A prospero-related homeobox gene Prox-1 is expressed during postnatal brain development as well as in the adult rodent brain.

Prox-1, a prospero-related homeobox gene, is known to be an important transcription factor during embryogenesis. However, very little is known about Prox-1 expression and functions in the adult nervous system. Here we have investigated the expression pattern of Prox-1 mRNA and protein during postnatal brain development and in adult rat and mouse brains using in situ hybridization (ISH), immunohistochemistry (IHC) and Western blotting. In the developing and adult brain, we found prominent, but restricted Prox-1 mRNA expression in the dentate gyrus of the hippocampus, in some thalamic nuclei, notably in the anterior thalamus, and in the cerebellar cortex. Other brain regions, such as the hypothalamus and nuclei belonging to the midbrain, revealed a moderate level of Prox-1 mRNA expression. In developing cerebral cortex, Prox-1 mRNA was seen only in the thin layer under the pial surface postnatally, and the signal almost disappeared by the 28th postnatal day (PD). Using IHC and ISH approaches, we demonstrated rather restricted, but intense Prox-1 labeling in adult brain of both rat and mouse species. During postnatal brain development Prox-1 proteins by IHC, were below the detection limit at PD 14, while Prox-1 mRNA remained at a high level. Western blotting demonstrated the existence of two different variants of Prox-1 protein, one of which was about 20 kDa larger than ordinary size. During the first PDs, the larger variant predominated. At PD 14, neither protein variant could be detected. From PD 16 onwards the smaller variant started to predominate and by PD 30 the larger size protein had almost disappeared. The prominent but limited distribution of Prox-1 in the brain suggests its potentially important role during postnatal brain development and in adult CNS, which remains to be ascertained in future studies.

Targeted Disruption of the Myocilin Gene (Myoc) Suggests that Human Glaucoma-Causing Mutations Are Gain of Function.

Glaucoma is a heterogeneous eye disease and a major cause of blindness worldwide. Recently, primary open angle glaucoma (POAG)-associated mutations have been found in the trabecular meshwork inducible glucocorticoid response gene (TIGR), also known as the myocilin gene (MYOC), at the GLC1A locus on chromosome 1q21-q31. These mutations occurred in a subset of patients with juvenile- and adult-onset POAG and exhibited autosomal dominant inheritance. Ocular expression and its involvement in POAG suggest that TIGR/MYOC may have a role(s) in regulating intraocular pressure (IOP). Here, we report the generation and analysis of mice heterozygous and homozygous for a targeted null mutation in Myoc. Our study shows that Myoc mutant mice are both viable and fertile. Our in vivo findings further demonstrate that Myoc is not required for normal IOP or normal ocular morphology. The lack of a discernable phenotype in both Myoc-heterozygous and Myoc-null mice suggests that haploinsufficiency is not a critical mechanism for POAG in individuals with mutations in MYOC. Instead, disease-causing mutations in humans likely act by gain of function.

Antagonistic action of Six3 and Prox1 at the gamma-crystallin promoter.

Gamma-crystallin genes are specifically expressed in the eye lens. Their promoters constitute excellent models to analyse tissue-specific gene expression. We investigated murine CRYGE/f promoters of different length in lens epithelial cell lines. The most active fragment extends from position -219 to +37. Computer analysis predicts homeodomain and paired-domain binding sites for all rodent CRYGD/e/f core promoters. As examples, we analysed the effects of Prox1 and Six3, which are considered important transcription factors involved in lens development. Because of endogenous Prox1 expression in N/N1003A cells, a weak stimulation of CRYGE/f promoter activity was found for PROX1. In contrast, PROX1 stimulated the CRYGF promoter 10-fold in CD5A cells without endogenous PROX1. In both cell lines Six3 repressed the CRYGF promoter to 10% of its basal activity. Our cell transfection experiments indicated that CRYG expression increases as Six3 expression decreases. Prox1 and Six3 act antagonistically on regulation of the CRYGD/e/f promoters. Functional assays using randomly mutated gammaF-crystallin promoter fragments define a Six3-responsive element between -101 and -123 and a Prox1-responsive element between -151 and -174. Since Prox1 and Six3 are present at the beginning of lens development, expression of CRYGD/e/f is predicted to remain low at this time. It increases as Six3 expression decreases during ongoing lens development.

LYVE-1 is not restricted to the lymph vessels: expression in normal liver blood sinusoids and down-regulation in human liver cancer and cirrhosis.

