Fgfr1 regulates patterning of the pharyngeal region

Development of the pharyngeal region depends on the interaction and integration of different cell populations, including surface ectoderm, foregut endoderm, paraxial mesoderm, and neural crest. Mice homozygous for a hypomorphic allele of Fgfr1 have craniofacial defects, some of which appeared to result from a failure in the early development of the second branchial arch. A stream of neural crest cells was found to originate from the rhombomere 4 region and migrate toward the second branchial arch in the mutants. Neural crest cells mostly failed to enter the second arch, however, but accumulated in a region proximal to it. Both rescue of the hypomorphic Fgfr1 allele and inactivation of a conditional Fgfr1 allele specifically in neural crest cells indicated that Fgfr1 regulates the entry of neural crest cells into the second branchial arch non-cell-autonomously. Gene expression in the pharyngeal ectoderm overlying the developing second branchial arch was affected in the hypomorphic Fgfr1 mutants at a stage prior to neural crest entry. Our results indicate that Fgfr1 patterns the pharyngeal region to create a permissive environment for neural crest cell migration.     

Multilineage differentiation of ectomesenchymal cells isolated from the first branchial arch

Cranial neural crest-derived ectomesenchymal cells may be pluripotent stem cells that are capable of generating a range of phenotypes. The fate of these cells appears to be determined in part by intrinsic genetic programs and also by the influence of extracellular signals in the local environment. The extent of lineage determination once neural crest cells have migrated to the first branchial arch is not clear, although branchial arch pattern is not thought to be the result of crest predetermination. The aim of the present study was to test the hypothesis that ectomesenchymal cells of the first branchial arch show properties of pluripotent stem cells, the lineage of which may be directed by specific molecular signaling. Ectomesenchymal cells were enzymatically isolated from the mandibular processes of BALB/c mice and maintained in an undifferentiated state while cultured with leukemia inhibitory factor or induced to differentiate by lineage-specific induction factors or growth conditions, including transforming growth factor beta, forskolin, and a mineralization-promoting medium. Morphological observations and immunocytochemistry demonstrated that cells could be induced to differentiate into smooth muscle cells, glial cells, and osteoblasts, respectively. In the presence of the mineralization-promoting medium, alkaline phosphatase activity increased significantly and mineralization nodules formed. The data reported support the concept that many, although not all, first branchial arch-derived ectomesenchymal cells show properties of multipotent stem cells, the subsequent fate of which can be influenced by induction factors and growth conditions. Some cells, however, showed a degree of commitment with respect to their fate. The possible application of first branchial arch-derived stem cells to tissue engineering of the orofacial tissues should involve consideration of the developmental stage of cell harvesting and the desired cell fate.     

Timing of odontogenic neural crest cell migration and tooth-forming capability in mice.

The mammalian tooth develops through sequential and reciprocal interactions between cranial neural crest (CNC)- derived ectomesenchymal cells and the stomadial epithelium. Classic tissue recombination studies demonstrated that premigratory CNC cells and CNC-derived ectomesenchymal cells possess odontogenic capacity and can respond to oral epithelial signals to form a tooth, suggesting that the CNC cells contributing to odontogenic tissue are not prespecified. Here we show that, in mice, CNC cells have populated the forming first branchial arch before the 9-somite stage and continue to migrate into the arch by the 13-somite stage. Grafts of the first arch from the 10-somite embryo or earlier yielded membranous bone and cysts but no teeth after subrenal culture. However, grafts of the first arch with its dorsally adjacent tissue containing migrating neural crest cells from the same age embryos gave rise to teeth. In contrast, teeth formed in first arch grafts that do not contain migrating neural crest cells from embryos with 12 or more somites. Interestingly, the acquisition of tooth forming capability in the first arch coincides with the onset of Fgf8 expression in the oral epithelium. These results suggest that there exists a population of odontogenic neural crest cells that migrates into the first arch between the 10- and 12-somite stages. These cells either possess odontogenic potential and are able to initiate tooth development, or can respond to odontogenic signals derived from the oral epithelium to support tooth formation.     

