RESUMO
The dentition is critical to animal survival and teeth are present in modern vertebrates including teleost fish, sharks, amphibians, mammals and reptiles. The developmental processes that give rise to teeth are not just preserved through evolution but also share high level of similarity with the embryogenesis of other ectodermal organs. In this review we go beyond the embryonic phase of tooth development to life-long tooth replacement. We will address the origins of successional teeth, the location of putative tissue-resident stem cells, how de novo tooth formation continues throughout life and how teeth are shed in a spatially and temporally controlled manner. We review the evidence that the dental epithelium, which is the earliest recognizable dental structure in the reptilian dentition, serves as a putative niche for tissue-resident epithelial stem cells and recent molecular findings from transcriptomics carried out in reptilian dentitions. We discuss how odontoclasts resorb the primary tooth allowing eruption of the successional tooth. The reptiles, particularly lizards, are emerging as some of the most accessible animals to study tooth replacement which has relevance to evolution of the dentition and human dental disorders.
RESUMO
In the face, symmetry is established when bilateral streams of neural crest cells leave the neural tube at the same time, follow identical migration routes and then give rise to the facial prominences. However, developmental instability exists, particularly surrounding the steps of lip fusion. The causes of instability are unknown but inability to cope with developmental fluctuations are a likely cause of congenital malformations, such as non-syndromic orofacial clefts. Here, we tracked cell movements over time in the frontonasal mass, which forms the facial midline and participates in lip fusion, using live-cell imaging of chick embryos. Our mathematical examination of cell velocity vectors uncovered temporal fluctuations in several parameters, including order/disorder, symmetry/asymmetry and divergence/convergence. We found that treatment with a Rho GTPase inhibitor completely disrupted the temporal fluctuations in all measures and blocked morphogenesis. Thus, we discovered that genetic control of symmetry extends to mesenchymal cell movements and that these movements are of the type that could be perturbed in asymmetrical malformations, such as non-syndromic cleft lip. This article has an associated 'The people behind the papers' interview.
Assuntos
Movimento Celular , Face/fisiologia , Mesoderma/crescimento & desenvolvimento , Crista Neural/fisiologia , Actomiosina , Animais , Encéfalo/anatomia & histologia , Encéfalo/crescimento & desenvolvimento , Divisão Celular , Proliferação de Células , Embrião de Galinha , Galinhas , Fenda Labial/genética , Fissura Palatina/genética , Olho/anatomia & histologia , Olho/crescimento & desenvolvimento , Face/anormalidades , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/anatomia & histologia , Morfogênese/genética , Crista Neural/anatomia & histologiaRESUMO
Heterozygous missense mutations in several genes in the WNT5A signaling pathway cause autosomal dominant Robinow syndrome 1 (DRS1). Our objective was to clarify the functional impact of a missense mutation in WNT5A on the skeleton, one of the main affected tissues in RS. We delivered avian replication competent retroviruses (RCAS) containing human wild-type WNT5A (wtWNT5A), WNT5AC83S variant or GFP/AlkPO4 control genes to the chicken embryo limb. Strikingly, WNT5AC83S consistently caused a delay in ossification and bones were more than 50% shorter and 200% wider than controls. In contrast, bone dimensions in wtWNT5A limbs were slightly affected (20% shorter, 25% wider) but ossification occurred on schedule. The dysmorphology of bones was established during cartilage differentiation. Instead of stereotypical stacking of chondrocytes, the WNT5AC83S-infected cartilage was composed of randomly oriented chondrocytes and that had diffuse, rather than concentrated Prickle staining, both signs of disrupted planar cell polarity (PCP) mechanisms. Biochemical assays revealed that C83S variant was able to activate the Jun N-terminal kinase-PCP pathway similar to wtWNT5A; however, the activity of the variant ligand was influenced by receptor availability. Unexpectedly, the C83S change caused a reduction in the amount of protein being synthesized and secreted, compared to wtWNT5A. Thus, in the chicken and human, RS phenotypes are produced from the C83S mutation, even though the variant protein is less abundant than wtWNT5A. We conclude the variant protein has dominant-negative effects on chondrogenesis leading to limb abnormalities.
