A study of Xlim1 function in the Spemann-Mangold organizer.

The Spemann-Mangold organizer is required in amphibian embryos to coordinate cell fate specification, differentiation of dorsal cell types and morphogenetic movements at early stages of development. A great number of genes are specifically expressed within the organizer, most of them encoding secreted proteins and transcription factors. The challenge is now to uncover genetic cascades and networks of interactions between these genes, in order to understand how the organizer functions. The task is immense and requires loss-of-function approaches to test the requirement for a given factor in a specific process. For transcription factors, it is possible to generate inhibitory molecules by fusing the DNA binding region to a repressor or activator domain, which should in principle antagonize the activity of the endogenous protein at the level of the DNA targets. We used this strategy to design activated and inhibitory forms of the LIM homeodomain transcription factor Lim1, which is encoded by an organizer gene involved in head development, as revealed by analyses of knockout mice. We found that Lim1 is a transcriptional activator, and can trigger dorso-anterior development upon ventral expression of hyperactive forms, in which Ldb1 is fused to Lim1. Using inhibitory Lim1 fusion proteins, we found that Lim1, or genes closely related to it, is required for head formation as well as for notochord development. Co-expression experiments revealed that Lim1 is required downstream of the early organizer factor Siamois, first, to establish the genetic program of the organizer and second, to mediate the action of organizer agents that are responsible for blocking ventralizing activities in the gastrula.

Xlim-1 and LIM domain binding protein 1 cooperate with various transcription factors in the regulation of the goosecoid promoter.

The homeobox genes Xlim-1 and goosecoid (gsc) are coexpressed in the Spemann organizer and later in the prechordal plate that acts as head organizer. Based on our previous finding that gsc is a possible target gene for Xlim-1, we studied the regulation of gsc transcription by Xlim-1 and other regulatory genes expressed at gastrula stages, by using gsc-luciferase reporter constructs injected into animal explants. A 492-bp upstream region of the gsc promoter responds to Xlim-1/3m, an activated form of Xlim-1, and to a combination of wild-type Xlim-1 and Ldb1, a LIM domain binding protein, supporting the view that gsc is a direct target of Xlim-1. Footprint and electrophoretic mobility shift assays with GST-homeodomain fusion proteins and embryo extracts overexpressing FLAG-tagged full-length proteins showed that the Xlim-1 homeodomain or Xlim-1/Ldb1 complex recognize several TAATXY core elements in the 492-bp upstream region, where XY is TA, TG, CA, or GG. Some of these elements are also bound by the ventral factor PV.1, whereas a TAATCT element did not bind Xlim-1 or PV.1 but did bind the anterior factors Otx2 and Gsc. These proteins modulate the activity of the gsc reporter in animal caps: Otx2 activates the reporter synergistically with Xlim-1 plus Ldb1, whereas Gsc and PV.1 strongly repress reporter activity. We show further, using animal cap assays, that the endogenous gsc gene was synergistically activated by Xlim-1, Ldb1, and Otx2 and that the endogenous otx2 gene was activated by Xlim-1/3m, and this activation was suppressed by the posterior factor Xbra. Based on these data, we propose a model for gene interactions in the specification of dorsoventral and anteroposterior differences in the mesoderm during gastrulation.

Expression pattern of the rat Lim-1 homeobox gene suggests a dual role during kidney development.

