Héctor Carrasco

Early requirement of Hyaluronan for tail regeneration in Xenopus tadpoles

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 GSK3β was sufficient to rescue proliferation and tail regeneration when HA synthesis was blocked, suggesting that GSK3β 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 GSK3β signaling during tail regeneration was demonstrated.

Non-canonical Wnt Signaling Induces Ubiquitination and Degradation of Syndecan4

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.

Syndecan-1 regulates BMP signaling and dorso-ventral patterning of the ectoderm during early Xenopus development

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 ventralized embryos. Experiments using chordin morpholinos established that xSyn1 mRNA can inhibit BMP 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.

Syndecan 4 interacts genetically with Vangl2 to regulate neural tube closure and planar cell polarity

Syndecan 4 (Sdc4) is a cell-surface heparan sulfate proteoglycan (HSPG) that regulates gastrulation, neural tube closure and directed neural crest migration in Xenopus development. To determine whether Sdc4 participates in Wnt/PCP signaling during mouse development, we evaluated a possible interaction between a null mutation of Sdc4 and the loop-tail allele of Vangl2. Sdc4 is expressed in multiple tissues, but particularly in the non-neural ectoderm, hindgut and otic vesicles. Sdc4;Vangl2Lp compound mutant mice have defective spinal neural tube closure, disrupted orientation of the stereocilia bundles in the cochlea and delayed wound healing, demonstrating a strong genetic interaction. In Xenopus, co-injection of suboptimal amounts of Sdc4 and Vangl2 morpholinos resulted in a significantly greater proportion of embryos with defective neural tube closure than each individual morpholino alone. To probe the mechanism of this interaction, we overexpressed or knocked down Vangl2 function in HEK293 cells. The Sdc4 and Vangl2 proteins colocalize, and Vangl2, particularly the Vangl2Lp mutant form, diminishes Sdc4 protein levels. Conversely, Vangl2 knockdown enhances Sdc4 protein levels. Overall HSPG steady-state levels were regulated by Vangl2, suggesting a molecular mechanism for the genetic interaction in which Vangl2Lp/+ enhances the Sdc4-null phenotype. This could be mediated via heparan sulfate residues, as Vangl2Lp/+ embryos fail to initiate neural tube closure and develop craniorachischisis (usually seen only in Vangl2Lp/Lp) when cultured in the presence of chlorate, a sulfation inhibitor. These results demonstrate that Sdc4 can participate in the Wnt/PCP pathway, unveiling its importance during neural tube closure in mammalian embryos.

Heparan sulfate proteoglycans exert positive and negative effects in Shh activity

Hedgehog (Hh) proteins are morphogens involved in short‐ and long‐range effects during early embryonic development. Genetic analysis in fly and vertebrate embryos showed that heparan sulfate proteoglycans (HSPGs) are required for Hh transport and signaling. To further understand how HSPGs regulate Sonic hedgehog (Shh), we performed experiments using cell culture and biochemical assays. When the synthesis of HSPGs was reduced, a decrease in Shh activity was observed. Contrary to that, addition of a peptide that competes the binding of Shh to HSPGs resulted in augmentation of Shh activity. From these results, we concluded that HSPGs exert positive and negative effects in Shh activity. This dual effect correlates with the finding that Shh interacts preferentially with two HSPGs. The current model for the role of HSPGs in Shh diffusion is discussed in view of our findings.

Syndecan-4 inhibits Wnt/β-catenin signaling through regulation of low-density-lipoprotein receptor-related protein (LRP6) and R-spondin 3.

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/β-catenin signaling. Gain- and loss-of-function experiments in mammalian cell lines and Xenopus embryos demonstrate that SDC4 and FN inhibit Wnt/β-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/β-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.

SHh activity and localization is regulated by perlecan

Proliferation and cell fate determination in the developing embryo are extrinsically regulated by multiple interactions among diverse secreted factors, such as Sonic Hedgehog (SHh), which act in a concentration-dependent manner. The fact that SHh is secreted as a lipid-modified protein suggests the existence of a mechanism to regulate its movement across embryonic fields. We have previously shown that heparan sulfate proteoglycans (HSPGs) are required for SHh binding and signalling. However, it was not determined which specific HSPG was responsible for these functions. Here we evaluated the contribution of perlecan on SHh localization and activity. To understand the mechanism of action of perlecan at the cellular level, we studied the role of perlecan-SHh interaction in SHh activity using both cell culture and biochemical assays. Our findings show that perlecan is a crucial anchor and modulator of SHh activity acting as an extracellular positive regulator of SHh.