RESUMO
Protocadherin 19 (Pcdh19) is a homophilic cell adhesion molecule and is involved in a variety of neuronal functions. Here, we tested whether Pcdh19 has a regulatory role in axon guidance using the developing Xenopus retinotectal system. We performed targeted microinjections of a translation blocking antisense morpholino oligonucleotide to knock down the expression of Pcdh19 selectively in the central nervous system. Knocking down Pcdh19 expression resulted in navigational errors of retinal ganglion cell (RGC) axons specifically at the optic chiasm. Instead of projecting to the contralateral optic tectum, RGC axons in the Pcdh19-depleted embryo misprojected ipsilaterally. Although incorrectly delivered into the ipsilateral brain hemisphere, these axons correctly reached the optic tectum. These data suggest that Pcdh19 has a critical role in preventing mixing of RGC axons originating from the opposite eyes at the optic chiasm, highlighting the importance of cell adhesion in bundling of RGC axons.
Assuntos
Orientação de Axônios , Axônios , Caderinas , Protocaderinas , Células Ganglionares da Retina , Proteínas de Xenopus , Xenopus laevis , Animais , Caderinas/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Células Ganglionares da Retina/metabolismo , Xenopus laevis/embriologia , Axônios/metabolismo , Retina/metabolismo , Retina/embriologia , Vias Visuais , Técnicas de Silenciamento de Genes , Quiasma Óptico/embriologia , Quiasma Óptico/metabolismo , Colículos Superiores/embriologia , Colículos Superiores/metabolismo , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
DNA-protein crosslinks (DPCs) are toxic lesions that inhibit DNA related processes. Post-translational modifications (PTMs), including SUMOylation and ubiquitylation, play a central role in DPC resolution, but whether other PTMs are also involved remains elusive. Here, we identify a DPC repair pathway orchestrated by poly-ADP-ribosylation (PARylation). Using Xenopus egg extracts, we show that DPCs on single-stranded DNA gaps can be targeted for degradation via a replication-independent mechanism. During this process, DPCs are initially PARylated by PARP1 and subsequently ubiquitylated and degraded by the proteasome. Notably, PARP1-mediated DPC resolution is required for resolving topoisomerase 1-DNA cleavage complexes (TOP1ccs) induced by camptothecin. Using the Flp-nick system, we further reveal that in the absence of PARP1 activity, the TOP1cc-like lesion persists and induces replisome disassembly when encountered by a DNA replication fork. In summary, our work uncovers a PARP1-mediated DPC repair pathway that may underlie the synergistic toxicity between TOP1 poisons and PARP inhibitors.
Assuntos
Reparo do DNA , Replicação do DNA , DNA Topoisomerases Tipo I , Poli(ADP-Ribose) Polimerase-1 , Poli ADP Ribosilação , Animais , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , DNA Topoisomerases Tipo I/metabolismo , Xenopus laevis , Ubiquitinação , Humanos , DNA/metabolismo , Dano ao DNA , Camptotecina/farmacologia , Processamento de Proteína Pós-Traducional , DNA de Cadeia Simples/metabolismo , Proteínas de Xenopus/metabolismoRESUMO
In response to DNA double-strand breaks or oxidative stress, ATM-dependent DNA damage response (DDR) is activated to maintain genome integrity. However, it remains elusive whether and how DNA single-strand breaks (SSBs) activate ATM. Here, we provide direct evidence in Xenopus egg extracts that ATM-mediated DDR is activated by a defined SSB structure. Our mechanistic studies reveal that APE1 promotes the SSB-induced ATM DDR through APE1 exonuclease activity and ATM recruitment to SSB sites. APE1 protein can form oligomers to activate the ATM DDR in Xenopus egg extracts in the absence of DNA and can directly stimulate ATM kinase activity in vitro. Our findings reveal distinct mechanisms of the ATM-dependent DDR activation by SSBs in eukaryotic systems and identify APE1 as a direct activator of ATM kinase.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , Quebras de DNA de Cadeia Simples , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Transdução de Sinais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Animais , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Humanos , Xenopus laevis , Reparo do DNARESUMO
