Section on Molecular Genetics

• Staff
• Selected Publications

Current Research

Introduction: The Section on Molecular Genetics (SMG) studies the tissue-preferred expression and function of genes known to be important in the eye, especially the lens and cornea. Both of these transparent tissues are responsible for proper refraction in the eye. The SMG pays particular attention to the major water-soluble proteins - known as crystallins - of the lens and cornea; the abundant lens and corneal crystallins account for up to ~90% and ~50% of the water-soluble protein, respectively, of these tissues. While the crystallins clearly play a pivotal role in proper refraction in the lens, their optical functions in the cornea remain obscure. Surprisingly, despite the similarity of lens and corneal function in different species, their abundant proteins often differ among species (known as taxon-specificity). The taxon-specific lens and corneal crystallins are multifunctional (generally metabolic enzymes or stress-related proteins) that are expressed in lesser amounts in other tissues. Selected examples of crystallins include small heat shock proteins/alpha-crystallins (all vertebrates), argininosuccinate lysase/delta-crystallin (birds, reptiles) and alpha-enolase/tau-crystallin (selected species; lamprey, mola-mola fish) in the lens, and aldehyde dehydrogenase 3 (mammals, especially rodents), transketolase (mammals) and gelsolin (fish) in the cornea. The SMG has coined the term 'gene sharing' to describe a single gene encoding a protein with different functions (i.e. metabolic enzyme or refractive protein) depending upon its expression in different tissues.

Gene sharing has many important implications. One is that gene duplication is not necessary for the innovation of new function; indeed, a change in expression pattern is sufficient. Another implication is that previous knowledge of a protein's function is not sufficient to know its role in any particular situation, tissue or disease state. The SMG explores the molecular basis for tissue-preferred gene expression of lens and corneal crystallins, the multiple functions of these versatile proteins, and the evolutionary history of their structure and their regulated pattern of gene expression.

Below is a brief summary of SMG research status as of March, 2004.

Lens Crystallin Gene Expression: The SMG has identified cis-control elements and transcription factors that govern the regulated expression of lens crystallin genes, and this is a continuing focus of research in this group. They were the first to demonstrate lens-specific activity of crystallin promoters in cultured cells and transgenic mice. Those experiments opened genetic engineering in the visual system, and spawned innumerable experiments throughout the world. Moreover, the SMG showed that Pax6, among other factors, is a critical factor for lens-specific promoter activity. The SMG has studied the molecular basis for expression of many different crystallin genes.

Emphasis has been and continues to be devoted to mouse small heat shock protein (shsp)/alpha-B -crystallin gene expression. The shsp/alpha-B-crystallin gene is stress-inducible, moderately expressed in numerous tissues (especially heart and skeletal muscle), and highly expressed in the lens. The SMG used transgenic mouse and transfection/mutagenesis experiments to identify an upstream multi-tissue enhancer and a proximal lens and corneal-specific promoter. The enhancer contains at least five tissue-specific cis-control elements. Two cis-acting elements (LSR1 and 2), each utilizing Pax6 and retinoic acid receptors for their activity, are critical for lens-specificity of the proximal promoter. Recently, the SMG discovered that the enhancer is orientation-dependent in its natural context (quite unusual for enhancers), and is bounded at its 5' border by an insulator with additional negative regulatory activity. This insulator/negative regulatory DNA sequence protects an upstream promoter of an alpha-B-crystallin sibling gene (known as myotonic dystrophy protein kinase binding protein, MKBP/HspB2) from being regulated by the powerful shsp/ alpha-B -crystallin enhancer. The SMG is continuing to explore the molecular parameters of the diverse expression patterns of the mouse shsp/alpha-B -crystallin gene.

The SMG is also investigating evolutionary changes in shsp/alpha-B-crystallin gene expression. They showed in transgenic mouse experiments that the shsp/alpha-B-crystallin enhancer/promoter from a blind mole rat (which has subcutaneous degenerate eyes) has adapted to subterranean existence of its host by losing lens-expression early in eye development and boosting skeletal muscle expression. The specific sequences responsible for this adaptive change are under investigation.

