Showing papers in "EMBO Reports in 2003"

Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector.

Abstract: We have designed a doxycycline-regulated form of the H1 promoter of RNA polymerase III that allows the inducible knockdown of gene expression by small interfering RNAs (siRNAs). As a proof-of-principle, we have targeted β-catenin in colorectal cancer (CRC) cells. T-cell factor (TCF) target-gene expression is induced by accumulated β-catenin, and is the main transforming event in these cells. We have shown previously that the disruption of β-catenin/TCF4 activity in CRC cells by the overexpression of dominant-negative TCF induces rapid G1 arrest and differentiation. Stable integration of our inducible siRNA vector allowed the rapid production of siRNAs on doxycycline induction, followed by specific downregulation of β-catenin. In these CRC cells, TCF reporter-gene activity was inhibited, and G1 arrest and differentiation occurred. The inhibition of two other genes using this vector system shows that it should be useful for the inducible knockdown of gene expression.

Dendritic‐cell‐specific ICAM3‐grabbing non‐integrin is essential for the productive infection of human dendritic cells by mosquito‐cell‐derived dengue viruses

Abstract: Dengue virus (DV) is a mosquito-borne flavivirus that causes haemorrhagic fever in humans. DV primarily targets immature dendritic cells (DCs) after a bite by an infected mosquito vector. Here, we analysed the interactions between DV and human-monocyte-derived DCs at the level of virus entry. We show that the DC-specific ICAM3-grabbing non-integrin (DC-SIGN) molecule, a cell-surface, mannose-specific, C-type lectin, binds mosquito-cell-derived DVs and allows viral replication. Conclusive evidence for the involvement of DC-SIGN in DV infection was obtained by the inhibition of viral infection by anti-DC-SIGN antibodies and by the soluble tetrameric ectodomain of DC-SIGN. Our data show that DC-SIGN functions as a DV-binding lectin by interacting with the DV envelope glycoprotein. Mosquito-cell-derived DVs may have differential infectivity for DC-SIGN-expressing cells. We suggest that the differential use of DC-SIGN by viral envelope glycoproteins may account for the immunopathogenesis of DVs.

A genomic overview of pyridoxal-phosphate-dependent enzymes.

Abstract: Enzymes that use the cofactor pyridoxal phosphate (PLP) constitute a ubiquitous class of biocatalysts. Here, we analyse their variety and genomic distribution as an example of the current opportunities and challenges for the study of protein families. In many free-living prokaryotes, almost 1.5% of all genes code for PLP-dependent enzymes, but in higher eukaryotes the percentage is substantially lower, consistent with these catalysts being involved mainly in basic metabolism. Assigning the function of PLP-dependent enzymes simply on the basis of sequence criteria is not straightforward because, as a consequence of their common mechanistic features, these enzymes have intricate evolutionary relationships. Thus, many genes for PLP-dependent enzymes remain functionally unclassified, and several of them might encode undescribed catalytic activities. In addition, PLP-dependent enzymes often show catalytic promiscuity (that is, a single enzyme catalyses different reactions), implying that an organism can have more PLP-dependent activities than it has genes for PLP-dependent enzymes. This observation presumably applies to many other classes of protein-encoding genes.

Regulating mammalian checkpoints through Cdc25 inactivation.

Abstract: Precise monitoring of DNA replication and chromosome segregation ensures that there is accurate transmission of genetic information from a cell to its daughters. Eukaryotic cells have developed a complex network of checkpoint pathways that sense DNA lesions and defects in chromosome segregation, spindle assembly and the centrosome cycle, leading to an inhibition of cell-cycle progression for the time required to remove the defect and thus preventing genomic instability. The activation of checkpoints that are responsive to DNA damage or incomplete DNA replication ultimately results in the inhibition of cyclin-dependent kinases. This review focuses on our understanding of the biochemical mechanisms that specifically inactivate Cdc25 (cell division cycle 25) phosphatases to achieve this. The evidence for links between checkpoint deregulation and oncogenesis is discussed.

Modification with SUMO. A role in transcriptional regulation.

Abstract: Small ubiquitin-related modifier (SUMO) is a protein moiety that is ligated to lysine residues in a variety of target proteins. The addition of SUMO can modulate the ability of proteins to interact with their partners, alter their patterns of subcellular localization and control their stability. It is clear that SUMO influences many different biological processes, but recent data suggest that it is particularly important in the regulation of transcription. Indeed, several transcription factors, such as Sp3, c-Jun, c-Myb and various nuclear receptors, have recently been shown to be subject to sumoylation and, although this modification can have a positive influence, a growing body of evidence highlights its role in the negative regulation of transcription. This review summarizes recent experiments focusing on sumoylation and transcriptional repression.

