|Molecular Vision 2002;
Received 4 May 2000 | Accepted 11 June 2002 | Published 12 June 2002
Temporal expression of three mouse lens fiber cell membrane protein genes during early development
Tong Chen, Robert
Department of Ophthalmology, Emory University School of Medicine, Atlanta, GA
Correspondence to: Correspondence to: Robert L. Church, Emory Eye Center, Room B5601, 1365B Clifton Rd, NE, Atlanta, GA, 30322; Phone: (404) 778-4101; FAX: (404) 778-2232; email: email@example.com
Purpose: Three different lens fiber cell intrinsic membrane proteins, MIP (Major Intrinsic Protein), MP19, and connexin50 (Cx50), have separately been implicated as causative candidates for congenital cataracts. The aim of this study was to determine gene transcript expression of these three proteins during successive stages of mouse embryonic development.
Methods: Total RNA was prepared from mouse embryos taken at days 9-10 (E9-E10) of gestation, heads of day 11-13 (E11-E13) embryos, and lenses of adult mice. Coupled reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine gene transcript expression of MIP, Cx50, and MP19 during embryonic development. The products of RT-PCR were further cloned into the TOPOTM TA vector, and further analyzed by double strand nucleotide sequencing.
Results: Cx50 gene expression was observed throughout the developmental period observed (E9-E13). MIP transcripts were first observed at mouse embryonic day 11.5 (E11.5) and synthesis continued throughout the developmental period observed. The gene for MP19 (Lim2) begins to express at mouse embryo day 12 (E12) and synthesis continued throughout the developmental period observed. mRNA levels for all three proteins appear to remain steady from these early embryonic stages through adulthood.
Conclusions: The identified early expression of Cx50, MIP, and Lim2 transcripts in mouse embryonic stages suggests that all three proteins play very important, probably quite different, roles in lens fiber cell differentiation. Variation in the temporal expression of these three genes during the course of development suggests a critical gene-coordinated regulation throughout lens fiber cell development. These three genes clearly play important roles in early normal lens development since it is known that mutations in the sequence of each membrane protein results in cataractogenesis.
Embryonic development of the mammalian lens is well known at the biochemical and histological level. However, little information is available at the molecular level concerning gene expression during development and differentiation of the lens. In the present study, we have investigated gene transcript expression of three major intrinsic proteins of the lens fiber cell membrane during early mouse lens development.
The first evidence of eye-related structures in the developing mouse embryo is found during Theiler  stage 14 (8.5-9.75 days post conception, dpc; with the morning after the vaginal plug is observed being designated 0.5 dpc, or E0.5) with the formation of the optic eminence and optic vesicle. By Theiler stage 15 (9.0-10.25 dpc) the lens placode forms, and the lens pit begins to develop during Theiler stages 16 and 17 (9.5-11.25 dpc). The lens vesicle appears late in Theiler stage 18 (10.5-11.25 dpc) and continues to develop throughout Theiler stages 19 (11.0-12.25 dpc) and 20 (11.5-13.0 dpc) with the formation of the lens anterior epithelium, lens cavity, and posterior epithelium. Finally, at Theiler stage 21 (12.5-14.0 dpc) the lens fibers begin to form and the lens cavity fills with primary lens fiber cells, essentially completing the initial development of the intact lens. The Edinburgh Mouse Atlas is an excellent internet-accessible database of mouse developmental anatomy .
The major intrinsic protein (MIP) of the lens is a 26-kDa protein that is exclusively expressed in lens fiber cells in high abundance (constituting more than 60% of the total membrane protein of lens fiber cells) and which forms specialized junctions between lens fiber cells [3,4]. When reconstituted into lipid bilayers, it is able to build up large, nonselective channels . These and other findings indicate that MIP may be crucial for water transport and maintenance of the transparency of the lens. Furthermore, MIP bears a potential relationship not only with increased water retention in the lens, a phenomenon frequently observed with age, but also with the development of cataracts. It has been reported that mutations in the MIP gene cause hereditary cataracts in mice [6,7].
MP19 (also referred to as MP17, MP18 and MP20 in the literature) is the second most abundant intrinsic membrane proteins of lens fiber cells. It was first described as a fiber-specific component of bovine lens membranes . Both the rat  and bovine  cDNAs and the entire human gene  have been cloned and the sequence reported. Unlike the other lens membrane proteins, MP19 bears no striking resemblance to any other reported protein family and has no defined structural or functional role. Like MIP, MP19 is uniformly distributed throughout fiber cell membranes, but MP19 also colocalizes with gap junctions in distinct regions of the lens [12,13], indicating that the function of these proteins is probably coordinated during lens fiber cell development. Coordinated regulation of the expression of these proteins would further indicate a possible coordination of their function. A single base-pair mutation in the coding region of the Lim2 gene has been shown to result in a heritable total cataract in the mouse, called the To3 cataract .
Cx50 was first identified in the ovine ocular lens, where its presence was noted to be coincident with fiber gap junctions, and it was referred to as MP70 (for Membrane Protein of 70,000 kDa molecular weight) . The Cx50 protein coding region was subsequently cloned from mouse genomic DNA and demonstrated to be a functional member of the gap junction family of proteins as well as the murine homologue of MP70 . The relatively simple gene structure of Cx50, like all known connexins , contains a single protein coding exon. This feature has facilitated the recent cloning of Cx50 from human  and sheep . The gene encoding Cx50 has also been mapped regionally to subchromosomal locations in both mouse  and human . Mutations in the Cx50 gene have been shown to result in cataract formation in both man  and mouse .
In order to further understand the expression of these three lens fiber cell membrane proteins in normal and diseased conditions, we have determined the start points of gene transcription for MIP, Cx50, and MP19 during mouse early embryonic development.
