Biomedical subjects

Zbynek Kozmik

Publications and source records attributed to Zbynek Kozmik.

12 recordsLinked to original sources

PubMed2006

Wnt-mediated down-regulation of Sp1 target genes by a transcriptional repressor Sp5.

Wnt/beta-catenin signaling regulates many processes during vertebrate development. To study transcriptional targets of canonical Wnt signaling, we used the conditional Cre/loxP system in mouse to ectopically activate beta-catenin during central nervous system development. We show that the activation of Wnt/beta-catenin signaling in the embryonic mouse telencephalon results in the up-regulation of Sp5 gene, which encodes a member of the Sp1 transcription factor family. A proximal promoter of Sp5 gene is highly evolutionarily conserved and contains five TCF/LEF binding sites that mediate direct regulation of Sp5 expression by canonical Wnt signaling. We provide evidence that Sp5 works as a transcriptional repressor and has three independent repressor domains, called R1, R2, and R3, respectively. Furthermore, we show that the repression activity of R1 domain is mediated through direct interaction with a transcriptional corepressor mSin3a. Finally, our data strongly suggest that Sp5 has the same DNA binding specificity as Sp1 and represses Sp1 target genes such as p21. We conclude that Sp5 transcription factor mediates the downstream responses to Wnt/beta-catenin signaling by directly repressing Sp1 target genes.

Amino Acid Sequence↗

PubMed2005

The cellular fate of cortical progenitors is not maintained in neurosphere cultures.

Neural progenitors of the mouse forebrain can be propagated in vitro as neurospheres in the presence of bFGF and EGF. However, less is understood whether regional characteristics or developmental stage properties of these cells are maintained in neurosphere cultures. Here we show that the original cell fate is lost in neurosphere cultures. We isolated neural progenitors from the dorsal telencephalon of D6-GFP mice and cultured them in vitro. The expression profile was specifically changed in cultured cells in just three passages. Markers of the dorsal forebrain were downregulated and several ventrally-expressed genes were induced. The altered gene expression led to a profound phenotypic change of cultured cells. D6-GFP positive cortical progenitors produce excitatory neurons in the cortex and few astrocytes in vivo but after culture in vitro, these cells differentiate into many astrocytes and also oligodendrocytes and inhibitory neurons. Wnt signaling in cultured neurospheres was downregulated in the same manner as other dorsal markers but dominant active Wnt signaling slowed down the loss of the dorsal identity in neurospheres.

Animals↗

PubMed2005

The Optimedin gene is a downstream target of Pax6.

The Optimedin gene, also known as Olfactomedin 3, encodes an olfactomedin domain-containing protein. There are two major splice variants of the Optimedin mRNA, Optimedin A and Optimedin B, transcribed from different promoters. The expression pattern of the Optimedin A variant in the eye and brain overlaps with that for Pax6, which encodes a protein containing the paired and homeobox DNA-binding domains. The Pax6 gene plays a critical role for the development of eyes, central nervous system, and endocrine glands. The proximal promoter of the Optimedin A variant contains a putative Pax6 binding site in position -86/-70. Pax6 binds this site through the paired domain in vitro as judged by electrophoretic mobility shift assay. Mutations in this site eliminate Pax6 binding as well as stimulation of the Optimedin promoter activity by Pax6 in transfection experiments. Pax6 occupies the binding site in the proximal promoter in vivo as demonstrated by the chromatin immunoprecipitation assay. Altogether these results identify the Optimedin gene as a downstream target regulated by Pax6. Although the function of optimedin is still not clear, it is suggested to be involved in cell-cell adhesion and cell attachment to the extracellular matrix. Pax6 regulation of Optimedin in the eye and brain may directly affect multiple developmental processes, including cell migration and axon growth.

Alternative Splicing↗

PubMed2005

Pax genes in eye development and evolution.