Assuntos
Condrócitos/citologia , Condrogênese , Anormalidades Craniofaciais/metabolismo , Nanismo/metabolismo , Extremidades/embriologia , Deformidades Congênitas dos Membros/metabolismo , Anormalidades Urogenitais/metabolismo , Proteína Wnt-5a/genética , Animais , Animais Geneticamente Modificados , Cartilagem/metabolismo , Polaridade Celular/fisiologia , Embrião de Galinha , Galinhas , Condrogênese/genética , Anormalidades Craniofaciais/genética , Modelos Animais de Doenças , Nanismo/genética , Células HEK293 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Deformidades Congênitas dos Membros/genética , Mutação de Sentido Incorreto , Fenótipo , Anormalidades Urogenitais/genética , Via de Sinalização Wnt , Proteína Wnt-5a/metabolismoRESUMO
BACKGROUND: The egg tooth is a vital structure allowing hatchlings to escape from the egg. In squamates (snakes and lizards), the egg tooth is a real tooth that develops within the oral cavity at the top of the upper jaw. Most squamates have a single large midline egg tooth at hatching, but a few families, such as Gekkonidae, have two egg teeth. In snakes the egg tooth is significantly larger than the rest of the dentition and is one of the first teeth to develop. RESULTS: We follow the development of the egg tooth in four snake species and show that the single egg tooth is formed by two tooth germs. These two tooth germs are united at the midline and grow together to produce a single tooth. In culture, this merging can be perturbed to give rise to separate smaller teeth, confirming the potential of the developing egg tooth to form two teeth. CONCLUSIONS: Our data agrees with previous hypotheses that during evolution one potential mechanism to generate a large tooth is through congrescence of multiple tooth germs and suggests that the ancestors of snakes could have had two egg teeth.
Assuntos
Serpentes/embriologia , Germe de Dente/embriologia , Animais , Dentição , DenteRESUMO
OBJECTIVES: Asymmetry has been noted in the human craniofacial region in several pathological conditional and growth abnormalities, often with a directional predilection. Physiological asymmetry has also been reported in normal adults and adolescents, with certain regions of the cranioskeleton, such as the mandible, displaying prevalent asymmetry. However, the timing at which such asymmetries arise has not been evaluated. The objectives of this study were to assess the degree of asymmetry in facial bones during the foetal stages of human development. MATERIAL AND METHODS: Twenty-one preserved conceptuses from the Congenital Anomaly Research Center at Kyoto University, between ages 15 and 20 weeks of gestation, were studied using high-resolution µCT imaging. Asymmetry analysis was performed on digitally segmented facial bone pairs, using geometric morphometric (GM) approaches as well as adapted deformation-based asymmetry (DBA) methods. RESULTS: GM analysis revealed that the developing facial bones display statistically significant fluctuating and directional asymmetry. DBA methods suggest that the magnitude of asymmetry in facial bones is low and does not appear to be correlated to the estimate of overall size of conceptus. Additionally, the patterns of asymmetry are highly variable between individual specimens. CONCLUSIONS: The developing foetal facial skeleton displays variable patterns of low magnitude asymmetry. GM and DBA methods offer unique advantages to assess facial asymmetry quantitatively and qualitatively.
Assuntos
Face , Assimetria Facial , Adolescente , Adulto , Ossos Faciais , Desenvolvimento Fetal , Humanos , Mandíbula , Adulto JovemRESUMO
We performed a test of how function impacts a genetically programmed process that continues into postnatal life. Using the dentition of the polyphyodont gecko as our model, tooth shedding was recorded longitudinally across the jaw. We compared two time periods: one in which teeth were patterned symmetrically in ovo and a later period when teeth were initiated post-hatching. By pairing shedding events on the right and left sides, we found the patterns of tooth loss are symmetrical and stable between periods, with only subtle deviations. Contralateral tooth positions shed within 3-4 days of each other in most animals (7/10). A minority of animals (3/10) had systematic tooth position shifts between right and left sides, likely due to changes in functional tooth number. Our results suggest that in addition to reproducible organogenesis of individual teeth, there is also a neotenic retention of jaw-wide dental patterning in reptiles. Finer analysis of regional asymmetries revealed changes to which contralateral position shed first, affecting up to one quarter of the jaw (10 tooth positions). Once established, these patterns were retained longitudinally. Taken together, the data support regional and global mechanisms of coordinating tooth cycling post-hatching.
