Search

The purpose of this clinical trial on possible effects of nocturnal MEL administration in perimenopausal women was to find if MEL by itself modifies levels of hormones and produces changes [...]

Glial cells provide physical and chemical support and protection for neurons and for the extracellular compartments of neural tissue through secretion of soluble factors, insoluble scaffolds, and vesicles. Additionally, glial cells have regenerative capacity by remodeling their physical microenvironment and changing physiological properties of diverse cell types in their proximity. Various types of aberrant glial and macrophage cells are associated with human diseases, disorders, and malignancy. We previously demonstrated that transmembrane protein, TMEM230 has tissue revascularization and regenerating capacity by its ability to secrete pro-angiogenic factors and metalloproteinases, inducing endothelial cell sprouting and channel formation. In healthy normal neural tissue, TMEM230 is predominantly expressed in glial and marcophate cells, suggesting a prominent role in neural tissue homeostasis. TMEM230 regulation of the endomembrane system was supported by co-expression with RNASET2 (lysosome, mitochondria, and vesicles) and STEAP family members (Golgi complex). Intracellular trafficking and extracellular secretion of glial cellular components are associated with endocytosis, exocytosis and phagocytosis mediated by motor proteins. Trafficked components include metalloproteins, metalloproteinases, glycans, and glycoconjugate processing and digesting enzymes that function in phagosomes and vesicles to regulate normal neural tissue microenvironment, homeostasis, stress response, and repair following neural tissue injury or degeneration. Aberrantly high sustained levels TMEM230 promotes metalloprotein expression, trafficking and secretion which contribute to tumor associated infiltration and hypervascularization of high tumor grade gliomas. Following injury of the central nervous or peripheral systems, transcient regulated upregulation of TMEM230 promotes tissue wound healing, remodeling and revascularization by activating glial and macrophage generated microchannels/microtubules (referred to as vascular mimicry) and blood vessel sprouting and branching. Our results support that TMEM230 may act as a master regulator of motor protein mediated trafficking and compartmentalization of a large class of metalloproteins in gliomas and gliosis., (Copyright © 2024. Published by Elsevier Inc.)

Glioblastomas (GBM) are the most aggressive tumors originating in the brain. Histopathologic features include circuitous, disorganized, and highly permeable blood vessels with intermittent blood flow. These features contribute to the inability to direct therapeutic agents to tumor cells. Known targets for anti-angiogenic therapies provide minimal or no effect in overall survival of 12-15 months following diagnosis. Identification of novel targets therefore remains an important goal for effective treatment of highly vascularized tumors such as GBM. We previously demonstrated in zebrafish that a balanced level of expression of the transmembrane protein TMEM230/C20ORF30 was required to maintain normal blood vessel structural integrity and promote proper vessel network formation. To investigate whether TMEM230 has a role in the pathogenesis of GBM, we analyzed its prognostic value in patient tumor gene expression datasets and performed cell functional analysis. TMEM230 was found necessary for growth of U87-MG cells, a model of human GBM. Downregulation of TMEM230 resulted in loss of U87 migration, substratum adhesion, and re-passaging capacity. Conditioned media from U87 expressing endogenous TMEM230 induced sprouting and tubule-like structure formation of HUVECs. Moreover, TMEM230 promoted vascular mimicry-like behavior of U87 cells. Gene expression analysis of 702 patients identified that TMEM230 expression levels distinguished high from low grade gliomas. Transcriptomic analysis of patients with gliomas revealed molecular pathways consistent with properties observed in U87 cell assays. Within low grade gliomas, elevated TMEM230 expression levels correlated with reduced overall survival independent from tumor subtype. Highest level of TMEM230 correlated with glioblastoma and ATP-dependent microtubule kinesin motor activity, providing a direction for future therapeutic intervention. Our studies support that TMEM230 has both glial tumor and endothelial cell intracellular and extracellular functions. Elevated levels of TMEM230 promote glial tumor cell migration, extracellular scaffold remodeling, and hypervascularization and abnormal formation of blood vessels. Downregulation of TMEM230 expression may inhibit both low grade glioma and glioblastoma tumor progression and promote normalization of abnormally formed blood vessels. TMEM230 therefore is both a promising anticancer and antiangiogenic therapeutic target for inhibiting GBM tumor cells and tumor-driven angiogenesis., Competing Interests: IZ and RR have a patent accepted concerning the use of Agents that modulate TMEM230 in tumor associated angiogenesis. Patent Application International Publication number: 20200247882. Agents that modulate TMEM230 as angiogenesis regulators and that detect TMEM230 AS markers of metastasis. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cocola, Magnaghi, Abeni, Pelucchi, Martino, Vilardo, Piscitelli, Consiglio, Grillo, Mosca, Gualtierotti, Mazzaccaro, La Sala, Di Pietro, Palizban, Liuni, DePedro, Morara, Nano, Kehler, Greve, Noghero, Marazziti, Bussolino, Bellipanni, D’Agnano, Götte, Zucchi and Reinbold.)

