Hyaluronan is required for cranial neural crest cells migration and craniofacial development

Background: Hyaluronan is a crucial glycosaminoglycan of the vertebrate embryonic extracellular matrix able to influence cell behaviour, both by assembling the pericellular matrices and by activating signal transducing receptors such as CD44. Results: We showed that the hyaluronan synthases, Has1 and Has2, and CD44 display a dynamic expression pattern during cranial neural crest cells (NCC) development. By knocking down Has1 and Has2 gene functions, we revealed that hyaluronan synthesized by Has1 and Has2 is necessary for the proper development of the visceral skeleton. Conclusions: The data suggest that hyaluronan helps to maintain the active migratory behaviour of cranial NCC, and that its presence around pre‐chondrogenic NCC is crucial for their survival. CD44 knock down also suggests that the role of hyaluronan in cranial NCC migration could be mediated, at least in part, by the activation of CD44. These findings contribute to the unveiling of the functional relation between NCC and their extracellular environment during craniofacial development. Developmental Dynamics 241:294–302, 2012. © 2011 Wiley Periodicals, Inc.     

Wnt11r is required for cranial neural crest migration

wnt11r is a recently identified member of the Wnt family of genes, which has been proposed to be the true Xenopus homologue to the mammalian wnt11 gene. In this study we have examined the role of wnt11r on neural crest development. Expression analysis of wnt11r and comparison with the neural crest marker snail2 and the noncanonical Wnt, wnt11, shows wnt11r is expressed at the medial or neural plate side of the neural crest while wnt11 is expressed at the lateral or epidermal side. Injection of wnt11r morpholino leads to strong inhibition of neural crest migration with no effect on neural crest induction or maintenance. This effect can be rescued by co‐injection of Wnt11r but not by Wnt11 mRNA, demonstrating the specificity of the loss of function treatment. Finally, neural crest graft experiments show that wnt11r is required in a non–cell‐autonomous manner to control neural crest migration. Developmental Dynamics 237:3404–3409, 2008. © 2008 Wiley‐Liss, Inc.     

Myosin‐X is required for cranial neural crest cell migration in Xenopus laevis

Myosin‐X (MyoX) belongs to a large family of unconventional, nonmuscle, actin‐dependent motor proteins. We show that MyoX is predominantly expressed in cranial neural crest (CNC) cells in embryos of Xenopus laevis and is required for head and jaw cartilage development. Knockdown of MyoX expression using antisense morpholino oligonucleotides resulted in retarded migration of CNC cells into the pharyngeal arches, leading to subsequent hypoplasia of cartilage and inhibited outgrowth of the CNC‐derived trigeminal nerve. In vitro migration assays on fibronectin using explanted CNC cells showed significant inhibition of filopodia formation, cell attachment, spreading and migration, accompanied by disruption of the actin cytoskeleton. These data support the conclusion that MyoX has an essential function in CNC migration in the vertebrate embryo. Developmental Dynamics 238:2522–2529, 2009. © Published 2009 Wiley‐Liss, Inc.^?     

The zebrafish homeobox gene irxl1 is required for brain and pharyngeal arch morphogenesis

Iroquois homeobox‐like 1 (irxl1) is a novel member of the TALE superfamily of homeobox genes that is most closely related to the Iroquois class. We have identified the zebrafish irxl1 gene and characterized its structure. The protein contains a homeodomain that shares 100% sequence identity with other vertebrate orthologs. During embryogenesis, irxl1 is expressed from 18 hours postfertilization onward and prominent expression is detected in the pharyngeal arches. Knockdown of irxl1 by morpholinos results in malformed brain and arch structures, which can be partially rescued by cRNA injection. The heads of the morphants become small and flat, and extensions along the anterior–posterior/dorso–ventral axes are reduced without affecting regional specification. Loss of irxl1 function also causes deficit in neural crest cells which consequently results in partial loss of craniofacial muscles and severe deformation of arch cartilages. These observations suggest that irxl1 may regulate factors involved in brain and pharyngeal arch development. Developmental Dynamics 239:639–650, 2010. © 2009 Wiley‐Liss, Inc.     

