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The neural crest (NC) is a transient embryonic cell population that migrates extensively during development. Ric-8A, a guanine nucleotide exchange factor (GEF) for different Gα subunits regulates cranial NC (CNC) cell migration in Xenopus through a mechanism that still remains to be elucidated. To properly migrate, CNC cells establish an axis of polarization and undergo morphological changes to generate protrusions at the leading edge and retraction of the cell rear. Here, we aim to study the role of Ric-8A in cell polarity during CNC cell migration by examining whether its signaling affects the localization of GTPase activity in Xenopus CNC using GTPase-based probes in live cells and aPKC and Par3 as polarity markers. We show that the levels of Ric-8A are critical during migration and affect the localization of polarity markers and the subcellular localization of GTPase activity, suggesting that Ric-8A, probably through heterotrimeric G-protein signaling, regulates cell polarity during CNC migration.

© The Author(s) 2020., The phytohormone jasmonoyl-isoleucine (JA-Ile) regulates fundamental plant processes as developmental and defense responses. JA-Ile mediates the interaction between the F-box protein COI1 (part of the SCF E3 ubiquitin ligase) and a JAZ repressor leading to early jasmonate responses. The Arabidopsis JAZ1 protein contains the canonical LPIARR degron sequence, which is responsible for the stabilization of the AtCOI1-JA-Ile-AtJAZ1 complex. In strawberry (Fragaria × ananassa) JAZ family was described at the transcriptional level during fruit development but the information about the interaction mode of this complex is still scarce at the molecular level. To gain insight into the strawberry JA-Ile receptor complex, we evaluated the interaction at the structural level, and protein models were built and analyzed for FaCOI1 and FaJAZ1, FaJAZ8.1, and FaJAZ10. The interaction between FaCOI1 and FaJAZ1, FaJAZ8.1 and FaJAZ10 were explored using several ligands, through molecular docking and molecular dynamics (MD) simulations, finding the strongest interaction with (+)-7-iso-JA-Ile than other ligands. Additionally, we tested interactions between FaCOI1 and FaJAZs by yeast two-hybrid assays in the presence of coronatine (COR, a JA-Ile mimic). We detected strong COR-dependent interactions between FaCOI1 and FaJAZ1. Interestingly, FaJAZ1 contains a new non-canonical (IPMQRK) functional degron sequence, in which Arg and Lys are the key residues for maintaining the interaction of the FaCOI1–COR–FaJAZ1 complex as we observed in mutated versions of the FaJAZ1 degron. Phylogenetic analysis showed that the IPMQRK degron is only present in orthologs belonging to the Rosoideae but not in other Rosaceae subfamilies. Together, this study uncovers a new degron sequence in plants, which could be required to make an alternative and functional JA-Ile perception complex in strawberry., This research was funded by the National Research and Development Agency (ANID, Chile), grant FONDECYT/Regular 1181310 to Carlos R. Figueroa. Adrián Garrido-Bigotes acknowledges ANID (grant FONDECYT/Postdoctorado 3190894). F.V-R acknowledges Universidad de Talca for a doctoral scholarship. Work in R. Solano’s laboratory was funded by the Spanish Ministry of Economy and Competitivity grant BIO2016-77216-R (MINECO/FEDER).

Diverse chromatin modifiers are involved in regulation of gene expression at the level of transcriptional regulation. Among these modifiers are ATP-dependent chromatin remodelers, where the SWI/SNF complex is the founding member. It has been observed that High Mobility Group (HMG) proteins can influence the activity of a number of these chromatin remodelers. In this context, we have previously demonstrated that the yeast HMG proteins Nhp6 and Hmo1 can stimulate SWI/SNF activity. Here, we studied the genome-wide binding patterns of Nhp6, Hmo1 and the SWI/SNF complex, finding that most of gene promoters presenting high occupancy of this complex also display high enrichment of these HMG proteins. Using deletion mutant strains we demonstrate that binding of SWI/SNF is significantly reduced at numerous genomic locations by deletion of NHP6 and/or deletion of HMO1. Moreover, alterations in the nucleosome landscape take place at gene promoters undergoing reduced SWI/SNF binding. Additional analyses show that these effects also correlate with alterations in transcriptional activity. Our results suggest that, besides the ability to stimulate SWI/SNF activity, these HMG proteins are able to assist the loading of this complex onto gene regulatory regions.

