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Coregulator proteins interact with signal-dependent transcription factors to modify their transcriptional activity. ZMIZ1 and ZMIZ2 (zinc finger MIZ-type containing 1 and 2) are coregulators with nonredundant functions that share unique structural characteristics. Among other interacting domains, they possess a MIZ (Msx-interacting zinc finger) that relates them to members of the protein inhibitor of activated STAT (PIAS) family and provides them the capacity to function as SUMO E3 ligases. The ZMIZ proteins stimulate the activity of various signaling pathways, including the androgen receptor (AR), P53, SMAD3/4, WNT/β-catenin, and NOTCH1 pathways, and interact with the BAF chromatin remodeling complex. Due to their molecular versatility, ZMIZ proteins have pleiotropic effects and thus are important for embryonic development and for human diseases. Both have been widely associated with cancer, and ZMIZ1 has been very frequently identified as a risk allele for several autoimmune conditions and other disorders. Moreover, mutations in the coding region of the ZMIZ1 gene are responsible for a severe syndromic neurodevelopmental disability. Because the actions of coregulators are highly gene-specific, a better knowledge of the associations that exist between the function of the ZMIZ coregulators and human pathologies is expected to potentiate the use of ZMIZ1 and ZMIZ2 as new drug targets for diseases such as hormone-dependent cancers.

ZMIZ1 is a transcriptional coactivator that is related to members of the protein inhibitor of activated STAT (PIAS) family. ZMIZ1 regulates the activity of various transcription factors including the androgen receptor, p53, and Smad3. ZMIZ1 also interacts with Notch1 and selectively regulates Notch1 target genes relevant for T cell development and leukemogenesis in mammals. Human ZMIZ1 is additionally characterized as a latitude-dependent autoimmune disease (LDAD) risk gene, as it is responsive to vitamin D and has been associated with at least eleven blood cell traits.To address the function of ZMIZ1 in fish, we introduced CRISPR/Cas9 mutations in the zmiz1a gene in zebrafish. We observed that inactivation of zmiz1a in developing zebrafish larvae results in lethality at 15 dpf and delayed erythroid maturation. Differential gene expression analysis indicated that 15 dpf zmiz1a-null larvae had altered expression of autophagy genes, and erythrocytes that lacked Zmiz1a function exhibited an accumulation of mitochondrial DNA. Furthermore, we observed that autophagy gene expression was dysregulated at earlier stages of development, which suggests the involvement of Zmiz1a in the regulation of autophagy genes beyond the process of red blood cell differentiation. Finally, we showed that the loss of Zmiz1a decreased the capacity of the embryos to respond to vitamin D, indicating additional participation of Zmiz1a as a mediator of vitamin D activity.

ZMIZ1 is a transcriptional coactivator that is related to members of the protein inhibitor of activated STAT (PIAS) family. ZMIZ1 regulates the activity of various transcription factors including the androgen receptor, p53, and Smad3. ZMIZ1 also interacts with Notch1 and selectively regulates Notch1 target genes relevant for T cell development and leukemogenesis in mammals. Human ZMIZ1 is additionally characterized as a latitude-dependent autoimmune disease (LDAD) risk gene, as it is responsive to vitamin D and has been associated with at least eleven blood cell traits. To address the function of ZMIZ1 in fish, we introduced CRISPR/Cas9 mutations in the zmiz1a gene in zebrafish. We observed that inactivation of zmiz1a in developing zebrafish larvae results in lethality at 15 days post fertilization (dpf) and delayed erythroid maturation. Differential gene expression analysis indicated that 15 dpf zmiz1a-null larvae had altered expression of autophagy genes, and erythrocytes that lacked Zmiz1a function exhibited an accumulation of mitochondrial DNA. Furthermore, we observed that autophagy gene expression was dysregulated at earlier stages of development, which suggests the involvement of Zmiz1a in the regulation of autophagy genes beyond the process of red blood cell differentiation. Finally, we showed that the loss of Zmiz1a decreased the capacity of the embryos to respond to vitamin D, indicating additional participation of Zmiz1a as a mediator of vitamin D activity.

Cell movements are essential for morphogenesis during animal development. Epiboly is the first morphogenetic process in zebrafish in which cells move en masse to thin and spread the deep and enveloping cell layers of the blastoderm over the yolk cell. While epiboly has been shown to be controlled by complex molecular networks, the contribution of reactive oxygen species (ROS) to this process has not previously been studied. Here, we show that ROS are required for epiboly in zebrafish. Visualization of ROS in whole embryos revealed dynamic patterns during epiboly progression. Significantly, inhibition of NADPH oxidase activity leads to a decrease in ROS formation, delays epiboly, alters E-cadherin and cytoskeleton patterns and, by 24 h post-fertilization, decreases embryo survival, effects that are rescued by hydrogen peroxide treatment. Our findings suggest that a delicate ROS balance is required during early development and that disruption of that balance interferes with cell adhesion, leading to defective cell motility and epiboly progression.