Spatiotemporally separated cardiac neural crest subpopulations that target the outflow tract septum and pharyngeal arch arteries

We used lacZ-retrovirus labeling combined with neural crest ablation in chick embryos to determine whether the cardiac neural crest cells constitute one group of multipotent cells, or they emigrate from the neural tube in time-dependent groups with different fates in the developing cardiovascular system. We demonstrated that early-migrating cardiac neural crest cells (HH9-10) massively target the aorticopulmonary septum and pharyngeal arch arteries, while the late-migrating cardiac neural crest cells (HH12) are restricted to the proximal part of the pharyngeal arch arteries. These results suggest a prominent role for early-migrating cells in outflow tract septation, and a function for late-migrating cells in pharyngeal arch artery remodeling. We demonstrated in cultures of neural tube explants an intrinsic difference between the early and late populations. However, by performing heterochronic transplantations we showed that the late-migrating cardiac neural crest cells were not developmentally restricted, and could contribute to the condensed mesenchyme of the aorticopulmonary septum when transplanted to a younger environment. Our findings on the exact timing and migratory behavior of cardiac neural crest cells will help narrow the range of factors and genes that are involved in neural crest-related congenital heart diseases.     

Formation and distribution of neural crest mesenchyme to the first pharyngeal arch region of the mouse embryo

The normal microscopic and submicroscopic structure of the lower respiratory tract of the budgerigar (Melopsittacus undulatus) is described and compared with other birds and mammals. Granular (type II) pneumocytes are confined to linings of air sacs, parabronchi, and their atria; however, their secretions (surfactant) cover the surfaces of the infundibula and respiratory space. Infundibula extend from the atria and give rise to the air capillaries, which branch and anastomose freely with those of adjacent infundibula and other parabronchi (interparabronchial septa are not found). Infundibula and the respiratory labyrinth are lined by a continuous epithelium of squamous pneumocytes, whose perikarya are concentrated in the infundibula and whose peripheral cytoplasm is markedly attenuated. The squamous pneumocytes of the respiratory labyrinth share a basal lamina with the blood capillaries that they envelop.     

Homocysteine inhibits cardiac neural crest cell formation and morphogenesis in vivo.

Elevated homocysteine increases the risk of neurocristopathies. Here, we determined whether elevating homocysteine altered the proliferation or number of chick neural crest cells that form between the midotic and third somite in vivo. Homocysteine increased the number of neural tube cells but decreased neural crest cell number. However, the sum total of cells was not different from controls. In controls, the 5-bromo-2'-deoxyuridine-labeling index was higher in newly formed neural crest cells than in their progenitors, paralleling reports showing these progenitors must pass the restriction point before undergoing epithelial-mesenchymal transition. Homocysteine decreased the labeling index of newly formed neural crest cells, suggesting that it inhibited cell cycle progression of neural crest progenitors or the S-phase entry of newly formed neural crest cells. Homocysteine also inhibited neural crest dispersal and decreased the distance they migrated from the neural tube. These results show neural crest morphogenesis is directly altered by elevated homocysteine in vivo. Developmental Dynamics 229:63-73, 2004.     

Expression of the homeobox gene Pitx2 in neural crest is required for optic stalk and ocular anterior segment development

Heterozygous mutations in the homeobox gene, PITX2, result in ocular anterior segment defects and a high incidence of early-onset glaucoma. Pitx2 is expressed in both the neural crest and the mesoderm-derived precursors of the periocular mesenchyme. Complete loss of function in mice results in agenesis or severe disruption of periocular mesenchyme structures and extrinsic defects in early optic nerve development. However, the specific requirements for Pitx2 in neural crest versus mesoderm could not be determined using these mice, and only roles in the initial stages of eye development could be assessed due to early embryonic lethality. To determine the specific roles of Pitx2 in the neural crest precursor pool, we generated neural crest-specific Pitx2 knockout mice (Pitx2-ncko). Because Pitx2-nkco mice are viable, we also analyzed gene function in later eye development. Pitx2 is intrinsically required in neural crest for specification of corneal endothelium, corneal stroma and the sclera. Pitx2 function in neural crest is also required for normal development of ocular blood vessels. Pitx2-ncko mice exhibit a unique optic nerve phenotype in which the eyes are progressively displaced towards the midline until they are directly attached to the ventral hypothalamus. As Pitx2 is not expressed in the optic stalk, an essential function of PITX2 protein in neural crest is to regulate an extrinsic factor(s) required for development of the optic nerve. We propose a revised model of optic nerve development and new mechanisms that may underlie the etiology of glaucoma in Axenfeld-Rieger patients.     

Bmp2 is required for migration but not for induction of neural crest cells in the mouse.

