Behavior Of Crest Cells Research Articles

Protein glycosylation in neural crest epithelial‐to‐mesenchymal transition and migration

Neural crest epithelial‐to‐mesenchymal transition (EMT) and migration involves coordinated remodeling of cell‐cell and cell‐matrix adhesions. During this cell phase transition event, interactions between adhesive molecules need to be dynamically regulated. We recently explored the involvement of protein glycosylation in this process. Protein glycosylation is one of the most complexed, yet least studied types of post‐translational modification. By attaching various forms of oligosaccharide (glycan) on different sites of a protein, glycosylation can regulate protein function by modulating its interaction with other molecules. Through the employment of chemical inhibitors for protein glycosylation, we demonstrated that glycosylation is required for neural crest EMT and migration in frog embryos. To gain a comprehensive understanding of how different proteins are glycosylated during this process, we performed a glycoproteomic analysis at different stages of neural crest development. Through this approach, we identified numerous membrane and secreted proteins that are differentially glycosylated, which may contribute to the dynamic changes in cell‐cell and cell‐matrix interactions during neural crest EMT and migration. We focused on E‐cadherin, a well‐known player in EMT and cell migration, to determine the function of specific protein glycosylation in protein activity and consequently in neural crest cell behavior. Our analysis identified two glycosylation linkage sites on E‐cadherin. The two glycosylation sites were mutated individually or in combination, and the function of mutant E‐cadherin was compared with that of wild type E‐cadherin. Our results showed that mutant E‐cadherins were less effective in promoting neural crest cell migration and this was partly due to an increase in the strength of cell‐cell adhesion mediated by the mutan t proteins. Therefore, our findings suggest a new mechanism to fine‐tune the activity of cell adhesive molecules during EMT and migration.Support or Funding InformationNIH R00 DE022796, Georgia Tech Startup Fund

Endogenous Natural Complement Inhibitor Regulates Cardiac Development.

Congenital heart defects are a major cause of perinatal mortality and morbidity, affecting >1% of all live births in the Western world, yet a large fraction of such defects have an unknown etiology. Recent studies demonstrated surprising dual roles for immune-related molecules and their effector mechanisms during fetal development and adult homeostasis. In this article, we describe the function of an endogenous complement inhibitor, mannan-binding lectin (MBL)-associated protein (MAp)44, in regulating the composition of a serine protease-pattern recognition receptor complex, MBL-associated serine protease (MASP)-3/collectin-L1/K1 hetero-oligomer, which impacts cardiac neural crest cell migration. We used knockdown and rescue strategies in zebrafish, a model allowing visualization and assessment of heart function, even in the presence of severe functional defects. Knockdown of embryonic expression of MAp44 caused impaired cardiogenesis, lowered heart rate, and decreased cardiac output. These defects were associated with aberrant neural crest cell behavior. We found that MAp44 competed with MASP-3 for pattern recognition molecule interaction, and knockdown of endogenous MAp44 expression could be rescued by overexpression of wild-type MAp44. Our observations provide evidence that immune molecules are centrally involved in the orchestration of cardiac tissue development.

The "domestication syndrome" in mammals: a unified explanation based on neural crest cell behavior and genetics.

Charles Darwin, while trying to devise a general theory of heredity from the observations of animal and plant breeders, discovered that domesticated mammals possess a distinctive and unusual suite of heritable traits not seen in their wild progenitors. Some of these traits also appear in domesticated birds and fish. The origin of Darwin’s “domestication syndrome” has remained a conundrum for more than 140 years. Most explanations focus on particular traits, while neglecting others, or on the possible selective factors involved in domestication rather than the underlying developmental and genetic causes of these traits. Here, we propose that the domestication syndrome results predominantly from mild neural crest cell deficits during embryonic development. Most of the modified traits, both morphological and physiological, can be readily explained as direct consequences of such deficiencies, while other traits are explicable as indirect consequences. We first show how the hypothesis can account for the multiple, apparently unrelated traits of the syndrome and then explore its genetic dimensions and predictions, reviewing the available genetic evidence. The article concludes with a brief discussion of some genetic and developmental questions raised by the idea, along with specific predictions and experimental tests.