The hematopoietic stem cell (HSC) is able to give rise to all blood cell lineages in vertebrates. HSCs are generated in the early embryo after two precedent waves of primitive hematopoiesis. Canonical Notch signaling is at the center of the complex mechanism that controls the development of the definitive HSC. The successful in vitro generation of hematopoietic cells from pluripotent stem cells with the capacity for multilineage hematopoietic reconstitution after transplantation requires the recapitulation of the most important process that takes place in the hemogenic endothelium during definitive hematopoiesis, that is the endothelial-to-hematopoietic transition (EHT). To meet this challenge, it is necessary to thoroughly understand the molecular mechanisms that modulate Notch signaling during the HSC differentiation process considering different temporal and spatial dimensions. In recent years, there have been important advances in this field. Here, we review relevant contributions describing different genes, factors, environmental cues, and signaling cascades that regulate the EHT through Notch interactions at multiple levels. The evolutionary conservation of the hematopoietic program has made possible the use of diverse model systems. We describe the contributions of the zebrafish model and the most relevant ones from transgenic mouse studies and from in vitro differentiated pluripotent cells.

Metamorphosis is a postembryonic developmental process that involves morphophysiological and behavioral changes, allowing organisms to adapt into a novel environment. In some amphibians, aquatic organisms undergo metamorphosis to adapt in a terrestrial environment. These organisms experience major changes in their circulatory, respiratory, digestive, excretory and reproductive systems. We performed a transcriptional global analysis of heart, lung and gills during diverse stages ofAmbystoma velascimetamorphosis. In our analyses, we identified eight gene clusters for each organ, according to the expression patterns of differentially expressed genes. We found 4,064 differentially expressed genes in the heart, 4,107 in the lung and 8,265 in the gills. Among the differentially expressed genes in the heart, we observed genes involved in the differentiation of cardiomyocytes in the interatrial zone, vasculogenesis and in the maturation of coronary vessels. In the lung, we found genes differentially expressed related to angiogenesis, alveolarization and synthesis of the surfactant protein. In the case of the gills, the most prominent biological processes identified are degradation of extracellular matrix, apoptosis and keratin production. Our study sheds light on the transcriptional responses and the pathways involved in the transformation of the facultative metamorphic salamanderA. velasciin an organ-specific manner.

Zebrafish germ plasm is composed of mRNAs such as vasa and nanos and of proteins such as Bucky ball, all of which localize symmetrically in four aggregates at the distal region of the first two cleavage furrows. The coordination of actin microfilaments, microtubules and kinesin is essential for the correct localization of the germ plasm. Rho-GTPases, through their effectors, coordinate cytoskeletal dynamics. We address the participation of RhoA and its effector ROCK in germ plasm localization during the transition from two- to eight-cell embryos. We found that active RhoA is enriched along the cleavage furrow during the first two division cycles, whereas ROCK localizes at the distal region of the cleavage furrows in a similar pattern as the germ plasm mRNAs. Specific inhibition of RhoA and ROCK affected microtubules organization at the cleavage furrow; these caused the incorrect localization of the germ plasm mRNAs. The incorrect localization of the germ plasm led to a dramatic change in the number of germ cells during the blastula and 24hpf embryo stages without affecting any other developmental processes. We demonstrate that the Rho/ROCK pathway is intimately related to the determination of germ cells in zebrafish embryos.

Characterization of a protein of interest during development is essential for functional studies. A general strategy for understanding the function of a particular protein involves the generation of null mutations, or treatment with drugs, that interfere with its activity. To demonstrate that the synthesis, stability, or activity of a protein has been affected, accurate and efficient detection of low amounts of protein is essential. This can be achieved by immunohistochemistry or by western blot. Here we describe a method for the detection of proteins from single de-yolked zebrafish embryos. This procedure includes a fixation step and the concomitant elimination of lipids from the yolk cell. We show that this approach allows the rapid analysis of proteins in embryos without having to manually remove the yolk. This method provides a convenient alternative for genotyping of mutant embryos as early as the 128 cell stage. In addition, in drug- or morpholino-treated embryos, the correlation between the penetrance of a phenotype and the concentration of a protein present may be established.