Invertebrate Lens Crystallins: The SMG showed that gene sharing and recruitment of multifunctional proteins as crystallins occur in invertebrates as well as vertebrates. They demonstrated that cephalopods (squid and octopus) have recruited glutathione S-transferase (S-crystallins) and aldehyde dehydrogenase (omega-crystallin) as lens crystallins; scallops use exclusively an apparently inactive aldehyde dehydrogenase as a lens crystallin. Even the transparent lens of the complex eyes of jellyfish was shown to use multifunctional proteins as crystallins. J3-crystallin of the jellyfish was found to show homology to saposins, a group of proteins that bridge membrane lipids and lysosomal hydrolases. Most interesting, is the finding that striking similarities exist between the mechanism of lens-specific gene expression among crystallin promoters of vertebrates and invertebrates, including the nature and arrangement of the cis-control elements. Pax proteins are a consistently important transcription factor. Recent studies showed that PaxB, an extant ancestral to Pax6, binds to and stimulates the jellyfish J3-crystallin promoter. The SMG hypothesized and has gathered evidence that convergent changes in promoters and enhancers unify the diverse crystallin genes at the level of their regulation. These experiments strongly support the link between the innovation of protein function by gene sharing and differential gene regulation.

A side issue of interest that has emerged from SMG studies of jellyfish crystallins is that the eye and ear may be evolutionarily related. Both lens crystallins and PaxB are expressed in the jellyfish statocyst, a balancing organ, as well as in the eye. Moreover, PaxB is a structural and functional chimera of Pax6 and Pax2, the two genes that have resulted from PaxB gene duplication during evolution. Both Pax2 and Pax6 are critical for eye development in higher species, while only Pax2 is important for ear development in mammals. Taken together, these findings from the SMG have opened a new path for exploration, linking the visual and auditory/balancing senses.

Corneal Crystallins: The SMG is investigating the abundant, water-soluble cytoplasmic corneal proteins. Particular attention has been given to mouse aldehyde dehydrogenase 3 (ALDH3), since it represents approximately 50% of the water-soluble protein of the corneal epithelial cells in this species. Amazingly, the mouse cornea remained clear and apparently normal after the single ALDH3 gene was eliminated by homologous recombination. No compensatory corneal protein was noted. Recent collaborative investigations, however, suggest that subtle changes in the structure of the corneal cell surface and extra cell layers in the stratified corneal epithelium may be present in the ALDH3 null mice. The SMG is also exploring the molecular basis of corneal-preferred expression of the ALDH3 gene in mice. The ALDH3 gene was the source of the first corneal-specific promoter in transgenic mice, and this gave rise to numerous genetic engineering experiments in the cornea. Continuing experiments by the SMG indicate the presence of positive and negative regulatory elements associated with the mouse ALDH3 promoter, and Pax6 appears to be important, as it is in lens-specific activity of crystallin promoters.

Other abundant proteins identified in mammalian corneas by the SMG are ALDH1 (specifically in rabbits), transketolase (TKT), and serum albumin. Rabbit ALDH1 comprises 15-20% of the water-soluble protein in the rabbit keratocytes and ~5% of the soluble protein in the corneal epithelium. Its regulated expression appears linked to hypoxic pathways. TKT accounts for 10-15% of the corneal epithelial cells in mice and other mammals. TKT null mice produced by homologous recombination died; by contrast, TKT heterozygote mice were viable, showed considerable reduction in body fat, and displayed female fertility problems. The potentially important role of TKT in obesity control warrants further study. Finally, SMG studies showed that serum albumin accounts for 10-15% of the water-soluble protein in the mouse and bovine corneal stroma. Other investigators have shown high serum albumin contents in human cornea. While its normal role in the stroma is not known, serum albumin's ability to bind drugs suggests that mice corneas could be exploited to study drug-serum albumin interactions in vivo and to test the usefulness of serum albumin as a drug carrier for corneal disorders.

The SMG discovered that zebrafish accumulate gelsolin, an actin binding protein that regulates the cytoskeleton, in the corneal epithelial cells instead of ALDH3. Additional studies showed that gelsolin is highly expressed in many fish corneas, including both the air and water cornea of the four-eyed surface fish, Anableps. The corneal gelsolin gene is also expressed at much lower levels in the lens and in very low amounts in the early cleavage stages of the zebrafish embryo. A variety of microinjection experiments in 1-2 cell stage embryos showed that the cornea/lens gelsolin participates in the dorsal-ventral signaling pathway during zebrafish development; indeed, gelsolin over expression even resulted in axis duplication of the developing embryo. Thus, although the corneal function of the abundant gelsolin (~50% of the water-soluble protein) is not known, it appears as if this ubiquitous cytoskeletal protein has become multifunctional through a gene sharing strategy.