Two complimentary approaches are widely used to study gene function in zebrafish: induction of genetic mutations, usually using targeted nucleases such as CRISPR/Cas9, and suppression of gene expression, typically using Morpholino oligomers. Neither method is perfect. Morpholinos (MOs) sometimes produce off-target or toxicity-related effects that can be mistaken for true phenotypes. Conversely, genetic mutants can be subject to compensation, or may fail to yield a null phenotype due to leakiness (e.g. use of cryptic splice sites or downstream AUGs). When discrepancy between mutant and morpholino-induced (morphant) phenotypes is observed, experimental validation of such phenotypes becomes very labor intensive. We have developed a simple genetic method to differentiate between genuine morphant phenotypes and those produced due to off-target effects. We speculated that indels within 5' untranslated regions would be unlikely to have a significant negative effect on gene expression. Mutations induced within a MO target site would result in a Morpholino-refractive allele thus suppressing true MO phenotypes whilst non-specific phenotypes would remain. We tested this hypothesis on one gene with an exclusively zygotic function, tbx5a, and one gene with strong maternal effect, ctnnb2. We found that indels within the Morpholino binding site are indeed able to suppress both zygotic and maternal morphant phenotypes. We also observed that the ability of such indels to suppress morpholino phenotypes does depend on the size and the location of the deletion. Nonetheless, mutating the morpholino binding sites in both maternal and zygotic genes can ascertain the specificity of morphant phenotypes.

Recent studies have suggested that microenvironmental stimuli play a significant role in regulating cellular proliferation and migration, as well as in modulating self-renewal and differentiation processes of mammary cells with stem cell (SCs) properties. Recent advances in micro/nanotechnology and biomaterial synthesis/engineering currently enable the fabrication of innovative tissue culture platforms suitable for maintenance and differentiation of SCs in vitro. Here, we report the design and fabrication of an open microfluidic device (OMD) integrating removable poly(ε-caprolactone) (PCL) based electrospun scaffolds, and we demonstrate that the OMD allows investigation of the behavior of human cells during in vitro culture in real time. Electrospun scaffolds with modified surface topography and chemistry can influence attachment, proliferation, and differentiation of mammary SCs and epigenetic mechanisms that maintain luminal cell identity as a function of specific morphological or biochemical cues imparted by tailor-made fiber post-treatments. Meanwhile, the OMD architecture allows control of cell seeding and culture conditions to collect more accurate and informative in vitro assays. In perspective, integrated systems could be tailor-made to mimic specific physiological conditions of the local microenvironment and then analyze the response from screening specific drugs for more effective diagnostics, long-term prognostics, and disease intervention in personalized medicine.

Histone deacetylase 8 (HDAC8) is a class 1 histone deacetylase and a member of the cohesin complex. HDAC8 is expressed in smooth muscles, but its expression in skeletal muscle has not been described. We have shown for the first time that HDAC8 is expressed in human and zebrafish skeletal muscles. Using RD/12 and RD/18 rhabdomyosarcoma cells with low and high differentiation potency, respectively, we highlighted a specific correlation with HDAC8 expression and an advanced stage of muscle differentiation. We inhibited HDAC8 activity through a specific PCI-34051 inhibitor in murine C2C12 myoblasts and zebrafish embryos, and we observed skeletal muscles differentiation impairment. We also found a positive regulation of the canonical Wnt signaling by HDAC8 that might explain muscle differentiation defects. These findings suggest a novel mechanism through which HDAC8 expression, in a specific time window of skeletal muscle development, positively regulates canonical Wnt pathway that is necessary for muscle differentiation., (© 2018 Wiley Periodicals, Inc.)