Heterotrimeric G protein signaling plays major roles during different cellular events. However, there is a limited understanding of the molecular mechanisms underlying G protein control during embryogenesis. G proteins are highly conserved and can be grouped into four subfamilies according to sequence homology and function. To further studies on G protein function during embryogenesis, the present analysis identified four Gα subunits representative of the different subfamilies and determined their spatiotemporal expression patterns during Xenopus tropicalis embryogenesis. Each of the Gα subunit transcripts was maternally and zygotically expressed, and, as development progressed, dynamic expression patterns were observed. In the early developmental stages, the Gα subunits were expressed in the animal hemisphere and dorsal marginal zone. While expression was observed at the somite boundaries, in vascular structures, in the eye, and in the otic vesicle during the later stages, expression was mainly found in neural tissues, such as the neural tube and, especially, in the cephalic vesicles, neural crest region, and neural crest-derived structures. Together, these results support the pleiotropism and complexity of G protein subfamily functions in different cellular events. The present study constitutes the most comprehensive description to date of the spatiotemporal expression patterns of Gα subunits during vertebrate development.

Proper regulation of gene expression is essential for normal development, cellular growth, and differentiation. Differential expression profiles of mRNA coding for vertebrate Ric-8B during embryo and adult stages have been observed. In addition, Ric-8B is expressed in few cerebral nuclei subareas. These facts point to a dynamic control of RIC8B gene expression. In order to understand the transcriptional regulation of this gene, we searched for cis-elements in the sequence of the human RIC8B promoter region, identifying binding sites for the basic/leucine zipper (bZip) CREB transcription factor family (CRE sites) and C/EBP transcription factor family (C/EBP sites). CRE sites were found clustered near the transcription start site, while the C/EBP sites were found clustered at around 300 bp upstream the CRE sites. Here, we demonstrate the ability of CREB1 and C/EBPβ to bind their respective elements identified in the RIC8B promoter. Comparative protein-DNA interaction analyses revealed only the proximal elements as high affinity sites for CREB1 and only the distal elements as high affinity sites for C/EBPβ. Chromatin immunoprecipitation analyses, carried out using a human neuroblastoma cell line, confirmed the preferential association of CREB to the proximal region of the RIC8B promoter. By performing luciferase reporter assays, we found the CRE sites as the most relevant elements for its transcriptional activity. Taken together, these data show the existence of functional CREB and C/EBP binding sites in the human RIC8B gene promoter, a particular distribution of these sites and demonstrate a relevant role of CREB in stimulating transcriptional activity of this gene. J. Cell. Biochem. 117: 1797-1805, 2016. © 2016 Wiley Periodicals, Inc.

Ric-8A is a pleiotropic guanine nucleotide exchange factor involved the activation of various heterotrimeric G protein pathways during adulthood and early development. Here, we sought to determine the downstream effectors of Ric-8A during the migration of the vertebrate cranial neural crest (NC) cells. We show that the Gα13 knockdown phenocopies the Ric-8A morphant condition, causing actin cytoskeleton alteration, protrusion instability and a strong reduction in the number and dynamics of focal adhesions. In addition, the overexpression of Gα13 is sufficient to rescue Ric-8A depleted cells. Ric-8A and Gα13 physically interact and co-localize in protrusions of the cells leading edge. The focal adhesion kinase FAK co-localizes and interacts with the endogenous Gα13, and a constitutively active form of Src efficiently rescues the Gα13 morphant phenotype in NC cells. We propose that Ric-8A-mediated Gα13 signaling is required for proper cranial NC cell migration by regulating focal adhesion dynamics and protrusion formation.