Lymphatic vessel endothelial hyaluronan receptor (LYVE)-1 is thought to be restricted to lymph vessels and has been used as such to show that tumor lymphangiogenesis occurs on overexpression of lymphangiogenic factors in mouse tumor models. However, these studies have not yet been corroborated in human tumors. Here we show, first, that LYVE-1 is not exclusive to the lymph vessels. Indeed, LYVE-1 is also present in normal hepatic blood sinusoidal endothelial cells in mice and humans. Surprisingly, LYVE-1 is absent from the angiogenic blood vessels of human liver tumors and only weakly present in the microcirculation of regenerative hepatic nodules in cirrhosis, though both vessels are largely derived from the liver sinusoids. Second, we propose a novel approach to identify lymphatics in human and murine liver. By combining LYVE-1 and Prox 1 (a transcription factor) immunohistochemistry, we demonstrate that lymphatics are abundant in cirrhosis. In contrast, in human hepatocellular carcinoma and liver metastases, they are restricted to the tumor margin and surrounding liver. The absence of intratumor lymphatics in hepatocellular carcinomas and liver metastases may impair molecular and cellular transport in these tumors. Finally, the presence of LYVE-1 in liver sinusoidal endothelia suggests that LYVE-1 has functions beyond the lymph vascular system.

Primary structure and lens-specific expression of genes for an intermediate filament protein and a beta-tubulin in cephalopods.

Intermediate filament (IF) protein and tubulin cDNAs of cephalopod eye lenses were cloned and sequenced. The rod regions of the deduced IF proteins of the squid and octopus were more similar (68% identical) than were head (33% identical) and tail (40% identical) regions. The rod sequences were closer to squid neuronal IF protein (39% identical) than to any other known IF protein. There was only 31% identity between the rod regions, 21-30% identity between the head regions and 23-32% identity between the tail regions of the present IF proteins of cephalopods and other invertebrates. The rod regions of the cephalopod IF proteins contained the 6 heptads characteristic of nuclear lamins, consistent with an evolutionary relationship between IF proteins and lamins. The present octopus alpha-tubulin was 93% and beta-tubulin was 87% identical to the corresponding tubulins of insects and vertebrates. SDS-PAGE and peptide sequencing indicated that the order of abundance of the cephalopod lens cytoskeletal proteins was IF proteins, actin and tubulins. Northern blot hybridization revealed a 4 kb mRNA for the octopus IF protein and 2.9 and 7.3 kb mRNAs for the squid IF protein; the alpha-tubulin mRNA was about 1.8 kb in the octopus and squid, and the beta-tubulin mRNA was about 2.8 kb in the octopus. The alpha-tubulin mRNA was present in all tissues examined; by contrast, the present beta-tubulin and IF protein mRNAs appeared specialized for lens expression.

O-Crystallin, arginine kinase and ferritin from the octopus lens.

Three proteins have been identified in the eye lens of the octopus, Octopus dofleini. A 22 kDa protein comprising 3-5% of the soluble protein of the lens is 35-43% identical to a family of phosphatidylethanolamine-binding proteins of vertebrates. Other members of this family include the immunodominant antigen of the filarial parasite, Onchocerca volvulus, putative odorant-binding proteins of Drosophila and a protein with unknown function of Caenorhabditis elegans. We have called this protein O-crystallin on the basis of its abundance in the transparent lens. O-Crystallin mRNA was detected only in the lens. Two tryptic peptides of another octopus lens protein, less abundant than O-crystallin, showed 80% identity to arginine kinase of invertebrates, a relative of creatine kinase of vertebrates. Finally, ferritin cDNA was isolated as an abundant cDNA from the octopus lens library. Northern blots showed that ferritin mRNA is not lens-specific.

Frog lens beta A1-crystallin: the nucleotide sequence of the cloned cDNA and computer graphics modelling of the three-dimensional structure.

Four recombinant cDNA clones coding for a 23 kDa beta-crystallin polypeptide of the frog (Rana temporaria) were identified in a collection of cloned cDNA and two of them were sequenced. The cDNA present in these clones codes for a polypeptide 198 amino-acid residues in length, which appears to be the frog beta A1-crystallin because of its high homology with the sequences of beta A1-crystallins from other species. Furthermore, the nucleotide sequence coding for the compact folded region of the protein is highly conserved. Virtually no homology was found in the 3' nontranslated regions of the mRNA. The amino-acid sequence of the Rana beta A1-crystallin was used to build a three-dimensional model based on the coordinates of the homologous bovine gamma II. An analysis of the model shows that the surface residues of the beta A1-crystallin (amphibian, mammalian and bird) are more highly conserved than the buried residues. It is suggested that this is related to the oligomeric nature of the lens beta-crystallins.

