Emergence and migration of trunk neural crest cells in a snake, the California Kingsnake (Lampropeltis getula californiae)
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The neural crest is a group of multipotent cells that emerges after an epithelial-to-mesenchymal transition from the dorsal neural tube early during development. These cells then migrate throughout the embryo, giving rise to a wide variety derivatives including the peripheral nervous system, craniofacial skeleton, pigment cells, and endocrine organs. While much is known about neural crest cells in mammals, birds, amphibians and fish, relatively little is known about their development in non-avian reptiles like snakes and lizards.
In this study, we show for the first time ever trunk neural crest migration in a snake by labeling it with DiI and immunofluorescence. As in birds and mammals, we find that early migrating trunk neural crest cells use both a ventromedial pathway and an inter-somitic pathway in the snake. However, unlike birds and mammals, we also observed large numbers of late migrating neural crest cells utilizing the inter-somitic pathway in snake.
We found that while trunk neural crest migration in snakes is very similar to that of other amniotes, the inter-somitic pathway is used more extensively by late-migrating trunk neural crest cells in snake.
KeywordsDorsal Root Ganglion Neural Tube Neural Crest Neural Crest Cell Dorsal Aorta
human natural killer-1 cell antibody, obtained from ATCC cell culture (Cat. No. TIB-200)
3H-Indolium, 5-[[4-(chloromethyl)benzoyl]amino]methyl]-2-[3-(1,3-dihydro-3,3-dimethyl-1-octadecyl-2H-indol-2-ylidene)-1-propenyl]-3,3-dimethyl-1-octadecyl-, chloride
green fluorescent protein
dorsal root ganglion
phosphate buffer saline, Dulbecco's recipe
Dulbecco minimal essential medium
fetal bovine serum
The neural crest is a group of multipotent cells that emerge after an epithelial-to-mesenchymal transition from the dorsal neural tube early after neural tube closure. These cells give rise to a wide variety of neuronal and glial derivatives in the peripheral nervous system, as well as parts of the head skeleton and endocrine organs [1, 2]. In jawed, anamniote vertebrates like sharks and teleosts, neural crest cells also give rise to electrosensory organs  and fin mesenchyme . The neural crest in the trunk portion of an embryo has been found to follow different migratory pathways in different organisms. In amniotes trunk neural crest cells will follow two main courses: a ventromedial pathway through the rostral part of somites, and a dorsolateral pathway between somites and ectoderm . In amphibians, trunk neural crest follows a dorsal pathway into the fin and a ventral pathway between the neural tube and the caudal portion of the somite . In zebrafish, trunk neural crest predominantly migrates between the neural tube and somites as in amphibians [7, 8].
The origin of the neural crest was an important event in vertebrate given that it forms most of the craniofacial skeleton . Agnathans (like lampreys) [10, 11], teleosts (bony fish) and amphibians clearly possess identifiable cranial neural crest streams that are similar to those observed in amniotes [8, 12]. However, the trunk neural crest is less prominent in anamniotes [13, 14], which appear to have fewer trunk neural crest cells than amniotes [15, 16, 17].
Among lepidosaurs, snakes provide an especially interesting case for evolutionary developmental studies as they display a mix of basal and derived amniote features. For example, the development of somites in snakes goes through a similar pattern as in other amniotes, albeit more rapid  generating in excess of 300 somites. It is unknown how the basal and derived features of snakes are reflected in the neural crest as neural crest development and migration is poorly described in lepidosaurs.
To better understand the evolution of neural crest migratory patterning in amniotes, we examined the neural crest in the trunk of snake embryos using vital dye labeling and fluorescent immunohistochemistry. We found that trunk neural crest migration in snakes closely follows the patterns observed in turtle, birds, and mammals, with most early-migrating trunk neural crest cells traveling through the anterior portion of the somites and a few migrating between somites. However, we also observed a large number of late-migrating trunk neural crest cells moving between the somites in snake, suggesting the prominence of this pathway may have been accentuated in snakes or reduced in other amniotes. Finally, we observed differences in the structure of a major trunk neural crest derivative, the dorsal root ganglia (DRGs) between non-avian reptiles, birds and mammals.