Injured cardiomyocytes promote dental pulp mesenchymal stem cell homing

BackgroundThe heart is unable to regenerate its tissues after severe injuries. Stem cell therapy appears to be one of the most promising approaches, though preclinical results are hitherto contradictory and clinical trials scanty and/or limited to phase-I. The limited knowledge about stem cell early homing in infarcted cardiac tissues can concur to this scenario. MethodsThe stem cell migration was assessed in in-vitro and ex-vivo models of heart ischemia, employing a rat dental pulp stem cell line (MUR-1) that shares the same ontogenic progenitors with portions of the heart, expresses markers typical of cardiac/vascular-like progenitors and is able to differentiate into cardiomyocytes in-vitro. ResultsHere, we demonstrated that the MUR-1 can reach the injured cells/tissue and make contacts with the damaged cardiomyocytes, likely through Connexin 43, N-cadherin and von Willebrand Factor mediated cell–cell interactions, both in in-vitro and ex-vivo models. Furthermore, we found that SDF-1, FGF-2 and HGF, but not VEGF are involved as chemotactic factors in MUR-1 migration, notifying a similarity with neural crest cell behavior during the organogenesis of both the splanchnocranium and the heart. ConclusionsHerein we found a similarity between what happens during the heart organogenesis and the early migration and homing of MUR-1 cells in ischemic models. General significanceThe comprehension of molecular aspects underlying the early phases of stem cell migration and interaction with damaged organ contributes to the future achievement of the coveted stem cell-mediated organ regeneration and function preservation in-vivo.

Serotonin 2B receptor signaling is required for craniofacial morphogenesis and jaw joint formation in Xenopus

Serotonin (5-HT) is a neuromodulator that plays many different roles in adult and embryonic life. Among the 5-HT receptors, 5-HT2B is one of the key mediators of 5-HT functions during development. We used Xenopus laevis as a model system to further investigate the role of 5-HT2B in embryogenesis, focusing on craniofacial development. By means of gene gain- and loss-of-function approaches and tissue transplantation assays, we demonstrated that 5-HT2B modulates, in a cell-autonomous manner, postmigratory skeletogenic cranial neural crest cell (NCC) behavior without altering early steps of cranial NCC development and migration. 5-HT2B overexpression induced the formation of an ectopic visceral skeletal element and altered the dorsoventral patterning of the branchial arches. Loss-of-function experiments revealed that 5-HT2B signaling is necessary for jaw joint formation and for shaping the mandibular arch skeletal elements. In particular, 5-HT2B signaling is required to define and sustain the Xbap expression necessary for jaw joint formation. To shed light on the molecular identity of the transduction pathway acting downstream of 5-HT2B, we analyzed the function of phospholipase C beta 3 (PLC) in Xenopus development and showed that PLC is the effector of 5-HT2B during craniofacial development. Our results unveiled an unsuspected role of 5-HT2B in craniofacial development and contribute to our understanding of the interactive network of patterning signals that is involved in the development and evolution of the vertebrate mandibular arch.

Loss of primary cilia affect neural crest cell behavior and leads to craniofacial defects

Loss of primary cilia affect neural crest cell behavior and leads to craniofacial defects

Behavior of enteric neural crest‐derived cells varies with respect to the migratory wavefront

Neural crest-derived cells colonize the entire gastrointestinal tract. The migration of these enteric neural crest-derived cells (ENCCs) occurs by their formation of cellular strands that extend into the intestinal mesenchyme. We have studied the behavior of crest cells that underlies the formation and extension of these strands by time-lapse microscopy. ENCCs expressing fluorescent marker molecules were visualized in situ in the embryonic mouse and chick gut. The major contributor to strand extension is from cells located within a region approximately 300 microm behind (rostral to) the most caudal cells in the migratory wavefront. Cells in the region immediately behind the leading cell of the strand either move intermittently in parallel with the leading cell, or advance caudally toward the wavefront over other ENCCs. Another addition to the strands arises from isolated cells located caudal to the wavefront. These cells showed a range of behavior including attachment and separation from the strands. The extending strands converged to form nodes, and then diverged along independent paths to form new strands, a behavior suggestive of attraction and repulsion. This behavior is probably responsible for the unique reticulated arrangement of ganglia in the enteric nervous system. As cells become positioned farther behind the wavefront, they exhibit more restricted movement and varied trajectories. We conclude that ENCCs exhibit different behaviors, depending on their position with respect to the wavefront. These different behaviors suggest a critical role for cell-cell interaction in the migratory process.