An essential function of the mitogen-activated protein kinase Erk2 in mouse trophoblast development.

Abstract: The closely related mitogen-activated protein kinase isoforms extracellular signal-regulated kinase 1 (ERK1) and ERK2 have been implicated in the control of cell proliferation, differentiation and survival. However, the specific in vivo functions of the two ERK isoforms remain to be analysed. Here, we show that disruption of the Erk2 locus leads to embryonic lethality early in mouse development after the implantation stage. Erk2 mutant embryos fail to form the ectoplacental cone and extra-embryonic ectoderm, which give rise to mature trophoblast derivatives in the fetus. Analysis of chimeric embryos showed that Erk2 functions in a cell-autonomous manner during the development of extra-embryonic cell lineages. We also found that both Erk2 and Erk1 are widely expressed throughout early-stage embryos. The inability of Erk1 to compensate for Erk2 function suggests a specific function for Erk2 in normal trophoblast development in the mouse, probably in regulating the proliferation of polar trophectoderm cells.

MYC recruits the TIP60 histone acetyltransferase complex to chromatin

Abstract: The transcription factor MYC binds specific DNA sites in cellular chromatin and induces the acetylation of histones H3 and H4. However, the histone acetyltransferases (HATs) that are responsible for these modifications have not yet been identified. MYC associates with TRRAP, a subunit of distinct macromolecular complexes that contain the HATs GCN5/PCAF or TIP60. Although the association of MYC with GCN5 has been shown, its interaction with TIP60 has never been analysed. Here, we show that MYC associates with TIP60 and recruits it to chromatin in vivo with four other components of the TIP60 complex: TRRAP, p400, TIP48 and TIP49. Overexpression of enzymatically inactive TIP60 delays the MYC-induced acetylation of histone H4, and also reduces the level of MYC binding to chromatin. Thus, the TIP60 HAT complex is recruited to MYC-target genes and, probably with other other HATs, contributes to histone acetylation in response to mitogenic signals.

An interaction between frataxin and Isu1/Nfs1 that is crucial for Fe/S cluster synthesis on Isu1.

Abstract: Depletion of the mitochondrial matrix protein frataxin is the molecular cause of the neurodegenerative disease Friedreich ataxia. The function of frataxin is unclear, although recent studies have suggested a function of frataxin (yeast Yfh1) in iron/sulphur (Fe/S) protein biogenesis. Here, we show that Yfh1 specifically binds to the central Fe/S-cluster (ISC)-assembly complex, which is composed of the scaffold protein Isu1 and the cysteine desulphurase Nfs1. Association between Yfh1 and Isu1/Nfs1 was markedly increased by ferrous iron, but did not depend on ISCs on Isu1. Functional analyses in vivo showed an involvement of Yfh1 in de novo ISC synthesis on Isu1. Our data demonstrate a crucial function of Yfh1 in Fe/S protein biogenesis by defining its function in an early step of this essential process. The iron-dependent binding of Yfh1 to Isu1/Nfs1 suggests a role of frataxin/Yfh1 in iron loading of the Isu scaffold proteins.

Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived small interfering RNAs

Abstract: Small interfering RNAs (siRNAs) efficiently inhibit gene expression by RNA interference. Here, we report efficient inhibition, by both synthetic and vector-derived siRNAs, of hepatitis C virus (HCV) replication, as well as viral protein synthesis, using an HCV replicon system. The siRNAs were designed to target the 5′ untranslated region (5′ UTR) of the HCV genome, which has an internal ribosomal entry site for the translation of the entire viral polyprotein. Moreover, the 5′ UTR is the most conserved region in the HCV genome, making it an ideal target for siRNAs. Importantly, we have identified an effective site in the 5′ UTR at which ∼80% suppression of HCV replication was achieved with concentrations of siRNA as low as 2.5 nM. Furthermore, DNA-based vectors expressing siRNA against HCV were also effective, which might allow the efficient delivery of RNAi into hepatocytes in vivo using viral vectors. Our results support the feasibility of using siRNA-based gene therapy to inhibit HCV replication, which may prove to be valuable in the treatment of hepatitis C.