Cranial neural crest ablation of Jagged1 recapitulates the craniofacial phenotype of Alagille syndrome patients

JAGGED1 mutations cause Alagille syndrome, comprising a constellation of clinical findings, including biliary, cardiac and craniofacial anomalies. Jagged1, a ligand in the Notch signaling pathway, has been extensively studied during biliary and cardiac development. However, the role of JAGGED1 during craniofacial development is poorly understood. Patients with Alagille syndrome have midface hypoplasia giving them a characteristic 'inverted V' facial appearance. This study design determines the requirement of Jagged1 in the cranial neural crest (CNC) cells, which encompass the majority of mesenchyme present during craniofacial development. Furthermore, with this approach, we identify the autonomous and non-autonomous requirement of Jagged1 in a cell lineage-specific approach during midface development. Deleting Jagged1 in the CNC using Wnt1-cre; Jag1 Flox/Flox recapitulated the midfacial hypoplasia phenotype of Alagille syndrome. The Wnt1-cre; Jag1 Flox/Flox mice die at postnatal day 30 due to inability to masticate owing to jaw misalignment and poor occlusion. The etiology of midfacial hypoplasia in the Wnt1-cre; Jag1 Flox/Flox mice was a consequence of reduced cellular proliferation in the midface, aberrant vasculogenesis with decreased productive vessel branching and reduced extracellular matrix by hyaluronic acid staining, all of which are associated with midface anomalies and aberrant craniofacial growth. Deletion of Notch1 from the CNC using Wnt1-cre; Notch1 F/F mice did not recapitulate the midface hypoplasia of Alagille syndrome. These data demonstrate the requirement of Jagged1, but not Notch1, within the midfacial CNC population during development. Future studies will investigate the mechanism in which Jagged1 acts in a cell autonomous and cell non-autonomous manner.     

Suppression of the melanogenic potential of migrating neural crest‐derived cells by the branchial arches

The development of melanocytes from neural crest‐derived precursors that migrate along the dorsolateral pathway has been attributed to the selection of this route by cells that are fate‐restricted to the melanocyte lineage. Alternatively, melanocytes could arise from nonspecified cells that develop in response to signals encountered while these cells migrate, or at their final destinations. In most animals, the bowel, which is colonized by crest‐derived cells that migrate through the caudal branchial arches, contains no melanocytes; however, the enteric microenvironment does not prevent melanocytes from developing from crest‐derived precursors placed experimentally into the bowel wall. To test the hypothesis that the branchial arches remove the melanogenic potential from the crest‐derived population that colonizes the gut, the Silky fowl (in which the viscera are pigmented) was studied. Sources of crest included Silky fowl and quail vagal and truncal neural folds/tubes, which were cultured or explanted to chorioallantoic membranes alone or together with branchial arches or limb buds from Silky fowl, White Leghorn, or quail embryos. Crest and mesenchyme‐derived cells were distinguished by using the quail nuclear marker. Melanocytes developed from Silky fowl and quail crest‐derived cells. Melanocyte development from both sources was inhibited by quail and White Leghorn branchial arches (and limb buds), but melanocyte development was unaffected by branchial arch (and limb buds) from Silky fowl. These observations suggest that a factor(s) that is normally expressed in the branchial arches, and is lacking in animals with the Silky mutation, prevents cells with a melanogenic potential from colonizing the bowel. Anat Rec 268:16–26, 2002. © 2002 Wiley‐Liss, Inc.     

Disruption of actin cytoskeleton and anchorage-dependent cell spreading induces apoptotic death of mouse neural crest cells cultured in vitro

In vertebrate embryos, neural crest cells emigrate out of the neural tube and contribute to the formation of a variety of neural and nonneural tissues. Some neural crest cells undergo apoptotic death during migration, but its biological significance and the underlying mechanism are not well understood. We carried out an in vitro study to examine how the morphology and survival of cranial neural crest (CNC) cells of the mouse embryo are affected when their actin cytoskeleton or anchorage-dependent cell spreading is perturbed. Disruption of actin fiber organization by cytochalasin D (1 microg/ml) and inhibition of cell attachment by matrix metalloproteinase-2 (MMP-2; 2.0 units/ml) were followed by morphologic changes and apoptotic death of cultured CNC cells. When the actin cytoskeleton was disrupted by cytochalasin D, the morphologic changes of cultured CNC cells preceded DNA fragmentation. These results indicate that the maintenance of cytoskeleton and anchorage-dependent cell spreading are required for survival of CNC cells. The spatially and temporally regulated expression of proteinases may be essential for the differentiation and migration of neural crest cells.     

Fluorescence-activated cell sorting of EGFP-labeled neural crest cells from murine embryonic 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. Crossing Wnt 1-Cre and Z/EG transgenic mouse lines resulted in offspring in which the Wnt 1-Cre transgene 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.     

Folic acid and homocysteine affect neural crest and neuroepithelial cell outgrowth and differentiation in vitro.