The ability to conditionally inactivate genes is instrumental for fine genetic analysis of all biological processes, but is especially important for studies of biological events, such as regeneration, which occur late in ontogenesis or in adult life. We have constructed and tested a fully conditional gene trap vector, and used it to inactivate tbx5a in the cardiomyocytes of larval and adult zebrafish. We observe that loss of tbx5a function significantly impairs the ability of zebrafish hearts to regenerate after ventricular resection, indicating that Tbx5a plays an essential role in the transcriptional program of heart regeneration., Competing Interests: The authors have declared that no competing interests exist.

During embryonic development, new arteries, and veins form from preexisting vessels in response to specific angiogenic signals. Angiogenic signaling is complex since not all endothelial cells exposed to angiogenic signals respond equally. Some cells will be selected to become tip cells and acquire migration and proliferation capacity necessary for vessel growth while others, the stalk cells become trailer cells that stay connected with pre-existing vessels and act as a linkage to new forming vessels. Additionally, stalk and tip cells have the capacity to interchange their roles. Stalk and tip cellular responses are mediated in part by the interactions of components of the Delta/Notch and Vegf signaling pathways. We have identified in zebrafish, that the transmembrane protein Tmem230a is a novel regulator of angiogenesis by its capacity to regulate the number of the endothelial cells in intersegmental vessels by co-operating with the Delta/Notch signaling pathway. Modulation of Tmem230a expression by itself is sufficient to rescue improper number of endothelial cells induced by aberrant expression or inhibition of the activity of genes associated with the Dll4/Notch pathway in zebrafish. Therefore, Tmem230a may have a modulatory role in vessel-network formation and growth. As the Tmem230 sequence is conserved in human, Tmem230 may represent a promising novel target for drug discovery and for disease therapy and regenerative medicine in promoting or restricting angiogenesis., (© 2017 Wiley Periodicals, Inc.)

Cellular reprogramming by epigenomic remodeling of chromatin holds great promise in the field of human regenerative medicine. As an example, human-induced Pluripotent Stem Cells (iPSCs) obtained by reprograming of patient somatic cells are sufficiently similar to embryonic stem cells (ESCs) and can generate all cell types of the human body. Clinical use of iPSCs is dependent on methods that do not utilize genome altering transgenic technologies that are potentially unsafe and ethically unacceptable. Transient delivery of exogenous RNA into cells provides a safer reprogramming system to transgenic approaches that rely on exogenous DNA or viral vectors. RNA reprogramming may prove to be more suitable for clinical applications and provide stable starting cell lines for gene-editing, isolation, and characterization of patient iPSC lines. The introduction and rapid evolution of CRISPR/Cas9 gene-editing systems has provided a readily accessible research tool to perform functional human genetic experiments. Similar to RNA reprogramming, transient delivery of mRNA encoding Cas9 in combination with guide RNA sequences to target specific points in the genome eliminates the risk of potential integration of Cas9 plasmid constructs. We present optimized RNA-based laboratory procedure for making and editing iPSCs. In the near-term these two powerful technologies are being harnessed to dissect mechanisms of human development and disease in vitro, supporting both basic, and translational research. J. Cell. Physiol. 232: 1262-1269, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Recent studies suggest that human tumors are generated from cancer cells with stem cell (SC) properties. Spontaneously occurring cancers in dogs contain a diversity of cells that like for human tumors suggest that certain canine tumors are also generated from cancer stem cells (CSCs). CSCs, like normal SCs, have the capacity for self-renewal as mammospheres in suspension cultures. To understand how cells with SC properties contribute to canine mammary gland tumor development and progression, comparative analysis between normal SCs and CSCs, obtained from the same individual, is essential. We have utilized the property of sphere formation to develop culture conditions for propagating stem/progenitor cells from canine normal and tumor tissue. We show that cells from dissociated mammospheres retain sphere reformation capacity for several serial passages and have the capacity to generate organoid structures ex situ. Utilizing various culture conditions for passaging SCs and CSCs, fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) were found to positively or negatively regulate mammosphere regeneration, organoid formation, and multi-lineage differentiation potential. The response of FGF2 and EGF on SCs and CSCs was different, with increased FGF2 and EGF self-renewal promoted in SCs and repressed in CSCs. Our protocol for propagating SCs from normal and tumor canine breast tissue will provide new opportunities in comparative mammary gland stem cell analysis between species and anticancer treatment and therapies for dogs. J. Cell. Biochem. 118: 570-584, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Many experimental techniques rely on specific recognition and stringent binding of proteins by antibodies. This can readily be achieved by introducing an epitope tag. We employed an approach that uses a relative lack of evolutionary conservation to inform epitope tag site selection, followed by integration of the tag-coding sequence into the endogenous locus in zebrafish. We demonstrate that an internal epitope tag is accessible for antibody binding, and that tagged proteins retain wild type function.