Animal eyes with widely different anatomical designs have long been thought to arise independently, multiple times during evolution. This view was challenged about a decade ago by the landmark discoveries that Pax6, a highly conserved transcription factor, plays a key role in eye morphogenesis in both flies and mammals. Since then, more evidence has emerged in favour of the redeployment of Pax6 and some other developmental control genes within the genetic program underlying eye formation throughout the animal kingdom. Recent work has indicated that other members of the Pax gene family play a pivotal role in eye morphogenesis. The Eye gone gene regulates eye growth in Drosophila, whereas the PaxB gene is implicated in visual system development in jellyfish, the most basal organism possessing eyes.

Animals↗

PubMed2004

Cubozoan jellyfish: an Evo/Devo model for eyes and other sensory systems.

Cnidaria are the most basal phylum containing a well-developed visual system located on specialized sensory structures (rhopalia) with eyes and statocyts. We have been exploring the cubozoan jellyfish, Tripedalia cystophora. In addition to containing simple photoreceptive ocelli, each rhopalium in Tridedalia has a large and small complex, camera-type eye with a cellular lens containing three distinct families of crystallins which apparently serve non-lenticular functions. Thus, Tridpedalia recruited crystallins by a gene sharing strategy as have mollusks and vertebrates. Tripedalia has a single Pax gene, PaxB, which encodes a structural and functional Pax 2/5/8-like paired domain as well as an octapeptide and Pax6-like homeodomain. PaxB binds to and activates Tripedalia crystallin promoters (especially J3-crystallin) and the Drosophila rhodopsin rh6 gene in transfection tests and induces ectopic eyes in Drosophila. In situ hybridization showed that PaxB and crystallin genes are expressed in the lens, retina and statocysts. We suggest from these results that an ancestral PaxB gene was a primordial gene in eye evolution and that eyes and ears (mechanoreceptors) may have had a common evolutionary origin. Thus, the numerous structural and molecular features of Tridpalia rhopalia indicate that ancient cubozoan jellyfish are fascinating models for evo/devo insights into eyes and other sensory systems.

Animals↗

PubMed2003

DACH1 inhibits transforming growth factor-beta signaling through binding Smad4.

The vertebrate homologues of Drosophila dachsund, DACH1 and DACH2, have been implicated as important regulatory genes in development. DACH1 plays a role in retinal and pituitary precursor cell proliferation and DACH2 plays a specific role in myogenesis. DACH proteins contain a domain (DS domain) that is conserved with the proto-oncogenes Ski and Sno. Since the Ski/Sno proto-oncogenes repress AP-1 and SMAD signaling, we hypothesized that DACH1 might play a similar cellular function. Herein, DACH1 was found to be expressed in breast cancer cell lines and to inhibit transforming growth factor-beta (TGF-beta)-induced apoptosis. DACH1 repressed TGF-beta induction of AP-1 and Smad signaling in gene reporter assays and repressed endogenous TGF-beta-responsive genes by microarray analyses. DACH1 bound to endogenous NCoR and Smad4 in cultured cells and DACH1 co-localized with NCoR in nuclear dotlike structures. NCoR enhanced DACH1 repression, and the repression of TGF-beta-induced AP-1 or Smad signaling by DACH1 required the DACH1 DS domain. The DS domain of DACH was sufficient for NCoR binding at a Smad4-binding site. Smad4 was required for DACH1 repression of Smad signaling. In Smad4 null HTB-134 cells, DACH1 inhibited the activation of SBE-4 reporter activity induced by Smad2 or Smad3 only in the presence of Smad4. DACH1 participates in the negative regulation of TGF-beta signaling by interacting with NCoR and Smad4.

Apoptosis↗

PubMed2003

Gelsolin is a dorsalizing factor in zebrafish.