Identification of the fertility restoration locus, Rfo, in radish, as a member of the pentatricopeptide-repeat protein family

Abstract: Ogura cytoplasmic male sterility (CMS) in radish (Raphanus sativus) is caused by an aberrant mitochondrial gene, Orf138, that prevents the production of functional pollen without affecting female fertility Rfo, a nuclear gene that restores male fertility, alters the expression of Orf138 at the post-transcriptional level The Ogura CMS/Rfo two-component system is a useful model for investigating nuclear-cytoplasmic interactions, as well as the physiological basis of fertility restoration Using a combination of positional cloning and microsynteny analysis of Arabidopsis thaliana and radish, we genetically and physically delimited the Rfo locus to a 15-kb DNA segment Analysis of this segment shows that Rfo is a member of the pentatricopeptide repeat (PPR) family In Arabidopsis, this family contains more than 450 members of unknown function, although most of them are predicted to be targeted to mitochondria and chloroplasts and are thought to have roles in organellar gene expression

Parallel analysis of transcript and metabolic profiles: a new approach in systems biology

Abstract: The past few years in the medical and biological sciences have been characterized by the advent of systems biology. However, despite the well-known connectivity between the molecules described by transcriptomic, proteomic and metabolomic approaches, few studies have tried to correlate parameters across the various levels of systemic description. When comparing the discriminatory power of metabolic and RNA profiling to distinguish between different potato tuber systems, using the techniques described here suggests that metabolic profiling has a higher resolution than expression profiling. When applying pairwise transcript–metabolite correlation analyses, 571 of the 26,616 possible pairs showed significant correlation, most of which was novel and included several strong correlations to nutritionally important metabolites. We believe this approach to be of high potential value in the identification of candidate genes for modifying the metabolite content of biological systems.

The MRN complex: coordinating and mediating the response to broken chromosomes

Abstract: The MRE11–RAD50–NBS1 (MRN) protein complex has been linked to many DNA metabolic events that involve DNA double-stranded breaks (DSBs). In vertebrate cells, all three components are encoded by essential genes, and hypomorphic mutations in any of the human genes can result in genome-instability syndromes. MRN is one of the first factors to be localized to the DNA lesion, where it might initially have a structural role by tethering together, and therefore stabilizing, broken chromosomes. This suggests that MRN could function as a lesion-specific sensor. As well as binding to DNA, MRN has other roles in both the processing and assembly of large macromolecular complexes (known as foci) that facilitate efficient DSB responses. Recently, a novel mediator protein, mediator of DNA damage checkpoint protein 1 (MDC1), was shown to co-immunoprecipitate with the MRN complex and regulate MRE11 foci formation. However, whether the initial recruitment of MRN to DSBs requires MDC1 is unclear. Here, we focus on recent developments in MRN research and propose a model for how DSBs are sensed and the cellular responses to them are mediated.

Fibulins: physiological and disease perspectives

Abstract: The fibulins are a family of proteins that are associated with basement membranes and elastic extracellular matrix fibres. This review summarizes findings from studies of animal models of fibulin deficiency, human fibulin gene mutations, human tumours and injury models that have advanced our understanding of the normal and pathological roles of members of this formerly obscure family.

IQGAP proteins are integral components of cytoskeletal regulation.

Abstract: IQGAP1 is a scaffolding protein that binds to a diverse array of signalling and structural molecules. By interacting with its target proteins, human IQGAP1 participates in multiple cellular functions, including Ca2+/calmodulin signalling, cytoskeletal architecture, CDC42 and Rac signalling, E-cadherin-mediated cell–cell adhesion and β-catenin-mediated transcription. Yeast IQGAP homologues are important regulators of cellular morphogenesis because they are required for budding and cytokinesis. Here we discuss the structure and function of IQGAP1 as a member of the family of IQGAP proteins and summarize the current knowledge about IQGAP1 and IQGAP2. Collectively, these data reveal that IQGAP1 is a fundamental regulator of cytoskeletal function.