DiI labeling of neural crest
Neural crest migration in vertebrate embryos progresses in a rostro-caudal manner, in other words, at a given stage of development, the cells emigrating caudally are the early-migrating cells while the cells emigrating rostrally correspond to later-emigrating cells. The first observation from the DiI labeled embryos was that snake neural crest migration followed this same pattern. At the caudal level, where neural crest cells are starting to emerge from the dorsal neural tube, neural crest cells were migrating as streams of cells avoiding the caudal third of the somite (Fig. 2E-F, H-I and 2L). At more rostral levels, usually by the 2nd coil, neural crest cells migrated as a small group of cells on what seemed to be the most rostral portion of the somites and the inter-somitic space (Fig. 2D, H-I, K, L).
Embryo No.1 had abundant delaminated DiI-labeled cells, although these cells did not enter the ventromedial pathway (Fig. 2A-C). Sections of these embryo showed that very few cells migrated into regions usually populated by neural crest cells, i.e. dorsal aorta, mesonephroi, sensory ganglia, heart (data not shown). Embryo No.2 presented extensive delaminated cells along the rostral somites, especially at the tail level (Fig. 2E, F), while at the level of first coil the migrated cells on the rostral third of the somites (Fig. 2D). Embryo No.4 showed the best labeling of all (Fig. 2G-I) followed by Embryo No.5. Both embryos showed extensive streams of cells along the rostro-caudal axis on the dorsal portion of the somites and by the mesonephroi (Fig. 2H, K). Higher power pictures showed that these DiI streams had punctuated labeling, suggestive of individual migrating cells (Fig. 2I, L).
HNK1 labeling of neural crest
The embryo pictured in Fig. 5 had three coils (st.20) and in each of these regions, it was possible to observe a different stage in the development of the neural crest. In coil 1, the first one posterior to the hindbrain, HNK1 labeled what are likely condensing dorsal root ganglia (DRG, Fig. 5C and 5D). In addition, there is a line of HNK1 positive cells along the same region where the sympathetic chain will form, and at the most ventral portion of the coil, a group of HNK1 positive cells that correspond to where mesonephroi will develop (Fig. 5D). At mid-trunk levels we observed HNK1 labeling by the sympathetic ganglia and mesonephroi areas as well (Fig. 5F). At the tail end of this embryo (still growing) we observed a similar pattern of HNK1 staining as when using DiI (Fig. 5E, G, H): neural crest cells migrating as a rostral stream through the somites towards the mesonephroi region.
Peripheral Nervous system development
In the present study we examined the migration of snake trunk neural crest during development by using live cell labeling and neural crest markers. We found that neural crest cells in the king snake (Lampropeltis getula California) follow what seem to be highly conserved migratory patterns among amniotes as shown by HNK1 staining in turtles and birds [17, 24].
It is known that neural crest cells move rapidly, and within 8 hrs after delamination from the neural tube they have reached their destinations in avian and mouse embryos [31, 32]. In our study we incubated the embryos after labeling the neural tube with DiI for 12 and 24 hrs, and found that after this period of time, large number of cells has reached the same locations as in other vertebrates including the dorsal aorta, mesonephros, and developing gut. We did not observe a large number of DiI neural crest cells in the developing gut, presumably because the labeling was done too late to mark the delaminating vagal neural crest .
Neural crest migration has been studied by using HNK1 in non-avian reptiles, including crocodile and turtle [23, 24]. Hou and Takeuchi found that the migratory pattern of cranial and trunk neural crest in turtles followed that of birds . In our present study we found that snake trunk neural crest followed the essentially same migratory pathways as described for turtles and and other non-avian reptiles [24, 35, 36] suggesting conservation of these core pathways among all reptiles and amniotes.