Assuntos
Arcada Osseodentária/fisiologia , Lagartos/crescimento & desenvolvimento , Odontogênese/fisiologia , Dente/crescimento & desenvolvimento , Animais , Padronização Corporal , Dentição , Lagartos/embriologia , Óvulo/crescimento & desenvolvimento , Dente/embriologiaRESUMO
Lipid nanoparticles (LNPs) containing distearoylphosphatidlycholine (DSPC), and ionizable amino-lipids such as dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA) are potent siRNA delivery vehicles in vivo. Here we explore the utility of similar LNP systems as transfection reagents for plasmid DNA (pDNA). It is shown that replacement of DSPC by unsaturated PCs and DLin-MC3-DMA by the related lipid DLin-KC2-DMA resulted in highly potent transfection reagents for HeLa cells in vitro. Further, these formulations exhibited excellent transfection properties in a variety of mammalian cell lines and transfection efficiencies approaching 90% in primary cell cultures. These transfection levels were equal or greater than achieved by Lipofectamine, with much reduced toxicity. Finally, microinjection of LNP-eGFP into the limb bud of a chick embryo resulted in robust reporter-gene expression. It is concluded that LNP systems containing ionizable amino lipids can be highly effective, non-toxic pDNA delivery systems for gene expression both in vitro and in vivo.
Assuntos
DNA/química , Sistemas de Liberação de Medicamentos , Lipídeos/química , Nanopartículas/química , Plasmídeos/química , Animais , Linhagem Celular Tumoral , Embrião de Galinha , Células HeLa , Humanos , Camundongos , TransfecçãoRESUMO
BACKGROUND: Pannexin 3 (PANX3) is a channel-forming protein capable of stimulating osteogenesis in vitro. Here, we studied the in vivo roles of PANX3 in the chicken embryo using the RCAS retroviral system to over-express and knockdown expression during endochondral bone formation. RESULTS: In the limbs, PANX3 RNA was first detected in the cartilage condensations and became restricted to the prehypertrophic cartilage of the epiphyses, diaphysis, and perichondrium. The increase in PANX3 was not sufficient to alter osteogenesis; however, knockdown with a virus containing an interference RNA construct caused a 20% reduction in bone volume. The control virus containing an shEGFP cassette did not affect development. Interestingly, the phenotype was restricted to later stages rather than to proliferation of the skeletogenic mesenchyme, formation of the cartilage condensation, or creation of the hypertrophic zones. In addition, there was also no change in readouts of Hedgehog, WNT, fibroblast growth factor, or bone morphogenetic protein signaling using either quantitative real-time polymerase chain reaction or radioactive in situ hybridization. CONCLUSIONS: Based on the normal expression domains of PANX3 and the relatively late manifestation of the phenotype, it is possible that PANX3 hemichannels may be required to facilitate the transition of hypertrophic chondrocytes to osteoblasts, thereby achieving final bone size. Developmental Dynamics 245:913-924, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Conexinas/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Animais , Desenvolvimento Ósseo/genética , Desenvolvimento Ósseo/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Embrião de Galinha , Condrogênese/genética , Condrogênese/fisiologia , Conexinas/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/genética , Osteogênese/fisiologia , Plasmídeos/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Lineage tracing has shown that most of the facial skeleton is derived from cranial neural crest cells. However, the local signals that influence postmigratory, neural crest-derived mesenchyme also play a major role in patterning the skeleton. Here, we study the role of BMP signaling in regulating the fate of chondro-osteoprogenitor cells in the face. RESULTS: A single Noggin-soaked bead inserted into stage 15 chicken embryos induced an ectopic cartilage resembling the interorbital septum within the palate and other midline structures. In contrast, the same treatment in stage 20 embryos caused a loss of bones. The molecular basis for the stage-specific response to Noggin lay in the simultaneous up-regulation of SOX9 and downregulation of RUNX2 in the maxillary mesenchyme, increased cell adhesiveness as shown by N-cadherin induction around the beads and increased RA pathway gene expression. None of these changes were observed in stage 20 embryos. CONCLUSIONS: These experiments demonstrate how slight changes in expression of growth factors such as BMPs could lead to gain or loss of cartilage in the upper jaw during vertebrate evolution. In addition, BMPs have at least two roles: one in patterning the skull and another in regulating the skeletogenic fates of neural crest-derived mesenchyme. Developmental Dynamics 245:947-962, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Mesoderma/citologia , Mesoderma/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas de Transporte/farmacologia , Embrião de Galinha , Face/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Mesoderma/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
