Browsing by Author "Larrain, Juan"

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    Analysis of the early response to spinal cord injury identified a key role for mTORC1 signaling in the activation of neural stem progenitor cells
    (2021) Penailillo, Johany; Palacios, Miriam; Mounieres, Constanza; Munoz, Rosana; Slater, Paula G.; De Domenico, Elena; Patrushev, Ilya; Gilchrist, Mike; Larrain, Juan
    Xenopus laevis are able to regenerate the spinal cord during larvae stages through the activation of neural stem progenitor cells (NSPCs). Here we use high-resolution expression profiling to characterize the early transcriptome changes induced after spinal cord injury, aiming to identify the signals that trigger NSPC proliferation. The analysis delineates a pathway that starts with a rapid and transitory activation of immediate early genes, followed by migration processes and immune response genes, the pervasive increase of NSPC-specific ribosome biogenesis factors, and genes involved in stem cell proliferation. Western blot and immunofluorescence analysis showed that mTORC1 is rapidly and transiently activated after SCI, and its pharmacological inhibition impairs spinal cord regeneration and proliferation of NSPC through the downregulation of genes involved in the G1/S transition of cell cycle, with a strong effect on PCNA. We propose that the mTOR signaling pathway is a key player in the activation of NPSCs during the early steps of spinal cord regeneration.
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    Cornifelin expression during Xenopus laevis metamorphosis and in response to spinal cord injury
    (2022) Torruella-Gonzalez, Sol; Slater, Paula G.; Lee-Liu, Dasfne; Larrain, Juan
    Background: In a high-throughput RNA sequencing analysis, comparing the transcriptional response between Xenopus laevis regenerative and non-regenerative stages to spinal cord injury, cornifelin was found among the most highly differentially expressed genes. Cornifelin is mainly expressed in stratified squamous epithelia, but its expression in the spinal cord and other central nervous structures has only been described during early development.Results: Here, we report cornifelin expression in the spinal cord, retina, and cornea throughout metamorphosis and in the spinal cord after injury. Cornifelin was detected in the grey matter and meninges of the spinal cord from NF-50 to NF-66, with decreased expression in the grey matter during metamorphosis. In the retina, cor-nifelin was expressed in the ganglion cell layer, the inner and outer nuclear layer, and the outer segment from NF-50 to NF-66. After spinal cord injury, we only observed cornifelin upregulation in NF-66 but no significant changes in NF-50. However, we found cornifelin positive cells in NF-50 meninges closing the spinal cord stumps 1 day after injury and delineating the borders of the spinal cord following the continuity of tissue regeneration in the following days after injury. Instead, in NF-66, cornifelin positive cells were distributed to the ventral side of the spinal cord at 6 days after injury, and at the injury gap at 10 days after injury.Conclusions: Cornifelin is expressed in the Xenopus laevis spinal cord and eye during metamorphosis and plays a role in the meningeal response to spinal cord injury.
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    Early requirement of Hyaluronan for tail regeneration in Xenopus tadpoles
    (COMPANY OF BIOLOGISTS LTD, 2009) Contreras, Esteban G.; Gaete, Marcia; Sanchez, Natalia; Carrasco, Hector; Larrain, Juan
    Tail regeneration in Xenopus tadpoles is a favorable model system to understand the molecular and cellular basis of tissue regeneration. Although turnover of the extracellular matrix (ECM) is a key event during tissue injury and repair, no functional studies to evaluate its role in appendage regeneration have been performed. Studying the role of Hyaluronan (HA), an ECM component, is particularly attractive because it can activate intracellular signaling cascades after tissue injury. Here we studied the function of HA and components of the HA pathway in Xenopus tadpole tail regeneration. We found that transcripts for components of this pathway, including Hyaluronan synthase2 (HAS2), Hyaluronidase2 and its receptors CD44 and RHAMM, were transiently upregulated in the regenerative bud after tail amputation. Concomitantly, an increase in HA levels was observed. Functional experiments using 4-methylumbelliferone, a specific HAS inhibitor that blocked the increase in HA levels after tail amputation, and transgenesis demonstrated that the HA pathway is required during the early phases of tail regeneration. Proper levels of HA are required to sustain proliferation of mesenchymal cells in the regenerative bud. Pharmacological and genetic inhibition of GSK3 beta was sufficient to rescue proliferation and tail regeneration when HA synthesis was blocked, suggesting that GSK3 beta is downstream of the HA pathway. We have demonstrated that HA is an early component of the regenerative pathway and is required for cell proliferation during the early phases of Xenopus tail regeneration. In addition, a crosstalk between HA and GSK3 beta signaling during tail regeneration was demonstrated.