SWI/SNF or BAF chromatin-remodeling complexes are polymorphic assemblies of homologous subunit families that remodel nucleosomes and facilitate tissue-specific gene regulation during development. BAF57/SMARCE1 is a BAF complex subunit encoded in animals by a single gene and is a component of all mammalian BAF complexes. In vivo, the loss of SMARCE1 would lead to the formation of deficient combinations of the complex which might present limited remodeling activities. To address the specific contribution of SMARCE1 to the function of the BAF complex, we generated CRISPR/Cas9 mutations of smarce1 in zebrafish. Smarce1 mutants showed visible defects at 72 hpf, including smaller eyes, abnormal body curvature and heart abnormalities. Gene expression analysis revealed that the mutant embryos displayed defects in endocardial development since early stages, which led to the formation of a misshapen heart tube. The severe morphological and functional cardiac problems observed at 4 dpf were correlated with the substantially increased expression of different cardiac transcription factors. Additionally, we showed that Smarce1 binds to cis-regulatory regions of the gata5 gene and is necessary for the recruitment of the BAF complex to these regions.

Although cell proliferation is an essential cell behavior for animal development, a detailed analysis of spatial and temporal patterns of proliferation in whole embryos are still lacking for most model organisms. Zebrafish embryos are particularly suitable for this type of analysis due to their transparency and size. Therefore, the main objective of the present work was to analyze the spatial and temporal patterns of proliferation during the first day of zebrafish embryo development by indirect immunofluorescence against phosphorylated histone H3, a commonly used mitotic marker. Several interesting findings were established. First, we found that mitosis metasynchrony among blastomeres could begin at the 2- to 4-cell stage embryos. Second, mitosis synchrony was lost before the midblastula transition (MBT). Third, we observed a novel pattern of mitotic clusters that coincided in time with the mitotic pseudo "waves" described to occur before the MBT. Altogether, our findings indicate that early development is less synchronic than anticipated and that synchrony is not a requirement for proper development in zebrafish.

Mammalian SWI/SNF or BAF chromatin-remodeling complexes are polymorphic assemblies of homologous subunit families that remodel nucleosomes. BAF57 is a subunit of the BAF complexes; it is encoded only in higher eukaryotes and is present in all mammalian assemblies. Its main structural feature is a high-mobility group domain, the DNA-binding properties of which suggest that BAF57 may play topological roles as the BAF complex enters or exits the nucleosome. BAF57 displays specific interactions with a number of proteins outside the BAF complex. Through these interactions, it can accomplish specific functions. In the embryo, BAF57 is responsible for the silencing of the CD4 gene during T-cell differentiation, and during the repression of neuronal genes in non-neuronal cells, BAF57 interacts with the transcriptional corepressor, Co-REST, and facilitates repression. Extensive work has demonstrated a specific role of BAF57 in regulating the interactions between BAF and nuclear hormone receptors. Despite its involvement in oncogenic pathways, new generation sequencing studies do not support a prominent role for BAF57 in the initiation of cancer. On the other hand, evidence has emerged to support a role for BAF57 as a metastasis factor, a prognosis marker and a therapeutic target. In humans, BAF57 is associated with disease, as mutations in this gene predispose to important congenital disorders, including menigioma disease or the Coffin-Siris syndrome. In this article, we present an exhaustive analysis of the BAF57 molecular and biochemical properties, cellular functions, loss-of-function phenotypes in living organisms and pathological manifestations in cases of human mutations.

Members of the PIAS (protein inhibitor of activated STAT) family perform essential functions in modulating the activity of transcriptional regulators. Zimp7 and Zimp10 are two proteins that together form a subfamily of the PIAS. Like the other members of this family, they contain the zinc-binding SP-RING/Miz domain, which confers SUMO-conjugating activity. Both proteins have been shown to stimulate androgen receptor-mediated transcription. Previously, we reported that both Zimp7 and Zimp10 genes are extensively expressed and dynamically regulated in the developing mouse embryo. In this work, we investigated the expression of these genes during gonadal development. We found that their expression is sex-specific. Both genes initiate their transcription at early stages in the embryonic male gonad, reaching their peak at 13.5 days post coitum, which coincides with the process of sex-specific germ cell mitotic arrest. Zimp7 is expressed in germ cells of the embryonic gonad and the adult testis. Immunofluorescence of spermatogenic cells revealed that Zimp7 protein localizes to nuclear territories in meiotic spermatocytes, including the XY bodies. On the other hand, Zimp10 is found in somatic cells, outside the testis cords and ceases to be expressed in the adult testis.

Kit is a receptor tyrosine kinase that plays a fundamental role during the development of germ cells. Additionally, a truncated product, tr-kit, expressed in haploid spermatids and mature spermatozoa can induce parthenogenetic activation when microinjected into mouse eggs, through the activation of PLCgamma-1. In this work, we induced ectopic expression of a mutated Kit protein, Kit(D814Y) during germ cell development. The in vivo expression of this mutant in spermatids produced malformations in mature spermatozoa, and in the most severe cases, sterility. Ultrastructural analysis indicated that condensing spermatids in the transgenic mouse presented a mislocalization of the manchette; a structure that has a crucial role during the elongation steps of spermiogenesis. This morphogenetic phenotype was accompanied by an increased phosphorylation of PLCgamma-1 in spermatogenic cells. Interestingly, we also found that, in wild-type testis, PLCgamma-1 is specifically phosphorylated in condensing spermatids, coincident with the timing of expression of tr-kit in spermiogenesis. We propose that alterations of PLCgamma-1 activity artificially promoted by ectopic Kit(D814Y) expression are related to the abnormalities of spermiogenesis. Our observations suggest that PLCgamma-1 activity could be involved in the shaping of spermatozoa.