Cranial neural crest cells form the majority of the facial skeleton. However exactly when the pattering information and hence jaw identity is established is not clear. We know that premigratory neural crest cells contain a limited amount of information about the lower jaw but the upper jaw and facial midline are specified later by local tissue interactions. The environmental signals leading to frontonasal identity have been explored by our group in the past. Altering the levels of two signaling pathways (Bone Morphogenetic Protein) and retinoic acid (RA) in the chicken embryo creates a duplicated midline on the side of the upper beak complete with egg tooth in place of maxillary derivatives (Lee et al., 2001). Here we analyze the transcriptome 16 h after bead placement in order to identify potential mediators of the identity change in the maxillary prominence. The gene list included RA, BMP and WNT signaling pathway genes as well as transcription factors expressed in craniofacial development. There was also cross talk between Noggin and RA such that Noggin activated the RA pathway. We also observed expression changes in several poorly characterized genes including the upregulation of Peptidase Inhibitor-15 (PI15). We tested the functional effects of PI15 overexpression with a retroviral misexpression strategy. PI15 virus induced a cleft beak analogous to human cleft lip. We next asked whether PI15 effects were mediated by changes in expression of major clefting genes and genes in the retinoid signaling pathway. Expression of TP63, TBX22, BMP4 and FOXE1, all human clefting genes, were upregulated. In addition, ALDH1A2, ALDH1A3 and RA target, RARß were increased while the degradation enzyme CYP26A1 was decreased. Together these changes were consistent with activation of the RA pathway. Furthermore, PI15 retrovirus injected into the face was able to replace RA and synergize with Noggin to induce beak transformations. We conclude that the microarrays have generated a rich dataset containing genes with important roles in facial morphogenesis. Moreover, one of these facial genes, PI15 is a putative clefting gene and is in a positive feedback loop with RA.
Assuntos
Bico/anormalidades , Bico/metabolismo , Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Animais , Animais Geneticamente Modificados , Padronização Corporal/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/metabolismo , Embrião de Galinha , Bases de Dados Genéticas , Face , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hibridização In Situ , Maxila/efeitos dos fármacos , Maxila/embriologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Secretadas Inibidoras de Proteinases/genética , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Controle de Qualidade , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tretinoína/metabolismo , Tretinoína/farmacologiaRESUMO
Wingless-related proteins (WNTs) regulate extension of the central axis of the vertebrate embryo (convergent extension) as well as morphogenesis of organs such as limbs and kidneys. Here, we asked whether WNT signaling directs facial morphogenesis using a targeted approach in chicken embryos. WNT11 is thought to mainly act via ß-catenin-independent pathways, and little is known about its role in craniofacial development. RCAS::WNT11 retrovirus was injected into the maxillary prominence, and the majority of embryos developed notches in the upper beak or the equivalent of cleft lip. Three-dimensional morphometric analysis revealed that WNT11 prevented lengthening of the maxillary prominence, which was due in part to decreased proliferation. We next determined, using a series of luciferase reporters, that WNT11 strongly induced JNK/planar cell polarity signaling while repressing the ß-catenin-mediated pathway. The activation of the JNK-ATF2 reporter was mediated by the DEP domain of Dishevelled. The impacts of altered signaling on the mesenchyme were assessed by implanted Wnt11- or Wnt3a-expressing cells (activates ß-catenin pathway) into the maxillary prominence or by knocking down endogenous WNT11 with RNAi. Host cells were attracted to Wnt11 donor cells. In contrast, cells exposed to Wnt3a or the control cells did not migrate. Cells in which endogenous WNT11 was knocked down were more oriented and shorter than those exposed to exogenous WNT11. The data suggest that JNK/planar cell polarity WNT signaling operates in the face to regulate several morphogenetic events leading to lip fusion.