Restricted expression of the homeobox gene prox 1 in developing zebrafish.

Prox 1 is a vertebrate homeobox gene which is homologous to the Drosophila transcription factor, prospero. We have isolated a prox 1 cDNA from zebrafish, which encodes a protein that has 82%, 84% and 83% amino acid identity with chicken, mouse and human Prox 1, respectively. Antibodies raised against human Prox 1 cross-react with zebrafish Prox 1 and are used here to determine the expression patterns of Prox 1 during zebrafish embryogenesis by whole-mount immunohistochemistry. In the 10-somite embryo, Prox 1 is expressed over the prospective lens placode and over a broad region of epithelium extending from the eye to the otic vesicle. As embryogenesis proceeds, Prox 1 expression in the eye lens becomes intense, and is detected in maturing muscle pioneer cells and superficial muscle cells. In the CNS, Prox 1 is expressed in a stripe along the forebrain-midbrain boundary, in a segmented pattern in the ventral hindbrain, and in selected cells of the ventral spinal cord. Additional sites of Prox 1 expression include the lateral line primordium, the trigeminal ganglia, the otic vesicle and occasional endodermal cells.

Pax6 in the sepiolid squid Euprymna scolopes: evidence for a role in eye, sensory organ and brain development.

The cloning of a Pax6 orthologue from the sepiolid squid Euprymna scolopes and its developmental expression pattern are described. The data are consistent with the presence of a single gene encoding a protein with highly conserved DNA-binding paired and homeodomains. A detailed expression analysis by in situ hybridization and immunodetection revealed Pax6 mRNA and protein with predominantly nuclear localization in the developing eye, olfactory organ, brain lobes (optic lobe, olfactory lobe, peduncle lobe, superior frontal lobe and dorsal basal lobe), arms and mantle, suggestive of a role in eye, brain, and sensory organ development.

Chicken Eyes absent 2 gene: isolation and expression pattern during development.

In all vertebrates studied (human, mouse, chicken), there are at least three genes related to Drosophila eyes absent (eya) gene. The chicken Eyes absent 2 (Eya2) cDNA was isolated from 14 day embryonic chicken lenses, and a complete open reading frame encoding a 59 kDa protein was elucidated. The chicken Eya2 protein is moderately conserved and 78-82% identical to the mouse and human Eya2. The Eya2 gene demonstrated a dynamic expression pattern in different tissues of diverse embryological origin. Expression of Eya2 was first detected at Hamburger and Hamilton stage 9 in the foregut. At later stages of development, Eya2 mRNA was detected in neural crest derivatives (dorsal root ganglia, branchial arches and cranial nerve ganglia). In the cranial placodes, expression of Eya2 was first detected in the nasal pit at stage 13. In the eye, expression of Eya2 was first convincingly detected in neural retina at stage 24 (day 4). The highest level of Eya2 mRNA in the lens was detected around day 9. Eya2 is also expressed in the cornea and iris. Therefore, chicken Eya2, as well as mouse Eya2, is expressed relatively early in the nasal (but not in the lens) placode and may mediate induction of the nasal placode. Expression of Eya2 in the wing and limb buds is consistent with its proposed role in the patterning of limb connective tissues.

Pax-6, eyes absent, and Prox 1 in eye development.

Eyes in different systematic groups including arthropods, molluscs and vertebrates probably have a common evolutionary origin. As a consequence of this, related genes are used for regulation of the early steps of eye development in different organisms. In this review, I briefly summarize data on three gene families which might be essential for eye development across species: Pax-6/eyeless, Eya/eyes absent and Prox/prospero with emphasis on our contribution here. Mechanisms of eye formation and the generation of different types of eyes in the course of evolution are discussed.

Abundant mRNAs in the squid light organ encode proteins with a high similarity to mammalian peroxidases.

A library derived from mRNA in the bacterial light organ of the squid, Euprymna scolopes, contained an unexpectedly high proportion of cDNAs that encode proteins with approximately 30% similarity to a family of mammalian peroxidases (PO) including myelo-PO, eosinophil PO, and thyroid PO (donor:hydrogen-peroxide oxidoreductase; EC 1.11.1.7). Two nearly full-length cDNAs were determined to encode putative PO of nearly 93 kDa each that are 97% identical in amino acid sequence to each other. Each contains four potential glycosylation sites, and His416, believed to be within the active site of the human PO, is conserved in the putative PO from the squid light organ. The mRNAs for the putative squid PO were approximately 250 times more abundant in the tissue housing the bacterial symbiont than in the ocular lens or mantle and were undetectable in the light organ lens. By analogy with the bacteriocidal function of PO in mammalian neutrophils, the putative squid PO may be important for modulating or limiting the population of bacteria within the light organ. The possibility that the squid light organ contains a high concentration of PO raises the possibility that the light organ lens is under oxidative stress, providing a possible rationale for the recruitment of its aldehyde dehydrogenase-like crystallin.