The neural crest (NC) is a transient embryonic structure induced at the border of the neural plate. NC cells extensively migrate towards diverse regions of the embryo, where they differentiate into various derivatives, including most of the craniofacial skeleton and the peripheral nervous system. The Ric-8A protein acts as a guanine nucleotide exchange factor for several Gα subunits, and thus behaves as an activator of signaling pathways mediated by heterotrimeric G proteins. Using in vivo transplantation assays, we demonstrate that Ric-8A levels are critical for the migration of cranial NC cells and their subsequent differentiation into craniofacial cartilage during Xenopus development. NC cells explanted from Ric-8A morphant embryos are unable to migrate directionally towards a source of the Sdf1 peptide, a potent chemoattractant for NC cells. Consistently, Ric-8A knock-down showed anomalous radial migratory behavior, displaying a strong reduction in cell spreading and focal adhesion formation. We further show that during in vivo and in vitro neural crest migration, Ric-8A localizes to the cell membrane, in agreement with its role as a G protein activator. We propose that Ric-8A plays essential roles during the migration of cranial NC cells, possibly by regulating cell adhesion and spreading.

The emergence of vertebrates is closely associated to the evolution of mineralized bone tissue. However, the molecular basis underlying the origin and subsequent diversification of the skeletal mineralized matrixisstillpoorlyunderstood. Oneefficientway totackle thisissueis tocomparethe expression, between vertebrate species, of osteoblastic genes coding for bone matrix proteins. In this work, wehave focusedon theevolution ofthenetwork formingcollagen familywhichcontains the Col8a1, Col8a2, and Col10a1 genes. Both phylogeny and synteny reveal that these three paralogues are vertebrate-specific and derive from two independent duplications in the vertebrate lineage. To shed light on the evolution of this family, we have analyzed the osteoblastic expression of the network forming collagens in endochondral and intramembraneous skeletal elements of the amphibian Xenopus tropicalis. Remarkably, we find that amphibian osteoblasts express Col10a1 ,a gene strongly expressed in osteoblasts in actinopterygians but not in amniotes. In addition, while Col8a1 is known to be robustly expressed in mammalian osteoblasts, the expression levels of its amphibian orthologue are dramatically reduced. Our work reveals that while a skeletal expression of network forming collagen members is widespread throughout vertebrates, osteoblasts from divergent vertebrate lineages express different combinations of network forming collagen paralogues. J. Exp. Zool. (Mol. Dev. Evol.) 9999B: 1-11, 2013. © 2013 Wiley Periodicals, Inc. How to cite this article: Aldea D, Hanna P, Munoz D, Espinoza J, Torrejon M, Sachs L, Buisine N, Oulion S, Escriva H, Marcellini S. 2013. Evolution of the vertebrate bone matrix: An expression analysisofthenetworkformingcollagenparaloguesinamphibianosteoblasts.J.Exp.Zool.(Mol. Dev. Evol.) 9999:1-11.

Extracellular matrix (ECM) molecules are pivotal for central nervous system development, facilitating cell migration, axonal growth, myelination, dendritic spine formation, and synaptic plasticity, among other processes. During axonal guidance, the ECM not only acts as a permissive or non-permissive substrate for navigating axons, but also modulates the effects of classical guidance cues, such as netrin or Eph/ephrin family members. Despite being highly important, little is known about the expression of ECM molecules during central nervous system development. Therefore, this study assessed the molecular expression patterns of tenascin, HNK-1, laminin, fibronectin, perlecan, decorin, and osteopontin along chick embryo prosomere 1 during posterior commissure development. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. Located in the dorso-caudal portion of prosomere 1, posterior commissure axons primarily arise from the neurons of basal pretectal nuclei that run dorsally to the roof plate midline, where some turn towards the ipsilateral side. Expressional analysis of ECM molecules in this area these revealed to be highly arranged, and molecule interactions with axon fascicles suggested involvement in processes other than structural support. In particular, tenascin and the HNK-1 epitope extended in ventro-dorsal columns and enclosed axons during navigation to the roof plate. Laminin and osteopontin were expressed in the midline, very close to axons that at this point must decide between extending to the contralateral side or turning to the ipsilateral side. Finally, fibronectin, decorin, and perlecan appeared unrelated to axonal pathfinding in this region and were instead restricted to the external limiting membrane. In summary, the present report provides evidence for an intricate expression of different extracellular molecules that may cooperate in guiding posterior commissure axons.