This study describes an in situ hybridization and immunohistochemical analysis of Lim-1 homeobox gene expression during kidney development in the rat. Lim-1 is expressed at all stages of mesonephric and metanephric kidney development. In the metanephros, Lim-1 gene mRNA is first found at day 13 in the ureteric bud, but not in uninduced mesenchyme. Expression in the mesenchyme can be seen only after mesenchymal cells have condensed around the ureteric bud tips and primary vesicles have formed. Experiments with mesenchymal explants induced to differentiate in vitro by high levels of basic FGF in the absence of ureteric bud also indicate that Lim-1 expression is correlated with tubulogenesis and this experimental model faithfully reproduces its expression in vivo. During mesenchymal differentiation Lim-1 protein and mRNA were found in comma- and S-shaped bodies, proximal and distal tubules, and collecting ducts. Lim-1 mRNA and Lim-1 protein were seen transiently at early stages of glomerulus formation. In the fully differentiated kidney Lim-1 gene products disappear from mesenchymal derivatives but persist in the collecting ducts which are derived from the ureteric bud. These data suggest a dual role for the Lim-1 homeobox gene in the developing kidney, a transient developmental function in the mesenchyme and a maintenance function in the ureteric bud and its derivatives. Further we suggest that Lim-1 is not directly involved in mesenchymal induction but may participate in its epithelial transformation at later stages as its expression in mesenchyme begins only after the formation of primary vesicle.

Neuronal and neuroendocrine expression of lim3, a LIM class homeobox gene, is altered in mutant zebrafish with axial signaling defects.

LIM class homeobox genes code for a family of transcriptional regulators that encode important determinants of cell lineage and cell type specificity. The lim3 gene from the zebrafish, Danio rerio, is highly conserved in sequence and expression pattern compared to its homologs in other vertebrates. In this paper we report immunocytochemical analysis of Lim3 protein expression in the pituitary, pineal, hindbrain, and spinal cord of the embryo, revealing an asymmetrical, lateral and late program of pituitary development in zebrafish, distinct from the pattern in other vertebrates. We studied Lim3 expression in no tail, floating head, and cyclops mutant embryos, all of which have midline defects, with special reference to spinal cord differentiation where Lim3 marks mostly motoneurons. cyclops embryos showed essentially normal Lim3 expression in the hindbrain and spinal cord despite the absence of the floor plate, while no tail mutant embryos, which lack a differentiated notochord, displayed an excess of Lim3-expressing cells in a generally normal pattern. In contrast, Lim3-positive cells largely disappeared from the posterior spinal cord in floating head mutants, except in patches that correlated with remnants of apparent floor plate cells. These results support the view that either notochord or floor plate signaling can specify Lim3-positive motoneurons in the spinal cord.

The LIM homeodomain protein Lim-1 is widely expressed in neural, neural crest and mesoderm derivatives in vertebrate development.

Polyclonal antibodies to Xlim-1 homeodomain protein of Xenopus laevis were used to study the developmental expression pattern of this protein in Xenopus, rat and mouse. Western blotting of embryo extracts injected with different Xlim-1 constructs confirmed the specificity of the antibody. Beginning at the gastrula stage, Xlim-1 protein was detected in three cell lineages: (i) notochord, (ii) pronephros and (iii) certain regions of the central nervous system, in agreement with earlier studies of the expression of Xlim-1 RNA (Taira et al., Development 120: 1525-1536, 1994a). In addition, several new locations of Xlim-1 expression were found, including the olfactory organ, retina, otic vesicle, dorsal root ganglia and adrenal gland. Similar expression patterns were seen for the Lim-1 protein in frog and rodent tissues. These observations implicate the Xlim-1 gene in the specification of multiple cell lineages, particularly within the nervous system, and emphasize the conserved nature of the role of this gene in different vertebrate animals.

Expression of mesoderm markers in Xenopus laevis Keller explants.

In an attempt to document at the molecular level the behavior of mesodermal cells in Keller explant preparation, we have analyzed the time course of expression of four molecular markers of mesoderm gsc, Xbra, Xnot and XLIM-1. Our findings demonstrate that, (i) all mesodermal markers tested were expressed in the explants, but patterning of the mesoderm appeared incomplete; (ii) during convergence and extension of the explants, mesodermal cells did not invade the ectodermal tissue at any time tested, supporting the view that mesoderm establishes exclusively planar contacts with the ectoderm in this preparation; (iii) planar contacts were not sufficient to promote the neural expression of XLIM-1 protein in these explants.

[Localization in the body of the mobile element MDG1 of 2 regions specifically binding with Drosophila nuclear proteins]. / Lokalizatsiia v tele mobil'nogo élementa MDG1 dvukh zon, spetsificheski sviazyvaiushchikhsia s iadernymi belkami drozofily.

