RESUMO
Contactins (CNTNs) are neural cell adhesion molecules that encode axon-target specificity during the patterning of the vertebrate visual and olfactory systems. Because CNTNs are tethered to the plasma membrane by a glycosylphosphatidylinositol anchor, they lack an intracellular region to communicate across the membrane. Instead, they form coreceptor complexes with distinct transmembrane proteins to transmit signals inside the cell. In particular, a complex of CNTN4 and amyloid precursor protein (APP) is known to guide the assembly of specific circuits in the visual system. Here, using in situ hybridization in zebrafish embryos, we show that CNTN4, CNTN5, and the APP homologs, amyloid beta precursor like protein 1 and amyloid beta precursor like protein 2, are expressed in olfactory pits, suggesting that these receptors may also function together in the organization of olfactory tissues. Furthermore, we use biochemical and structural approaches to characterize interactions between members of these two receptor families. In particular, APP and amyloid beta precursor like protein 1 interact with CNTN3-5, whereas amyloid beta precursor like protein 2 only binds to CNTN4 and CNTN5. Finally, structural analyses of five CNTN-amyloid pairs indicate that these proteins interact through a conserved interface involving the second fibronectin type III repeat of CNTNs and the copper-binding domain of amyloid proteins. Overall, this work sets the stage for analyzing CNTN-amyloid-mediated connectivity in vertebrate sensory circuits.
Assuntos
Peptídeos beta-Amiloides , Precursor de Proteína beta-Amiloide , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Axônios/metabolismo , Contactinas/química , Contactinas/metabolismo , Peixe-ZebraRESUMO
Canonical Wnt signaling plays crucial roles during development and disease. How Wnt signaling is modulated in different in vivo contexts is currently not well understood. Here, we investigate the modulation of Wnt signaling in the posterior lateral line primordium (pLLP), a cohort of ~100 cells that collectively migrate along the trunk of the zebrafish embryo. The pLLP comprises proliferative progenitor cells and organized epithelial cells that will form the mechanosensory organs of the posterior lateral line. Wnt signaling is active in the leading progenitor zone of the pLLP and restricted from the trailing zone through expression of the secreted Wnt inhibitors dkk1b and dkk2. We have identified a zebrafish strain, krm1(nl10), which carries a mutation in the kremen1 gene, a non-obligate co-receptor for the Dkk family of proteins. Previous studies have shown that Kremen1 inhibits Wnt signaling by facilitating internalization of the Kremen1-Dkk-Lrp5/6 complex. Surprisingly, we found that disruption of Kremen1 in the pLLP exhibited molecular and cellular phenotypes associated with a decrease rather than overactivation of Wnt signaling. Transplantation of wild-type cells into the mutant primordia failed to rescue the krm1(nl10) phenotype, thus revealing that the effects of Kremen1 loss are non-cell-autonomous. Finally, ectopic expression of Dkk1b-mTangerine protein revealed larger spread of the fusion protein in the mutant primordia compared with the wild type. Based on our data, we propose a novel mechanism in which Kremen1 modulates Wnt activity by restricting the range of secreted Dkk proteins during collective cell migration in the pLLP.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Sistema da Linha Lateral/embriologia , Proteínas de Membrana/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Movimento Celular , Proliferação de Células , Clonagem Molecular , Células Epiteliais/citologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Mutação , Fenótipo , Transdução de Sinais , Células-Tronco/citologia , Proteínas Wnt/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Multicellular rosettes have recently been appreciated as important cellular intermediates that are observed during the formation of diverse organ systems. These rosettes are polarized, transient epithelial structures that sometimes recapitulate the form of the adult organ. Rosette formation has been studied in various developmental contexts, such as in the zebrafish lateral line primordium, the vertebrate pancreas, the Drosophila epithelium and retina, as well as in the adult neural stem cell niche. These studies have revealed that the cytoskeletal rearrangements responsible for rosette formation appear to be conserved. By contrast, the extracellular cues that trigger these rearrangements in vivo are less well understood and are more diverse. Here, we review recent studies of the genetic regulation and cellular transitions involved in rosette formation. We discuss and compare specific models for rosette formation and highlight outstanding questions in the field.