Efficient transgenesis in farm animals by lentiviral vectors

Abstract: Microinjection of DNA is now the most widespread method for generating transgenic animals, but transgenesis rates achieved this way in higher mammals are extremely low. To address this longstanding problem, we used lentiviral vectors carrying a ubiquitously active promoter (phosphoglycerate kinase, LV-PGK) to deliver transgenes to porcine embryos. Of the 46 piglets born, 32 (70%) carried the transgene DNA and 30 (94%) of these pigs expressed the transgene (green fluorescent protein, GFP). Direct fluorescence imaging and immunohistochemistry showed that GFP was expressed in all tissues of LV-PGK transgenic pigs, including germ cells. Importantly, the transgene was transmitted through the germ-line. Tissue-specific transgene expression was achieved by infecting porcine embryos with lentiviral vectors containing the human keratin K14 promoter (LV-K14). LV-K14 transgenic animals expressed GFP specifically in basal keratinocytes of the skin. Finally, infection of bovine oocytes after and before in vitro fertilization with LV-PGK resulted in transgene expression in 45% and 92% of the infected embryos, respectively.

Stimulation of c-MYC transcriptional activity and acetylation by recruitment of the cofactor CBP

Abstract: The c-MYC oncoprotein regulates various aspects of cell behaviour by modulating gene expression. Here, we report the identification of the cAMP-response-element-binding protein (CBP) as a novel c-MYC binding partner. The two proteins interact both in vitro and in cells, and CBP binds to the carboxy-terminal region of c-MYC. Importantly, CBP, as well as p300, is associated with E-box-containing promoter regions of genes that are regulated by c-MYC. Furthermore, c-MYC and CBP/p300 function synergistically in the activation of reporter-gene constructs. Thus, CBP and p300 function as positive cofactors for c-MYC. In addition, c-MYC is acetylated in cells. This modification does not require MYC box II, suggesting that it is independent of TRRAP complexes. Instead, CBP acetylates c-MYC in vitro, and co-expression of CBP with c-MYC stimulates in vivo acetylation. Functionally, this results in a decrease in ubiquitination and stabilization of c-MYC proteins. Thus, CBP and p300 are novel functional binding partners of c-MYC.

X‐ray structure of human acid‐β‐glucosidase, the defective enzyme in Gaucher disease

Abstract: Gaucher disease, the most common lysosomal storage disease, is caused by mutations in the gene that encodes acid-β-glucosidase (GlcCerase). Type 1 is characterized by hepatosplenomegaly, and types 2 and 3 by early or chronic onset of severe neurological symptoms. No clear correlation exists between the ∼200 GlcCerase mutations and disease severity, although homozygosity for the common mutations N370S and L444P is associated with non- neuronopathic and neuronopathic disease, respectively. We report the X-ray structure of GlcCerase at 2.0 A resolution. The catalytic domain consists of a (β/α)8 TIM barrel, as expected for a member of the glucosidase hydrolase A clan. The distance between the catalytic residues E235 and E340 is consistent with a catalytic mechanism of retention. N370 is located on the longest α-helix (helix 7), which has several other mutations of residues that point into the TIM barrel. Helix 7 is at the interface between the TIM barrel and a separate immunoglobulin-like domain on which L444 is located, suggesting an important regulatory or structural role for this non-catalytic domain. The structure provides the possibility of engineering improved GlcCerase for enzyme-replacement therapy, and for designing structure-based drugs aimed at restoring the activity of defective GlcCerase.

Parkin binds the Rpn10 subunit of 26S proteasomes through its ubiquitin-like domain

Abstract: Parkin, a product of the causative gene of autosomal-recessive juvenile parkinsonism (AR-JP), is a RING-type E3 ubiquitin ligase and has an amino-terminal ubiquitin-like (Ubl) domain. Although a single mutation that causes an Arg to Pro substitution at position 42 of the Ubl domain (the Arg 42 mutation) has been identified in AR-JP patients, the function of this domain is not clear. In this study, we determined the three-dimensional structure of the Ubl domain of parkin by NMR, in particular by extensive use of backbone 15N-1H residual dipolar-coupling data. Inspection of chemical-shift-perturbation data showed that the parkin Ubl domain binds the Rpn10 subunit of 26S proteasomes via the region of parkin that includes position 42. Our findings suggest that the Arg 42 mutation induces a conformational change in the Rpn10-binding site of Ubl, resulting in impaired proteasomal binding of parkin, which could be the cause of AR-JP.

Otubains: a new family of cysteine proteases in the ubiquitin pathway.

