RESUMO
In anurans, the vertebral column diverges widely from that of other tetrapods; yet the molecular mechanisms underlying its morphogenesis remain largely unexplored. In this study, we investigate the role of the homeologous uncx.L and uncx.S genes in the vertebral column morphogenesis of the allotetraploid frog Xenopus laevis. We initiated our study by cloning the uncx orthologous genes in the anuran Xenopus and determining their spatial expression patterns using in situ hybridization. Additionally, we employed gain-of-function and loss-of-function approaches through dexamethasone-inducible uncx constructs and antisense morpholino oligonucleotides, respectively. Comparative analysis of the messenger RNA sequences of homeologous uncx genes revealed that the uncx.L variant lacks the eh1-like repressor domain. Our spatial expression analysis indicated that in the presomitic mesoderm and somites, the transcripts of uncx.L and uncx.S are located in overlapping domains. Alterations in the function of uncx genes significantly impact the development and differentiation of the sclerotome and myotome, resulting in axial skeleton malformations. Our findings suggest a scenario where the homeologous genes uncx.L and uncx.S exhibit antagonistic functions during somitogenesis. Specifically, uncx.S appears to be crucial for sclerotome development and differentiation, while uncx.L primarily influences myotome development. Postallotetraploidization, the uncx.L gene in X. laevis evolved to lose its eh1-like repressor domain, transforming into a "native dominant negative" variant that potentially competes with uncx.S for the same target genes. Finally, the histological analysis revealed that uncx.S expression is necessary for the correct formation of pedicles and neural arch of the vertebrae, and uncx.L is required for trunk muscle development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio , Proteínas de Xenopus , Xenopus laevis , Animais , Evolução Biológica , Somitos/metabolismo , Coluna Vertebral/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismoRESUMO
The nephron, functional unit of the vertebrate kidney, is specialized in metabolic wastes excretion and body fluids osmoregulation. Given the high evolutionary conservation of gene expression and segmentation patterning between mammalian and amphibian nephrons, the Xenopus laevis pronephric kidney offers a simplified model for studying nephrogenesis. The Lhx1 transcription factor plays several roles during embryogenesis, regulating target genes expression by forming multiprotein complexes with LIM binding protein 1 (Ldb1). However, few Lhx1-Ldb1 cofactors have been identified for kidney organogenesis. By tandem- affinity purification from kidney-induced Xenopus animal caps, we identified single-stranded DNA binding protein 2 (Ssbp2) interacts with the Ldb1-Lhx1 complex. Ssbp2 is expressed in the Xenopus pronephros, and knockdown prevents normal morphogenesis and differentiation of the glomus and the convoluted renal tubules. We demonstrate a role for a member of the Ssbp family in kidney organogenesis and provide evidence of a fundamental function for the Ldb1-Lhx1-Ssbp transcriptional complexes in embryonic development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Pronefro , Animais , Xenopus laevis/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Rim/metabolismo , Desenvolvimento Embrionário/genética , Morfogênese/genética , Pronefro/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Mamíferos/metabolismoRESUMO
Development of the central nervous system in amphibians has called attention from scientists for over a century. Interested in the matter of embryonic inductions, Hans Spemann and Hilde Mangold found out that the dorsal blastopore lip of the salamander's embryo has organizer properties. Such an ectopic graft could induce structures in the host embryo, including a neural tube overlying the notochord of a perfect secondary body axis. A couple of decades later, the frog Xenopus laevis emerged as an excellent embryological experimental model and seminal concepts involving embryonic inductions began to be revealed. The so-called primary induction is, in fact, a composition of signaling and inductive events that are triggered as soon as fertilization takes place. In this regard, since early 1990s an intricate network of signaling pathways has been built. The Wnt pathway, which began to be uncovered in cancer biology studies, is crucial during the establishment of two signaling centers in Xenopus embryogenesis: Nieuwkoop center and the blastula chordin noggin expression center (BCNE). Here we will discuss the historical events that led to the discovery of those centers, as well as the molecular mechanisms by which they operate. This chapter highlights the cooperation of both signaling centers with potential to be further explored in the future. We aim to address the essential morphological transformation during gastrulation and neurulation as well as the role of Wnt signaling in patterning the organizer and the neural plate.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Via de Sinalização Wnt , Animais , Xenopus laevis , Indução Embrionária , Gastrulação , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Padronização CorporalRESUMO
Gastrulation is a key event in animal embryogenesis during which germ layer precursors are rearranged and the embryonic axes are established. Cell polarization is essential during gastrulation, driving asymmetric cell division, cell movements, and cell shape changes. The furry (fry) gene encodes an evolutionarily conserved protein with a wide variety of cellular functions, including cell polarization and morphogenesis in invertebrates. However, little is known about its function in vertebrate development. Here, we show that in Xenopus, Fry plays a role in morphogenetic processes during gastrulation, in addition to its previously described function in the regulation of dorsal mesoderm gene expression. Using morpholino knock-down, we demonstrate a distinct role for Fry in blastopore closure and dorsal axis elongation. Loss of Fry function drastically affects the movement and morphological polarization of cells during gastrulation and disrupts dorsal mesoderm convergent extension, responsible for head-to-tail elongation. Finally, we evaluate a functional interaction between Fry and NDR1 kinase, providing evidence of an evolutionarily conserved complex required for morphogenesis.