The Cell Adhesion Molecule L1 Is Required for Chain Migration of Neural Crest Cells in the Developing Mouse Gut

During development, the enteric nervous system is derived from neural crest cells that emigrate from the hindbrain, enter the foregut, and colonize the gut. Defects in neural crest migration can result in intestinal aganglionosis. Hirschsprung's disease (congenital aganglionosis) is a human condition in which enteric neurons are absent from the distal bowel. A number of clinical studies have implicated the cell adhesion molecule L1 in Hirschsprung's disease. We examined the role of L1 in the migration of neural crest cells through the developing mouse gut. A variety of in vitro and in vivo assays were used to examine: (1) the effect of L1 blocking antibodies or exogenous soluble L1 protein known to compromise L1 function on the rate of crest cell migration, (2) the effect of blocking L1 activity on the dynamic behavior of crest cells using time-lapse microscopy, and (3) whether the colonization of the gut by crest cells in L1-deficient mice differs from control mice. We show that L1 is expressed by neural crest cells as they colonize the gut. Perturbation studies show that disrupting L1 activity retards neural crest migration and increases the number of solitary neural crest cells. L1-deficient mice show a small but significant reduction in neural crest cell migration at early developmental stages, but the entire gastrointestinal tract is colonized. L1 is important for the migration of neural crest cells through the developing gut and is likely to be involved in the etiology of Hirschsprung's disease.

Conotruncal anomalies in the trisomy 16 mouse: an immunohistochemical analysis with emphasis on the involvement of the neural crest.

The trisomy 16 (Ts16) mouse is generally considered a model for human Down's syndrome (trisomy 21). However, many of the cardiac defects in the Ts16 mouse do not reflect the heart malformations seen in patients suffering from this chromosomal disorder. In this study we describe the conotruncal malformations in mice with trisomy 16. The development of the outflow tract was immunohistochemically studied in serially sectioned hearts from 34 normal and 26 Ts16 mouse embryos ranging from 8.5 to 14.5 embryonic days. Conotruncal malformations observed in the Ts 16 embryos included double outlet right ventricle, persistent truncus arteriosus, Tetralogy of Fallot, and right-sided aortic arch. This spectrum of malformations is remarkably similar to that seen in humans suffering from DiGeorge syndrome (DGS). As perturbation of neural crest development has been proposed in the pathogenesis of DGS we specifically focussed on the fate of neural crest derived cells during outflow tract development of the Ts16 mouse using an antibody that enabled us to trace these cells during development. Severe perturbation of the neural crest-derived cell population was observed in each trisomic specimen. The abnormalities pertained to: 1) the size of the columns of neural crest-derived cells (or prongs); 2) the spatial orientation of these prongs within the mesenchymal tissues of the outflow tract; and 3) the location in which the neural crest cells interact with the myocardium. The latter abnormality appeared to be responsible for ectopic myocardialization found in trisomic embryos. Our observations strongly suggest that abnormal neural crest cell behavior is involved in the pathogenesis of the conotruncal malformations in the Ts16 mouse.

Isolation and Characterization of Chondroitin Sulfate Proteoglycans from Embryonic Quail That Influence Neural Crest Cell Behavior

The movement of neural crest cells is controlled in part by extracellular matrix. Aggrecan, the chondroitin sulfate proteoglycan from adult cartilage, curtails the ability of neural crest cells to adhere, spread, and move across otherwise favorable matrix substratesin vitro.Our aim was to isolate, characterize, and compare the structure and effect on neural crest cells of aggrecan and proteoglycans purified from the tissues through which neural crest cells migrate. We metabolically radiolabeled proteoglycans in E2.5 quail embryos and isolated and characterized proteoglycans from E3.3 quail trunk and limb bud. The major labeled proteoglycan was highly negatively charged, similar in hydrodynamic size to chick limb bud versican/PG-M, smaller than adult cartilage aggrecan but larger than reported for embryonic sternal cartilage aggrecan. The molecular weight of the iodinated core protein was about 400 kDa, which is more than reported for aggrecan but less than that of chick versican/PG-M. The proteoglycan bore chondroitin sulfate glycosaminoglycan chains of 45 kDa, which is larger than those of aggrecan. It lacked dermatan sulfate, heparan sulfate, or keratan sulfate chains. It bound to collagen type I, like aggrecan, but not to fibronectin (unlike versican/PG-M), collagen type IV, or laminin-1 in solid-phase assays and it bound to hyaluronate in gel-shift assays. When added at concentrations between 10 and 30 μg/ml to substrates of fibronectin, trunk proteoglycan inhibited neural crest cell spreading and migration. Attenuation of cell spreading was shown to be the most sensitive and titratable measure of the effect on neural crest cells. This effect was sensitive to digestion with chondroitinase ABC. Similar cell behavior was also produced by aggrecan and the small dermatan sulfate proteoglycan decorin; however, 30-fold more aggrecan was required to produce an effect of similar magnitude. When added in solution to neural crest cells which were already spread and migrating on fibronectin, the embryonic proteoglycan rapidly and reversibly caused complete rounding of the cells, being at least 30-fold more potent than aggrecan in this activity.