Abstract: The modification of cellular proteins by ubiquitin (Ub) is an important event that underlies protein stability and function in eukaryotes. Protein ubiquitylation is a dynamic and reversible process; attached Ub can be removed by deubiquitylating enzymes (DUBs), a heterogeneous group of cysteine proteases that cleave proteins precisely at the Ub–protein bond. Two families of DUBs have been identified previously. Here, we describe new, highly specific Ub iso-peptidases, that have no sequence homology to known DUBs, but which belong to the OTU (ovarian tumour) superfamily of proteins. Two novel proteins were isolated from HeLa cells by affinity purification using the DUB-specific inhibitor, Ub aldehyde (Ubal). We have named these proteins otubain 1 and otubain 2, for OTU-domain Ubal-binding protein. Functional analysis of otubains shows that the OTU domain contains an active cysteine protease site.

Regulating histone acetyltransferases and deacetylases

Abstract: Histone acetyltransferases and histone deacetylases regulate the acetylation of histones and transcription factors, and in doing so have major roles in the control of cell fate. Many recent results have indicated that their function is strictly regulated in cells through the modulation of their levels, activity and availability for interaction with specific transcription factors. In this review, we present the various molecular mechanisms that bring about this tight regulation and discuss how these regulatory events influence cellular responses to environmental changes.

Gli proteins and the control of spinal‐cord patterning

Abstract: The secreted protein sonic hedgehog (Shh) is crucial for the specification of neuronal subtype identity in the vertebrate neural tube. Zinc-finger proteins of the Gli family are known to be transcriptional mediators of Shh signalling, and to coordinately pattern the dorsal–ventral axis of the spinal cord. Recent studies indicate that additional signals may provide positional information in parallel to Shh to specify neuronal fate in this tissue. We review the role of Gli proteins in spinal-cord development and propose that various upstream patterning signals may be integrated by the Gli proteins to direct a coherent programme of neurogenesis.

Yeast histone 2A serine 129 is essential for the efficient repair of checkpoint-blind DNA damage

Abstract: Cells maintain genomic stability by the coordination of DNA-damage repair and cell-cycle checkpoint control. In replicating cells, DNA damage usually activates intra-S-phase checkpoint controls, which are characterized by delayed S-phase progression and increased Rad53 phosphorylation. We show that in budding yeast, the intra-S-phase checkpoint controls, although functional, are not activated by the topoisomerase I inhibitor camptothecin (CPT). In a CPT-hypersensitive mutant strain that lacks the histone 2A (H2A) phosphatidylinositol-3-OH kinase (PI(3)K) motif at Ser 129 (h2a-s129a), the hypersensitivity was found to result from a failure to process full-length chromosomal DNA molecules during ongoing replication. H2A Ser 129 is not epistatic to the RAD24 and RAD9 checkpoint genes, suggesting a non-checkpoint role for the H2A PI(3)K site. These results suggest that H2A Ser 129 is an essential component for the efficient repair of DNA double-stranded breaks (DSBs) during replication in yeast, particularly of those DSBs that do not induce the intra-S-phase checkpoint.

Priming the nucleosome: a role for HMGB proteins?

Abstract: The high-mobility-group B (HMGB) chromosomal proteins are characterized by the HMG box, a DNA-binding domain that both introduces a tight bend into DNA and binds preferentially to a variety of distorted DNA structures. The HMGB proteins seem to act primarily as architectural facilitators in the manipulation of nucleoprotein complexes; for example, in the assembly of complexes involved in recombination and transcription. Recent genetic and biochemical evidence suggests that these proteins can facilitate nucleosome remodelling. One mechanism by which HMGB proteins could prime the nucleosome for migration is to loosen the wrapped DNA and so enhance accessibility to chromatin-remodelling complexes and possibly also to transcription factors. By constraining a tight loop of untwisted DNA at the edge of a nucleosome, an HMGB protein could induce movements in the contacts between certain core histones that would result in an overall change in nucleosome structure.

Monitoring global messenger RNA changes in externally controlled microarray experiments

Abstract: Expression profiling is a universal tool, with a range of applications that benefit from the accurate determination of differential gene expression. To allow normalization using endogenous transcript levels, current microarray analyses assume that relatively few transcripts vary, or that any changes that occur are balanced. When normalization using endogenous genes is carried out, changes in expression levels are calculated relative to the behaviour of most of the transcripts. This does not reflect absolute changes if global shifts in messenger RNA populations occur. Using external RNA controls, we have set up microarray experiments to monitor global changes. The levels of most mRNAs were found to change during yeast stationary phase and human heat shock when external controls were included. Even small global changes had a significant effect on the number of genes reported as being differentially expressed. This suggests that global mRNA changes occur more frequently than is assumed at present, and shows that monitoring such effects may be important for the accurate determination of changes in gene expression.