Two regions in mdg1 mobile element's body can specifically bind nuclear proteins of Drosophila melanogaster, as demonstrated by the method of retention of DNA-protein complexes of nitrocellulose filters. The first region is situated in the 5'-end part of mdg1, 1 kb downstream the site of initiation of transcription and contains long oligo (A) blocks (from 14 to 30 nucleotides) in the coding chain. The second region is localized near the 3'-LTR and consists of tandem 14-nucleotide repeats and a palindrome, destruction of which leads to weaker binding. There is no competition between the two regions for proteins, which evidence that they are recognized by the different proteins. The binding with the first region can be suppressed by adding the 412 mobile element DNA. These regions are supposed to take place in the regulation of mdg1 transcription.

[Interaction of products of su(Hw) and su(f) genes with MDG4 region regulating its transcription suppresses mutations in Drosophila caused by insertion of this gene]. / Vzaimodeistvie produktov genov su(Hw) i su(f) s oblast'iu MDG4, reguliruiushchei ego transkriptsiiu, supressiruet mutatsii u drozofily, vyzvannye vstraivaniem étogo gena.

The mdg4 (gypsy) mobile element of Drosophila contains two closely situated regions binding to proteins from nuclear extracts. One of these is an imperfect palindrome having homology with the lac operator of Escherichia coli, the other contains a reiterated sequence (5'PyPuTCTGCATACTPyPy) homologous to the octamer which is the core of many enhancers and upstream promoter elements. The transient expression of deletion mutants has shown that these DNA regions are negative and positive regulators, respectively, of mdg4 transcription. As was demonstrated earlier, mutations induced by the presence of mdg4 at different loci are suppressed, owing to either repression or activation of mdg4 transcription in Drosophila lines carrying unlinked mutations in su(Hw) or su(f) genes. We have shown that binding to a negative regulator (silencer) is weakened in nuclear extracts isolated from cell lines carrying su(f) mutations which activate mdg4 transcription; therefore, the su(f) gene codes for a protein capable of mdg4 repression. Furthermore, binding to a positive regulator is weakened in nuclear extracts isolated from cell lines carrying su(Hw) gene mutations which decrease the level of mdg4 transcription; hence, the su(Hw) gene encodes a protein which activates mdg4 transcription.

Suppression in Drosophila: su(Hw) and su(f) gene products interact with a region of gypsy (mdg4) regulating its transcriptional activity.

The gypsy (mdg4) mobile element of Drosophila contains two closely spaced regions which bind proteins from nuclear extracts. One of these is an imperfect palindrome having homology with the lac-operator of Escherichia coli; the other contains a reiterated sequence (5'PyPuT/C TGCATAC/TPyPy) homologous to the octamer that is the core of many enhancers and upstream promoter elements. Transient expression of deletion mutants has shown that these DNA regions are negative and positive regulators of transcription. As was demonstrated earlier by other authors, mutations induced by the presence of gypsy in different loci are suppressed owing to either repression or activation of gypsy transcription in Drosophila strains carrying unlinked mutations in su(Hw) or su(f) genes. We have shown that binding to a negative regulator (silencer) is weakened in nuclear extracts isolated from fly stocks carrying su(f) mutations which activate gypsy transcription; therefore the su(f) gene seems to code for a protein capable of gypsy repression. Furthermore, binding to a positive regulator is weakened in nuclear extracts isolated from fly stocks carrying su(Hw) gene mutations which decrease the level of gypsy transcription; therefore, the su(Hw) gene most likely encodes a protein which activates gypsy transcription.

Cell differentiation as assayed by the topography and number of ribosomal genes.

