The Mechanism of σ-Crystallin Gene Regulation: Cooperation of Lens-Specific and Non-Specific Elements1

Employing δ-crystallin gene as a model, we have investigated tissue-specific gene regulation. Our approach was to analyze regulatory elements associated with the gene utilizing gene transfer techniques. Introduction of the chicken δ1-crystallin gene into the genome of developing mouse embryos resulted in lens-specific expression, indicating that the elements governing the tissue specificity are located in the DNA sequence introduced. Through analysis of various regions of the δ1-crystallin gene and the associated DNA sequences, we identified a lens-specific enhancer in the third intron of the gene. It was demonstrated that this enhancer alone is sufficient to account for lens specificity of the δ1-crystallin gene. Dissection of the δ1-crystallin enhancer and functional assessment by multiplication of enhancer fragments demonstrated the cooperative interaction of lens-specific and nonspecific elements in the enhancer. The mechanism by which heterologous elements cooperate in generating enhancer activity unquestionably provides great flexibility to the regulatory system, and may account for developmental modulation of gene activity superimposed on tissue specificity.     

Developmental regulation of the chicken δ1-crystallin gene: analysis by transgenesis and gene dissection

We previously reviewed what we had learned about the regulation of the δ1-crystallin gene through experiments using gene transfer techniques [Kondoh et al. (1986) Cell Differ. 19, 151–160]. It was concluded then that regulatory genetic elements for the lens-specific expression are associated with the δ1-crystallin gene, and that these chicken elements properly function in mammalian cells. In the last couple of years, we have made significant progress in the understanding of lens-specific δ-crystallin expression. This is owing to success in transgenesis of mouse with the δ1-crystallin gene and in functional dissection of the gene which led us to the discovery of an intragenic enhancer as the major determinant for lens-specific expression. In this article, we summarize these recent advances.     

The control of δ-crystallin gene expression during lens cell development: Dissociation of cell elongation,cell division, δ-crystallin synthesis,and δ-crystallin mRNA accumulation

Previous studies have shown that freshly explanted 6-day-old embryonic chick lens epithelial cells elongate, differentially increase their synthesis of δ-crystallin, and accumulate δ-crystallin mRNA when cultured with fetal calf serum; in contrast, precultured serum-starved 6-day-old and freshly explanted 19-day-old embryonic epithelial cells divide when treated with fetal calf serum. We have explored whether the stimulation of δ-crystallin gene expression (as measured by δ-crystallin synthesis and δ-crystallin mRNA accumulation) is affected by inhibiting lens cell elongation with colchicine, and whether δ-crystallin gene expression is increased in lens epithelial cells stimulated to divide by treatment with fetal calf serum, as it is in those stimulated to elongate by treatment with serum. Three new findings were made in this study. First, the stimulation of δ-crystallin gene expression does not require elongation of the cultured lens cells. Second, a decreased proportion of δ-crystallin synthesis is observed in lens epithelial cells during normal development and during serum starvation; in neither case is this decrease associated with a reduction in the number of δ-crystallin mRNA sequences per cell. Finally, serum stimulation of lens cell division does not increase the proportion of δ-crystallin synthesis, but can promote the accumulation of δ-crystallin mRNA. Thus, the relative proportion of δ-crystallin synthesized during chick lens development is not solely a function of the number of δ-crystallin mRNA sequences in the lens cells.     

Degradation of δ-crystallin mRNA in the lens fiber cells of the chicken

δ-Crystallin is the principal protein synthesized in the embryonic chicken lens. After hatching δ-crystallin synthesis decreases and eventually ceases. We have determined when the δ-crystallin messenger RNA (mRNA) disappears from the lens fiber cells during the first year of age by cell-free translation of lens RNA in a reticulocyte lysate, RNA blot (Northern) hybridization, and in situ hybridization. The hybridization was performed with a nick-translated, cloned δ-crystallin cDNA (pδCr2). δ-Crystallin mRNA was present in the lens until 3 months of age and disappeared between the third and fifth month after hatching. The in situ hybridization experiments indicated that the δ-crystallin mRNA was present throughout the lens fiber mass until 1 month after hatching and was greatly reduced in the cortical fiber cells thereafter. In contrast to earlier stages, then, the cortical fiber cells differentiating at the lens equator after about 1 month of age do not accumulate δ-crystallin mRNA. The data also indicate that the maximal half-life of functional δ-crystallin mRNA in the posthatched chicken lens is about 2 months.     