Proper regulation of gene expression is essential for normal development, cellular growth, and differentiation. Differential expression profiles of mRNA coding for vertebrate Ric-8B during embryo and adult stages have been observed. In addition, Ric-8B is expressed in few cerebral nuclei subareas. These facts point to a dynamic control of RIC8B gene expression. In order to understand the transcriptional regulation of this gene, we searched for cis-elements in the sequence of the human RIC8B promoter region, identifying binding sites for the basic/leucine zipper (bZip) CREB transcription factor family (CRE sites) and C/EBP transcription factor family (C/EBP sites). CRE sites were found clustered near the transcription start site, while the C/EBP sites were found clustered at around 300 bp upstream the CRE sites. Here, we demonstrate the ability of CREB1 and C/EBPβ to bind their respective elements identified in the RIC8B promoter. Comparative protein-DNA interaction analyses revealed only the proximal elements as high affinity sites for CREB1 and only the distal elements as high affinity sites for C/EBPβ. Chromatin immunoprecipitation analyses, carried out using a human neuroblastoma cell line, confirmed the preferential association of CREB to the proximal region of the RIC8B promoter. By performing luciferase reporter assays, we found the CRE sites as the most relevant elements for its transcriptional activity. Taken together, these data show the existence of functional CREB and C/EBP binding sites in the human RIC8B gene promoter, a particular distribution of these sites and demonstrate a relevant role of CREB in stimulating transcriptional activity of this gene. J. Cell. Biochem. 117: 1797-1805, 2016. © 2016 Wiley Periodicals, Inc.

The origin of bone and cartilage, and their subsequent diversification in specific vertebrate lineages, is intimately linked to the precise transcriptional control of genes involved in matrix mineralization. It is not yet clear, however, to which extent the osteoblasts, osteocytes, and chondrocytes of each of the major vertebrate groups express similar sets of genes. In this study we have focused on the evolution of two independent families of genes that code for extracellular matrix components of the skeleton and that include secreted protein, acidic, cysteine-rich (SPARC), bone sialoprotein (BSP) and dentin matrix protein 1 (DMP1) paralogues, and the osteocalcin (OC) and matrix gla protein (MGP) paralogues. Analyzing developing Xenopus tropicalis skeletal elements, we show that the expression patterns of these genes are well conserved with mammals. The fact that only a few osteoblasts express DMP1, while only some osteocytes express SPARC and BSP, reveals a significant degree of molecular heterogeneity for these two populations of X. tropicalis cells, similarly to what has been described in mouse. Although the cis-regulatory modules (CRM) of the mammalian OC, DMP1, and BSP orthologs have been functionally characterized, we found no evidence of sequence similarity between these regions and the X. tropicalis genome. Furthermore, these regulatory elements evolve rapidly, as they are only poorly conserved between human and rodents. Therefore, the SPARC/DMP1/BSP and the OC/MGP families provide a good paradigm to study how transcriptional output can be maintained in skeletal cells despite extensive sequence divergence of CRM.

Immature stage VI Xenopus oocytes are arrested at the G2/M border of meiosis I until exposed to progesterone, which induces meiotic resumption through a non-genomic mechanism. One of the earliest events produced by this hormone is inhibition of the plasma membrane enzyme adenylyl cyclase (AC), with the concomitant drop in intracellular cAMP levels and reinitiation of the cell cycle. Recently Gsα and Gβγ have been shown to play an important role as positive regulators of Xenopus oocyte AC, maintaining the oocyte in the arrested state. However, a question that still remains unanswered, is how the activated state of Gsα and Gβγ is achieved in the immature oocyte, since no receptor or ligand have been found to be required. Here we provide evidence that xRic-8 can act in vitro and in vivo as a GEF for Gsα. Overexpression of xRic-8, through mRNA injection, greatly inhibits progesterone induced oocyte maturation and endogenous xRic-8 mRNA depletion, through siRNA microinjection, induces spontaneous oocyte maturation. These results suggest that xRic-8 is participating in the immature oocyte by keeping Gsα-Gβγ-AC signaling complex in an activated state and therefore maintaining G2 arrest. J. Cell. Physiol. 214: 673–680, 2008. © 2007 Wiley-Liss, Inc.