Oct4 is a transcription factor that has been associated with pluripotency and fate determination in the initial cell lineages of mammals. On the other hand, Pou2, the ortholog of Oct4 in zebrafish, serves additional later functions during brain development acting as a differentiation switch. In mice, Oct4 is expressed throughout the neural plate of embryos until embryonic day (E) 8.0. In this study, we produced transgenic mouse embryos that ubiquitously express Oct4 and analyzed the consequences during development. We show that, at E8.0, a higher dosage of Oct4 in the neuroectoderm is sufficient to transiently alter mid– hindbrain patterning and produced a strong up-regulation of Pax2, indicating that Oct4 can regulate this gene in vivo. After E9.5, ectopic Oct4 in this region produced cell death and affected the development of the forebrain, suggesting that, at these later stages, Oct4 down-regulation is necessary for normal development to proceed. The phenotype of the transgenic embryos was also accompanied with an increase of Fgf8 expression in several of its endogenous domains, suggesting the possibility that Oct4 can participate in the regulation of expression of this ligand. Our observations support the hypothesis that Oct4, like zebrafish Pou2, has a conserved function during early brain patterning in mouse. Developmental Dynamics 232:180 –190, 2005. © 2004 Wiley-Liss, Inc.

The tumor suppressor gene PTEN, which is frequently mutated in human cancers, encodes a lipid phosphatase for phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3] and antagonizes phosphatidylinositol 3 kinase. Primordial germ cells (PGCs), which are the embryonic precursors of gametes, are the source of testicular teratoma. To elucidate the intracellular signaling mechanisms that underlie germ cell differentiation and proliferation, we have generated mice with a PGC-specific deletion of the Pten gene. Male mice that lacked PTEN exhibited bilateral testicular teratoma, which resulted from impaired mitotic arrest and outgrowth of cells with immature characters. Experiments with PTEN-null PGCs in culture revealed that these cells had greater proliferative capacity and enhanced pluripotent embryonic germ (EG) cell colony formation. PTEN appears to be essential for germ cell differentiation and an important factor in testicular germ cell tumor formation.

The XPD/ERCC2/Rad3 gene is required for excision repair of UV-damaged DNA and is an important component of nucleotide excision repair. Mutations in the XPD gene generate the cancer-prone syndrome, xeroderma pigmentosum, Cockayne’s syndrome, and trichothiodystrophy. XPD has a 5′- to 3′-helicase activity and is a component of the TFIIH transcription factor, which is essential for RNA polymerase II elongation. We present here the characterization of the Drosophila melanogaster XPD gene (DmXPD). DmXPD encodes a product that is highly related to its human homologue. The DmXPD protein is ubiquitous during development. In embryos at the syncytial blastoderm stage, DmXPD is cytoplasmic. At the onset of transcription in somatic cells and during gastrulation in germ cells, DmXPD moves to the nuclei. Distribution analysis in polytene chromosomes shows that DmXPD is highly concentrated in the interbands, especially in the highly transcribed regions known as puffs. UV-light irradiation of third-instar larvae induces an increase in the signal intensity and in the number of sites where the DmXPD protein is located in polytene chromosomes, indicating that the DmXPD protein is recruited intensively in the chromosomes as a response to DNA damage. This is the first time that the response to DNA damage by UV-light irradiation can be visualized directly on the chromosomes using one of the TFIIH components.

Programmed cell death or apoptosis is an essential process during the morphogenesis of a large number of structures. Evidence obtained over the past few years indicates that, in some cases, the generation of reactive oxygen species (ROS) is an important event during the course of apoptosis. Using an in vitro culture system in which digit individualization of developing limbs normally occurs, we assayed the effect of different antioxidants on the cell death that takes place at interdigits. The addition of phenol, dimethyl sulfoxide, or 2',7'-dichlorodihydrofluorescein diacetate (DCDHF-DA) to murine developing limbs in culture prevented digit individualization as well as the typical interdigital cell death. Two ROS-sensitive dyes, 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide and DCDHF-DA, stained interdigits and the so-called "necrotic zones," implying that they contain cells under oxidative stress. Very few interdigital cells were doubly stained with the ROS probes and two cell death indicators (i.e., acridine orange and propidium iodide), suggesting that they detect a different stage during the course of apoptosis. Furthermore, we found cells stained for ROS that did not express a specific macrophage marker and in a few cases were seen surrounded by a macrophage. Surprisingly, many regions of the midgestation mouse embryo that are undergoing cell death correlated with those that have a markedly higher level of ROS. Our data suggest that the generation of oxidative stress is a common requirement for cell death that occurs during mouse embryonic development.