Xenopus embryos provide a favorable material to dissect the sequential steps that lead to dorsal-ventral (D-V) and anterior-posterior (A-P) cell differentiation. Here, we analyze the signaling pathways involved in this process using loss-of-function and gain-of-function approaches. The initial step was provided by Hwa, a transmembrane protein that robustly activates early ß-catenin signaling when microinjected into the ventral side of the embryo leading to complete twinned axes. The following step was the activation of Xenopus Nodal-related growth factors, which could rescue the depletion of ß-catenin and were themselves blocked by the extracellular Nodal antagonists Cerberus-Short and Lefty. During gastrulation, the Spemann-Mangold organizer secretes a cocktail of growth factor antagonists, of which the BMP antagonists Chordin and Noggin could rescue simultaneously D-V and A-P tissues in ß-catenin-depleted embryos. Surprisingly, this rescue occurred in the absence of any ß-catenin transcriptional activity as measured by ß-catenin activated Luciferase reporters. The Wnt antagonist Dickkopf (Dkk1) strongly synergized with the early Hwa signal by inhibiting late Wnt signals. Depletion of Sizzled (Szl), an antagonist of the Tolloid chordinase, was epistatic over the Hwa and Dkk1 synergy. BMP4 mRNA injection blocked Hwa-induced ectopic axes, and Dkk1 inhibited BMP signaling late, but not early, during gastrulation. Several unexpected findings were made, e.g., well-patterned complete embryonic axes are induced by Chordin or Nodal in ß-catenin knockdown embryos, dorsalization by Lithium chloride (LiCl) is mediated by Nodals, Dkk1 exerts its anteriorizing and dorsalizing effects by regulating late BMP signaling, and the Dkk1 phenotype requires Szl.
Assuntos
Padronização Corporal , Peptídeos e Proteínas de Sinalização Intercelular , Transdução de Sinais , Proteínas de Xenopus , beta Catenina , Animais , Padronização Corporal/genética , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , beta Catenina/metabolismo , beta Catenina/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Xenopus laevis/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Gastrulação , Proteína Nodal/metabolismo , Proteína Nodal/genética , Embrião não Mamífero/metabolismo , Embrião não Mamífero/embriologia , Organizadores Embrionários/metabolismo , GlicoproteínasRESUMO
The SRY HMG box transcription factor Sox21 plays multiple critical roles in neurogenesis, with its function dependent on concentration and developmental stage. In the allotetraploid Xenopus laevis, there are two homeologs of sox21, namely sox21.S and sox21.L. Previous studies focused on Sox21.S, but its amino acid sequence is divergent, lacking conserved poly-A stretches and bearing more similarity with ancestral homologs. In contrast, Sox21.L shares higher sequence similarity with mouse and chick Sox21. To determine if Sox21.S and Sox21.L have distinct functions, we conducted gain and loss-of-function studies in Xenopus embryos. Our studies revealed that Sox21.S and Sox21.L are functionally redundant, but Sox21.L is more effective at driving changes than Sox21.S. These results also support our earlier findings in ectodermal explants, demonstrating that Sox21 function is dose-dependent. While Sox21 is necessary for primary neuron formation, high levels prevent their formation. Strikingly, these proteins autoregulate, with high levels of Sox21.L reducing sox21.S and sox21.L mRNA levels, and decreased Sox21.S promoting increased expression of sox21.L. Our findings shed light on the intricate concentration-dependent roles of Sox21 homeologs in Xenopus neurogenesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Neurogênese , Proteínas de Xenopus , Xenopus laevis , Animais , Neurogênese/genética , Xenopus laevis/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Neurônios/metabolismo , Fatores de Transcrição SOXB2/genética , Fatores de Transcrição SOXB2/metabolismoRESUMO
Peptidoglycan recognition proteins (PGRPs) are a family of multifunctional proteins playing vital roles in PGN metabolism and antibacterial defense, and their functions have been well-characterized in mammals, bony fishes, and insects. However, the information about the functions of amphibian long-type PGRP is rather limited. Here, we identified and cloned a long-type PGRP gene (named Xl-PGRP-L) from African clawed frog, Xenopus laevis. Xl-PGRP-L gene was detected in all orangs/tissues examined, and was rapidly induced in intestine, liver, and lung following the stimulation of PGN. Sequence analysis showed that Xl-PGRP-L possesses four Zn2+-binding residues (His358, Tyr395, His470, and Cys478) required for amidase activity of catalytic PGRPs, and assays for amidase activity revealed that recombinant Xl-PGRP-L cloud degrade PGN in a Zn2+-dependent manner, indicating that Xl-PGRP-L is belonging to catalytic PGRPs. In addition, Xl-PGRP-L have antibacterial activity against Gram-negative bacteria Edwardsiella tarda and Gram-positive bacteria Streptococcus agalactiae. The present investigation represents the first characterization regarding the biological activities of amphibian long-type PGRPs, thus contributes to a better understanding of the functions of tetrapod PGRPs and the molecular mechanisms of amphibian antibacterial defense.