The SMG has also identified a second gelsolin gene in zebrafish. Unlike the cornea/lens gelsolin gene, the second gene is expressed ubiquitously in low amounts, and does not participate in dorsal-ventral signaling. The SMG continues to explore cornea/lens gelsolin's participation in the dorsal-ventral signaling pathway as well as the molecular basis of corneal-preferred gene expression of the two gelsolin genes. In addition, attention will be given to the putative role of the abundant corneal/lens gelsolin to corneal wound healing in zebrafish.

Thus, it appears that the cornea accumulates ubiquitous proteins by a gene sharing strategy. In contrast to the lens, the optical functions of the so-called 'corneal crystallins' are not known yet. Since both lens and cornea are transparent, important for refraction in the eye, and contain high levels of multifunctional proteins by gene sharing, the SMG has put forward the 'refracton' hypothesis linking these two tissues functionally and evolutionarily.

Corneal Development (Pax6 and SAGE): The SMG is exploring corneal development by analyzing the role of Pax6 and by mapping all the genes expressed during development through serial analysis of gene expression (SAGE). Analysis of the Small eye mouse that carries a single wildtype Pax6 allele indicated that Pax6 haploinsufficiency results in poorly stratified corneal epithelial cells. ALDH3 and other abundant corneal epithelial cell proteins (cytokeratin 12, transketolase) are limited in quantity in the Small eye mouse cornea, and the epithelial cells show adhesive deficiencies. This is being investigated further by the creation of Cre/lox corneal-specific Pax6 knockouts. The availability of transgenic mice that express the Cre recombinase specifically in the cornea will open a host of additional experiments as well.

The SMG has undertaken an extensive SAGE analysis of corneal development. Approximately 9000 genes were found expressed in the mouse cornea; one-third of these were confined to the non-stratified 9-day-old cornea, one-third to the stratified six-week-old adult cornea, and one-third overlapped the two stages. Another SAGE corneal library was made at birth, and this is in the process of being analyzed. Among the interesting findings so far are a select group of expressed transcription factors, specific genes differentially expressed within the layers of the stratified epithelium, and the suggestion of significant translational or posttranslational control mechanisms to account for disparities between specific mRNA levels and relative abundance of their encoded protein (especially ALDH3).

SAGE analysis has also been performed on both the limbal stem cells surrounding the cornea and the central corneal epithelial cells of the adult rat. Relatively few striking differences were found between the limbal stem cells and the central corneal epithelial cells. However, several gene products were enriched in the limbal stem cells (a cytokeratin, a trypsin-like serine protease, and a protease inhibitor); these have been cloned and are the subject of further study. Finally, the SMG is performing SAGE analysis on normal adult human cornea to compare corneal gene expression profiles between mice and humans. The wealth of data derived from SAGE indicate dynamic changes in gene expression after eye opening (~13 days after birth) in mice and provide new probes for exploring corneal epithelial cell stratification, development and function. The data also point to specific genes as candidates for the isolation of promoters/enhancers that can be used for targeting genes to specific regions within the cornea. The extensive SAGE analysis in progress in the SMG will lead to a molecular signature for cornea and provide the foundation for understanding the normal and diseased tissue.

Staff

Selected Publications (2000-Present)

Duncan, M.K., Cvekl, A., Li, X., and Piatigorsky, J.: Truncated forms of Pax-6 disrupt lens morphology in transgenic mice, Invest. Ophthalmol. Vis. Sci. 41: 464-473, 2000. PubMed

Piatigorsky, J.: A case for corneal crystallins. J. Ocular Pharmacol. Therapeutics 16: 173-180, 2000. PubMed

Gopal-Srivastava, R., Kays, W.T., and Piatigorsky, J.: Enhancer-independent promoter activity of the mouse aB-crystallin/small heat shock protein gene in the lens and cornea of transgenic mice. Mech. Develop. 92: 125-134, 2000. PubMed