The beneficial effect of additional folic acid in the periconceptional period to prevent neural tube defects, orofacial clefts, and conotruncal heart defects in the offspring has been shown. Folate shortage results in homocysteine accumulation. Elevated levels of homocysteine have been related to neural tube defects. We studied the behavior of neuroepithelial cells and cranial and cardiac neural crest cells in vitro. Neural tube explants were cultured for 24 and 48 hr in medium after addition of folic acid and/or homocysteine. Folic acid addition increased neuroepithelial cell outgrowth and increased neural crest cell differentiation into nerve and smooth muscle cells. Addition of homocysteine increased neural crest cell outgrowth and migration from the neural tube and inhibited neural crest cell differentiation. Our findings suggest that neural tube defects caused by folate deficiency and hyperhomocysteinemia develop due to increased neuroepithelial to neural crest cell transformation. This increased transformation leads to a shortage of neuroepithelial cells in the neural tube. Defects in orofacial and conotruncal development are explained by abnormal differentiation of neural crest cells in the presence of high homocysteine concentrations. Our findings supports a critical role for folic acid and homocysteine in the development of neural tube defects and neural crest related heart malformations.     

Role of cranial neural crest cells in visceral arch muscle positioning and morphogenesis in the Mexican axolotl, Ambystoma mexicanum.

The role of cranial neural crest cells in the formation of visceral arch musculature was investigated in the Mexican axolotl, Ambystoma mexicanum. DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine, perchlorate) labeling and green fluorescent protein (GFP) mRNA injections combined with unilateral transplantations of neural folds showed that neural crest cells contribute to the connective tissues but not the myofibers of developing visceral arch muscles in the mandibular, hyoid, and branchial arches. Extirpations of individual cranial neural crest streams demonstrated that neural crest cells are necessary for correct morphogenesis of visceral arch muscles. These do, however, initially develop in their proper positions also in the absence of cranial neural crest. Visceral arch muscles forming in the absence of neural crest cells start to differentiate at their origins but fail to extend toward their insertions and may have a frayed appearance. Our data indicate that visceral arch muscle positioning is controlled by factors that do not have a neural crest origin. We suggest that the cranial neural crest-derived connective tissues provide directional guidance important for the proper extension of the cranial muscles and the subsequent attachment to the insertion on the correct cartilage. In a comparative context, our data from the Mexican axolotl support the view that the cranial neural crest plays a fundamental role in the development of not only the skeleton of the vertebrate head but also in the morphogenesis of the cranial muscles and that this might be a primitive feature of cranial development in vertebrates.     

Isolation of multipotent neural crest-derived stem cells from the adult mouse cornea

We report the presence of neural crest-derived corneal precursors (COPs) that initiate spheres by clonal expansion from a single cell. COPs expressed the stem cell markers nestin, Notch1, Musashi-1, and ABCG2 and showed the side population cell phenotype. COPs were multipotent with the ability to differentiate into adipocytes, chondrocytes, as well as neural cells, as shown by the expression of beta-III-tubulin, glial fibrillary acidic protein, and neurofilament-M. COP spheres prepared from E/nestin-enhanced green fluorescent protein (EGFP) mice showed induction of EGFP expression that was not originally observed in the cornea, indicating activation of the neural-specific nestin second intronic enhancer in culture. COPs were Sca-1(+), CD34(+), CD45(-), and c-kit(-). Numerous GFP(+) cells were observed in the corneas of mice transplanted with whole bone marrow of transgenic mice ubiquitously expressing GFP; however, no GFP(+) COP spheres were initiated from these mice. On the other hand, COP spheres from transgenic mice encoding P0-Cre/Floxed-EGFP as well as Wnt1-Cre/Floxed-EGFP were GFP(+), indicating the neural crest origin of COPs, which was confirmed by the expression of the embryonic neural crest markers Twist, Snail, Slug, and Sox9. Taken together, these data indicate the existence of neural crest-derived, multipotent stem cells in the adult cornea.     

The differentiation and morphogenesis of craniofacial muscles.