Assuntos
Proteínas de Transporte , Proteínas de Xenopus , Xenopus laevis , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Clonagem Molecular , Sequência de Aminoácidos , Peptidoglicano/metabolismo , Amidoidrolases/genética , Amidoidrolases/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Zinco/metabolismo , Filogenia , Streptococcus agalactiae/genéticaRESUMO
The Daam1 protein regulates Wnt-induced cytoskeletal changes during vertebrate gastrulation though its full mode of action and binding partners remain unresolved. Here we identify Reversion Induced LIM domain protein (RIL) as a new interacting protein of Daam1. Interaction studies uncover binding of RIL to the C-terminal actin-nucleating portion of Daam1 in a Wnt-responsive manner. Immunofluorescence studies showed subcellular localization of RIL to actin fibers and co-localization with Daam1 at the plasma membrane. RIL gain- and loss-of-function approaches in Xenopus produced severe gastrulation defects in injected embryos. Additionally, a simultaneous loss of Daam1 and RIL synergized to produce severe gastrulation defects indicating RIL and Daam1 may function in the same signaling pathway. RIL further synergizes with another novel Daam1-interacting protein, Formin Binding Protein 1 (FNBP1), to regulate gastrulation. Our studies altogether show RIL mediates Daam1-regulated non-canonical Wnt signaling that is required for vertebrate gastrulation.
Assuntos
Citoesqueleto de Actina , Gastrulação , Proteínas dos Microfilamentos , Via de Sinalização Wnt , Proteínas de Xenopus , Xenopus laevis , Animais , Feminino , Humanos , Ratos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas com Domínio LIM/metabolismo , Proteínas com Domínio LIM/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Ligação Proteica , Via de Sinalização Wnt/fisiologia , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genéticaRESUMO
The initiation of DNA replication is tightly controlled by the licensing system that loads replicative DNA helicases onto replication origins to form pre-replicative complexes (pre-RCs) once per cell cycle. Cdc10-dependent transcript 1 (Cdt1) plays an essential role in the licensing reaction by recruiting mini-chromosome maintenance (MCM) complexes, which are eukaryotic replicative DNA helicases, to their origins via direct protein-protein interactions. Cdt1 interacts with other pre-RC components, the origin recognition complex, and the cell division cycle 6 (Cdc6) protein; however, the molecular mechanism by which Cdt1 functions in the MCM complex loading process has not been fully elucidated. Here, we analyzed the protein-protein interactions of recombinant Cdt1 and observed that Cdt1 self-associates via the central region of the molecule, which is inhibited by the endogenous licensing inhibitor, geminin. Mutation of two ß-strands of the winged-helix domain in the central region of Cdt1 attenuated its self-association but could still interact with other pre-RC components and DNA similarly to wild-type Cdt1. Moreover, the Cdt1 mutant showed decreased licensing activity in Xenopus egg extracts. Together, these results suggest that the self-association of Cdt1 is crucial for licensing.