Despite conservation of the main trunk neural crest migratory pathways, we did observe some differences in how these pathways were utilized. Snakes have the ability to grow their tail extensively (up to 350 somites length) allowing the simultaneous observation of the newly delaminated neural crest cells at the tail end  as well as the beginning of differentiating neural crest in the first coils. Our DiI labeling in a colubridae snake showed cell streams in what we term a 'dual pathway' of migration. The first group of delaminating neural crest cells followed very much the migratory pathways observed in avian and mammalian embryos; cells avoiding the caudal portion of the somites and moving along the ventromedial path towards the dorsal aorta region [5, 37]. However, at more rostral axial levels, a second large group of late-migrating trunk neural crest cells was observed moving as narrow streams between the somites. Such intersomitic migration has been observed for chicken as an early event in the first delaminating cells [5, 38], but not in late migrating neural crest cells. Interestingly, no intersomitic migration has been observed in non-avian reptiles . These findings suggest heavy utilization of the intersomitic pathway by late-migrating neural crest cells may be a derived feature of snakes, or an aspect of development lost in birds and mammals. More detailed studies of trunk neural crest migration in other non-avian reptiles will be needed to determine which scenario is more likely.
Recently, it was shown that mice neural crest cells will migrate along the intersomitic space when both Sema3A, F and their receptors (Neuropilin 1 and 2) are absent . This finding reinforces the importance of such inhibitory molecules (somite environment) in controlling neural crest migration and suggests they may play important roles in altering migratory patterns during evolution.
In addition to differences in the migration of late-emerging neural crest cells, between snake and other amniotes, HNK1 staining revealing a difference in the shape of dorsal root ganglia (DRGs) between non-avian reptiles, mouse, and chick. Whereas mouse and chick DRGs are triangular, those of snake, gecko and turtle are smaller and spindle-shaped. This likely reflects some level of convergence between the structure of bird and mammalian DRGs related to the evolution of a more active lifestyle in these two warm-blooded groups. Such convergence has been reported in evolution of the bird and mammalian hearts .
Another unexpected result from this study was the large proportion of trunk neural crest cells found in the mesonephric region by using DiI and HNK1 labeling. We found a considerable number of neural crest cells that migrated beyond the sympathetic ganglia region towards the mesonephroi. These cells do not correspond to the ones reported previously around the aorta . The mesonephroi in birds are derived from mesodermal tissues. The adrenal cortex primordia in snakes align as a strand between the mesonephroi and the dorsal aorta, although later it will lose contact with the kidneys . It is assumed that snake neural crest cells migrate towards the mesonephroi region and give rise to the chromaffin adrenal cells and even that snake mesonephroi could be partly crest derived [41, 42]. The present study supports a crest derived origin for the chromaffin cells, though not so for mesonephroi itself. Our results show DiI and HNK1 labeled cells around developing tubules, not as part of the tubules themselves. Therefore, snake trunk neural crest likely gives rise to the adrenal chromaffin cells, although we could not determine the ultimate fate of all those cells migrating towards the developing kidney. Future studies looking more closely, and after longer labeling period, will address this issue.
In summary, our study is the first description of trunk neural crest cell migration in snakes, using both vital dye labeling, and immunofluorescence. The pattern of migration in this organism initially follows the rostral ventromedial pathway typical of model amniotes and then switches to a late inter-somitic pathway. This late dependence on the intersomitic pathway appears may be unique to snakes, or may have been lost in other amniotes. Our results highlight both conserved and divergent features of snake trunk neural crest migration.
Collection and Staging of Embryos
Eggs of Lampropeltis getula californiae (Blainville, 1835), the common California king snake, were gathered from the herpetarium collection at CSUN two to three days after oviposition and embryos were collected within the next three days. Animal use and up keeping was according to approved protocols by the IACUC board of CSUN (Protocol #0506-012c). The embryos were staged according to Zehr's normal table .
Embryos were removed from egg cases by incision on one of the top edges (embryos are usually located in the midsection of the soft eggshell), the contents were emptied onto a Petri dish and after locating the embryo the shell was fully opened. Embryos were fixed in Carnoy's (70% ethanol, 20% formaldehyde and 10% glacial acetic acid) overnight for 24 hrs at 4°C, and then stored in 100% methanol at -20°C until histology preparation. Embryos went through prolonged (several hours each) dehydration steps in alcohol series and then placed in histosol for clearing. The tissues were then immersed in hot paraffin (McCormick Scientific Paraplast Plus) in a vacuum oven for two days before preparing the blocks and sectioning. Embryos were sectioned (10-12 μm) with a microtome, placed on Super-Frost slides and dried overnight at 37°C on a slide warmer.