In situ hybridization, Ag-staining and electron microscopy were used to study the distribution of ribosomal genes in isolated nuclei of rat cerebellar cells and the correspondence of the ribosomal genome topography to the nucleolar structure. rDNA-DNA autoradiography revealed clusters of silver grains, as well as diffuse groups and rows. The cluster frequencies corresponded to the frequencies of nucleoli on Ag-stained slides. Competitive hybridization in situ using unlabelled rat rRNA and hybridization with a nonspacer rDNA fragment showed that the diffuse groups and rows of grains also correspond to the ribosomal genes. Spatial organization of the ribosomal genome in the Purkinje cells differs from that in the other cerebellar neurons and glial cells. A 1.5-fold redundancy of the ribosomal genes was found in some Purkinje cells, while most of these as well as microneurons contained the diploid value of the genes.

[Intra- and interpopulation heterogeneity of the cerebellar nerve cells in topography and ribosomal gene content]. / Vnutri- i mezhpopuliatsionnaia geterogennost' nervnykh kletok mozzhechka po topografii i soderzhaniiu ribosomnykh genov.

Using the method of in situ hybridization of ribosomal DNA with DNA of isolated nuclei, a study was made of localization and relative content of ribosomal genes in the Purkinje cells and other cells of the rat cerebellum. It has been shown that various cells of cerebellum have, on the average, the same number of ribosomal genes. A subpopulation with amplified ribosomal genes have been found among the nuclei of Purkinje's cells.

Nonhistone protein with high affinity for histone H1 and HMG 14 protein.

Specific interaction of a nonhistone protein from mouse spleen chromatin with histones HI, H2A and HMG 14 protein is shown. Some implications of these findings are briefly discussed.

[Transcription-active portions of the chromatin]. / Transkriptsionno-aktivnye uchastki khromatina.

A review of data on transcriptionally active chromatin obtained during the last 5--7 years. The role of hypersensitive regions, of non-histone proteins, of DNA undermethylation and the participation of Hl histone in the organization of active chromatin are considered. The role of some of these processes in the mechanisms determining cell differentiation during ontogenesis is discussed.

[Non-histone chromatin proteins]. / Negistonovye belki khromatina.

Recent data on non-histone chromatin proteins are reviewed from the point of their possible participation in the regulation of differential genetic expression. Individual proteins as well as defined groups are considered. The existence of non-specific structural regulators of transcription, i.e. of proteins common to the structure of all active chromatin regions, is proposed.

[Isoelectric focusing with silver impregnation of proteins]. / Izoélektrofokusirovanie s impregnatsiei belkov serebrom.

Silver impregnation of polyacrylamide gel sheets after isoelectrofocusing is described. This method of staining allows to increase the sensitivity of the traditional isoelectrofocusing 5 to 10 times, as compared with staining of gels by Coumassi brilliant-blue R-250 (in case of analysis of complex protein mixtures). When analyzing individual protein preparations, the sensitivity of isoelectrofocusing with silver staining is within the range of several dozens of ng per sample. A special attention is paid to methods permitting to decrease the non-specific background staining of gel by silver.

[Interaction of the non-histone protein PS1 with some chromatin components]. / Bzaimodeistvie negistonovogo belka PS1 a nekotorymi komponentami khromatina.

The binding of non-histone protein from mouse spleen chromatin located in the sites highly sensitive to micrococcal nuclease and DNA-ase I, to DNA and histones was studied. The binding of the DNA-protein complexes to nitrocellulose filters demonstrated the absence of protein binding to DNA. A highly selective binding of protein PS1 to histones H1 and H2A and to one of the non-histone proteins (presumably HMG 14) was revealed. It is concluded that protein PS1 is incorporated into chromatin by the protein-protein interactions.

[Use of polyethylene glycol for the microinjection of exogenous protein into cultured murine cells]. / Ispol'zovanie poliétilenglikolia dlia mikroin"ektsii ékzogennogo belka v kul'tiviruemye kletki myshi.

Polyethylene glycol was applied to microinject two exogenous proteins: bovine serum albumin and non-histone protein derived from mouse spleen chromatin, into the mouse L-cells. The effectiveness of fusion of mammal (human, in the given case) erythrocytes in which hemoglobin is substituted for the protein under study was shown to be higher than when Sendai virus was used. The microinjected proteins preserve their specificity to subcellular structures.