Assuntos
Polaridade Celular , Face , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Morfogênese , Transdução de Sinais , Proteínas Wnt/metabolismo , Animais , Sequência de Bases , Embrião de Galinha , Primers do DNA , Reação em Cadeia da Polimerase em Tempo RealRESUMO
It is essential to complete palate closure at the correct time during fetal development, otherwise a serious malformation, cleft palate, will ensue. The steps in palate formation in humans take place between the 7th and 12th week and consist of outgrowth of palatal shelves from the paired maxillary prominences, reorientation of the shelves from vertical to horizontal, apposition of the medial surfaces, formation of a bilayered seam, degradation of the seam and bridging of mesenchyme. However, in the soft palate, the mechanism of closure is unclear. In previous studies it is possible to find support for both fusion and the alternative mechanism of merging. Here we densely sample the late embryonic-early fetal period between 54 and 74 days post-conception to determine the timing and mechanism of soft palate closure. We found the epithelial seam extends throughout the soft palates of 57-day specimens. Cytokeratin antibody staining detected the medial edge epithelium and distinguished clearly that cells in the midline retained their epithelial character. Compared with the hard palate, the epithelium is more rapidly degraded in the soft palate and only persists in the most posterior regions at 64 days. Our results are consistent with the soft palate following a developmentally more rapid program of fusion than the hard palate. Importantly, the two regions of the palate appear to be independently regulated and have their own internal clocks regulating the timing of seam removal. Considering data from human genetic and mouse studies, distinct anterior-posterior signaling mechanisms are likely to be at play in the human fetal palate.
Assuntos
Feto/embriologia , Morfogênese/fisiologia , Palato Mole/embriologia , Epitélio/embriologia , Humanos , Mesoderma/embriologia , Estudos RetrospectivosRESUMO
Transplantation experiments have shown that a true organizer provides instructive signals that induce and pattern ectopic structures in the responding tissue. Here, we review craniofacial experiments to identify tissues with organizer properties and signals with organizer properties. In particular, we evaluate whether transformation of identity took place in the mesenchyme. Using these stringent criteria, we find the strongest evidence for the avian foregut ectoderm. Transplanting a piece of quail foregut endoderm to a host chicken embryo caused ectopic beaks to form derived from chicken mesenchyme. The beak identity, whether upper or lower as well as orientation, was controlled by the original anterior-posterior position of the donor endoderm. There is also good evidence that the nasal pit is necessary and sufficient for lateral nasal patterning. Finally, we review signals that have organizer properties on their own without the need for tissue transplants. Mouse germline knockouts of the endothelin pathway result in transformation of identity of the mandible into a maxilla. Application of noggin-soaked beads to post-migratory neural crest cells transforms maxillary identity. This suggests that endothelin or noggin rich ectoderm could be organizers (not tested). In conclusion, craniofacial, neural crest-derived mesenchyme is competent to respond to tissues with organizer properties, also originating in the head. In future, we can exploit such well defined systems to dissect the molecular changes that ultimately lead to patterning of the upper and lower jaw.