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    Exploration and perspectival modelling with model organisms: developmental biology as a case study
    (2024) Larrain, Juan
    Model organisms are at the centre of progress in biology but attributing them an excessive representational power and concentrating on a limited group of them, although efficient for research, can have negative consequences, mainly of epistemic nature. Here, I argue that model organisms are exploratory models with a perspectival modelling function, and that a deflated representational power is needed for their proper use. In support of this argument, I will analyse developmental biology as a case study. Firstly, I show that model organisms in developmental biology are not selected because of their representational capabilities, but mainly based on practical criteria. Secondly, I defend that the epistemic organization of developmental biology around questions fosters exploration and perspectival modelling and I propose that developmental biology is a 'model organism situated knowledge'. Lastly, I use the study of the mechanisms of cell fate acquisition during early embryonic development in C. elegans and mice as a case study to illustrate how a plurality of model organisms allows exploration and perspectival modelling. The use of model organisms for exploration and perspectival modelling, with a limited representational power, should allow more adequate inferences about human embryonic development and encourage the introduction of more model organisms for a comprehensive navigation of the space of possibilities.
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    Mitochondrial function in spinal cord injury and regeneration
    (SPRINGER BASEL AG, 2022) Slater, Paula G.; Dominguez-Romero, Miguel E.; Villarreal, Maximiliano; Eisner, Veronica; Larrain, Juan
    Many people around the world suffer from some form of paralysis caused by spinal cord injury (SCI), which has an impact on quality and life expectancy. The spinal cord is part of the central nervous system (CNS), which in mammals is unable to regenerate, and to date, there is a lack of full functional recovery therapies for SCI. These injuries start with a rapid and mechanical insult, followed by a secondary phase leading progressively to greater damage. This secondary phase can be potentially modifiable through targeted therapies. The growing literature, derived from mammalian and regenerative model studies, supports a leading role for mitochondria in every cellular response after SCI: mitochondrial dysfunction is the common event of different triggers leading to cell death, cellular metabolism regulates the immune response, mitochondrial number and localization correlate with axon regenerative capacity, while mitochondrial abundance and substrate utilization regulate neural stem progenitor cells self-renewal and differentiation. Herein, we present a comprehensive review of the cellular responses during the secondary phase of SCI, the mitochondrial contribution to each of them, as well as evidence of mitochondrial involvement in spinal cord regeneration, suggesting that a more in-depth study of mitochondrial function and regulation is needed to identify potential targets for SCI therapeutic intervention.
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    Non-canonical Wnt Signaling Induces Ubiquitination and Degradation of Syndecan
    (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010) Carvallo, Loreto; Munoz, Rosana; Bustos, Francisco; Escobedo, Noelia; Carrasco, Hector; Olivares, Gonzalo; Larrain, Juan
    Dynamic regulation of cell adhesion receptors is required for proper cell migration in embryogenesis, tissue repair, and cancer. Integrins and Syndecan4 (SDC4) are the main cell adhesion receptors involved in focal adhesion formation and are required for cell migration. SDC4 interacts biochemically and functionally with components of the Wnt pathway such as Frizzled7 and Dishevelled. Non-canonical Wnt signaling, particularly components of the planar cell polarity branch, controls cell adhesion and migration in embryogenesis and metastasic events. Here, we evaluate the effect of this pathway on SDC4. We have found that Wnt5a reduces cell surface levels and promotes ubiquitination and degradation of SDC4 in cell lines and dorsal mesodermal cells from Xenopus gastrulae. Gain-and loss-of-function experiments demonstrate that Dsh plays a key role in regulating SDC4 steady-state levels. Moreover, a SDC4 deletion construct that interacts inefficiently with Dsh is resistant to Wnt5a-induced degradation. Non-canonical Wnt signaling promotes monoubiquitination of the variable region of SDC4 cytoplasmic domain. Mutation of these specific residues abrogates ubiquitination and results in increased SDC4 steady-state levels. This is the first example of a cell surface protein ubiquitinated and degraded in a Wnt/Dsh-dependent manner.
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    Sonic hedgehog is basolaterally sorted from the TGN and transcytosed to the apical domain involving Dispatched-1 at Rab11-ARE
    (2022) Sandoval, Lisette; Labarca, Mariana; Retamal, Claudio; Sanchez, Paula; Larrain, Juan; Gonzalez, Alfonso
    Hedgehog proteins (Hhs) secretion from apical and/or basolateral domains occurs in different epithelial cells impacting development and tissue homeostasis. Palmitoylation and cholesteroylation attach Hhs to membranes, and Dispatched-1 (Disp-1) promotes their release. How these lipidated proteins are handled by the complex secretory and endocytic pathways of polarized epithelial cells remains unknown. We show that polarized Madin-Darby canine kidney cells address newly synthesized sonic hedgehog (Shh) from the TGN to the basolateral cell surface and then to the apical domain through a transcytosis pathway that includes Rab11-apical recycling endosomes (Rab11-ARE). Both palmitoylation and cholesteroylation contribute to this sorting behavior, otherwise Shh lacking these lipid modifications is secreted unpolarized. Disp-1 mediates first basolateral secretion from the TGN and then transcytosis from Rab11-ARE. At the steady state, Shh predominates apically and can be basolaterally transcytosed. This Shh trafficking provides several steps for regulation and variation in different epithelia, subordinating the apical to the basolateral secretion.