The gene for gelsolin (an actin-binding, cytoskeletal regulatory protein) was shown earlier to be specialized for high corneal expression in adult zebrafish. We show here that zebrafish gelsolin is required for proper dorsalization during embryogenesis. Inhibition of gelsolin expression by injecting fertilized eggs with a specific morpholino oligonucleotide resulted in a range of concentration-dependent ventralized phenotypes, including those lacking a brain and eyes. These were rescued by coinjection of zebrafish gelsolin or chordin (a known dorsalizing agent) mRNAs, or human gelsolin protein. Moreover, injection of gelsolin mRNA or human gelsolin protein by itself dorsalized the developing embryos, often resulting in axis duplication. Injection of the gelsolin-specific morpholino oligonucleotide enhanced the expression of Vent mRNA, a ventral marker downstream of bone morphogenetic proteins, whereas injection of gelsolin mRNA enhanced the expression of chordin and goosecoid mRNAs, both dorsal markers. Our results indicate that gelsolin also modulates embryonic dorsalventral pattern formation in zebrafish.

Animals↗

PubMed2003

Role of Pax genes in eye evolution: a cnidarian PaxB gene uniting Pax2 and Pax6 functions.

PaxB from Tripedalia cystophora, a cubomedusan jellyfish possessing complex eyes (ocelli), was characterized. PaxB, the only Pax gene found in this cnidarian, is expressed in the larva, retina, lens, and statocyst. PaxB contains a Pax2/5/8-type paired domain and octapeptide, but a Pax6 prd-type homeodomain. Pax2/5/8-like properties of PaxB include a DNA binding specificity of the paired domain, activation and inhibitory domains, and the ability to rescue spa(pol), a Drosophila Pax2 eye mutant. Like Pax6, PaxB activates jellyfish crystallin and Drosophila rhodopsin rh6 promoters and induces small ectopic eyes in Drosophila. Pax6 has been considered a "master" control gene for eye development. Our data suggest that the ancestor of jellyfish PaxB, a PaxB-like protein, was the primordial Pax protein in eye evolution and that Pax6-like genes evolved in triploblasts after separation from Cnidaria, raising the possibility that cnidarian and sophisticated triploblastic eyes arose independently.

Amino Acid Sequence↗

PubMed2003

Cloning and developmental expression of amphioxus Dachschund.

The nuclear factor dachshund (dac) is a key regulator of eye and leg development in Drosophila. We have cloned a Dachshund homologue from an invertebrate chordate amphioxus (Branchiostoma floridae). Sequence comparison reveals a high degree of similarity of amphioxus Dachshund (AmphiDach) to the known vertebrate and Drosophila dachshund genes. AmphiDach is first expressed in the prospective paraxial mesoderm at the gastrula stage. At the early neurula stage, expression is detected in developing somites and anterior endoderm, but in late neurula transcripts are present exclusively in the most posterior region of the cerebral vesicle and the anterior pharynx endoderm. Then, in larva, AmphiDach is localized in photoreceptive neurons of the frontal eye, infundibular organ, and endostyle as well as in Hesse organs and in nerve cells scattered along the nerve cord. Comparison of Dach expression in amphioxus and vertebrates suggests that such patterns are relatively similar (because they are expressed in somites, photoreceptor cells and CNS), even if expression of AmphiDach in the endostyle has no counterparts in its vertebrate homolog, the thyroid.

Amino Acid Sequence↗

PubMed2002

Complex regulatory element within the gammaE- and gammaF-crystallin enhancers mediates Pax6 regulation and is required for induction by retinoic acid.