Genetic variants within components of the cohesin complex (NIPBL, SMC1A, SMC3, RAD21, PDS5, ESCO2, HDAC8) are believed to be responsible for a spectrum of human syndromes known as "cohesinopathies" that includes Cornelia de Lange Syndrome (CdLS). CdLS is a multiple malformation syndrome affecting almost any organ and causing severe developmental delay. Cohesinopathies seem to be caused by dysregulation of specific developmental pathways downstream of mutations in cohesin components. However, it is still unclear how mutations in different components of the cohesin complex affect the output of gene regulation. In this study, zebrafish embryos and SMC1A-mutated patient-derived fibroblasts were used to analyze abnormalities induced by SMC1A loss of function. We show that the knockdown of smc1a in zebrafish impairs neural development, increases apoptosis, and specifically down-regulates Ccnd1 levels. The same down-regulation of cohesin targets is observed in SMC1A-mutated patient fibroblasts. Previously, we have demonstrated that haploinsufficiency of NIPBL produces similar effects in zebrafish and in patients fibroblasts indicating a possible common feature for neurological defects and mental retardation in cohesinopathies. Interestingly, expression analysis of Smc1a and Nipbl in developing mouse embryos reveals a specific pattern in the hindbrain, suggesting a role for cohesins in neural development in vertebrates., (© 2015 Wiley Periodicals, Inc.)

The Coiled-Coil Domain Containing 80 (CCDC80) gene has been identified as strongly induced in rat thyroid PC CL3 cells immortalized by the adenoviral E1A gene. In human, CCDC80 is a potential oncosoppressor due to its down-regulation in several tumor cell lines and tissues and it is expressed in almost all tissues. CCDC80 has homologous in mouse, chicken, and zebrafish. We cloned the zebrafish ccdc80 and analyzed its expression and function during embryonic development. The in-silico translated zebrafish protein shares high similarity with its mammalian homologous, with nuclear localization signals and a signal peptide. Gene expression analysis demonstrates that zebrafish ccdc80 is maternally and zygotically expressed throughout the development. In particular, ccdc80 is strongly expressed in the notochord and it is under the regulation of the Hedgehog pathway. In this work we investigated the functional effects of ccdc80-loss-of-function during embryonic development and verified its interaction with gadd45β2 in somitogenesis., (© 2014 Wiley Periodicals, Inc.)

β-Catenin is a central effector of the Wnt pathway and one of the players in Ca(+)-dependent cell-cell adhesion. While many wnts are present and expressed in vertebrates, only one β-catenin exists in the majority of the organisms. One intriguing exception is zebrafish that carries two genes for β-catenin. The maternal recessive mutation ichabod presents very low levels of β-catenin2 that in turn affects dorsal axis formation, suggesting that β-catenin1 is incapable to compensate for β-catenin2 loss and raising the question of whether these two β-catenins may have differential roles during early axis specification. Here we identify a specific antibody that can discriminate selectively for β-catenin1. By confocal co-immunofluorescent analysis and low concentration gain-of-function experiments, we show that β-catenin1 and 2 behave in similar modes in dorsal axis induction and cellular localization. Surprisingly, we also found that in the ich embryo the mRNAs of the components of β-catenin regulatory pathway, including β-catenin1, are more abundant than in the Wt embryo. Increased levels of β-catenin1 are found at the membrane level but not in the nuclei till high stage. Finally, we present evidence that β-catenin1 cannot revert the ich phenotype because it may be under the control of a GSK3β-independent mechanism that required Axin's RGS domain function., (© 2014 Wiley Periodicals, Inc.)