Although cell proliferation is an essential cell behavior for animal development, a detailed analysis of spatial and temporal patterns of proliferation in whole embryos are still lacking for most model organisms. Zebrafish embryos are particularly suitable for this type of analysis due to their transparency and size. Therefore, the main objective of the present work was to analyze the spatial and temporal patterns of proliferation during the first day of zebrafish embryo development by indirect immunofluorescence against phosphorylated histone H3, a commonly used mitotic marker. Several interesting findings were established. First, we found that mitosis metasynchrony among blastomeres could begin at the 2- to 4-cell stage embryos. Second, mitosis synchrony was lost before the midblastula transition (MBT). Third, we observed a novel pattern of mitotic clusters that coincided in time with the mitotic pseudo "waves" described to occur before the MBT. Altogether, our findings indicate that early development is less synchronic than anticipated and that synchrony is not a requirement for proper development in zebrafish.

The mammalian SWI/SNF chromatin remodeling complexes play essential roles in cell cycle control through the transcriptional regulation of cell-cycle-specific genes. These complexes depend on the energy of ATP hydrolysis provided by the BRG1 or BRM catalytic subunit. They contain seven or more noncatalytic subunits, some being constitutive components, with others having paralogs that assemble in a combinatory manner producing different SWI/SNF-related complexes with specific functions. ARID1A and ARID1B are mutually exclusive subunits of the BAF complex. The specific presence of these subunits in the complex has been demonstrated to determine whether SWI/SNF functions as a corepressor (ARID1A) or as a coactivator (ARID1B) of the cell cycle genes. Our aim has been to analyze the relevance of the ARID1 subunits in development. We have compared the patterns of expression of these two genes through various mouse embryonic stages. Arid1a is expressed widely and intensively, whereas Arid1b is poorly transcribed and expressed in selected regions. Moreover, ARID1A and ARID1B present different kinetics of expression in the cell cycle. ARID1A accumulates in G0 and is downregulated throughout the cell cycle phases but is completely eliminated during mitosis, whereas ARID1B is expressed at comparable levels at all phases, even during mitosis. These kinetics probably affect the incorporation patterns of the ARID1 proteins to the complex and hence modulate SWI/SNF activity during proliferation and arrest.

The N-methyl D-aspartate (NMDA) receptor subtype of glutamate-gated ion channels possesses high calcium permeability and unique voltage-dependent sensitivity to magnesium and is modulated by glycine. Molecular cloning identified three complementary DNA species of rat brain, encoding NMDA receptor subunits NMDAR2A (NR2A), NR2B, and NR2C, which are 55 to 70% identical in sequence. These are structurally related, with less than 20% sequence identity, to other excitatory amino acid receptor subunits, including the NMDA receptor subunit NMDAR1 (NR1). Upon expression in cultured cells, the new subunits yielded prominent, typical glutamate- and NMDA-activated currents only when they were in heteromeric configurations with NR1. NR1-NR2A and NR1-NR2C channels differed in gating behavior and magnesium sensitivity. Such heteromeric NMDA receptor subtypes may exist in neurons, since NR1 messenger RNA is synthesized throughout the mature rat brain, while NR2 messenger RNA show a differential distribution.

Background Members of the Snail gene family, which encode zinc finger proteins that function as transcriptional repressors, play essential roles during embryonic development in vertebrates. Mouse embryos with conditional deletion of the Snail1 (Snai1) gene in the epiblast, but not in most extraembryonic membranes, exhibit defects in left-right asymmetry specification and migration of mesoderm cells through the posterior primitive streak. Here we describe phenotypic defects that result in death of the mutant embryos by 9.5 days of gestation. Findings Endothelial cells differentiated in epiblast-specific Snai1-deficient embryos, but formation of an interconnected vascular network was abnormal. To determine whether the observed vascular defects were dependent on disruption of blood flow, we analyzed vascular remodeling in cultured allantois explants from the mutant embryos. Similar vascular defects were observed in the mutant allantois explants. Conclusion These studies demonstrate that lethality in the Snai1-conditional mutant embryos is caused by multiple defects in the cardiovascular system.