During early stages of development, encephalic vesicles are composed by a layer of neuroepithelial cells surrounding a central cavity filled with embryonic cerebrospinal fluid (eCSF). This fluid contains several morphogens that regulate proliferation and differentiation of neuroepithelial cells. One of these neurogenic factors is SCO-spondin, a giant protein secreted to the eCSF from early stages of development. Inhibition of this protein in vivo or in vitro drastically decreases the neurodifferentiation process. Other important neurogenic factors of the eCSF are low density lipoproteins (LDL), the depletion of which generates a 60% decrease in mesencephalic explant neurodifferentiation. The presence of several LDL receptor class A (LDLrA) domains (responsible for LDL binding in other proteins) in the SCO-spondin sequence suggests a possible interaction between both molecules. This possibility was analyzed using three different experimental approaches: 1) Bioinformatics analyses of the SCO-spondin region, that contains eight LDLrA domains in tandem, and of comparisons with the LDL receptor consensus sequence; 2) Analysis of the physical interactions of both molecules through immunohistochemical colocalization in embryonic chick brains and through the immunoprecipitation of LDL with anti-SCO-spondin antibodies; and 3) Analysis of functional interactions during the neurodifferentiation process when these molecules were added to a culture medium of mesencephalic explants. The results revealed that LDL and SCO-spondin interact to form a complex that diminishes the neurogenic capacities that both molecules have separately. Our work suggests that the embryonic cerebrospinal fluid is an active signaling center with a complex regulation system that allows for correct brain development.

A coupled finite/boundary element method to approximate the free vibration modes of an elastic structure containing an incompressible fluid is analyzed in this paper. The effect of the fluid is taken into account by means of one of the most usual procedures in engineering practice: an added mass formulation, which is posed in terms of boundary integral equations. Piecewise linear continuous elements are used to discretize the solid displacements and the fluid-solid interface variables. Spectral convergence is proved and error estimates are settled for the approximate eigenfunctions and their corresponding vibration frequencies. Implementation issues are also discussed and numerical experiments are reported.

Bilaterally symmetric organisms need to exchange information between the two sides of their bodies in order to integrate sensory inputs and coordinate motor control. This exchange occurs through commissures formed by neurons that project axons across the midline to the contralateral side of the central nervous system. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. It is located in the dorsal portion of the prosomere 1, at the caudal diencephalon. The axons of the posterior commissure principally come from neurons of ventrolateral and dorsolateral pretectal nuclei (parvocellular and magnocellular nucleus of the posterior commissure, respectively) that extend their axons toward the dorsal region. The trajectory of these axons can be divided into the following three stages: (1) dorsal axon extension towards the lateral roof plate; (2) fasciculation in the lateral roof plate; and (3) midline decision of turning to the ipsilateral side or continuing to the opposite side. The mechanisms and molecules that guide the axons during these steps are unknown. In the present work, immunohistochemical and in situ hybridization analyses were performed, with results suggesting the participation of EphA7 in guiding axons from the ventral to the dorsal region of the prosomere 1 through the generation of an axonal corridor limited by repulsive EphA7 walls. At the lateral roof plate, the axons became fasciculated in presence of SCO-spondin until reaching the midline. Finally, EphA7 expression was observed in the diencephalic midline roof plate, specifically in the region where some axons turn to the ipsilateral side, suggesting its participation in this decision. In summary, the present work proposes a mechanism of posterior commissure formation orchestrated by the complementary expression of the axon guidance cues SCO-spondin and EphA7.

The Galpha subunits of heterotrimeric G proteins are constituted by a conserved GTPase "Ras-like" domain (RasD) and by a unique alpha-helical domain (HD). Upon GTP binding, four regions, called switch I, II, III, and IV, have been identified as undergoing structural changes. Switch I, II, and III are located in RasD and switch IV in HD. All Galpha known functions, such as GTPase activity and receptor, effector, and Gbetagamma interaction sites have been found to be localized in RasD, but little is known about the role of HD and its switch IV region. Through the construction of chimeras between human and Xenopus Gsalpha we have previously identified a HD region, encompassing helices alphaA, alphaB, and alphaC, that was responsible for the observed functional differences in their capacity to activate adenylyl cyclase (Antonelli et al. [1994]: FEBS Lett 340:249-254). Since switch IV is located within this region and contains most of the nonconservative amino acid differences between both Gsalpha proteins, in the present work we constructed two human Gsalpha mutant proteins in which we have changed four and five switch IV residues for the ones present in the Xenopus protein. Mutants M15 (hGsalphaalphaS133N, M135P, P138K, P143S) and M17 (hGsalphaalphaS133N, M135P, V137Y, P138K, P143S) were expressed in Escherichia coli, purified, and characterized by their ability to bind GTPgammaS, dissociate GDP, hydrolyze GTP, and activate adenylyl cyclase. A decreased rate of GDP release, GTPgammaS binding, and GTP hydrolysis was observed for both mutants, M17 having considerably slower kinetics than M15 for all functions tested. Reconstituted adenylyl cyclase activity with both mutants showed normal activation in the presence of AlF(4)(-), but a decreased activation with GTPgammaS, which is consistent with the lower GDP dissociating rate they displayed. These data provide new evidence on the role that HD is playing in modulating the GDP/GTP exchange of the Gsalpha subunit.