Malignant pleural effusions (MPEs) are a common manifestation found in patients with lung cancer. After cytological and histological confirmation of malignancy, talc pleurodesis still remains the treatment of choice in patients with MPEs resistant to chemotherapy. Despite this, primary challenges include reduced quality of life and life expectancy in general. Therefore, a better understanding of the cell biology of MPEs, along with improvements in treatment is greatly needed. It has recently been demonstrated that MPEs may represent an excellent source for identification of molecular mechanisms within the tumor and its environment. The present review summarizes the current understanding of MPEs cells and tumor microenvironment, and particularly focuses on dissecting the cross-talk between MPEs and epithelial to mesenchymal transition (EMT), inflammation and cancer stem cells., (© 2014 Wiley Periodicals, Inc.)

Chordoma is a rare malignant tumor that recapitulates the notochord phenotype and is thought to derive from notochord remnants not correctly regressed during development. Apoptosis is necessary for the proper notochord development in vertebrates, and the apoptotic pathway mediated by Fas and Fasl has been demonstrated to be involved in notochord cells regression. This study was conducted to investigate the expression of FAS/FASL pathway in a cohort of skull base chordomas and to analyze the role of fas/fasl homologs in zebrafish notochord formation. FAS/FASL expression was found to be dysregulated in chordoma leading to inactivation of the downstream Caspases in the samples analyzed. Both fas and fasl were specifically expressed in zebrafish notochord sorted cells. fas and fasl loss-of-function mainly resulted in larvae with notochord multi-cell-layer jumps organization, larger vacuolated notochord cells, defects in the peri-notochordal sheath structure and in vertebral mineralization. Interestingly, we observed the persistent expression of ntla and col2a1a, the zebrafish homologs of the human T gene and COL2A1 respectively, which are specifically up-regulated in chordoma. These results demonstrate for the first time the dysregulation of FAS/FASL in chordoma and their role in notochord formation in the zebrafish model, suggesting their possible implication in chordoma onset.

Exposing tissues to extreme high or low temperature leads to burns. Burned animals sustain several types of damage, from the disruption of the tissue to degeneration of axons projecting through muscle and skin. Such damage causes pain due to both inflammation and axonal degeneration (neuropathic-like pain). Thus, the approach to cure and alleviate the symptoms of burns must be twofold: rebuilding the tissue that has been destroyed and alleviating the pain derived from the burns. While tissue regeneration techniques have been developed, less is known on the treatment of the induced pain. Thus, appropriate animal models are necessary for the development of the best treatment for pain induced in burned tissues. We have developed a methodology in the zebrafish aimed to produce a new animal model for the study of pain induced by burns. Here, we show that two events linked to the onset of burn-induced inflammation and neuropathic-like pain in mammals, degeneration of axons innervating the affected tissues and over-expression of specific genes in sensory tissues, are conserved from zebrafish to mammals., (© 2013 Wiley Periodicals, Inc.)