ZIMP7 and ZIMP10 are two novel human PIAS-like proteins that share a similarity beyond the SP-RING Zn-finger domain that characterizes the PIAS family. This extended similarity is conserved in proteins of several other species and define an independent subfamily. ZIMP10 has been shown to increase the sumoylation of the androgen receptor (AR) leading to a stimulation of AR-mediated transcription. The Drosophila tonalli (tna) is the ortholog gene of ZIMP7 and ZIMP10 and presents genetic interactions with the SWI–SNF complex. Mutations in the tna gene produce flies with homeotic phenotypes. In this study, we determined the spatial–temporal expression pattern of Zimp7 and Zimp10 in mouse embryos from embryonic day 7.5 (E7.5), to mid-gestation. We found that these two genes are extensively expressed during these embryonic days and present partially overlapping patterns with a predomination of the transcripts in the neural tissues at early stages and a drop of expression at E12.5. Unlike other PIAS proteins, the tonalli-related Zimp genes might be essential for development. Comparison of conserved motifs in Zimp7 and Zimp10 protein sequences identified characteristic family domains that might be related to their specific biological roles, besides their common role previously identified in the sumoylation pathway.

Kit is a receptor tyrosine kinase that plays a fundamental role during the development of germ cells. Additionally, a truncated product, tr-kit, expressed in haploid spermatids and mature spermatozoa can induce parthenogenetic activation when microinjected into mouse eggs, through the activation of PLCgamma-1. In this work, we induced ectopic expression of a mutated Kit protein, Kit(D814Y) during germ cell development. The in vivo expression of this mutant in spermatids produced malformations in mature spermatozoa, and in the most severe cases, sterility. Ultrastructural analysis indicated that condensing spermatids in the transgenic mouse presented a mislocalization of the manchette; a structure that has a crucial role during the elongation steps of spermiogenesis. This morphogenetic phenotype was accompanied by an increased phosphorylation of PLCgamma-1 in spermatogenic cells. Interestingly, we also found that, in wild-type testis, PLCgamma-1 is specifically phosphorylated in condensing spermatids, coincident with the timing of expression of tr-kit in spermiogenesis. We propose that alterations of PLCgamma-1 activity artificially promoted by ectopic Kit(D814Y) expression are related to the abnormalities of spermiogenesis. Our observations suggest that PLCgamma-1 activity could be involved in the shaping of spermatozoa.

Oct4 is a transcription factor that has been associated with pluripotency and fate determination in the initial cell lineages of mammals. On the other hand, Pou2, the ortholog of Oct4 in zebrafish, serves additional later functions during brain development acting as a differentiation switch. In mice, Oct4 is expressed throughout the neural plate of embryos until embryonic day (E) 8.0. In this study, we produced transgenic mouse embryos that ubiquitously express Oct4 and analyzed the consequences during development. We show that, at E8.0, a higher dosage of Oct4 in the neuroectoderm is sufficient to transiently alter mid-hindbrain patterning and produced a strong up-regulation of Pax2, indicating that Oct4 can regulate this gene in vivo. After E9.5, ectopic Oct4 in this region produced cell death and affected the development of the forebrain, suggesting that, at these later stages, Oct4 down-regulation is necessary for normal development to proceed. The phenotype of the transgenic embryos was also accompanied with an increase of Fgf8 expression in several of its endogenous domains, suggesting the possibility that Oct4 can participate in the regulation of expression of this ligand. Our observations support the hypothesis that Oct4, like zebrafish Pou2, has a conserved function during early brain patterning in mouse.

Previous studies have shown that Oct4 has an essential role in maintaining pluripotency of cells of the inner cell mass (ICM) and embryonic stem cells. However, Oct4 null homozygous embryos die around the time of implantation, thus precluding further analysis of gene function during development. We have used the conditional Cre/loxP gene targeting strategy to assess Oct4 function in primordial germ cells (PGCs). Loss of Oct4 function leads to apoptosis of PGCs rather than to differentiation into a trophectodermal lineage, as has been described for Oct4-deficient ICM cells. These new results suggest a previously unknown function of Oct4 in maintaining viability of mammalian germline.

The Cre/loxP site-specific recombination system combined with embryonic stem cell-mediated technologies has greatly expanded our capability to address normal and disease development in mammals using genetic approaches. The success of this emerging technology hinges on the production of Cre-expressing transgenic lines that provide cell type-, tissue-, or developmental stage-specific recombination between loxP sites placed in the genome. Here we describe and characterize the production of a double-reporter mouse line that provides a convenient and reliable readout of Cre recombinase activity. Throughout all embryonic and adult stages, the transgenic animal expresses the lacZ reporter gene before Cre-mediated excision occurs. Cre excision, however, removes the lacZ gene, allowing expression of the second reporter, the human alkaline phosphatase gene. This double-reporter transgenic line is able to indicate the occurrence of Cre excision in an extremely widespread manner from early embryonic to adult lineages. It will be a valuable reagent for the increasing number of investigators taking advantage of the powerful tools provided by the Cre/loxP site-specific recombinase system.

AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor channels mediate the fast component of excitatory postsynaptic currents in the central nervous system. Site-selective nuclear RNA editing controls the calcium permeability of these channels, and RNA editing at a second site is shown here to affect the kinetic aspects of these channels in rat brain. In three of the four AMPA receptor subunits (GluR-B, -C, and -D), intronic elements determine a codon switch (AGA, arginine, to GGA, glycine) in the primary transcripts in a position termed the R/G site, which immediately precedes the alternatively spliced modules "flip" and "flop." The extent of editing at this site progresses with brain development in a manner specific for subunit and splice form, and edited channels possess faster recovery rates from desensitization.

The ligand-gated receptors for L-glutamate play a central role in acute neuronal degeneration. Recently cDNAs have been isolated for subunits of several glutamate receptor subtypes. By sequence homology all these subunits clearly belong to one large gene family. Several subfamilies exist and match roughly previously pharmacologically and electrophysiologically defined subtypes of glutamate receptors. Currently four genes (GluR A, B, C and D) are known that code for the AMPA subtypes of glutamate receptors. Recombinant expression of wild type and mutated sequences identified a critical residue in the putative TM2 channel-lining segment that controls Ca2+ ion permeability. The arginine (R) found in GluR B subunits at that position renders AMPA channels impermeable for Ca2+ ions, whereas glutamine (Q) containing GluR A, C and D subunits give rise to Ca2+ permeable channels. RNA editing converts the genomically encoded glutamine codon into the arginine codon found in GluR B cDNAs for the Q/R site. NMDA subtypes of glutamate receptors are formed after coexpression of the NR1 cDNA with a cDNA of the NR2 family. Depending on the member of the NR2 family used, NMDA receptors with different kinetical and pharmacological properties are generated. Common to all channels of these NMDA receptors is a high permeability for Ca2+ ions and a voltage dependent block by Mg2+ ions. All currently known NMDA receptor subunits have an asparagine at the Q/R homologous position. We found that this residue is critical for Mg2+ block and Ca2+ permeability of NMDA receptor channels.

We have characterized a second member (delta-2) of a new class of subunits for the ligand-gated excitatory amino acid receptor superfamily. The sequence of delta-2 exhibits an average identity of 25% and 18.5% to the non-NMDA and NMDA receptor subunits, respectively. The rat delta-2 gene is expressed predominantly in Purkinje cells of the cerebellum whereas only low levels of delta-1 transcripts are found in the adult brain. However, delta-1 gene expression undergoes a pronounced developmental peak, with particularly high mRNA levels in the caudate putamen of late embryonic/early postnatal stages.

system are a powerful approach to study thefunction of genes in particular cell lineages and forwhich generalized null mutations result in early lethalphenotypes (Lobe and Nagy, 1998). Here we report thecreation of a primarily PGC (primordial germ cell) spe-cific Cre recombinase transgenic line, designated TNAP-Cre. In this strain, the Cre recombinase has beenknocked into the locus of the TNAP (Tissue Non-SpecificAlkaline Phosphatase) gene.The structure of the TNAP-Cre targeting construct isdepicted in Figure 1a. After electroporation into R1 ES(Nagy

Many diseases are characterized by a long incubation period during which the patient is asymptomatic and has a low titer of the infectious agent, yet throughout this period the patient is infectious. It is desirable to detect these agents when they are present in a low titer in order to treat infected individuals and to prevent the spread of the disease to other people. Assays that utilize nucleic acid hybridization probes (Gillespie and Spiegelman 1965) offer the promise of sufficient sensitivity to detect these agents at an early stage. Single-stranded oligonucleotide probes can seek out and bind to unique complementary nucleotide sequences from the infectious agent. The resulting probe-target hybrids are the strongest and most specific intermolecular complexes known. Moreover, these complexes form rapidly in solution. The problem with using oligonucleotide probes is that when the number of targets is small, it is very difficult to detect the correspondingly small number of probes that are bound to the targets. The traditional solution to this problem is to tag the probes with radioactive groups or fluorescent moieties, or to link the probes to enzymes and then incubate them with colorless substrates that are converted to colored products. However, the limit of detection with these schemes is 200 000 target molecules. It is desirable to improve the sensitivity so that as few as 2000 target molecules can be detected. This would, for example, enable the detection of a single, infected cell in a blood sample from a patient exposed to human immunodeficiency virus (Pelligrino et al. 1987).