Sax, C.M., Kays, W. T., Salamon, C., Chervenak, M.M., Xu, Y.-S., and Piatigorsky, J.: Transketolase gene expression in the cornea is influenced by environmental factors and developmentally controlled events. Cornea 19: 833-841, 2000. PubMed

Duncan, M.K., Kozmik, Z., Cveklova, K., Piatigorsky, J., and Cvekl, A.: Overexpression of PAX6(5a) in lens fiber cells results in cataract and upregulation in (alpha)5(beta)1 integrin expression. J. Cell Sci. 113, 3173-3185, 2000. PubMed

Xu, Y.-S., Kantorow, M., Davis, J., and Piatigorsky, J.: Evidence for gelsolin as a corneal crystallin in zebrafish. J. Biol. Chem. 27, 24645-24652, 2000. PubMed

Magabo, K.S., Horwitz, J., Piatigorsky, J., and Kantarow, M..: Expression of ßB2-crystallin protein in brain, testis and retina. Invest. Ophthalmol. Vis. Sci. 41, 3056-3060, 2000. PubMed

Piatigorsky, J., Kozmik, Z., Horwitz, J., Ding, L., Carosa, E., Robison, W.G., Steinbach, P.J., and Tamm, E.R.: O-Crystallin of the scallop lens: a dimeric aldehyde dehydrogenase class 1/2 enzyme-crystallin. J. Biol. Chem. 275, 41064-41073, 2000. PubMed

Hejtmancik, J.F., Kaiser, M.I., and Piatigorsky, J.: Molecular Biology and Inherited Disorders of Lens. In: The Metabolic & Molecular Bases of Inherited Disease. Vol. IV. Eighth Edition (Scriver, C.R., Beaudet, A.L., Sly, W.S., and Valle, D., editors). Pp. 6033-6061. McGraw-Hill Medical Publishing Division, New York, 2001.

Piatigorsky, J.: Dual use of the transcriptional repressor (CtBP2)/ribbon synapse (RIBEYE) gene: how prevalent are multifunctional genes? Trends Neurosci. 24, 555-557, 2001. PubMed

Piatigorsky, J: The enigma of the abundant water-soluble cytoplasmic proteins of the cornea: the "refracton" hypothesis. Cornea 20, 853-858, 2001. PubMed

Chen, W.V., Hejtmancik, J.F., Piatigorsky, J., and Duncan, M.K.: The mouse ßB1-crystallin promoter: strict regulation of lens fiber cell specificity. Biochim. Biophys. Acta 93522, 1-9, 2001. PubMed

Piatigorsky, J., Norman, B., Dishaw, L., Kos, L., Horwitz, J., Steinbach, P., and Kozmik, Z.: J3-crystallin of the jellyfish lens: similarity to saposins. Proc. Natl. Acad. Sci. USA 98, 12362-12367, 2001. PubMed

Carosa, E., Kozmik, Z., Rall, J.E., and Piatigorsky, J.: Structure and expression of the scallop O-crystallin gene: evidence for convergent evolution of promoter sequences. J. Biol. Chem. 277: 656-664, 2002. PubMed

Nees, D.W., Wawrousek, E., Robison, W.G., Jr., and Piatigorsky, J.: Structurally normal cornea in aldehyde dehydrogenase 3a1-deficient mice. Mol. Cell. Biol. 22: 849-855, 2002. PubMed

Xu, Z-P., Wawrousek, E.F. and Piatigorsky, J.: Transketolase haploinsufficiency reduces adipose tissue and female fertility in mice. Mol. Cell. Biol. 22: 6142-6147, 2002. PubMed

Flugel-Koch, C., Ohlmann, A., Piatigorsky, J. and Tamm, E.R.: Disruption of anterior segment development by TGF-ß1 overexpression in the eyes of transgenic mice. Dev. Dyn. 225: 111-125, 2002. PubMed

Hough, R.B., Avivi, A., Davis, J., Joel, A., Nevo, E. and Piatigorsky, J.: Adaptive evolution of small heat shock protein/aB-crystallin promoter activity of the blind subterranean mole rat, Spalax ehrenbergi. Proc. Natl. Acad. Sci. USA 99: 8145-8150, 2002. PubMed