Multiple genes coding for the frog eye lens gamma-crystallins.

Three new recombinant cDNA clones coding for gamma-crystallins have been identified in the frog (Rana temporaria) clonotheque by hybrid-selected translation/immunoprecipitation experiments, in addition to the gamma-1-crystallin clone that was isolated and sequenced previously [ Tomarev et al., Gene 17 (1982) 131-138; FEBS Letters 146 (1982) 315-318]. mRNA species coding for all these gamma-crystallins are about 650 nucleotides in length, but differ in structure, as follows from restriction and sequence analysis of the cloned cDNAs. The conclusion is that the R. temporaria genome contains a family of at least four similar but not identical gamma-crystallin genes. The complete nucleotide sequence has been determined for the cDNA of one of these clones coding for gamma-2-crystallin. It is 69% homologous with that of R. temporaria gamma-1-crystallin and contains four regions of partial internal homology corresponding to the four structural folding units of the gamma-crystallin molecules. An unusual feature of the gamma-2-crystallin amino acid sequence is the high lysine/arginine ratio equal to 1.1, in contrast to 0.05-0.16 for other known gamma-crystallins.

Molecular cloning of double-stranded cDNA from the eye lens of the frog Rana temporaria: construction of the cDNA clonotheque and identification of a clone containing the nucleotide sequences of the lambda-crystallin gene.

Poly(A)+ RNA from the lens of the frog Rana temporaria contains three components (1200 +/- 50, 1000 +/- 50, and 900 +/- 50 bp in size) and a more heterogeneous RNA species with a length of 650-750 nucleotides. This RNA was used as a template for the AMV reverse transcriptase and Escherichia coli DNA-polymerase I and the total cDNA obtained was cloned in the PstI site of the pBR322 plasmid vector. Recombinant plasmids corresponding to abundant poly(A)+ RNA classes contain cDNA inserts from less than or equal to 200 to 1200 nucleotides in length. Part of the library (clonotheque) was divided into classes differing in the presence of absence of the restriction sites for BamHI, EcoRI and HindIII restriction endonucleases. The clones belonging to each of the five classes were characterized by the hybridization-translation test. The translation product of mRNA hybridizing with the clone pRT(1)294 has an M4 of about 22 000 and is specifically precipitated by the antiserum to lambda-crystallins of Rana temporaria. The size of the cDNA present in pRT(1)294, equal to 580 +/- 20 bp, is sufficient for coding the greater part of the lambda-crystallin amino acid sequence. On the basis of these data, we conclude that the clone pRT(1)294 codes for one of the frog lambda-crystallins.

A computer graphics model of frog gamma-crystallin based on the three-dimensional structure of calf gamma-II crystallin.

Molecular models for Rana gamma-1 and gamma-2 crystallins have been constructed using computer graphics on the basis of the protein primary structure derived from the complementary DNA sequence and the three-dimensional structure of calf gamma-II crystallin that has been defined at high resolution by X-ray analysis. The models show that the cores of the two domains are conserved as hydrophobic, with the polypeptide chain arranged as a four Greek-key motif structure. Although many lysines replace arginines at equivalent positions in mammalian proteins, the Rana crystallins also have an extensive series of ion pairs on their surface; these are strongly implicated in their function as stable structural molecules, which are highly conserved in the evolution of the vertebrate eye lens.

The absence of the long 3'-non-translated region in mRNA coding for eye lens alpha A2-crystallin of the frog (Rana temporaria).

The nucleotide sequence of a cloned cDNA (clone pRt(1)297; GENE (1982) 17, 131) coding for a 18 kDa polypeptide of the frog eye lens has been determined. The sequence, 791 nucleotide in length has only one long open reading frame (447 nucleotides). The derived amino acid sequence in this frame has greater than 90% homology with the region 25-173 of alpha A2-crystallin amino acid sequence from a related frog species Rana pipiens. The 5'-terminal part of mRNA corresponding to the first 24 amino acids of alpha A2-crystallin has been lost in cloning and substituted by an artefactual sequence. The 3'-terminal part appears to be intact as follows from the presence of the universal poly(A) addition site and poly(A) tract. The 3'-nontranslated region present in frog alpha A2-crystallin mRNA (130 nucleotides) is about 4-times shorter than in mammalian alpha A2-crystallin mRNA. Intact alpha A2-crystallin mRNA with a size of about 700 nucleotides as determined by Northern blot hybridization is about twice smaller than corresponding mammalian mRNAs.