The paired domain, DNA-binding domain of Pax6 and other Pax transcription factors, is composed of two subdomains (PAI and RED), each recognizing distinct half-sites of the bipartite binding site in adjacent major grooves of the DNA helix. The alternatively spliced Pax6(5a) isoform containing 14 extra amino acids within the PAI domain recognizes the 5aCON sequence consisting of four interdigitated 5' half-sites of the bipartite consensus sequence. A genome database search for similar tetrameric Pax6(A) recognition sequences led to the identification of a Pax6-binding site in the lens-specific enhancer of the mouse E- and F-crystallin genes. This binding site combines the properties of bipartite and tetrameric recognition sequences and, by mutational analysis, is shown to mediate Pax6-dependent regulation of the E- and F-crystallin promoter constructs both in primary chicken lens cells and in chicken embryo fibroblasts. The Pax6-binding site is adjacent to a previously identified retinoic acid response element and is itself required for retinoic acid induction of the F- and E-crystallin genes, suggesting that Pax proteins and retinoic acid receptors cooperate in transcriptional regulation. In summary, our protein-DNA binding and transactivation studies suggest that -crystallin genes are under the control of a multifunctional enhancer element that mediates Pax6 regulation as well as retinoic acid-mediated induction.

Animals↗

PubMed2002

Functional equivalency of amphioxus and vertebrate Pax258 transcription factors suggests that the activation of mid-hindbrain specific genes in vertebrates occurs via the recruitment of Pax regulatory elements.

Pax genes encode transcription factors that control key developmental decisions in various animal phyla. The Pax2/5/8 subfamily plays a key role in specification and/or maintenance of vertebrate mid-hindbrain boundary (MHB) region by directly regulating expression of other genes, most notably En2. In the invertebrate chordate amphioxus, expression of AmphiPax2/5/8 is found in many sites that are homologous to the regions of the vertebrate embryo expressing orthologous genes Pax2, Pax5 or Pax8. However, no co-expression of AmphiPax2/5/8 and AmphiEn is detected in the region of the neural tube that might correspond to the vertebrate MHB. Based on this observation and the absence of AmphiWnt expression in this region it appears that amphioxus does not have a MHB. Here we investigated the possibility that the AmphiPax2/5/8, as a key component of MHB development, has lost some of the properties of its vertebrate counterparts. We have analyzed both the DNA-binding and transactivation properties of AmphiPax2/5/8 as well as its ability to interact with the groucho co-repressor. In all these assays AmphiPax2/5/8 is indistinguishable from the human Pax5. In addition, we found two alternatively spliced AmphiPax2/5/8 isoforms that function similarly to the alternatively spliced isoforms of human Pax8. Analysis of the AmphiEn regulatory region provided no evidence for AmphiPax2/5/8 binding and transactivation. Therefore, in amphioxus, AmphiPax2/5/8, although capable of performing all the necessary functions has not been recruited for a developmental mechanism which usually sets up MHB development in vertebrates.

3T3 Cells↗

PubMed2001

Structure and expression of the scallop Omega-crystallin gene. Evidence for convergent evolution of promoter sequences.

Omega-crystallin of the scallop lens is an inactive aldehyde dehydrogenase (1A9). Here we have cloned the scallop Omega-crystallin gene. Except for an extra novel first exon, its 14-exon structure agrees well with that of mammalian aldehyde dehydrogenases 1, 2, and 6. The -2120/+63, -714/+63, and -156/+63 Omega-crystallin promoter fragments drive the luciferase reporter gene in transfected alphaTN4-1 lens cells and L929 fibroblasts but not in Cos7 cells. Putative binding sequences for cAMP-responsive element-binding protein (CREB)/Jun, alphaACRYBP1, AP-1, and PAX-6 in the Omega-crystallin promoter are surprisingly similar to the cis-elements used for lens promoter activity of the mouse and chicken alphaA-crystallin genes, which encode proteins homologous to small heat shock proteins. Site-specific mutations in the overlapping CREB/Jun and Pax-6 sites abolished activity of the Omega-crystallin promoter in transfected cells. Gel shift experiments utilizing extracts from the alphaTN4-1, L929, and Cos7 cells and the scallop stomach and oligonucleotides derived from the putative binding sites of the Omega-crystallin promoter showed complex formation. Gel shift experiments showed binding of recombinant Pax-6 and CREB to their respective sites. Our data suggest convergent evolutionary adaptations that underlie the preferential expression of crystallin genes in the lens of vertebrates and invertebrates.

Animals↗