Conditional transgene expression mediated by the mouse beta-actin locus

Transgenic mice are an effective model to study gene function in vivo; however, position effects can complicate tissue-specific transgene analysis. To facilitate precise targeting of a transgenic construct into the mouse genome, we combined the Cre/lox and Flp/FRT recombination systems to allow for rapid transgene replacement and conditional transgene expression from the endogenous beta-actin locus. Flp/FRT recombination was used to rapidly exchange FRT-flanked transgene cassettes by recombinase-mediated cassette exchange in embryonic stem cells, while transgene expression can be activated in mice after Cre-mediated excision of a floxed STOP cassette. To validate our system, we analyzed the expression profile of an EGFP reporter gene after integration into the beta-actin locus and Cre-mediated excision of the floxed STOP cassette. Breeding of EGFP reporter mice with various Cre mouse lines resulted in the expected expression profiles, demonstrating the feasibility of the model to facilitate predictable and strong transgene expression in a spatially and temporally controlled manner.     

Dual Reporter Genes Enabling Cell Tracing with Viable and Reliable Selection of Various Cell Types

Dual reporter genes driven by either a ubiquitous cytomegalovirus (CMV) or a neuro-specific tubulin α1 promoter (Tα1) were constructed. The new genes, CMV (pCMV-GL) or Tα1 promoter-driven GFP–LacZ (pTα1-GL), robustly expressed the fused GFP–LacZ protein reporting constitutive expressions in various cell types including CHO cells, loach and chicken embryos, and neuro-specific expression in differentiating mouse embryonic stem cells, respectively. The dual reporter genes thus provide a versatile tool for the studies of gene expression, cell lineage within the embryo and possibly the fate of stem cells in transplantation experiment, thus facilitating different analyses depending on the experimental purposes. Revisions requested 11 October 2005; Revisions received 25 November 2005     

Delta-crystallin mRNA in chick lens cells: mRNA accumulates during differential stimulation of delta-crystallin synthesis in cultured cells.

Increasing specialization for δ-crystallin synthesis is a prominent feature of the differentiation of chick lens epithelial cells into lens fiber cells and can be studied in cultured embryonic lens epithelia. Quantitation of δ-crystallin mRNA by molecular hybridizaton to a [³H]DNA complementary to δ-crystallin mRNA demonstrates that differentiation, both in ovo and in tissue culture, is associated with the accumulation of δ-crystallin mRNA. In the cultures, there is an overall stimulation of protein synthesis, including δ-crystallin mRNA during the first 5 hr in vitro. Between 5 and 24 hr in vitro there is a differential stimulation of δ-crystallin synthesis and an accumulation of δ-crystallin mRNA that can quantitatively account for this stimulation.     

Developmental changes in proteins of purified membranes of chicken lenses and evidence for contamination by cytoplasmic δ-crystallin

Urea-washed membranes from embryonic chick lenses (15 days old) and from the cortical and nuclear regions of adult chicken lenses (1 year) have been prepared by repeated centrifugation through discontinuous density gradients. The protein components of the isolated membranes have been examined by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate and urea. Proteins with molecular weights of 75 000, 56 000, 54 000, 48 000, 34 000, 32 000, 25 000, and 22 000 were present in all the membrane preparations, although their proportions changed during development. One additional protein, molecular weight 70 000, was seen only in the embryonic lens membranes. The greatest developmental change was the increase in 25 000 molecular weight protein from 12% in the embryonic lens to about 45% in the adult lens. Since it has been suggested that this protein is associated with gap junctions, its increase during development may reflect a corresponding increase in the number of gap junctions in the lens.The 50 000 molecular weight protein of embryonic lens membranes and membranes of adult nuclear lens fibers consisted at least partly of δ-crystallin, since δ-crystallin peptides could be identified in tryptic pepetide maps of the isolated protein after in vitro radioiodination. Peptide maps of the 50 000 molecular weight protein of cortical lens fiber membranes contained no identifiable δ-crystallin peptides, although it is possible that modified δ-crystallin peptides may be present. The level of cytoplasmic contamination of the membrane fraction was estimated by preparing lens membranes in the presence of added δ-[³⁵S]crystallin. The results indicated that cytoplasmic contamination contributes significantly to the presence of δ-crystallin in lens membrane preparations.     

Derivation and characterization of pluripotent embryonic germ cells in chicken

Embryonic germ (EG) cell lines established from primordial germ cells (PGCs) are undifferentiated and pluripotent stem cells. To date, EG cells with proven germ-line transmission have been completely established only in the mouse with embryonic stem (ES) cells. We isolated PGCs from 5.5-day-old (stage 28) chicken embryonic gonads and established a putative chicken EG cell line with EG culture medium supplemented with stem cell factor (SCF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), interleukin-11 (IL-11), and insulin-like growth factor-I (IGF-I). These cells grew continuously for ten passages (4 months) on a feeder layer of mitotically active chicken embryonic fibroblasts. After several passages, these cells were characterized by screening with the periodic acid-Schiff reaction, anti-SSEA-1 antibody, and a proliferation assay. The chicken EG cells maintained characteristics of gonadal PGCs and undifferentiated stem cells. When cultured in suspension, the chicken EG cells successfully formed an embryoid body and differentiated into a variety of cell types. The chicken EG cells were injected into stage X blastodermal layer and produced chimeric chickens with various differentiated tissues derived from the EG cells. Chicken EG cells will be useful for the production of transgenic chickens and for studies of germ cell differentiation and genomic imprinting.     