Using the yeast two-hybrid system, we studied the physical interaction between the complete C1 and C2 cytosolic domains of Xenopus laevis type 9 (xl9C1, xl9C2) and the C2 domain of rat type 6 (r6C2) adenylyl cyclase (AC). Heterodimerization between xl9C1 and xl9C2 and homodimerization between C2 (but not C1) domains was observed. Interaction between C2 and human G alpha s (hG alpha s) was also detected and was dependent on G alpha s activation. In contrast X. laevis G alpha s (xlG alpha s), which is 92% identical to hG alpha s, was unable to interact with any of the three AC cytosolic domains tested, corroborating previous findings that showed no effector activation. Through the construction of chimeras, we demonstrated that the amino-terminal half of xlG alpha s was responsible for the lack of interaction with AC. Chimeras between mouse G alpha i2 and G alpha s (N-mG alpha i2/C-G alpha s), that have previously shown to activate AC to a higher extent than wild-type G alpha s, also interacted with the C2 cytosolic domain and with a higher affinity. Interestingly, N-mG alpha i2/C-xlG alpha s chimera was not only able to interact with C2 but also with the C1 cytosolic domain.

We have cloned a cDNA that encodes a novel Xenopus laevis oocyte adenylyl cyclase (xlAC) using oligonucleotides against conserved mammalian adenylyl cyclase regions. The isolated cDNA is 4372 bp long with an open reading frame of 4065 nucleotides which encodes a protein of 1355 amino acids. Comparison of the deduced amino acid sequence with previously cloned mammalian adenylyl cyclases shows a low identity, 19.7% with type 2 rat adenylyl cyclase and 24.2% with type 4 rat adenylyl cyclase, indicating that this Xenopus isoform represents a new member of this protein family. Gene expression studies of the xlAC by reverse PCR showed that this gene is expressed in all oogenesis stages but not during early embryogenesis. Expression of the xlAC in COS-7 cells resulted in increased basal AC activity, that was stimulated by forskolin, Gpp(NH)p and aluminium fluoride, and was insensitive to calcium and calcium-calmodulin (Ca2(+)-CaM).

The central nervous system (CNS) develops from the neural tube, a hollow structure filled with embryonic cerebrospinal fluid (eCSF) and surrounded by neuroepithelial cells. Several lines of evidence suggest that the eCSF contains diffusible factors regulating the survival, proliferation, and differentiation of the neuroepithelium, although these factors are only beginning to be uncovered. One possible candidate as eCSF morphogenetic molecule is SCO-spondin, a large glycoprotein whose secretion by the diencephalic roof plate starts at early developmental stages. In vitro, SCO-spondin promotes neuronal survival and differentiation, but its in vivo function still remains to be elucidated. Here we performed in vivo loss of function experiments for SCO-spondin during early brain development by injecting and electroporating a specific shRNA expression vector into the neural tube of chick embryos. We show that SCO-spondin knock down induces an increase in neuroepithelial cells proliferation concomitantly with a decrease in cellular differentiation toward neuronal lineages, leading to hyperplasia in both the diencephalon and the mesencephalon. In addition, SCO-spondin is required for the correct morphogenesis of the posterior commissure and pineal gland. Because SCO-spondin is secreted by the diencephalon, we sought to corroborate the long-range function of this protein in vitro by performing gain and loss of function experiments on mesencephalic explants. We find that culture medium enriched in SCO-spondin causes an increased neurodifferentiation of explanted mesencephalic region. Conversely, inhibitory antibodies against SCO-spondin cause a reduction in neurodifferentiation and an increase of mitosis when such explants are cultured in eCSF. Our results suggest that SCO-spondin is a crucial eCSF diffusible factor regulating the balance between proliferation and differentiation of the brain neuroepithelial cells.