Assuntos
Proteínas de Ciclo Celular , Geminina , Animais , Geminina/metabolismo , Geminina/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Replicação do DNA , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis , Domínios Proteicos , Xenopus , Humanos , Proteínas de Ligação a DNARESUMO
The tRNA-histidine guanylyltransferase 1-like (THG1L), also known as induced in high glucose-1 (IHG-1), encodes for an essential mitochondria-associated protein highly conserved throughout evolution, that catalyses the 3'-5' addition of a guanine to the 5'-end of tRNA-histidine (tRNAHis). Previous data indicated that THG1L plays a crucial role in the regulation of mitochondrial biogenesis and dynamics, in ATP production, and is critically involved in the modulation of apoptosis, cell-cycle progression and survival, as well as in cellular stress responses and redox homeostasis. Dysregulations of THG1L expression play a central role in various pathologies, including nephropathies, and neurodevelopmental disorders often characterized by developmental delay and cerebellar ataxia. Despite the essential role of THG1L, little is known about its expression during vertebrate development. Herein, we examined the detailed spatio-temporal expression of this gene in the developing Xenopus laevis. Our results show that thg1l is maternally inherited and its temporal expression suggests a role during the earliest stages of embryogenesis. Spatially, thg1l mRNA localizes in the ectoderm and marginal zone mesoderm during early stages of development. Then, at tadpole stages, thg1l transcripts mostly localise in neural crests and their derivatives, somites, developing kidney and central nervous system, therefore largely coinciding with territories displaying intense energy metabolism during organogenesis in Xenopus.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Nucleotidiltransferases , Proteínas de Xenopus , Xenopus laevis , Animais , Embrião não Mamífero/metabolismo , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário/genética , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Xenopus laevis/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
The zinc finger and BTB domain-containing 11 gene (zbtb11) is expressed in the Xenopus anterior neuroectoderm, but the molecular nature of the Zbtb11 protein during embryonic development remains to be elucidated. Here, we show the role of Zbtb11 in anterior patterning of the neuroectoderm and the cooperative action with the transcription factor Otx2. Both overexpression and knockdown of zbtb11 caused similar phenotypes: expanded expression of the posterior gene gbx2 in the neural plate, and later microcephaly with reduced eyes, suggesting that a proper level of zbtb11 expression is necessary for normal patterning of the neuroectoderm, including eye formation. Co-immunoprecipitation assays showed that Zbtb11 formed a complex with itself and with a phosphomimetic and repressive form of Otx2, suggesting that Zbtb11 forms a dimer or oligomer and interacts with Otx2 in a phosphorylation-dependent manner. Reporter analysis further showed that Zbtb11 enhanced the activity of the phosphomimetic Otx2 to repress a silencer element of the posterior gene meis3. These data suggest that Zbtb11 coordinates with phosphorylated Otx2 to specify the anterior neuroectoderm by repressing posterior genes.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Placa Neural , Fatores de Transcrição Otx , Proteínas de Xenopus , Animais , Fatores de Transcrição Otx/metabolismo , Fatores de Transcrição Otx/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Placa Neural/metabolismo , Placa Neural/embriologia , Xenopus laevis , Ligação Proteica , Fosforilação , Padronização Corporal/genéticaRESUMO
The formation of dynamic protein filaments contributes to various biological functions by clustering individual molecules together and enhancing their binding to ligands. We report such a propensity for the BTB domains of certain proteins from the ZBTB family, a large eukaryotic transcription factor family implicated in differentiation and cancer. Working with Xenopus laevis and human proteins, we solved the crystal structures of filaments formed by dimers of the BTB domains of ZBTB8A and ZBTB18 and demonstrated concentration-dependent higher-order assemblies of these dimers in solution. In cells, the BTB-domain filamentation supports clustering of full-length human ZBTB8A and ZBTB18 into dynamic nuclear foci and contributes to the ZBTB18-mediated repression of a reporter gene. The BTB domains of up to 21 human ZBTB family members and two related proteins, NACC1 and NACC2, are predicted to behave in a similar manner. Our results suggest that filamentation is a more common feature of transcription factors than is currently appreciated.