Correlation between Musashi-1 and c-hairy-1 expression and cell proliferation activity in the developing intestine and stomach of both chicken and mouse

Musashi-1 (Msi-1) is an RNA-binding protein that plays key roles in the maintenance of neural stem cell states and in their differentiation into neural cells. Msi-1 has also been proposed as a candidate marker gene of mammalian intestinal stem cells and their immediate lineages. In this study, we examined Msi-1 expression in the small intestine and the stomach of both chicken and mouse during embryonic, fetal and postnatal development. In addition, we analyzed the expression of c-hairy-1, a chicken homologue of mouse Hes1, and assessed the proliferative activity of the cells expressing both of these factors. Significantly, during the development of these digestive organs in both species Msi-1 expression showed dynamic changes, suggesting that it is important for digestive organ development, particularly for epithelial differentiation. Based on our observations of the expression patterns of Msi-1 and c-hairy-1 in the adult small intestine, we speculate that Msi-1 is also a stem cell marker of the chicken small intestinal epithelium.     

Chicken leukemia inhibitory factor maintains chicken embryonic stem cells in the undifferentiated state

Mouse embryonic stem (ES) cells can be maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF), a member of the interleukin-6 cytokine family. In other mammals, this is not possible with LIF alone. Chicken ES-like cells (blastodermal cells) have only been cultured with mouse LIF because chicken LIF was not available. However the culture system is imperfect and chicken ES-like cells equivalent to mouse ES cells were not observed. In the present study, we cloned the cDNA-encoding chicken LIF using mRNA subtraction and RACE methodology. The chicken LIF cDNA encodes a protein with approximately 40% sequence identity to mouse LIF. It has 211 amino acids including a putative N-terminal signal peptide of 24 residues. Chicken blastodermal cells were cultured in the presence of bacterially expressed chicken LIF or mouse LIF. The expression of alkaline phosphatase and embryonal carcinoma cell monoclonal antibody-1 and stage-specific embryonic antigen-1 and the activation of STAT3 were examined, all of which are indices of the undifferentiated state. Exposure in the blastodermal cells to recombinant chicken LIF but not to mouse LIF maintained the expression of these various markers. After 9 days of incubation, the blastodermal cells formed cystic embryoid bodies in the presence of mouse LIF but not in the presence of recombinant chicken LIF. We conclude that chicken LIF is able to maintain chicken ES cell cultures in the undifferentiated state.     

The clinical potential of stem cells

Stem cells are defined by their capacity for self-renewal and multilineage differentiation, making them uniquely situated to treat a broad spectrum of human diseases. For example, because hematopoietic stem cells can reconstitute the entire blood system, bone marrow transplantation has long been used in the clinic to treat various diseases. Similarly, the transplantation of other tissue-specific stem cells, such as stem cells isolated from epithelial and neural tissues, can treat mouse disease models and human patients in which epithelial and neural cells are damaged. An alternative to tissue-specific stem cell therapy takes advantage of embryonic stem cells, which are capable of differentiating into any tissue type. Furthermore, nuclear transfer, the transfer of a post-mitotic somatic cell nucleus into an enucleated oocyte, creates a limitless source of autologous cells that, when combined with gene therapy, can serve as a powerful therapeutic tool.     

Expression and evolutionary conservation of the tescalcin gene during development

The tescalcin gene (Tesc) encodes an EF-hand calcium-binding protein that interacts with the sodium/hydrogen exchanger, NHE1. Previous studies indicated that Tesc was expressed in mouse embryonic testis, but not in ovary, during the critical period of testis and ovary determination. In this paper we compared the expression of Tesc in embryonic tissues of chicken and mouse. Tesc expression was sexually dimorphic in the embryonic gonads of both mouse and chicken. Tescalcin (TESC) was detected in both Sertoli cells and germ cells. In the embryonic brain of both mouse and chicken, Tesc was highly expressed in the nasal placode and in fibers extending from the olfactory epithelium to the primordial olfactory bulb. Tesc was expressed in the embryonic heart of both chicken and mouse. In mouse Tesc expression was also detected in embryonic adrenal. These studies indicate very specific expression of Tesc in various tissues in chicken and mouse during embryologic development, and conservation of Tesc expression in both species.