF. We establish that full derepression of the Hh pathway in ptc1;ptc2 mutants converts the posterior cardinal vein into a second arterial vessel that manifests intact arterial gene expression, intersegmental vessel sprouting, and HSC gene expression. Importantly, although VEGF was thought to be absolutely essential for arterial fates, we find that normal and ectopic arterial differentiation can occur without VEGF signaling in ptc1;ptc2 mutants. Furthermore, Hh is able to bypass VEGF to induce arterial differentiation in ECs via the calcitonin receptor-like receptor, thus revealing a surprising complexity in the interplay between Hh and VEGF signaling during arteriovenous specification. Finally, our experiments establish a dual function of Hh during induction of runx1+ HSCs