Assuntos
Domínio BTB-POZ , Fatores de Transcrição , Proteínas de Xenopus , Animais , Humanos , Núcleo Celular/metabolismo , Núcleo Celular/genética , Cristalografia por Raios X , Células HEK293 , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Xenopus laevis , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/químicaRESUMO
The actin-based cytoskeleton is considered a fundamental driving force for cell differentiation and development. Destrin (Dstn), a member of the actin-depolymerizing factor family, regulates actin dynamics by treadmilling actin filaments and increasing globular actin pools. However, the specific developmental roles of dstn have yet to be fully elucidated. Here, we investigated the physiological functions of dstn during early embryonic development using Xenopus laevis as an experimental model organism. dstn is expressed in anterior neural tissue and neural plate during Xenopus embryogenesis. Depleting dstn promoted morphants with short body axes and small heads. Moreover, dstn inhibition extended the neural plate region, impairing cell migration and distribution during neurulation. In addition to the neural plate, dstn knockdown perturbed neural crest cell migration. Our data suggest new insights for understanding the roles of actin dynamics in embryonic neural development, simultaneously presenting a new challenge for studying the complex networks governing cell migration involving actin dynamics.
Assuntos
Movimento Celular , Destrina , Desenvolvimento Embrionário , Xenopus laevis , Animais , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Destrina/metabolismo , Destrina/genética , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Crista Neural/metabolismo , Crista Neural/embriologia , Crista Neural/citologia , Neurogênese , Placa Neural/metabolismo , Placa Neural/embriologia , Actinas/metabolismo , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Neural crest cells are a stem cell population unique to vertebrate embryos that retains broad multi-germ layer developmental potential through neurulation. Much remains to be learned about the genetic and epigenetic mechanisms that control the potency of neural crest cells. Here, we examine the role that epigenetic readers of the BET (bromodomain and extra terminal) family play in controlling the potential of pluripotent blastula and neural crest cells. We find that inhibiting BET activity leads to loss of pluripotency at blastula stages and a loss of neural crest at neurula stages. We compare the effects of HDAC (an eraser of acetylation marks) and BET (a reader of acetylation) inhibition and find that they lead to similar cellular outcomes through distinct effects on the transcriptome. Interestingly, loss of BET activity in cells undergoing lineage restriction is coupled to increased expression of genes linked to pluripotency and prolongs the competence of initially pluripotent cells to transit to a neural progenitor state. Together these findings advance our understanding of the epigenetic control of pluripotency and the formation of the vertebrate neural crest.
Assuntos
Crista Neural , Animais , Crista Neural/citologia , Crista Neural/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Blástula/metabolismo , Blástula/citologia , Diferenciação Celular , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Transcriptoma/genéticaRESUMO
SoxB1 transcription factors (Sox2/3) are well known for their role in early neural fate specification in the embryo, but little is known about functional roles for SoxB1 factors in non-neural ectodermal cell types, such as the neural plate border (NPB). Using Xenopus laevis, we set out to determine whether SoxB1 transcription factors have a regulatory function in NPB formation. Here, we show that SoxB1 factors are necessary for NPB formation, and that prolonged SoxB1 factor activity blocks the transition from a NPB to a neural crest state. Using ChIP-seq, we demonstrate that Sox3 is enriched upstream of NPB genes in early NPB cells and in blastula stem cells. Depletion of SoxB1 factors in blastula stem cells results in downregulation of NPB genes. Finally, we identify Pou5f3 factors as potential Sox3 partners in regulating the formation of the NPB and show that their combined activity is needed for normal NPB gene expression. Together, these data identify a role for SoxB1 factors in the establishment and maintenance of the NPB, in part through partnership with Pou5f3 factors.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Crista Neural , Placa Neural , Fatores de Transcrição SOXB1 , Proteínas de Xenopus , Xenopus laevis , Animais , Placa Neural/metabolismo , Placa Neural/embriologia , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Crista Neural/metabolismo , Crista Neural/citologia , Blástula/metabolismo , Embrião não Mamífero/metabolismoRESUMO
The Formin protein Daam1 is required for Wnt-induced cytoskeletal changes during gastrulation, though how it accomplishes this remains unresolved. Here we report the characterization of Formin Binding Protein 1 (FNBP1) as a binding partner of Daam1. The interaction of Daam1 with FNBP1 and its domains required for this interaction were delineated. Immunofluorescence studies showed FNBP1 co-localizes with Daam1, and is an integral component of the actin cytoskeletal complex that is responsive to Wnt stimulation. Specifically, FNBP1 can induce intracellular tubule-like structures and localize to focal adhesions suggesting a role for FNBP1 in cell migration. Functional FNBP1 studies in Xenopus embryos uncover a critical role for FNBP1 in regulating vertebrate gastrulation. Additionally, suboptimal doses of Daam1 and FNBP1 synergize to produce severe gastrulation defects, indicating FNBP1 and Daam1 may function within the same signaling pathway. These results together show FNBP1 is an integral component of Daam1-regulated non-canonical Wnt signaling required for vertebrate gastrulation.