Assuntos
Padronização Corporal/fisiologia , Adesão Celular/fisiologia , Células Epiteliais/fisiologia , Modelos Biológicos , Morfogênese/fisiologia , Animais , Citoesqueleto/fisiologia , Drosophila melanogaster , Humanos , Túbulos Renais/embriologia , Sistema da Linha Lateral/embriologia , Tubo Neural/embriologia , Pâncreas/embriologia , Células Fotorreceptoras de Invertebrados/fisiologia , Xenopus laevis , Peixe-ZebraRESUMO
The multipotent cells of the vertebrate neural crest (NC) arise at the dorsal aspect of the neural tube, then migrate throughout the developing embryo and differentiate into diverse cell types, including the sensory neurons and glia of the dorsal root ganglia (DRG). As multiple cell types are derived from this lineage, it is ideal for examining mechanisms of fate restriction during development. We have isolated a mutant, ouchless, that specifically fails to develop DRG neurons, although other NC derivatives develop normally. This mutation affects the expression of Sorbs3, a scaffold protein known to interact with proteins involved in focal adhesions and several signaling pathways. ouchless mutants share some phenotypic similarities with mutants in ErbB receptors, EGFR homologs that are implicated in diverse developmental processes and associated with several cancers; and ouchless interacts genetically with an allele of erbb3 in DRG neurogenesis. However, the defect in ouchless DRG neurogenesis is distinct from ErbB loss of function in that it is not associated with a loss of glia. Both ouchless and neurogenin1 heterozygous fish are sensitized to the effects of ErbB chemical inhibitors, which block the development of DRG in a dose-dependent manner. Inhibitors of MEK show similar effects on DRG neurogenesis. We propose a model in which Sorbs3 helps to integrate ErbB signals to promote DRG neurogenesis through the activation of MAPK and upregulation of neurogenin1.
Assuntos
Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Genes erbB/genética , Genes erbB/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Crista Neural/citologia , Crista Neural/metabolismo , Neurogênese/genética , Neurogênese/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
The neural crest is a migratory, multipotent cell lineage that contributes to myriad tissues, including sensory neurons and glia of the dorsal root ganglia (DRG). To identify genes affecting cell fate specification in neural crest, we performed a forward genetic screen for mutations causing DRG deficiencies in zebrafish. This screen yielded a mutant lacking all DRG, which we named sensory deprived (sdp). We identified a total of four alleles of sdp, all of which possess lesions in the gene coding for reversion-inducing cysteine-rich protein containing Kazal motifs (Reck). Reck is an inhibitor of metalloproteinases previously shown to regulate cell motility. We found reck function to be both necessary for DRG formation and sufficient to rescue the sdp phenotype. reck is expressed in neural crest cells and is required in a cell-autonomous fashion for appropriate sensory neuron formation. In the absence of reck function, sensory neuron precursors fail to migrate to the position of the DRG, suggesting that this molecule is crucial for proper migration and differentiation.
Assuntos
Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Gânglios Espinais/embriologia , Metaloproteases/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Diferenciação Celular/genética , Movimento Celular/genética , Proteínas Ligadas por GPI/biossíntese , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Metaloproteases/biossíntese , Metaloproteases/metabolismo , Crista Neural/citologia , Crista Neural/metabolismo , Neurogênese , Polimorfismo de Nucleotídeo Único , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/biossínteseRESUMO
The zebrafish posterior lateral line (pLL) is a sensory system that comprises clusters of mechanosensory organs called neuromasts (NMs) that are stereotypically positioned along the surface of the trunk. The NMs are deposited by a migrating pLL primordium, which is organized into polarized rosettes (proto-NMs). During migration, mature proto-NMs are deposited from the trailing part of the primordium, while progenitor cells in the leading part give rise to new proto-NMs. Wnt signaling is active in the leading zone of the primordium and global Wnt inactivation leads to dramatic disorganization of the primordium and a loss of proto-NM formation. However, the exact cellular events that are regulated by the Wnt pathway are not known. We identified a mutant strain, lef1(nl2), that contains a lesion in the Wnt effector gene lef1. lef1(nl2) mutants lack posterior NMs and live imaging reveals that rosette renewal fails during later stages of migration. Surprisingly, the overall primordium patterning, as assayed by the expression of various markers, appears unaltered in lef1(nl2) mutants. Lineage tracing and mosaic analyses revealed that the leading cells (presumptive progenitors) move out of the primordium and are incorporated into NMs; this results in a decrease in the number of proliferating progenitor cells and eventual primordium disorganization. We concluded that Lef1 function is not required for initial primordium organization or migration, but is necessary for proto-NM renewal during later stages of pLL formation. These findings revealed a novel role for the Wnt signaling pathway during mechanosensory organ formation in zebrafish.