Mammalian craniofacial embryology in vitro.

Our review demonstrates that the whole embryo culture system established by New and his colleagues, in combination with beneficial fluorescent dye cell-tracing techniques, has greatly contributed to many advancements in the field of mammalian craniofacial embryology, especially with regard to elucidating the developmental behavior of cephalic crest cells. In addition, based on recent results, further combining whole embryo culture with mandibular organ culture methods has allowed us to trace cranial crest cells for a much longer developmental period, i.e., presently up to the cap stage in odontogenesis.

Patterning of the cranial nerves in the chick embryo is dependent on cranial mesoderm and rhombomeric metamerism.

The vertebrate peripheral nervous system (PNS) consists of two groups of nerves that have a metamerical series of proximal roots along the body axis: the branchial and spinal nerves. Spinal nerve metamerism is brought about by the presence of somites, while that of the branchial nerves is, in part, intrinsic to rhombomeres, the segmental compartments of the hind-brain. As the distribution pattern of neural crest cells prefigures the morphology of the PNS, we constructed tissue-recombinant chick embryos in order to determine factors that might regulate the crest cell distribution pattern. When the segmental plate was transplanted between the hind-brain and the head mesoderm before crest cell emigration, it developed into ectopic somites that inhibited the dorsolateral migration of crest cells such that formation of the cranial nerve trunks was disturbed. Even so, proximal portions of the nerve roots were intact. An ectopic graft of lateral mesoderm did not inhibit the directional migration of the crest cells, but allowed their ectopic distribution, resulting in the fusion of cranial nerve trunks. When spinal neurectoderm was transplanted into the hind-brain, the graft behaved like an even-numbered rhombomere and caused the fusion of cranial nerve roots. The identity of the spinal neurectoderm was preserved in the ectopic site analyzed by the immunolocalization of Hoxb-5 protein, a spinal cord marker. We conclude that the spatial distribution of cephalic crest cells is regulated by successive processes that act on their proximal and distal distribution. The migratory behavior of crest cells is achieved partly by an embryonic environment that is dependent upon the presence of somitomeres, which do not epithelialize as somites, in the trunk.

Transforming growth factor-β alters differentiation in cultures of avian neural crest-derived cells: Effects on cell morphology, proliferation, fibronectin expression, and melanogenesis

Neural crest cell differentiation is responsive to a variety of extrinsic signals that include extracellular matrix (ECM) molecules and growth factors. Transforming growth factor-β (TGF-β) has diverse, cell type-specific effects, many of which involve regulation of synthesis of ECM molecules and their cell surface receptors. We are studying both separate and potentially interrelated influences of ECM and growth factors on crest differentiation and report here that TGF-β alters several aspects of crest cell behavior in vitro. Clusters of quail neural crest cells were cultured in the presence and absence of 400 p M TGF-β1 and examined at 1, 3, and 5 days. When examined at 5 days, there was a dramatic decrease in the number of melanocytes in treated cultures, regardless of the onset or duration of TGF-β treatment. With continuous TGF-β treatment, or with treatment only during crest cluster formation on explanted neural tubes, many cells increased in area, becoming extremely flat. These changes were evident beginning on Day 3. While quantitative analyses of video images documented the size increase, several aspects of motility were relatively unchanged. Synthesis of fibronectin (FN) by approximately 11% of cells on Day 3 and 31% of cells on Day 5 was demonstrated by immunocytochemistry and was associated with a sixfold increase in FN mRNA by Day 5. Experiments which correlated FN immunoreactivity with incorporation of bromodeoxyuridine suggested that the population of large, flat, FN-positive cells did not proliferate selectively and that there was a slower rate of proliferation in TGF-β-treated cultures than in untreated cultures. The large FN-immunoreactive cells resemble cells derived from cepalic neural crest and raise interesting questions concerning potential roles for TGF-β in regulating crest differentiation in vivo.