Assuntos
Gastrulação , Via de Sinalização Wnt , Proteínas de Xenopus , Xenopus laevis , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Movimento Celular , Embrião não Mamífero/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Ligação Proteica , Proteínas rho de Ligação ao GTP , Via de Sinalização Wnt/fisiologia , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , FemininoRESUMO
Primordial germ cells (PGCs) are the precursors of sperms and oocytes. Proper development of PGCs is crucial for the survival of the species. In many organisms, factors responsible for PGC development are synthesized during early oogenesis and assembled into the germ plasm. During early embryonic development, germ plasm is inherited by a few cells, leading to the formation of PGCs. While germline development has been extensively studied, how components of the germ plasm regulate PGC development is not fully understood. Here, we report that Dzip1 is dynamically expressed in vertebrate germline and is a novel component of the germ plasm in Xenopus and zebrafish. Knockdown of Dzip1 impairs PGC development in Xenopus embryos. At the molecular level, Dzip1 physically interacts with Dazl, an evolutionarily conserved RNA-binding protein that plays a multifaced role during germline development. We further showed that the sequence between amino acid residues 282 and 550 of Dzip1 is responsible for binding to Dazl. Disruption of the binding between Dzip1 and Dazl leads to defective PGC development. Taken together, our results presented here demonstrate that Dzip1 is dynamically expressed in the vertebrate germline and plays a novel function during Xenopus PGC development.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas , Proteínas de Ligação a RNA , Proteínas de Xenopus , Xenopus laevis , Animais , Feminino , Células Germinativas/metabolismo , Células Germinativas/citologia , Oogênese/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Xenopus laevis/genética , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
The signals that regulate peripheral blood vessel formation during development are still under investigation. The hormone leptin promotes blood vessel formation, adipose tissue establishment and expansion, tumor growth, and wound healing, but the underlying mechanisms for these actions are currently unknown. We investigated whether leptin promotes angiogenesis in the developing tail fin using embryonic transgenic xflk-1:GFP Xenopus laevis, which express a green fluorescent protein on vascular endothelial cells to mark blood vessels. We found that leptin protein is expressed in endothelial cells of developing blood vessels and that leptin treatment via injection increased phosphorylated STAT3 signaling, which is indicative of leptin activation of its receptor, in blood vessels of the larval tail fin. Leptin administration via media increased vessel length, branching, and reconnection with the cardinal vein, while decreased leptin signaling via immunoneutralization had an opposing effect on vessel development. We also observed disorganization of major vessels and microvessels of the tail fin and muscle when leptin signaling was decreased. Reduced leptin signaling lowered mRNA expression of cenpk, gpx1, and mmp9, markers for cell proliferation, antioxidation, and extracellular matrix remodeling/cell migration, respectively, in the developing tail, providing insight into three possible mechanisms underlying leptin's promotion of angiogenesis. Together these results illustrate that leptin levels are correlated with embryonic angiogenesis and that leptin coordinates multiple aspects of blood vessel growth and development, showing that leptin is an important morphogen during embryonic development.