Signaling via heterotrimeric G-proteins is evoked by agonist-mediated stimulation of seven transmembrane spanning receptors (GPCRs). During the last decade it has become apparent that Gα subunits can be activated by receptor-independent mechanisms. Ric-8 belongs to a highly conserved protein family that regulates heterotrimeric G-protein function, acting as a non-canonical guanine nucleotide exchange factors (GEF) over a subset of Gα subunits. In this review we discuss the roles of Ric-8 in the regulation of diverse cell functions, emphasizing the contribution of its multiple domain protein structure in these diverse functions. J. Cell. Biochem. 113: 2797–2805, 2012. © 2012 Wiley Periodicals, Inc.

RIC-8 is a highly conserved protein that promotes G protein signaling as it acts as a Guanine nucleotide Exchanging Factor (GEF) over a subset of Gα subunits. In invertebrates, RIC-8 plays crucial roles in synaptic transmission as well as in asymmetric cell division. As a first step to address further studies on RIC-8 function in vertebrates, here we have cloned a ric-8 gene from Xenopus tropicalis (xtric-8) and determined its spatiotemporal expression pattern throughout embryogenesis. The xtric-8 transcript is expressed maternally and zygotically and, as development proceeds, it shows a dynamic expression pattern. At early developmental stages, xtric-8 is expressed in the animal hemisphere, whereas its expression is later restricted to neural tissues, such as the neural tube and the brain, as well as in the eye and neural crest-derived structures, including those of the craniofacial region. Together, our findings suggest that RIC-8 functions are related to the development of the nervous system.

The non-canonical Wnt/Ca2+ signaling pathway has been implicated in the regulation of axis formation and gastrulation movements during early Xenopus laevis embryo development, by antagonizing the canonical Wnt/β-catenin dorsalizing pathway and specifying ventral cell fate. However, the molecular mechanisms involved in this antagonist crosstalk are not known. Since Gαq is the main regulator of Ca2+ signaling in vertebrates and from this perspective probably involved in the events elicited by the non-canonical Wnt/Ca2+ pathway, we decided to study the effect of wild-type Xenopus Gq (xGαq) in dorso-ventral axis embryo patterning. Overexpression of xGαq or its endogenous activation at the dorsal animal region of Xenopus embryo both induced a strong ventralized phenotype and inhibited the expression of dorsal-specific mesoderm markers goosecoid and chordin. Dorsal expression of an xGαq dominant-negative mutant reverted the xGαq-induced ventralized phenotype. Finally, we observed that the Wnt8-induced secondary axis formation is reverted by endogenous xGαq activation, indicating that it is negatively regulating the Wnt/β-catenin pathway. J. Cell. Physiol. 214: 483–490, 2008. © 2007 Wiley-Liss, Inc.

The vertebrates share the ability to produce a skeleton made of mineralized extracellular matrix. However, our understanding of the molecular changes that accompanied their emergence remains scarce. Here, we describe the evolutionary history of the SPARC (secreted protein acidic and rich in cysteine) family, because its vertebrate orthologues are expressed in cartilage, bones and teeth where they have been proposed to bind calcium and act as extracellular collagen chaperones, and because further duplications of specific SPARC members produced the small calcium-binding phosphoproteins (SCPP) family that is crucial for skeletal mineralization to occur. Both phylogeny and synteny conservation analyses reveal that, in the eumetazoan ancestor, a unique ancestral gene duplicated to give rise toSPARCandSPARCBdescribed here for the first time. Independent losses have eliminated one of the two paralogues in cnidarians, protostomes and tetrapods. Hence, only non-tetrapod deuterostomes have conserved both genes. Remarkably,SPARCandSPARCBparalogues are still linked in the amphioxus genome. To shed light on the evolution of the SPARC family members in chordates, we performed a comprehensive analysis of their embryonic expression patterns in amphioxus, tunicates, teleosts, amphibians and mammals. Our results show that in the chordate lineage SPARC and SPARCB family members were recurrently recruited in a variety of unrelated tissues expressing collagen genes. We propose that one of the earliest steps of skeletal evolution involved the co-expression of SPARC paralogues with collagenous proteins.