A novel type of crystallin in the frog eye lens. 35-kDa polypeptide is not homologous to any of the major classes of lens crystallins.

The nucleotide sequence of a cloned DNA coding for the 35-kDa polypeptide of the eye lens of the frog (Rana temporaria) has been determined. The sequence without connectors and poly(A) tract is 889 nucleotides in length and shows no homology with sequences coding for other classes of crystallins: alpha-, beta-, gamma- or delta-crystallins. The sequence contains one reading frame 675 nucleotides in length, an apparently intact 3'-non-translated region with the polyadenylation signal sequence and a poly(A) tract; the 5'-non-translated region is lost along with part of the coding region; this accounts for about 1/4 of the total mRNA length. The secondary structure prediction according to the Ptitsin - Finkelstein method shows the presence of predominantly beta-strands with only a few alpha-helical regions. We conclude that the 35-kDa polypeptide from the frog eye lens belongs to a new class of eye lens crystallins for which we propose the name epsilon-crystallin.

Regulation of Prox 1 during lens regeneration.

PURPOSE: To determine the expression pattern of Prox 1 during the process of lens regeneration in the urodele Notophthalmus viridescens. METHODS: Polymerase chain reaction was performed to amplify a partial newt Prox 1 sequence. In situ hybridization and immunodetection methods were used to detect the Prox 1 mRNA and the Prox 1 protein, respectively. RESULTS: Prox 1 mRNA was present in the retina and in the lens (in the epithelium and bow region) of the intact eye. Prox 1 protein was found to be predominantly present in the lens and dorsal iris of the intact eye, although some trace levels of Prox 1 protein were detected in the ventral iris as well. After lentectomy, expression of the mRNA was also pronounced in the dorsal dedifferentiating iris and the regenerating lens. The ventral iris also expressed Prox 1 but seemingly at lower levels. Although Prox 1 protein showed upregulation in the dorsal iris during the process of lens regeneration, trace levels were also detected in the ventral iris. In the retina, Prox 1 protein was distributed in horizontal cells of the inner nuclear layer, whereas the mRNA was expressed in all layers of the retina. CONCLUSIONS: Prox 1 was unevenly distributed in the intact cells of the newt iris, with significantly higher levels of Prox 1 protein present in the dorsal versus the ventral margin. This protein was differentially regulated during the process of lens regeneration, with obvious upregulation in the dorsal iris. Prox 1 is the first transcriptional factor to be shown to be regulated in the dorsal versus ventral iris during the process of lens regeneration.

Changes in mRNA levels of the Myoc/Tigr gene in the rat eye after experimental elevation of intraocular pressure or optic nerve transection.

PURPOSE: To isolate the rat Myoc/Tigr gene and investigate changes in its expression pattern in normal eyes and in eyes with either pressure-induced optic nerve damage or optic nerve transection. METHODS: Expression pattern of the rat Myoc/Tigr gene was investigated by Northern blot hybridization. Optic nerve damage and death of ganglion cells in the retina were induced unilaterally, by injection of hypertonic saline solution, episcleral vein cauterization, or optic nerve transection. The levels of mRNA for Myoc/Tigr were compared between several tissues of the control and surgically altered eyes, by using semiquantitative RT-PCR, real-time PCR, and Northern blot analysis. RESULTS: The rat Myoc/Tigr gene is 10 kb long and contains three exons. Among the eye tissues analyzed, Myoc/Tigr mRNA was detected in the combined tissues of the eye angle, sclera, cornea, retina, and optic nerve head. With pressure-induced optic nerve degeneration, the level of Myoc/Tigr mRNA decreased in the retina and the combined tissues of the eye angle, but increased in the optic nerve head. After optic nerve transection, the level of Myoc/Tigr mRNA increased in the retina, but did not change in the combined tissues of the eye angle. CONCLUSIONS: The decreased level of Myoc/Tigr mRNA in the retina after induction of elevated intraocular pressure compared with that in the control retina cannot be explained by ganglion cell death alone. Differences in Myoc/Tigr mRNA levels in eye tissues after elevation of intraocular pressure or optic nerve transection may reflect the activation of different signaling pathways involved in regulation of this gene.