6 Sequential Segregation and Fate of Developmentally Restricted Intermediate Cell Populations in the Neural Crest Lineage

This chapter discusses the evidence that neural crest cells begin to diversify before they disperse and that developmentally restricted intermediate subpopulations arise sequentially within the crest lineage. It discusses the results, indicating that some of these developmentally distinct subpopulations respond differentially to specific developmental cues that they encounter on their migration pathways. Finally, the chapter discusses how the recent results with cultured crest cells can be reconciled, with the early morphogenetic and differentiative behavior of crest cells in vivo , how developmental cues are normally presented to the responsive crest-derived populations, and how differentially the responsive crest populations might arise during development. The neural crest of vertebrate embryos has been usefully exploited to learn how the differences in developmental ability are established in vertebrate embryonic cell lineages. Neural crest cells disperse along characteristic paths through embryonic interstitial spaces, where they encounter a variety of different environmental cues and ultimately produce a remarkable diversity of cellular phenotypes. A variety of heterotopic grafting experiments have shown that the neural crest from every axial level has a wide range of developmental potentialities.

Neural crest cell behavior analyzed by using the thiebaud marker: [formula omitted], and [formula omitted], Texas Christian University, Forth Worth, Texas 76129 U.S.A.

Neural crest cell behavior analyzed by using the thiebaud marker: [formula omitted], and [formula omitted], Texas Christian University, Forth Worth, Texas 76129 U.S.A.

Neural crest cell behavior in white and dark larvae of Ambystoma mexicanum: differences in cell morphology, arrangement, and extracellular matrix as related to migration.

Melanocytes of white (d/d) larvae of the Mexican axolotl (Ambystoma mexicanum) are confined to the dorsal midline of the trunk region, whereas in dark (D/-) larvae they are spread laterally on the flank as well, where they contribute to the normal pigment pattern of the trunk. Pigment cell migration in the subepidermal space of white larvae is inhibited by the white epidermis (Dalton '50; Keller et al., '82). The present scanning electron microscopic study describes a well-defined sequence of changes in shape and arrangement of neural crest cells during and after their segregation from the neural tube in both dark and white axolotls. The morphology of the neural crest cells migrating in the subepidermal pathway of dark larvae is correlated with their motile behavior and pattern of migration in vivo, as described by time-lapse cinemicrography (Keller and Spieth, '83). Also, the structures of the matrix material in the subepidermal space of dark and white axolotls differ in ways that may be related to the epidermal inhibition of migration in the latter. Numerous possibilities for contact guidance offered by the structure and topography of the substrata, neighboring cells, and the extracellular matrix in the migration path are described and discussed.

Neural crest cell behavior in white and dark larvae of Ambystoma mexicanum: time-lapse cinemicrographic analysis of pigment cell movement in vivo and in culture.

The pattern of migration and motile activity of developing pigment cells of the Mexican axolotl, Ambystoma mexicanum, were analyzed by time-lapse cinemicrography in vivo and in culture. In vivo, melanocytes of dark (D/-) larvae migrate from dorsal to ventral in a highly directional manner. They are elongated and aligned parallel to the direction of migration. Nearly all protrusive activity occurs at their ventral, leading edges. Translocation occurs at a mean rate of 0.7 micron/min and involves alternate or simultaneous advance of the leading and trailing edges of the cell. Indirect evidence suggests that cytoplasmic flow is common. Directional migration occurs in apparent absence of contact between melanocytes. In white (d/d) larvae, protrusive activity is infrequent and the melanocytes move slowly or not at all. Explanted neural crest cells of dark and white larvae attach, spread, and differentiate into melanophores and xanthophores in culture. Individual cultured cells are unbiased in direction of protrusive activity and path of migration. Centrifugal spreading occurs by contacting inhibition of movement. Distribution of protrusive activity, polarity, and contact behavior changes with developmental age in vivo and in culture in ways that may be important in establishing the pigment pattern.

Neural crest cell behavior in white and dark embryos of Ambystoma mexicanum: Epidermal inhibition of pigment cell migration in the white axolotl

Melanophores in larvae of the white ( dd) strain of the Mexican axolotl ( Ambystoma mexicanum) are confined to the dorsal midline of the trunk and dorsal posterior part of the head, whereas those in dark larvae ( D -) are distributed over the flank as well. Our results show that this phenotype of white larvae is the result of the failure of the melanophores or their neural crest precursor cells to migrate laterally due to an inhibition of or a failure in the support of their migration in the subepidermal space by the overlying epidermis. Correlated light and scanning electron microscopy of dissected larvae showed melanophores occupying the subepidermal space on the flank of dark larvae, whereas these cells were restricted to the dorsal midline of white larvae. Grafting experiments in which patches of epidermis, the underlying mesoderm, or both, were exchanged between dark and white embryos suggested that white epidermis alone can prevent the integration of pigment cells on the flank of dark larvae and, conversely, that grafts of dark epidermis alone can support their migration on the flank of white larvae. Mesoderm, when grafted alone, could not be shown to have similar effects.