Nociception is the sensory mechanism used to detect cues that can harm an organism. The understanding of the neural networks and molecular controls of the reception of pain remains an ongoing challenge for biologists. While we have made significant progress in identifying a number of molecules and pathways that are involved in transduction of noxious stimuli, from the skin through the sensory receptor cell and from this to the spinal cord on into the central nervous system, we still lack a clear understanding of the perceptual processes, the responses to pain and the regulation of pain perception. Mice and rat animal models have been extensively used for nociception studies. However, the study of pain and noiception in these organisms can be rather laborious, costly and time consuming. Conversely, the use of Drosophila and Caenorhabditis elegans may be affected by the large evolutionary distance between these animals and humans. We outline here the reasons why zebrafish presents a new and attractive model for studying pain reception and responses and the most interesting findings in the study of nociception that have been obtained using the zebrafish model., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: External development and optical transparency of embryos make zebrafish exceptionally suitable for in vivo insertional mutagenesis using fluorescent proteins to visualize expression patterns of mutated genes. Recently developed Gene Breaking Transposon (GBT) vectors greatly improve the fidelity and mutagenicity of transposon-based gene trap vectors., Results: We constructed and tested a bipartite GBT vector with Gal4-VP16 as the primary gene trap reporter. Our vector also contains a UAS:eGFP cassette for direct detection of gene trap events by fluorescence. To confirm gene trap events, we generated a UAS:mRFP tester line. We screened 270 potential founders and established 41 gene trap lines. Three of our gene trap alleles display homozygous lethal phenotypes ranging from embryonic to late larval: nsf( tpl6), atp1a3a(tpl10) and flr(tpl19). Our gene trap cassette is flanked by direct loxP sites, which enabled us to successfully revert nsf( tpl6), atp1a3a(tpl10) and flr(tpl19) gene trap alleles by injection of Cre mRNA. The UAS:eGFP cassette is flanked by direct FRT sites. It can be readily removed by injection of Flp mRNA for use of our gene trap alleles with other tissue-specific GFP-marked lines. The Gal4-VP16 component of our vector provides two important advantages over other GBT vectors. The first is increased sensitivity, which enabled us to detect previously unnoticed expression of nsf in the pancreas. The second advantage is that all our gene trap lines, including integrations into non-essential genes, can be used as highly specific Gal4 drivers for expression of other transgenes under the control of Gal4 UAS., Conclusions: The Gal4-containing bipartite Gene Breaking Transposon vector presented here retains high specificity for integrations into genes, high mutagenicity and revertibility by Cre. These features, together with utility as highly specific Gal4 drivers, make gene trap mutants presented here especially useful to the research community.

Distinct molecular pathways could be constitutively active in mouse T-Antigen positive and T-Antigen negative medulloblastoma cell lines, contributing to their phenotypic differences as well as to cellular responses, cell cycle progression, cell death and survival. The diversity of these responses may be due, at least in part, to distinct activities of Rb2/p130, CTCF and BORIS proteins in response to an altered network of signaling evoked by the T-Ag presence. Here, we provided evidence supporting a role for the T-Antigen in causing chronic endoplasmic reticulum (ER) stress and aberrant Caspase-12 expression and activation, subsequently driving to both massive cell death, and perhaps selection of cells with a higher malignant phenotype. Furthermore, we observed that the endoplasmic stress, either chronically caused by T-Ag or transiently induced by glucose deprivation, is accompanied by the formation of complexes between the retinoblastoma related protein Rb2/p130 and the chromatin insulator CCCTC-binding factor CTCF, or the CTCF-paralogue BORIS. Our study represents the first evidence supporting a role of the T-Antigen in inducing/maintaining chronic ER-stress, as well as, indicating a role of Rb2/p130, CTCF and BORIS as potential mediators of non-canonical ER-dependent death pathway in mouse medulloblastoma.

Otx proteins are involved in the induction of neurectoderm patterning and morphogenetic movements, leading to the formation of the vertebrate central nervous system. Despite lack of homology of sequence outside the homeodomain, a large body of evidence has shown that the Otx/Otd class of proteins has similar functions in many animal phyla. Thus, characterization of functional domains in proteins of this family would help in understanding how this functional equivalence operates. Our previous analysis using the zebrafish embryo (Bellipanni et al., 2000, Dev Biol 223:339-353), has suggested that induction of cell aggregation is a morphoregulatory role of Otx/Otd factors in embryonic development. We now use the induction of cell aggregation as an in vivo assay to examine the functional requirement for particular domains of the zOtx1 protein. We demonstrate that zOtx1 induces cell aggregation by acting as a transcriptional activator through its C-terminal region. Further, we show that a region of 37 amino acids in the C-terminal third of zOtx1 is necessary but not sufficient for this activation potential. The effects of selective deletion of each of the three homeodomain alpha-helices of zOtx1 on cell aggregation were also tested. Surprisingly, we find that helix 3, which is required for binding to DNA, is dispensable for stimulation of cell aggregation. Our results suggest that for transcriptional activation of at least one gene in the cell aggregation pathway, zOtx1 need not bind directly to DNA, but does require helix 1 and 2 of its homeodomain to interact with an as yet undefined DNA binding protein., ((c) 2009 Wiley-Liss, Inc.)