Xu, Z-P., Dutra, A., Stellrecht, C.M., Wu, C., Piatigorsky, J. and Saunders, G.F.: Functional and structural characterization of the human gene BHLHB5, encoding a basic helix-loop-helix transcription factor. Genomics 80: 311-318, 2002. PubMed

Swamynathan, S.K. and Piatigorsky, J.: Orientation-dependent influence of an intergenic enhancer on the promoter activity of the divergently transcribed mouse Shsp/aB-crystallin and Mkbp/HspB2 genes. J. Biol. Chem. 277: 49700-49706, 2002. PubMed

Piatigorsky, J.: Crystallin Genes: Specialization by changes in gene regulation may precede gene duplication. In: (Genomic Duplications in Evolution), Jacques Monod Conference, Aussois France, Academic Press. 2003. Also in Piatigorsky, J. J. Struct. Funct. Genomics 3: 131-137, 2003. PubMed

Kanungo, K. Kozmik, Z., Swamynathan, S.K. and Piatigorsky, J: Gelsolin is a dorsalizing factor in zebrafish. Proc. Natl. Acad. Sci. USA 100: 3287-3292, 2003. PubMed

Davis, J. Duncan, M.K., Robison, W.G., Jr.and Piatigorsky, J.: Requirement for Pax6 in corneal morphogenesis: a role in adhesion. J. Cell Sci. 116: 2157-2167, 2003. PubMed

Nees, D., Fariss, R.N. and Piatigorsky, J.: Serum albumin in mammalian cornea: implications for clinical application. Invest. Ophthal. Vis. Sci. 44: 3339-3345, 2003. PubMed

Kozmik, Z., Daube, M., Frei, E., Norman, B., Kos, L., Dishaw, L.J., Noll, M. and Piatigorsky, J.: Role of Pax genes in eye evolution: a cnidarian PaxB gene uniting Pax2 and Pax6 functions. (Featured article: Cover photo). Dev. Cell. 5: 773-785, 2003. PubMed

Swamynathan, S.K. Crawford, M.A., Robison, W.G., Jr., Kanungo, J. and Piatigorsky, J.: Adaptive differences in the structure and macromolecular compositions of the air and water corneas of the "four-eyed" fish (Anableps anableps). FASEB J. 17: 1996-2005, 2003. PubMed

Kostrouschova, M., Kostrouch, Z., Saudek, V., Piatigorsky, J. and Rall, J.E.: BIR-1, a Caenorhabditis elegans homologue of Survivin, regulates transcription and development. Proc. Natl. Acad. Sci. USA 100: 5240-5245, 2003. PubMed

Piatigorsky, J.: Gene sharing, lens crystallins and speculations on an eye/ear evolutionary relationship. Int. Comp. Biol. 43: 492-499, 2003.

Norman, B., Davis, J. and Piatigorsky, J.: Postnatal gene expression in the normal mouse cornea: a SAGE analysis. Invest. Ophthalmol. Vis. Sci. 45: 429-440, 2004. PubMed

Hough, R.B. and Piatigorsky, J.: Preferential transcription of rabbit ALDH1a1 in the cornea: implication of hypoxia-related pathways. Mol. Cell. Biol. 24: 1324-1340, 2004. PubMed

Cui, W., Tomarev, S.I., Piatigorsky, J., Chepelinksy, A.B. and Duncan, M.K.: Mafs, Prox1 and Pax6 can regulate chicken ßB1-crystallin gene expression. J. Biol. Chem. (in press). PubMed

Kanungo, J. Swamynathan, S.K. and Piatigorsky, J.: Abundant corneal gelsolin in zebrafish and the 'four-eyed' fish, Anableps anableps: possible analogy with multifunctional lens crystallins. Exp. Eye Res. (in press).

Piatigorsky, J. and Kozmik, Z.: Cubozoan jellyfish: an evo/devo model for eyes and other sensory systems. Int. J. Dev. Biol. (in press).

Duncan, M.K., Cvekl, A., Kantorow, M., and Piatigorsky, J.: Lens crystallins. In: Development of the Ocular Lens. (Robinson, M.L. and Lovicu, F.J., editors). Cambridge University Press. (in press).