Bone morphogenetic protein (BMP) signaling is essential for neural crest development in several vertebrates. Genetic experiments in the mouse have shown that Bmp2 is essential for the genesis of migratory neural crest cells. Using several markers and a transgenic reporter approach, we now show that neural crest cells are induced in Bmp2 null mutant embryos, but that these cells fail to migrate out of the neural tube. The absence of migratory neural crest cells in these mutants is not due to their elimination by cell death. The neuroectoderm of Bmp2-/- embryos fail to close and create abnormal folds both along the anterior-posterior and medio-lateral axes, which are associated with an apparent medio-lateral expansion of the neural tube. Finally, our data suggest that the molecular cascade downstream of BMP signaling in early neural crest development may be different in mouse and avian embryos.     

Cre-mediated gene inactivation demonstrates that FGF8 is required for cell survival and patterning of the first branchial arch

In mammals, the first branchial arch (BA1) develops into a number of craniofacial skeletal elements including the jaws and teeth. Outgrowth and patterning of BA1 during early embryogenesis is thought to be controlled by signals from its covering ectoderm. Here we used Cre/loxP technology to inactivate the mouse Fgf8 gene in this ectoderm and have obtained genetic evidence that FGF8 has a dual function in BA1: it promotes mesenchymal cell survival and induces a developmental program required for BA1 morphogenesis. Newborn mutants lack most BA1-derived structures except those that develop from the distal-most region of BA1, including lower incisors. The data suggest that the BA1 primordium is specified into a large proximal region that is controlled by FGF8, and a small distal region that depends on other signaling molecules for its outgrowth and patterning. Because the mutant mice resemble humans with first arch syndromes that include agnathia, our results raise the possibility that some of these syndromes are caused by mutations that affect FGF8 signaling in BA1 ectoderm.     

Notch pathway regulation of neural crest cell development in vivo

Background : The function of Notch signaling in murine neural crest–derived cell lineages in vivo was examined. Results : Conditional gain (Wnt1Cre;Rosa^Notch) or loss (Wnt1Cre;RBP‐J^f/f) of Notch signaling in neural crest cells (NCCs) in vivo results in craniofacial, cardiac, and trunk abnormalities. Severe craniofacial malformations are apparent in Wnt1Cre;Rosa^Notch embryos, while less severe skull abnormalities are evident in Wnt1Cre;RBP‐J^f/f mice. Deficient cardiac neural crest migration, resulting in cardiac outflow tract malformations, occurs with increased or decreased Notch signaling in NCCs. Smooth muscle cell differentiation also is impaired in pharyngeal NCC derivatives in both Wnt1Cre;Rosa^Notch and Wnt1Cre;RBP‐J^f/f embryos. Neurogenesis is absent and gliogenesis is increased in the dorsal root ganglia of Wnt1Cre;Rosa^Notch embryos, while neurogenesis is increased and gliogenesis is decreased in Wnt1Cre;RBP‐J^f/f embryos. Conclusions : Together, these studies demonstrate essential cell‐autonomous roles for appropriate levels of Notch signaling during NCC migration, proliferation, and differentiation with critical implications in craniofacial, cardiac, and neurogenic development and disease. Developmental Dynamics 241:376–389, 2012. © 2011 Wiley Periodicals, Inc.     

Effect of neural crest ablation on development of the heart and arch arteries in the chick

Mesenchymal derivatives of the neural crest contribute to the connective tissues and blood vessels of the pharyngeal arches, and participate in the septation of the outflow tract of the heart. The present study was designed to determine the nature and timing of alterations in the development of the heart and arch arteries subsequent to diminished neural crest contributions. The neural crest contributing to the three caudalmost pharyngeal arches was ablated bilaterally in chick embryos and compared with sham or unoper-ated controls. Heart development was studied by scanning electron microscopy. Arch artery development was studied microscopically after intravascular injection of India ink and clearing of the specimen. Neural crest ablation caused morphological changes in most hearts. Hearts in experimental animals commonly were elongate and were subject to inappropriate development of ventricular and atrial areas. A surgical effect delayed the disappearance of arch arteries one and two, and removal of neural crest produced an additional delay. Neural crest ablation caused failure of arch arteries three, four (right), and six to develop to the proper size in some animals. Survival of those whose sixth arch arteries achieved the proper size caused group measurements to reach normal values again by stage 32. Closure of arch arteries in some animals and maintenance in others produced greater variability in experimental animals than in controls. It is significant that heart morphology was altered before septation of the outflow tract normally occurs. This indicates at the least that another factor, such as altered blood flow, contributes to the abnormal development. Altered flow may result from changes in pharyngeal arch mesenchyme and arch artery endothelium.     