Post-translational stabilization of beta-catenin is a key step in Wnt signaling, but the features of beta-catenin required for stabilization are incompletely understood. We show that forms of beta-catenin lacking the unstructured C-terminal domain (CTD) show faster turnover than full-length or minimally truncated beta-catenins. Mutants that exhibit faster turnover show enhanced association with axin in co-transfected cells, and excess CTD polypeptide can compete binding of the beta-catenin armadillo (arm) repeat domain to axin in vitro, indicating that the CTD may restrict beta-catenin binding to the axin-scaffold complex. Fluorescent resonance energy transmission (FRET) analysis of cyan fluorescent protein (CFP)-arm-CTD-yellow fluorescent protein beta-catenin reveals that the CTD of beta-catenin can become spatially close to the N-terminal arm repeat region of beta-catenin. FRET activity is strongly diminished by the coexpression of beta-catenin binding partners, indicating that an unliganded groove is absolutely required for an orientation that allows FRET. Amino acids 733-759 are critical for beta-catenin FRET activity and stability. These data indicate that an N-terminal orientation of the CTD is required for beta-catenin stabilization and suggest a model where the CTD extends toward the N-terminal arm repeats, shielding these repeats from the beta-catenin destruction complex.

Using embryos transgenic for the TOP-GFP reporter, we show that the two zebrafish beta-catenins have different roles in the organizer and germ-ring regions of the embryo. beta-Catenin-activated transcription in the prospective organizer region specifically requires beta-catenin-2, whereas the ventrolateral domain of activated transcription is abolished only when both beta-catenins are inhibited. chordin expression during zebrafish gastrulation has been previously shown in both axial and paraxial domains, but is excluded from ventrolateral domains. We show that this gene is expressed in paraxial territories adjacent to the domain of ventrolateral beta-catenin-activated transcription, with only slight overlap, consistent with the now well-known inhibitory effects of Wnt8 on dorsal gene expression. Eliminating both Wnt8/beta-catenin signaling and organizer activity by inhibition of expression of the two beta-catenins results in massive ectopic circumferential expression of chordin and later, by formation of a distinctive embryonic phenotype ('ciuffo') that expresses trunk and anterior neural markers with correct relative anteroposterior patterning. We show that chordin expression is required for this neural gene expression. The Nodal gene squint has been shown to be necessary for optimal expression of chordin and is sufficient in some contexts for its expression. However, chordin is not normally expressed in the ventrolateral germ-ring despite robust expression of squint in this domain. We show the ectopic circumferential expression of chordin and other dorsal genes to be completely dependent on Nodal and FGF signaling, and to be independent of a functional organizer. We propose that whereas the axial domain of chordin expression is formed by cells that are derived from the organizer, the paraxial domain is the result of axial-derived anti-Wnt signals, which relieve the repression that otherwise is set by the Wnt8/beta-catenin/vox,vent pathway on latent germ-ring Nodal/FGF-activated expression.

In Xenopus, Wnt signals and their transcriptional effector beta-catenin are required for the development of dorsal axial structures. In zebrafish, previous loss-of-function studies have not identified an essential role for beta-catenin in dorsal axis formation, but the maternal-effect mutation ichabod disrupts beta-catenin accumulation in dorsal nuclei and leads to a reduction of dorsoanterior derivatives. We have identified and characterized a second zebrafish beta-catenin gene, beta-catenin-2, located on a different linkage group from the previously studied beta-catenin-1, but situated close to the ichabod mutation on LG19. Although the ichabod mutation does not functionally alter the beta-catenin-2 reading frame, the level of maternal beta-catenin-2, but not beta-catenin-1, transcript is substantially lower in ichabod, compared with wild-type, embryos. Reduction of beta-catenin-2 function in wild-type embryos by injection of morpholino antisense oligonucleotides (MOs) specific for this gene (MO2) results in the same ventralized phenotypes as seen in ichabod embryos, and administration of MO2 to ichabod embryos increases the extent of ventralization. MOs directed against beta-catenin-1 (MO1), by contrast, had no ventralizing effect on wild-type embryos. beta-catenin-2 is thus specifically required for organizer formation and this function is apparently required maternally, because the ichabod mutation causes a reduction in maternal transcription of the gene and a reduced level of beta-catenin-2 protein in the early embryo. A redundant role of beta-catenins in suppressing formation of neurectoderm is revealed when both beta-catenin genes are inhibited. Using a combination of MO1 and MO2 in wild-type embryos, or by injecting solely MO1 in ichabod embryos, we obtain expression of a wide spectrum of neural markers in apparently appropriate anteroposterior pattern. We propose that the early, dorsal-promoting function of beta-catenin-2 is essential to counteract a later, dorsal- and neurectoderm-repressing function that is shared by both beta-catenin genes.