Unraveling the complex tissue interactions necessary to generate the structural and functional diversity present among craniofacial muscles is challenging. These muscles initiate their development within a mesenchymal population bounded by the brain, pharyngeal endoderm, surface ectoderm, and neural crest cells. This set of spatial relations, and in particular the segmental properties of these adjacent tissues, are unique to the head. Additionally, the lack of early epithelialization in head mesoderm necessitates strategies for generating discrete myogenic foci that may differ from those operating in the trunk. Molecular data indeed indicate dissimilar methods of regulation, yet transplantation studies suggest that some head and trunk myogenic populations are interchangeable. The first goal of this review is to present key features of these diversities, identifying and comparing tissue and molecular interactions regulating myogenesis in the head and trunk. Our second focus is on the diverse morphogenetic movements exhibited by craniofacial muscles. Precursors of tongue muscles partly mimic migrations of appendicular myoblasts, whereas myoblasts destined to form extraocular muscles condense within paraxial mesoderm, then as large cohorts they cross the mesoderm:neural crest interface en route to periocular regions. Branchial muscle precursors exhibit yet another strategy, establishing contacts with neural crest populations before branchial arch formation and maintaining these relations through subsequent stages of morphogenesis. With many of the prerequisite stepping-stones in our knowledge of craniofacial myogenesis now in place, discovering the cellular and molecular interactions necessary to initiate and sustain the differentiation and morphogenesis of these neglected craniofacial muscles is now an attainable goal.     

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.     

Engraftable neural crest stem cells derived from cynomolgus monkey embryonic stem cells

Neural crest stem cells (NCSCs), a population of multipotent cells that migrate extensively and give rise to diverse derivatives, including peripheral and enteric neurons and glia, craniofacial cartilage and bone, melanocytes and smooth muscle, have great potential for regenerative medicine. Non-human primates provide optimal models for the development of stem cell therapies. Here, we describe the first derivation of NCSCs from cynomolgus monkey embryonic stem cells (CmESCs) at the neural rosette stage. CmESC-derived neurospheres replated on polyornithine/laminin-coated dishes migrated onto the substrate and showed characteristic expression of NCSC markers, including Sox10, AP2α, Slug, Nestin, p75, and HNK1. CmNCSCs were capable of propagating in an undifferentiated state in vitro as adherent or suspension cultures, and could be subsequently induced to differentiate towards peripheral nervous system lineages (peripheral sympathetic neurons, sensory neurons, and Schwann cells) and mesenchymal lineages (osteoblasts, adipocytes, chondrocytes, and smooth muscle cells). CmNCSCs transplanted into developing chick embryos or fetal brains of cynomolgus macaques survived, migrated, and differentiated into progeny consistent with a neural crest identity. Our studies demonstrate that CmNCSCs offer a new tool for investigating neural crest development and neural crest-associated human disease and suggest that this non-human primate model may facilitate tissue engineering and regenerative medicine efforts.     

Comparative embryology of the carotid body

Vertebrate carotid bodies and related structures (branchial arch oxygen chemoreceptors in fishes, carotid labyrinth in amphibians, chemoreceptors in the wall of the common carotid and its branches in birds) develop in embryos when neural crest cells, blood vessels, and nerve fibers from sympathetic and cranial nerve ganglia invade mesenchymal primordia in the wall of the 3rd branchial arch. This review focuses on literature published since the 1970s investigating similarities and differences in the embryological development of 3rd arch oxygen chemoreceptors, especially between mammals and birds, but also considering reptiles, amphibians and fishes.     

神经嵴细胞和胰岛因子1阳性细胞与小鼠胚胎心流出道发育的关系

目的 探讨迁移中的细胞视黄酸结合蛋白1(CRABP1)阳性神经嵴细胞、胰岛因子1(ISL-1)、阳性心肌前体细胞与小鼠胚胎心流出道发育的关系.方法 36只胚龄8.5～13d小鼠胚胎心连续石蜡切片,选用抗α-平滑肌肌动蛋白(α-SMA)、抗心肌肌球蛋白重链(MHC)、抗转录因子ISL-1、抗CRABP1和抗磷酸化组蛋白H3(PHH3)抗体,进行免疫组织化学及免疫荧光染色.结果 胚龄8.5～10d,ISL-1阳性心肌前体细胞相继出现在心背系膜、原始咽两侧、头面部、鳃弓核心间充质和心包腔背侧壁间充质,构成心管流出道发育的第二生心区.胚龄11～13d,ISL-1阳性细胞在咽前方聚集,形成特征性锥体形结构,并向升主动脉、肺动脉干及主肺动脉隔延伸.胚龄9d前,神经嵴细胞散在分布于ISL-1阳性细胞之间,流出道远侧端可见少量CRABP1和ISL-1双阳性细胞.胚龄10d,CRABP1阳性神经嵴细胞分布在ISL-1阳性鳃弓核心间充质周围.随着发育,弓动脉等处的神经嵴细胞逐渐失去CRABP1表达,开始表达α-SMA.结论 ISL-1阳性第二生心区是动态结构,胚龄8.5～10d时,在神经嵴细胞配合下,向心管动脉端添加心肌细胞;胚龄11d后,开始向平滑肌方向分化,参与升主动脉、肺动脉干和主肺动脉隔的发育.