Human arteries engineered in vitro.

Abstract: There is a pressing need to develop methods to engineer small-calibre arteries for bypass surgery. We hypothesized that the rate-limiting step that has thwarted previous attempts to engineer such vessels from non-neonatal tissues is the limited proliferative capacity of smooth muscle cells (SMCs), which are the main cellular component of these vessels. Ectopic expression of the human telomerase reverse transcriptase subunit (hTERT) has been shown recently to extend the lifespan of certain human cells. We therefore introduced hTERT into human SMCs and found that the resulting cells proliferated far beyond their normal lifespan but retained characteristics of normal control SMCs. Importantly, using these non-neonatal SMCs, we were able to engineer mechanically robust human vessels, a crucial step towards creating arteries of clinical value for bypass surgery.

Finding the right organelle

Abstract: The mitochondrial outer membrane contains a diverse set of proteins that includes enzymes, components of the preprotein translocation machinery, pore-forming proteins, regulators of programmed cell death, and those that control the morphology of the organelle. All these proteins, like the vast majority of mitochondrial proteins, are encoded in the nucleus, so they are synthesized in the cytosol and contain signals that are essential for their subsequent import into mitochondria. This review summarizes our current knowledge of the signals that target mitochondrial outer-membrane proteins to their correct intracellular location. In addition, the mechanisms by which these signals are decoded by the mitochondria are discussed.

RAL GTPases are linchpin modulators of human tumour-cell proliferation and survival.

Abstract: The monomeric RAL (RAS-like) GTPases have been indirectly implicated in mitogenic regulation and cell transformation. Here, we show that RALA and RALB collaborate to maintain tumorigenicity through regulation of both proliferation and survival. Remarkably, this task is divided between these highly homologous isoforms. RALB is specifically required for survival of tumour cells but not normal cells. RALA is dispensable for survival, but is required for anchorage-independent proliferation. Reducing the 'oncogenic burden' in human tumour cells relieves the sensitivity to loss of RALB. These observations establish RAL GTPases as crucial components of the cellular machinery that are exploited by factors that drive oncogenic transformation.

Righting the wrongs.

Abstract: DNA and protein sequence databases are increasingly useful research tools. But to maximize their potential, the errors in them need to be addressed

Integrin‐linked kinase regulates chondrocyte shape and proliferation

Abstract: The interaction of chondrocytes with the extracellular-matrix envi- ronment is mediated mainly by integrins. Ligated integrins are recruited to focal adhesions (FAs) together with scaffolding proteins and kinases, such as integrin-linked kinase (Ilk). Ilk binds the cyto- plasmic domain of β1-, β2- and β3-integrins and recruits adaptors and kinases, and is thought to stimulate downstream signalling events through phosphorylation of protein kinase B/Akt (Pkb/Akt) and glycogen synthase kinase 3-β (GSK3-β). Here, we show that mice with a chondrocyte-specific disruption of the gene encoding Ilk develop chondrodysplasia, and die at birth due to respiratory dis- tress. The chondrodysplasia was characterized by abnormal chon- drocyte shape and decreased chondrocyte proliferation. In addition, Ilk-deficient chondrocytes showed adhesion defects, failed to spread and formed fewer FAs and actin stress fibres. Surprisingly, phosphorylation of Pkb/Akt and GSK3-β is unaffected in Ilk- deficient chondrocytes. These findings suggest that Ilk regulates actin reorganization in chondrocytes and modulates chondrocyte growth independently of phosphorylation of Pkb/Akt and GSK3-β.

Loss of eyes in zebrafish caused by mutation of chokh/rx3.

Abstract: The vertebrate eye forms by specification of the retina anlage and subsequent morphogenesis of the optic vesicles, from which the neural retina differentiates. chokh (chk) mutant zebrafish lack eyes from the earliest stages in development. Marker gene analysis indicates that retinal fate is specified normally, but optic vesicle evagination and neuronal differentiation are blocked. We show that the chk gene encodes the homeodomain-containing transcription factor, Rx3. Loss of Rx3 function in another teleost, medaka, has also been shown to result in an eyeless phenotype. The medaka rx3 locus can fully rescue the zebrafish mutant phenotype. We provide evidence that the regulation of rx3 is evolutionarily conserved, whereas the downstream cascade contains significant differences in gene regulation. Thus, these mutations in orthologous genes allow us to study the evolution of vertebrate eye development at the molecular level.