Transcription factors of the bHLH class play crucial roles in neurogenesis by controlling the location and timing of neuronal commitment and differentiation, as well as influencing neuronal identity. Proneural bHLH factors belong to the Olig, Neurogenin, NeuroD, Achaete-scute and Atonal subfamilies, and are expressed in partially overlapping or complementary patterns within the vertebrate embryonic neural tube. The combinatorial expression of these factors likely drives the generic and cell type-specific properties of neurogenesis throughout the nervous system. As an approach towards identifying a complete set of vertebrate proneural factors, we report here the isolation of two new zebrafish neural bHLH gene members, atoh1.2 and beta3.1. Among other sites, both are expressed in the late embryonic and early larval anterior hindbrain. In this territory we demonstrate that atoh1.2 and beta3.1 are transcribed in distinct precursors, further highlighting the subdivision of anterior zebrafish hindbrain into subdomains of bHLH expression.

The monoamine serotonin (5-HT) exerts key neuromodulatory activities in all animal phyla, but the development and function of the serotonergic system is still incompletely understood. The zebrafish Danio rerio is an excellent model to approach this question since it is amenable to a combination of genetic, molecular and embryological studies. In order to characterize the organization of serotonergic neurons in the zebrafish we cloned two cDNAs encoding distinct forms of tryptophan hydroxylase (Tph), the rate-limiting enzyme in serotonin synthesis. We report here the pattern of expression of these two genes in relation with immunoreactive TH and 5-HT nuclei in the developing zebrafish embryo and early larva. tphD1 expression starts at 22 h post-fertilization (hpf) in the epiphysis and in basal spinal cells. Expression persists in the epiphysis until at least 4 days (dpf). Between 48 hpf and 3 dpf, tphD1 expression is initiated in retinal amacrine cells and in restricted preoptic and posterior tubercular nuclei within the basal diencephalon. At 3 and 4 dpf, tphD1 expression is newly initiated in the caudal hypothalamus and in branchial arches-associated neurons. tphD2 mRNA is detected transiently (between 30 somites and 32 hpf) in a restricted preoptic nucleus. All sites of tphD1 or D2 expression within the anterior central nervous system are also immunoreactive for 5-HT, but are not positive for TH. However, neither tphD gene is expressed in raphe nuclei, suggesting that additional tph gene(s) exist in zebrafish to account for 5-HT synthesis in that location. The co-expression of tphD1, tphD2 and 5-HT in the zebrafish diencephalon appears in striking contrast to the situation in mammals, where diencephalic serotonin results from re-uptake rather than from local production.

In the zebrafish embryo, cells fated to give rise to the rostral brain move in a concerted fashion and retain tissue coherence during morphogenesis. We demonstrate here that Otx proteins have a dramatic effect on cell-cell interactions when expressed ectopically in the zebrafish embryo. Injection of zebrafish Otx1 or Drosophila otd RNAs into a single cell at the 16-cell stage results in aggregation of descendants of the injected cell. The Otx/Otd homeodomain is necessary for aggregation and appears to be sufficient for the effect when substituted for the homeodomain of an unrelated homeodomain protein. When cells containing injected zOtx1 RNA are limited to the area that is normally fated to become the anterior brain and neural retina, the induced aggregates contribute to anterior brain and retina tissues. In many other embryonic regions, which do not express endogenous zOtx1, the aggregates appear to be incompatible with normal development and do not integrate into developing tissues. By using an activatable Otx1-glutocorticoid receptor fusion protein that results in the stimulation of cell association, we demonstrate that cell aggregates can form as a result of Otx1 activity even after gastrulation is completed. Time-lapse analysis of cell movements show that cell aggregation occurs with only a slight inhibition of the rate of convergence. These results suggest that promotion of cell adhesion or mediation of cell repulsion may be one of the normal functions of the Otx proteins in the establishment of the anterior brain., (Copyright 2000 Academic Press.)