Lizard embryos (Banded gecko, Coleonyx variegatus, Baird, 1859) were collected from three eggs from the herpetarium collection at CSUN after oviposition. Turtle embryos (Red-eared slider Trachemys scripta elegans, Wied-Neuwied, 1839) were collected from a freshly laid clutch in a California pond and embryos were fixed two weeks after oviposition. Chicken embryos were obtained from a Los Angeles purveyor of fertilized farm after incubating them to HH17 .
Snake tissue sections were re-hydrated in histosol and a graded series of ethanol washes (histosol, 100, 90, 70, 50 and 25% ethanol washes in water) and then equilibrated in PBS (Dulbecco's) before blocking in PBS containing 10% expired FBS and 1% Triton X-100 for 12 hrs. Primary antibodies were added in a 1:100 (or 1:1 for hybridoma supernatants) dilution in PBS and slides were incubated for two days at 4°C. After washing the sections in PBS for at least 20 minutes, secondary antibodies (Alexa fluoroprobes conjugated to anti-rabbit or anti-mouse IgG, Invitrogen) were added for 30 min and washed in PBS for immuno-fluorescence visualization and cover-slipped with Permount. Pictures of sections were taken using Axiovision LE software (Zeiss™) with an AxioCam black and white camera attached to a Zeiss AxioimagerA1 upright fluorescent microscope and assembled into figures using Adobe Photoshop 7. Primary antibodies were: HNK1 hybridoma collected at Caltech from a supernatant prepared following ATCC (Cat. No. TIB-200) instructions for HNK1, Tuj1 from Sigma and GFP antibody from Molecular Probes (Invitrogen).
Embryos were blocked overnight in blocking buffer (Phosphate Buffered Saline (PBS) containing 10% expired FBS and 1% Triton X-100 for 12 hrs, and then incubated with primary antibodies in PBS overnight at 4°C. The next day, embryos were extensively washed with PBS and incubated with secondary antibodies (anti-mouse or anti-rabbit-Alexa 488/594, Invitrogen, Molecular Probes). The following day the embryos were washed extensively for at least 4 hrs in PBS and photographed with either a Zeiss A-1 AxioImager or a LUMAR.
We tested two strategies at the beginning of these experiments: in the first, three snake embryos at st.18-19  were collected and while still alive injected with DiI (cell tracker CM-DiI, C-7001, Invitrogen/Molecular Probes) (diluted 1:10 in ethanol in 10% sucrose) inside the neural tube along its length and hindbrain regions (Additional file 2). The first embryo was then placed on a Petri dish after rinsing in Ringer solution and incubated with 5 ml of DMEM, 10% FBS, penicillin and streptomycin at 37°C for 24 hrs. The second and third embryos were cultured in 3 ml of a 1:1:1 mix of: a) DMEM/10%FBS, b) equal amounts of snake egg white and yolk, and c) 100% FBS at 25°C and 37°C for 12 hrs. In the second strategy, we cut the embryos leaving as much as possible its surrounding membranes and cultured them in DMEM, 20% FBS, penicillin and streptomycin at 25°C for 24 hrs. All embryos survived after DiI injection. At the end of incubation, the surviving cultured embryos were fixed for 1 hr to keep morphology, then placed in a vial for overnight fixing at 4°C and kept there until analysis. In both strategies we injected DiI with a mouth pipette after the hindbrain blowing gently to fill the neural tube all the way until the tail. In embryos that were st.20 we needed to inject around the beginning of second coil to make the DiI reach until the tail, thus these embryos had two points of injection (see Additional file 2).
We thank Daniel Meulemans Medeiros for invaluable help in writing this manuscript. David Arce for helping with tissue sectioning, Bobby Espinoza for kindly allowing us to collect the snake eggs from his reptilian collection, Chris Waldheim for technical assistance, Tony Milanes for help with the tissue preparation. Clare Baker and Vivian Lee provided valuable comments during all phases of the work and Scott Gilbert, Jack Sechrist, and Jerry Springer critically reviewed the manuscript. We would like to express our thanks to an anonymous reviewer that provided valuable insights and comments that significantly improved this paper. This work was partly supported by an NIH/NINDS AREA grant 1R15-NS060099-01 and NIH-MBRS SCORE-5S06GM048680-13 to MEdB.
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