Assuntos
Movimento Celular , Gastrulação , Proteínas Repressoras/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Feminino , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/genética , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
G protein-activated inward-rectifying potassium (K+ ) channels (Kir3/GIRK) participate in cell excitability. The GIRK5 channel is present in Xenopus laevis oocytes. In an attempt to investigate the physiological role of GIRK5, we identified a noncanonical di-arginine endoplasmic reticulum (ER) retention motif (KRXY). This retention motif is located at the N-terminal region of GIRK5, coded by two small exons found only in X. laevis and X. tropicalis. These novel exons are expressed through use of an alternative transcription start site. Mutations in the sequence KRXY produced functional channels and induced progesterone-independent oocyte meiotic progression. The chimeric proteins enhanced green fluorescent protein (EGFP)-GIRK5-WT and the EGFP-GIRK5K13AR14A double mutant, were localized to the ER and the plasma membrane of the vegetal pole of the oocyte, respectively. Silencing of GIRK5 or blocking of this channel by external barium prevented progesterone-induced meiotic progression. The endogenous level of GIRK5 protein decreased through oocyte stages in prophase I augmenting by progesterone. In conclusion, we have identified a unique mechanism by which the expression pattern of a K+ channel evolved to control Xenopus oocyte maturation.
Assuntos
Motivos de Aminoácidos , Sequência de Aminoácidos , Retículo Endoplasmático/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/química , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Oócitos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Animais , Sequência Conservada , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Humanos , Oócitos/efeitos dos fármacos , Filogenia , Ligação Proteica , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
Geoffroea decorticans (chañar) is commonly used for culinary and medicinal purposes in rural communities. The aim of this work was to chemically characterize three Geoffroea decorticans extracts and determine their capacity to modulate the wnt/ß-catenin pathway. This signaling pathway plays a key role in embryonic development but its overactivation leads to cancer cell growth. Phytochemical analysis of extracts showed presence of major classes of phytochemicals. Gas chromatography-mass spectrometry results revealed the presence of acids, esters and furanic compounds. Using Xenopus embryos as in vivo model organisms, we found that the extracts modulated dorso-ventral axis formation and rescued hyperdorsalized phenotypes produced by LiCl treatment. In agreement with these findings, Geoffroea decorticans extracts decreased ß-catenin levels and suppressed the expression of wnt target genes such as xnr3 and chordin, thus demonstrating an inhibitory regulation of the wnt/ß-catenin signaling pathway. All these results support a new role for Geoffroea decorticans fruit derivatives with possible anti-carcinogenic actions.
Assuntos
Fabaceae/química , Frutas/química , Terapia de Alvo Molecular , Neoplasias/metabolismo , Extratos Vegetais/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Xenopus laevis , beta Catenina/antagonistas & inibidores , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glicoproteínas/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Cloreto de Lítio/farmacologia , Masculino , Neoplasias/tratamento farmacológico , Extratos Vegetais/química , Fator de Crescimento Transformador beta/genética , Via de Sinalização Wnt/genética , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
This review highlights the work that my research group has been developing, together with international collaborators, during the last decade. Since we were able to establish the Xenopus laevis experimental model in Brazil, we have been focused on understanding early embryonic patterns regarding neural induction and axes establishment. In this context, the Wnt pathway appears as a major player and has been much explored by us and other research groups. Here, we chose to review three published works which we consider to be landmarks within the course of our research and also within the history of modern findings regarding neural induction and patterning. We intend to show how our series of discoveries, when painted together, tells a story that covers crucial developmental windows of early differentiation paths of anterior neural tissue: 1. establishing the head organizer in contrast to the trunk organizer in the early gastrula; 2. deciding between neural ectoderm and epidermis ectoderm at the blastula/gastrula stages, and 3. the gathering of prechordal unique properties in the late gastrula/early neurula.