Assuntos
Sistema da Linha Lateral/citologia , Sistema da Linha Lateral/embriologia , Células-Tronco/fisiologia , Fatores de Transcrição/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Padronização Corporal/fisiologia , Desenvolvimento Ósseo/genética , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células , Embrião não Mamífero , Sistema da Linha Lateral/metabolismo , Mutação/fisiologia , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Transativadores/genética , Transativadores/metabolismo , Transativadores/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteínas Wnt/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The zebrafish lateral line mechanosensory system shares considerable morphological and molecular similarities with the inner ear. In particular, mechanosensory hair cells are responsible for transducing sensory stimuli in both structures. The epithelia cell adhesion molecule (EpCAM) is expressed in the cells of the inner ear of mammals and in the lateral lines system of fish. EpCAM regulates the many cellular functions including adhesion, migration, proliferation, and differentiation. In this study, we use the epcam jh79 mutant zebrafish line to determine that EpCAM function is required for proper development and regeneration of posterior lateral line hair cells.
RESUMO
Mechanosensory hair cells in the inner ear mediate the sensations of hearing and balance, and in the specialized lateral line sensory system of aquatic vertebrates, the sensation of water movement. In mammals, hair cells lack the ability to regenerate following damage, resulting in sensory deficits. In contrast, non-mammalian vertebrates, such as zebrafish, can renew hair cells throughout their lifespan. Wnt signaling is required for development of inner ear and lateral line hair cells and regulates regeneration. Kremen1 inhibits Wnt signaling and hair cell formation, though its role in regeneration is unknown. We used a zebrafish kremen1 mutant line to show overactive Wnt signaling results in supernumerary support cells and hair cell regeneration without increased proliferation, in contrast with the previously described role of Wnt signaling during hair cell regeneration. This work allows us to understand the biology of mechanosensory hair cells and how regeneration might be promoted following damage.
RESUMO
Mechanosensory hair cells located in the inner ear mediate the sensations of hearing and balance. If damaged, mammalian inner ear hair cells are unable to regenerate, resulting in permanent sensory deficits. Aquatic vertebrates like zebrafish (Danio rerio) have a specialized class of mechanosensory hair cells found in the lateral line system, allowing them to sense changes in water current. Unlike mammalian inner ear hair cells, lateral line hair cells can robustly regenerate following damage. In mammalian models, the transcription factor Foxg1 functions to promote normal development of the inner ear. Foxg1a is expressed in lateral line sensory organs in zebrafish larvae, but its function during lateral line development and regeneration has not been investigated. We find that loss of Foxg1a function results in reduced hair cell development and regeneration, as well as decreased cellular proliferation in the lateral line system. These data suggest that Foxg1 may be a valuable target for investigation of clinical hair cell regeneration. Summary statement: Our work demonstrates a role for Foxg1a in developing and regenerating new sensory cells through proliferation.
RESUMO
Mechanosensory hair cells in the inner ear mediate the sensations of hearing and balance, and in a specialize lateral line sensory system of aquatic vertebrates, the sensation of water movement. In mammals, hair cells lack the ability of regenerate following damage, resulting in sensory deficits. In contrast, non-mammalian vertebrates, such zebrafish, can renew hair cells throughout the life of the animal. Wnt signaling is required for development of inner ear and lateral line hair cells and regulates regeneration. Kremen1 inhibits Wnt signaling and hair cell formation, though its role in regeneration has not been established. We use a zebrafish kremen1 mutant line, to show that when Wnt signaling is overactivated in the lateral line, excessive regeneration occurs in the absence of increased proliferation, due to an increase in support cells. This contrasts with the previously described role of Wnt signaling during hair cell regeneration. This work will allow us to understand the biology of mechanosensory hair cells, and how regeneration might be promoted following damage.
RESUMO
The proneural transcription factor neurogenin 1 (neurog1) has been shown to be a key regulator of dorsal root ganglion (DRG) neuron development. Here we use a novel transgenic zebrafish line to demonstrate that the neural crest population that gives rise to DRG neurons becomes fate restricted to a neuronal/glial precursor before the onset of neurog1 function. We generated a stable transgenic zebrafish line that carries a modified bacterial artificial chromosome that expresses green fluorescent protein (GFP) under the control of the neurog1 promoter [Tg(neurog1:EGFP)]. In contrast to previously described neurog1 transgenic lines, Tg(neurog1:EGFP) expresses GFP in DRG neuronal precursors cells as they migrate ventrally and after their initial differentiation as neurons. Using this line, we are able to track the fate of DRG neuronal precursor cells during their specification. When Neurog1 function is blocked, either by neurog1 morpholino antisense oligonucleotide injection or in neurog1 mutants, GFP expression initiates in neural crest cells, although they fail to form DRG neurons. Rather, these cells take on a glial-like morphology, retain proliferative capacity, and express glial markers and become associated with the ventral motor root. These results suggest that, within the zebrafish neural crest, there is a fate-restricted lineage that is limited to form either sensory neurons or glia in the developing DRG. Neurog1 acts as the key factor in this lineage to direct the formation of sensory neurons.