Assuntos
Galinhas , Ectoderma , Embrião de Galinha , Animais , Camundongos , Arcada Osseodentária , Crista Neural , Endotelinas , Padronização CorporalRESUMO
Robinow syndrome is a rare disease caused by variants of seven WNT pathway genes. Craniofacial features include widening of the nasal bridge and jaw hypoplasia. We used the chicken embryo to test whether two missense human FZD2 variants (1301G>T, p.Gly434Val; 425C>T, p.Pro142Lys) were sufficient to change frontonasal mass development. In vivo, the overexpression of retroviruses with wild-type or variant human FZD2 inhibited upper beak ossification. In primary cultures, wild-type and variant human FZD2 significantly inhibited chondrogenesis, with the 425C>T variant significantly decreasing activity of a SOX9 luciferase reporter compared to that for the wild type or 1301G>T. Both variants also increased nuclear shuttling of ß-catenin (CTNNB1) and increased the expression of TWIST1, which are inhibitory to chondrogenesis. In canonical WNT luciferase assays using frontonasal mass cells, the variants had dominant-negative effects on wild-type FZD2. In non-canonical assays, the 425C>T variant failed to activate the reporter above control levels and was unresponsive to exogenous WNT5A. This is the first single amino acid change to selectively alter ligand binding in a FZD receptor. Therefore, FZD2 missense variants are pathogenic and could lead to the altered craniofacial morphogenesis seen in Robinow syndrome.
Assuntos
Condrogênese , Anormalidades Craniofaciais , Receptores Frizzled , Animais , Embrião de Galinha , Humanos , Bico , beta Catenina/metabolismo , Núcleo Celular/metabolismo , Condrogênese/genética , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/patologia , Nanismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Deformidades Congênitas dos Membros , Crânio/patologia , Crânio/embriologia , Proteína 1 Relacionada a Twist/metabolismo , Proteína 1 Relacionada a Twist/genética , Anormalidades Urogenitais , Via de Sinalização WntRESUMO
The chondrocranium provides the key initial support for the fetal brain, jaws and cranial sensory organs in all vertebrates. The patterns of shaping and growth of the chondrocranium set up species-specific development of the entire craniofacial complex. The 3D development of chondrocranium have been studied primarily in animal model organisms, such as mice or zebrafish. In comparison, very little is known about the full 3D human chondrocranium, except from drawings made by anatomists many decades ago. The knowledge of human-specific aspects of chondrocranial development are essential for understanding congenital craniofacial defects and human evolution. Here advanced microCT scanning was used that includes contrast enhancement to generate the first 3D atlas of the human fetal chondrocranium during the middle trimester (13 to 19 weeks). In addition, since cartilage and bone are both visible with the techniques used, the endochondral ossification of cranial base was mapped since this region is so critical for brain and jaw growth. The human 3D models are published as a scientific resource for human development.
Assuntos
Imageamento Tridimensional , Humanos , Feto/diagnóstico por imagem , Feminino , Microtomografia por Raio-X , Crânio/diagnóstico por imagem , Crânio/embriologia , Gravidez , Cartilagem/diagnóstico por imagem , Cartilagem/embriologiaRESUMO
Most dentate vertebrates, including humans, replace their teeth and yet the process is poorly understood. Here, we investigate whether dental epithelial stem cells exist in a polyphyodont species, the leopard gecko (Eublepharis macularius). Since the gecko dental epithelium lacks a histologically distinct site for stem cells analogous to the mammalian hair follicle bulge, we performed a pulse-chase experiment on juvenile geckos to identify label-retaining cells (LRCs). We detected LRCs exclusively on the lingual side of the dental lamina, which exhibits low proliferation rates and is not involved in tooth morphogenesis. Lingual LRCs were organized into pockets of high density close to the successional lamina. A subset of the LRCs expresses Lgr5 and other genes that are markers of adult stem cells in mammals. Also similar to mammalian stem cells, the LRCs appear to proliferate in response to gain of function of the canonical Wnt pathway. We suggest that the LRCs in the lingual dental lamina represent a population of stem cells, the immediate descendents of which form the successional lamina and, ultimately, the replacement teeth in the gecko. Furthermore, their location on the non-tooth-forming side of the dental lamina implies that dental stem cells are sequestered from signals that might otherwise induce them to differentiate.