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    Spinal Cord Transection In Xenopus laevis Tadpoles
    (2021) Slater, Paula G.; Larrain, Juan
    Spinal cord injury (SCI) is a permanent affliction, which affects the central nervous system (CNS) motor and sensory nerves, resulting in paralysis beneath the injury site. To date, there is no functional recovery therapy for SCI, and there is a lack of clarity regarding the many complexes and dynamic events occurring after SCI. Many non-mammalian organisms can regenerate after severe SCI, such as teleost fishes, urodele amphibians, and larval stages of anuran amphibians, including Xenopus laevis tadpoles. These are bona fide model organisms to study and understand the response to SCI and the mechanisms underlying successful regenerative processes. This type of research can lead to the identification of potential targets for SCI therapeutic intervention. This article describes how to perform Xenopus laevis tadpole spinal cord transection, including husbandry, surgery, postsurgery care, and functional test evaluation. This injury method can be applied for elucidating the different steps of spinal cord regeneration by studying the cellular, molecular, and genetic mechanisms, as well as histological and functional evolution after SCI and during spinal cord regeneration.
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    Syndecan-1 regulates BMP signaling and dorso-ventral patterning of the ectoderm during early Xenopus development
    (ACADEMIC PRESS INC ELSEVIER SCIENCE, 2009) Olivares, Gonzalo H.; Carrasco, Hector; Aroca, Francisco; Carvallo, Loreto; Segovia, Fabian; Larrain, Juan
    Extracellular regulation of growth factor signaling is a key event for embryonic patterning. Heparan sulfate proteoglycans (HSPG) are among the molecules that regulate this signaling during embryonic development. Here we study the function of syndecan1 (Syn1), a cell-surface HSPG expressed in the non-neural ectoderm during early development of Xenopus embryos. Overexpression of Xenopus Syn1 (xSyn1) mRNA is Sufficient to reduce BMP signaling, induce chordin expression and rescue dorso-ventral patterning in ventrailized embryos. Experiments using chordin morpholinos established that xSyn1 mRNA can inhibit BM P signaling in the absence of chordin. Knockdown of xSyn1 resulted in a reduction of BMP signaling and expansion of the neural plate with the concomitant reduction of the non-neural ectoderm. Overexpression of xSyn1 mRNA in xSyn1 morphant embryos resulted in a biphasic effect, with BMP being inhibited at high concentrations and activated at low concentrations of xSyn1. Interestingly, the function of xSyn1 on dorso-ventral patterning and BMP signaling is specific for this HSPG. In summary, we report that xSyn1 regulates dorso-ventral patterning of the ectoderm through modulation of BMP signaling. (C) 2009 Elsevier Inc. All rights reserved.
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    Syndecan-4 inhibits Wnt/β-catenin signaling through regulation of low-density-lipoprotein receptor-related protein (LRP6) and R-spondin 3
    (2014) Astudillo, Pablo; Carrasco, Hector; Larrain, Juan
    Regulation of Wnt signaling is crucial for embryonic development and adult homeostasis. Here we study the role of Syndecan-4 (SDC4), a cell-surface heparan sulphate proteoglycan, and Fibronectin (FN), in Wnt/beta-catenin signaling. Gain- and loss-of-function experiments in mammalian cell lines and Xenopus embryos demonstrate that SDC4 and FN inhibit Wnt/beta-catenin signaling. Epistatic and biochemical experiments show that this inhibition occurs at the cell membrane level through regulation of LRP6. R-spondin 3, a ligand that promotes canonical and non-canonical Wnt signaling, is more prone to potentiate Wnt/beta-catenin signaling when SDC4 levels are reduced, suggesting a model whereby SDC4 tunes the ability of R-spondin to modulate the different Wnt signaling pathways. Since SDC4 has been previously related to non-canonical Wnt signaling, our results also suggest that this proteoglycan can be a key component in the regulation of Wnt signaling. (C) 2013 Elsevier Ltd. All rights reserved.
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    Transcriptome analysis of the response to thyroid hormone in Xenopus neural stem and progenitor cells
    (2023) Cordero-Veliz, Camila; Larrain, Juan; Faunes, Fernando
    Background The thyroid hormones-thyroxine (T4) and 3,5,3 ' triiodothyronine (T3)-regulate the development of the central nervous system (CNS) in vertebrates by acting in different cell types. Although several T3 target genes have been identified in the brain, the changes in the transcriptome in response to T3 specifically in neural stem and progenitor cells (NSPCs) during the early steps of NSPCs activation and neurogenesis have not been studied in vivo. Here, we characterized the transcriptome of FACS-sorted NSPCs in response to T3 during Xenopus laevis metamorphosis. Results We identified 1252 upregulated and 726 downregulated genes after 16 hours of T3 exposure. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that T3-upregulated genes were significantly enriched in rRNA processing and maturation, protein folding, ribosome biogenesis, translation, mitochondrial function, and proteasome. These results suggest that NSPCs activation induced by T3 is characterized by an early proteome remodeling through the synthesis of the translation machinery and the degradation of proteins by the proteasome. Conclusion This work provides new insights into the dynamics of activation of NPSCs in vivo in response to T3 during a critical period of neurogenesis in the metamorphosis.