We have synthesized recombinant RNA molecules that function both as hybridization probes and as templates for exponential amplification by Qβ replicase. Each recombinant consists of a sequence specific for the protozoan parasite, Plasmodium falciparum, embedded within the sequence of MDV-1 RNA, which is a natural template for Qβ replicase. The probe sequence was inserted within a hairpin loop that occurs on the exterior of MDV-1 RNA. The recombinant RNAs hybridize specifically to complementary DNA, despite topological constraints on the probe domain, are replicated at the same rate as MDV-1 RNA, despite their additional length, and are able to serve as templates for the synthesis of a large number of RNA copies. A Qβ replicase reaction initiated with only 0.14 femtograms of recombinant RNA (1,000 molecules) can produce 129 nanograms of recombinant RNA product in 30 minutes. This represents a one-billion fold amplification. Our results demonstrate the feasibility of employing exponentially replicatable RNAs in bioassays, where they would serve the dual role of specific probe and amplifiable reporter.

Mammalian brain expresses receptors which bind the potent neurotoxins, kainate and domoate, with high affinity, and which form a subclass of ionotropic glutamate receptors. A new member of these receptors, expressed in both adult a embryonic CNS is compared in its ligand binding properties to its closely sequence-related homologs.

The Zmiz2 (Zimp7) protein and its homolog Zmiz1 (Zimp10) were initially identified in humans as androgen receptor co-activators. Sequence analysis revealed the presence of an SP-RING/Miz domain, which is highly conserved in members of the PIAS family and confers SUMO-conjugating activity. Zimp7 has been shown to interact with components of the Wnt/β-Catenin signaling pathway and with Brg1 and BAF57, components of the ATP-dependent mammalian SWI/SNF-like BAF chromatin-remodeling complexes.In this work, we analyze the role of zygotic Zimp7 in zebrafish development. We describe evidence indicating that Zimp7 is required for mesoderm development and dorsoventral patterning. Morpholino-mediated reduction of zygotic Zimp7 produced axial mesodermal defects that were preceded by up-regulation of organizer genes such as bozozok, goosecoid and floating head at the onset of gastrulation and by down-regulation of the ventral markers vox, vent and eve1 indicating loss of the ventrolateral mesoderm. Consistently, embryos overexpressing zimp7 RNA exhibited midline defects such as loss of forebrain and cyclopia accompanied by transcriptional changes directly opposite of those found in the morphants. In addition, the patterning of ventralized embryos produced by the overexpression of vox and vent was restored by a reduction of Zimp7 activity. Altogether, our findings indicate that Zimp7 is involved in transcriptional regulation of factors that are essential for patterning in the dorsoventral axis.

The plasmid pBR322 was one of the first EK2 multipurpose cloning vectors to be designed and constructed (ten years ago) for the efficient cloning and selection of recombinant DNA molecules in Escherichia coli . This 4363-bp DNA molecule has been extensively used as a cloning vehicle because of its simplicity and the availability of its nucleotide sequence. The widespread use of pBR322 has prompted numerous studies into its molecular structure and function. These studies revealed two features that detract from the plasmid's effectiveness as a cloning vector: (a) plasmid instability in the absence of selection and, (b) the lack of a direct selection scheme for recombinant DNA molecules. Several vectors based on pBR322 have been constructed to overcome these limitations and to extend the vector's versatility to accomodate special cloning purposes. The objective of this review is to provide a survey of these derivative vectors and to summarize information currently available on pBR322.

During spermatogenesis the activity of intracellular Ca2+-release channels is likely to play an important role in different specific cellular functions. Accordingly, messenger RNAs for the three inositol 1,4,5-trisphosphate receptor (IP3R) subtypes were found to be present throughout spermatogenesis. Immunocytochemical analysis revealed distinct distribution patterns of the mature IP3Rs during sperm differentiation. At early stages, IP3Rs are distributed throughout the cytoplasm, and as differentiation proceeds they become selectively localised to the Golgi complex. Consistently, spermatogonia underwent large intracellular Ca2+ release in response to thapsigargin (TG), while smaller responses were detected in late spermatocytes and spermatids. The distribution of IP3Rs and the larger Ca2+-release responses found in spermatogonia, suggest that IP3Rs may be involved in cell proliferation at this stage. This notion is supported by our observations in a spermatogenic cell line that depletion of intracellular Ca2+ pools using TG inhibits cell division, and that incubation with an IP3R-I antisense oligonucleotide completely inhibited proliferation. Furthermore, the three genes encoding ryanodine receptor proteins (RyRs) are expressed at all stages of spermatogenesis. However, immunocytochemical studies with specific antibodies against each of the RyR subtypes detected types 1 and 3 in spermatogenic cells and only type 3 in mature sperm. In contrast to IP3Rs, RyRs remain scattered in the cytoplasm throughout differentiation. Functional responses to caffeine and ryanodine were absent in spermatogenic cells and in mature sperm. These findings suggest that IP3Rs have significantly more important roles in spermatogenesis than RyRs, and that one of these roles is crucial for cell proliferation.