Assuntos
Células-Tronco Adultas/fisiologia , Diferenciação Celular , Células Epiteliais/fisiologia , Lagartos/fisiologia , Regeneração/fisiologia , Dente/fisiologia , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Biomarcadores/metabolismo , Ciclo Celular/fisiologia , Proliferação de Células , Células Cultivadas , Simulação por Computador , Embrião não Mamífero , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Expressão Gênica , Lagartos/embriologia , Lagartos/genética , Lagartos/crescimento & desenvolvimento , Modelos Biológicos , Dente/citologia , Dente/crescimento & desenvolvimentoRESUMO
The study of rare genetic diseases provides valuable insights into human gene function. The autosomal dominant or autosomal recessive forms of Robinow syndrome are genetically heterogeneous, and the common theme is that all the mutations lie in genes in Wnt signaling pathways. Cases diagnosed with Robinow syndrome do survive to adulthood with distinct skeletal phenotypes, including limb shortening and craniofacial abnormalities. Here, we focus on mutations in dishevelled 1 (DVL1), an intracellular adaptor protein that is required for both canonical (ß-catenin-dependent) or non-canonical (requiring small GTPases and JNK) Wnt signaling. We expressed human wild-type DVL1 or DVL1 variants alongside the endogenous genome of chicken and Drosophila. This design is strategically suited to test for functional differences between mutant and wild-type human proteins in relevant developmental contexts. The expression of variant forms of DVL1 produced a major disorganization of cartilage and Drosophila wing morphology compared to expression of wild-type DVL1. Moreover, the variants caused a loss of canonical and gain of non-canonical Wnt signaling in several assays. Our data point to future therapies that might correct the levels of Wnt signaling, thus improving skeletal growth.
Assuntos
Galinhas , Anormalidades Craniofaciais , Proteínas Desgrenhadas , Drosophila , Animais , Humanos , Galinhas/metabolismo , Anormalidades Craniofaciais/genética , Proteínas Desgrenhadas/genética , Proteínas Desgrenhadas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Via de Sinalização Wnt/genéticaRESUMO
Here we report that highly concentrated cationic lipid/helper lipid-nucleic acid complexes (lipoplexes) can facilitate reproducible delivery of a variety of oligonucleotides and plasmids to chicken embryos or to mouse embryonic mesenchyme. Specifically, liposomes composed of N,N-dioleyl-N,N-dimethylammonium chloride (DODAC)/1,2 dioleoyl glycero-3-phosphorylethanolamine (DOPE) prepared at 18-mM concentrations produced high levels of transfection of exogenous genes in vivo and in vitro. Furthermore, we report sufficient uptake of plasmids expressing interference RNA to decrease expression of both exogenous and endogenous genes. The simplicity of preparation, implementation, and relatively low toxicity of this transfection reagent make it an attractive alternative for developmental studies in post-gastrulation vertebrate embryos.
Assuntos
Lipossomos/química , Transfecção/métodos , Animais , Embrião de Galinha , Embrião de Mamíferos , Lipossomos/administração & dosagem , Mesoderma/metabolismo , Camundongos , Fosfatidiletanolaminas/química , Compostos de Amônio Quaternário/química , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/químicaRESUMO
Dental patterns in vertebrates range from absence of teeth to multiple sets of teeth that are replaced throughout life. Despite this great variation, most of our understanding of tooth development is derived from studies on just a few model organisms. Here we introduce the reptile as an excellent model in which to study the molecular basis for early dental specification and, most importantly, for tooth replacement. We review recent snake studies that highlight the conserved role of Shh in marking the position of the odontogenic band. The distinctive molecular patterning of the dental lamina in the labial-lingual and oral-aboral axes is reviewed. We explain how these early signals help to specify the tooth-forming and non-tooth forming sides of the dental lamina as well as the presumptive successional lamina. Next, the simple architecture of the reptilian enamel organ is contrasted with the more complex, mammalian tooth bud and we discuss whether or not there is an enamel knot in reptilian teeth. The role of the successional lamina during tooth replacement in squamate reptiles is reviewed and we speculate on the possible formation of a vestigial, post-permanent dentition in mammals. In support of these ideas, we present data on agamid teeth in which development of a third generation is arrested. We suggest that in diphyodont mammals, similar mechanisms may be involved in reducing tooth replacement capacity. Finally, we review the location of label-retaining cells and suggest ways in which these putative dental epithelial stem cells contribute to continuous tooth replacement.