Assuntos
Padronização Corporal , Via de Sinalização Wnt , Animais , Ectoderma/metabolismo , Indução Embrionária , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Xenopus/genética , Xenopus laevis/metabolismoRESUMO
Cation-coupled chloride cotransporters (CCC) play a role in modulating intracellular chloride concentration ([Cl-]i) and cell volume. Cell shrinkage and cell swelling are accompanied by an increase or decrease in [Cl-]i, respectively. Cell shrinkage and a decrease in [Cl-]i increase the activity of NKCCs (Na-K-Cl cotransporters: NKCC1, NKCC2, and Na-Cl) and inhibit the activity of KCCs (K-Cl cotransporters: KCC1 to KCC4), wheras cell swelling and an increase in [Cl-]i activate KCCs and inhibit NKCCs; thus, it is unlikely that the same kinase is responsible for both effects. WNK1 and WNK4 are chloride-sensitive kinases that modulate the activity of CCC in response to changes in [Cl-]i. Here, we showed that WNK3, another member of the serine-threonine kinase WNK family with known effects on CCC, is not sensitive to [Cl-]i but can be regulated by changes in extracellular tonicity. In contrast, WNK4 is highly sensitive to [Cl-]i but is not regulated by changes in cell volume. The activity of WNK3 toward NaCl cotransporter is not affected by eliminating the chloride-binding site of WNK3, further confirming that the kinase is not sensitive to chloride. Chimeric WNK3/WNK4 proteins were produced, and analysis of the chimeras suggests that sequences within the WNK's carboxy-terminal end may modulate the chloride affinity. We propose that WNK3 is a cell volume-sensitive kinase that translates changes in cell volume into phosphorylation of CCC.
Assuntos
Tamanho Celular , Proteínas Serina-Treonina Quinases/genética , Simportadores de Cloreto de Sódio/metabolismo , Proteínas de Xenopus/genética , Animais , Cloretos/química , Cloretos/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Humanos , Oócitos/química , Oócitos/metabolismo , Fosforilação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores de Cloreto de Sódio/química , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
BACKGROUND: In most vertebrates, the segmentation of the paraxial mesoderm involves the formation of metameric units called somites through a mesenchymal-epithelial transition. However, this process is different in Xenopus laevis because it does not form an epithelial somite. Xenopus somitogenesis is characterized by a complex cells rearrangement that requires the coordinated regulation of cell shape, adhesion, and motility. The molecular mechanisms that control these cell behaviors underlying somite formation are little known. Although the Paraxis has been implicated in the epithelialization of somite in chick and mouse, its role in Xenopus somite morphogenesis has not been determined. RESULTS: Using a morpholino and hormone-inducible construction approaches, we showed that both gain and loss of function of paraxis affect somite elongation, rotation and alignment, causing a severe disorganization of somitic tissue. We further found that depletion or overexpression of paraxis in the somite led to the downregulation or upregulation, respectively, of cell adhesion expression markers. Finally, we demonstrated that paraxis is necessary for the proper expression of myotomal and sclerotomal differentiation markers. CONCLUSIONS: Our results demonstrate that paraxis regulates the cell rearrangements that take place during the somitogenesis of Xenopus by regulating cell adhesion. Furthermore, paraxis is also required for somite differentiation.
Assuntos
Diferenciação Celular/fisiologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Adesão Celular/genética , Adesão Celular/fisiologia , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/genética , Somitos/embriologia , Somitos/metabolismo , Fatores de Transcrição/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Spinal cord regeneration is very inefficient in humans, causing paraplegia and quadriplegia. Studying model organisms that can regenerate the spinal cord in response to injury could be useful for understanding the cellular and molecular mechanisms that explain why this process fails in humans. Here, we use Xenopus laevis as a model organism to study spinal cord repair. Histological and functional analyses showed that larvae at pre-metamorphic stages restore anatomical continuity of the spinal cord and recover swimming after complete spinal cord transection. These regenerative capabilities decrease with onset of metamorphosis. The ability to study regenerative and non-regenerative stages in Xenopus laevis makes it a unique model system to study regeneration. We studied the response of Sox2(/)3 expressing cells to spinal cord injury and their function in the regenerative process. We found that cells expressing Sox2 and/or Sox3 are present in the ventricular zone of regenerative animals and decrease in non-regenerative froglets. Bromodeoxyuridine (BrdU) experiments and in vivo time-lapse imaging studies using green fluorescent protein (GFP) expression driven by the Sox3 promoter showed a rapid, transient and massive proliferation of Sox2(/)3(+) cells in response to injury in the regenerative stages. The in vivo imaging also demonstrated that Sox2(/)3(+) neural progenitor cells generate neurons in response to injury. In contrast, these cells showed a delayed and very limited response in non-regenerative froglets. Sox2 knockdown and overexpression of a dominant negative form of Sox2 disrupts locomotor and anatomical-histological recovery. We also found that neurogenesis markers increase in response to injury in regenerative but not in non-regenerative animals. We conclude that Sox2 is necessary for spinal cord regeneration and suggest a model whereby spinal cord injury activates proliferation of Sox2/3 expressing cells and their differentiation into neurons, a mechanism that is lost in non-regenerative froglets.