Avian transitin expression mirrors glial cell fate restrictions during neural crest development.

During development, trunk neural crest cells give rise to three primary classes of derivatives: glial cells, melanocytes, and neurons. As part of an effort to learn how neural crest diversification is regulated, we have produced monoclonal antibodies (MAbs) that recognize antigens expressed by neural crest cells early in development. One of these, MAb 7B3 (7B3), was found to recognize an avian transitin-like protein by co-immunostaining with a series of transitin-specific monoclonal antibodies and by Western blot analysis. In neural crest cell cultures, we found that 7B3 initially recognizes the majority of neural crest cells as they emerge from the neural tube. Subsequently, 7B3-immunoreactivity (IR) is progressively restricted to a smaller subpopulation of cells. In fully differentiated trunk neural crest cell cultures, 7B3-IR is expressed only by cells that do not express neuronal markers and lack melanin granules. During development in vivo, 7B3-IR is evident in neural crest cells on the medial, but not the lateral migration pathway, suggesting that it is not expressed by melanocyte precursors. Later, the antigen is detected in non-neuronal, presumptive glial cells in dorsal root ganglia (DRG) and sympathetic ganglia, as well as along ventral roots. Cultures of E5 DRG confirm that 7B3-IR is restricted to non-neuronal cells of ganglia, many of which closely associate with neuronal processes. Therefore, of the three major classes of differentiated trunk neural crest derivatives, 7B3 exclusively recognizes glial cells, including both satellite glia and Schwann cells. Since the pattern of 7B3 expression in vitro mirrors the pattern of glial cell fate-restrictions in the trunk neural crest lineage, and is expressed by neural crest-derived glia in vivo, we conclude that 7B3 is an early pan-glial marker for neural crest-derived glial cells and their precursors.     

Sonic hedgehog from pharyngeal arch 1 epithelium is necessary for early mandibular arch cell survival and later cartilage condensation differentiation

Background: Morphogenesis of vertebrate craniofacial skeletal elements is dependent on a key cell population, the cranial neural crest cells (NCC). Cranial NCC are formed dorsally in the cranial neural tube and migrate ventrally to form craniofacial skeletal elements as well as other tissues. Multiple extracellular signaling pathways regulate the migration, survival, proliferation, and differentiation of NCC. Results: In this study, we demonstrate that Shh expression in the oral ectoderm and pharyngeal endoderm is essential for mandibular development. We show that a loss of Shh in these domains results in increased mesenchymal cell death in pharyngeal arch 1 (PA1) after NCC migration. This increased cell death can be rescued in utero by pharmacological inhibition of p53. Furthermore, we show that epithelial SHH is necessary for the early differentiation of mandibular cartilage condensations and, therefore, the subsequent development of Meckel's cartilage, around which the dentary bone forms. Nonetheless, a rescue of the cell death phenotype does not rescue the defect in cartilage condensation formation. Conclusions: Our results show that SHH produced by the PA1 epithelium is necessary for the survival of post‐migratory NCC, and suggests a key role in the subsequent differentiation of chondrocytes to form Meckel's cartilage. Developmental Dynamics 244:564–576, 2015. © 2015 Wiley Periodicals, Inc.     

Lineage specification in neural crest cell pathfinding.

There are two principal models to explain neural crest patterning. One assumes that neural crest cells are multipotent precursors that migrate throughout the embryo and differentiate according to cues present in the local environment. A second proposes that the neural crest is a population of cells that becomes restricted to particular fates early in its existence and migrates along particular pathways dependent on unique cell-autonomous properties. Although it is now evident that the neural crest cell population, as a whole, is actually heterogenous (composed of both multipotent and restricted progenitors), evidence supporting the model of prespecification has increased over the past few years. This review will begin by telling the story of melanoblasts: a neural crest subpopulation that is biased toward a single fate and subsequently acquires intrinsic properties that guide cells of this lineage to their final destination. The remainder of this review will explore whether this model is exclusive to melanoblasts or if it can also be used to explain the patterning of other neural crest cells like those of the sensory, sympathoadrenal, and enteric lineages.