Assuntos
Larva , Leptina , Neovascularização Fisiológica , Transdução de Sinais , Cauda , Xenopus laevis , Animais , Leptina/metabolismo , Cauda/irrigação sanguínea , Cauda/embriologia , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Larva/metabolismo , Vasos Sanguíneos/embriologia , Vasos Sanguíneos/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Animais Geneticamente Modificados , Fator de Transcrição STAT3/metabolismo , Embrião não Mamífero/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Motile cilia on the cell surface produce fluid flows in the body and abnormalities in motile cilia cause primary ciliary dyskinesia. Dynein axonemal assembly factor 6 (DNAAF6), a causative gene of primary ciliary dyskinesia, was isolated as an interacting protein with La ribonucleoprotein 6 (LARP6) that regulates ciliogenesis in multiciliated cells (MCCs). In MCCs of Xenopus embryos, LARP6 and DNAAF6 were colocalized in biomolecular condensates termed dynein axonemal particles and synergized to control ciliogenesis. Moreover, tubulin alpha 1c-like mRNA encoding α-tubulin protein, that is a major component of ciliary axoneme, was identified as a target mRNA regulated by binding LARP6. While DNAAF6 was necessary for high α-tubulin protein expression near the apical side of Xenopus MCCs during ciliogenesis, its mutant, which abolishes binding with LARP6, was unable to restore the expression of α-tubulin protein near the apical side of MCCs in Xenopus DNAAF6 morphant. These results indicated that the binding of LARP6 and DNAAF6 in dynein axonemal particles regulates highly expressed α-tubulin protein near the apical side of Xenopus MCCs during ciliogenesis.
Assuntos
Cílios , Ribonucleoproteínas , Tubulina (Proteína) , Proteínas de Xenopus , Xenopus laevis , Cílios/metabolismo , Animais , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Tubulina (Proteína)/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Humanos , Antígeno SS-B , Autoantígenos/metabolismo , Autoantígenos/genética , Ligação Proteica , Axonema/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genéticaRESUMO
Deubiquitylating enzymes (DUBs) remove ubiquitin from proteins thereby regulating their stability or activity. Our understanding of DUB-substrate specificity is limited because DUBs are typically not compared to each other against many physiological substrates. By broadly inhibiting DUBs in Xenopus egg extract, we generated hundreds of ubiquitylated proteins and compared the ability of 30 DUBs to deubiquitylate them using quantitative proteomics. We identified five high-impact DUBs (USP7, USP9X, USP36, USP15, and USP24) that each reduced ubiquitylation of over 10% of the isolated proteins. Candidate substrates of high-impact DUBs showed substantial overlap and were enriched for disordered regions, suggesting this feature may promote substrate recognition. Other DUBs showed lower impact and non-overlapping specificity, targeting distinct non-disordered proteins including complexes such as the ribosome or the proteasome. Altogether our study identifies candidate DUB substrates and defines patterns of functional redundancy and specificity, revealing substrate characteristics that may influence DUB-substrate recognition.
Assuntos
Ubiquitina , Especificidade por Substrato , Animais , Ubiquitina/metabolismo , Ubiquitinação , Enzimas Desubiquitinantes/metabolismo , Xenopus laevis , Proteínas de Xenopus/metabolismo , Xenopus , Proteômica , Humanos , Proteases Específicas de Ubiquitina/metabolismoRESUMO
The coordinated movement of germ layer progenitor cells reaches its peak at the dorsal side, where the Bmp signaling gradient is low, and minimum at the ventral side, where the Bmp gradient is high. This dynamic cell movement is regulated by the interplay of various signaling pathways. The noncanonical Wnt signaling cascade serves as a pivotal regulator of convergence and extension cell movement, facilitated by the activation of small GTPases such as Rho, Rab, and Rac. However, the underlying cause of limited cell movement at the ventral side remains elusive. To explore the functional role of a key regulator in constraining gastrulation cell movement at the ventral side, we investigated the Bmp4-direct target gene, sizzled (szl), to assess its potential role in inhibiting noncanonical Wnt signaling. In our current study, we demonstrated that ectopic expression of szl led to gastrulation defects in a dose-dependent manner without altering cell fate specification. Overexpression of szl resulted in decreased elongation of Activin-treated animal cap and Keller explants. Furthermore, our immunoprecipitation assay unveiled the physical interaction of Szl with noncanonical Wnt ligand proteins (Wnt5 and Wnt11). Additionally, the activation of small GTPases involved in Wnt signaling mediation (RhoA and Rac1) was diminished upon szl overexpression. In summary, our findings suggest that Bmp4 signaling negatively modulates cell movement from the ventral side of the embryo by inducing szl expression during early Xenopus gastrulation.