BALB/cAnNHsd mice were obtained from Harlan-Sprague Dawley. Pregnant mice were sacrificed by cervical dislocation at different time periods of gestation (days of gestation, dpc). Female and male mice were placed together and the females were observed twice per day for the presence of a vaginal plug. The gestational age was determined by counting the days following appearance of the vaginal plug (the morning following the appearance of a vaginal plug was called 0.5 dpc [E0.5]). Embryos were surgically removed, and at stages from E9.0 through E10.0 entire embryos were collected due to the very small size of the embryo. At stages E10.5 through E13.0, head regions were collected. In addition, lens tissue from adult mice were collected and used as positive controls in these studies. Tissues from the various developmental stages and adult lenses were collected and immediately homogenized in RNAzolTM B (TEL-TEST, Inc., Friendswood, TX) solution for RNA isolation.
Tissues were collected at 0.5 day periods from E9.0 through E13.0. The litter size at each stage was 5-7 pups. RNA was extracted from each pup separately and each time point was from a single pup. Each time point was repeated at least 3 times, using different litters.
Mice were housed and cared for in the Emory Eye Center's animal facility, which is supervised and maintained by the Emory University Animal Facilities group. All care and research handing of the mice comply with the "Principles of Laboratory Animal Care" and the guide for the "Care and the Use of Laboratory Animals" (NIH publications No. 80-23, revised 1978).
Total RNA from the collected tissues was extracted individually using the RNAzolTM B method (TEL-TEST). The concentration and purity of the extracted RNA were measured by optical density at 260 and 280 nm. Polyadenylated RNA was further purified by oligo (dT) cellulose chromatography using the Oligotex mRNA Midi Kit (QIAGEN, Valencia, CA).
Display of RNA transcripts
MIP-, Cx50-, and MP19-specific oligonucleotide primer pairs were synthesized. Poly (A) mRNA was used as template in coupled RT-PCR reactions, using the Superscript One-Step RT-PCR System (Life Technologies, Gaithersburg, MD). RT-PCR was carried out in a PTC-100 DNA Engine (MJ Research, Watertown, MA). Each reaction was in a total volume of 50 μl. Each 50 μl reaction contained 25 μl of reaction buffer, 2 μl of template RNA (100 ng of RNA), 1 μl of sense primer (10 μM), 1 μl of antisense primer (10 μM), 1 μl of RT/Platinum Taq mix, and autoclaved distilled water to 50 μl. The following cycling conditions were used: 20 min reverse transcription at 50 °C followed by PCR amplification consisting of an initial denaturation at 94 °C for 120 s followed by 60 cycles of 30 s at 94 °C, 30 s at 59 °C for the MIP primer set, 61 °C for the Cx50 primer, and 62 °C for the MP19 primer set, 30 s at 70 °C followed by 5 min final extension at 72 °C.
The MIP primer set; sense: 5'-GGG CCA TAT TTG CTG AGT TCT; antisense: 5'-CCA TTC CGC CTC TCG TCG TA; which spanned across an intronic region. These primers would amplify a 436 base pair fragment if only mRNA was present. Figure 1 shows a schematic of the area amplified in the MIP gene. If contaminating DNA was present, a 934 bp product would also be observed.
The Cx50 primer set; sense: 5'-ATG GGC GAC TGG AGT TTC CTG; antisense: 5'-CAG CTT CAC GGT CCT TTC GCT; these primers would be expected to amplify a 334 base pair product. Since no intron was spanned with these primers, the 334 bp product would be observed with mRNA or contaminating DNA. Figure 2 shows a schematic of the area amplified in the cx50 gene.
The MP19 primer set; sense: 5'-ATG TAC AGC TTC ATG GGA GGC; antisense: 5'-GAG GCG AAA AAC ATG ATG CCT; which spanned across three introns and would amplify a 611 base pair fragment if only mRNA was present. Figure 3 shows a schematic of the area amplified in the Lim2 gene. If contaminating DNA was present, a 5,652 bp product would also be observed.
Total RNA from the adult lens served as positive control tissue. The poly(A) mRNAs from E9.0 to E13.0 have been tested in these particular experiments.
Negative control RT-PCR reaction
The primer sets for both MIP and MP19 both span intronic regions, and from the deduced size of the RT-PCR product, one can easily determine that only mRNA was amplified. However, the Cx50 primers do not span an intronic region and thus it would be difficult to determine whether contaminating genomic DNA was present in the RNA preparation. Therefore, we carried out negative reverse transcription controls on each of the three genes. RT-PCR preparations were set up for each of the three genes as outlined above, from E13 eyes. Then each of the samples was divided into two aliquots. One aliquot is put into the thermal cycler running the normal RT-PCR cycle. The other aliquot was immediately placed at 95 °C for 10 min, then put on ice for 5 min, and then back into the thermal cycler when it starts the first cycle of the PCR, ie, the first 94 °C step of the RT-PCR cycling. This treatment will kill the reverse transcription enzyme. Thus, if product is observed, it will be due to contaminating DNA being amplified, not RNA. This experiment would demonstrate that there was no DNA contamination. If the RNA preparation had been contaminated with DNA, we would have observed a product in the boiled samples.
Sequencing of RT-PCR products
Each of the RT-PCR products (Cx50, MIP, and MP19) was further cloned into the TA plasmid pCRtm2.1-TOPOTM for further sequence analysis. The TOPOTM cloning reactions and transformations were carried out as outlined in the manual included in the TA cloning kit (Invitrogen, Carlsbad, CA). Both M13 reverse and forward (-40) sequencing primers were used to sequence both directions.
This DNA was used as a template in cycle sequencing reactions using Cy5-labeled oligonucleotide primers and the Cy5 AutoCycle Sequencing Kit (Pharmacia Biotech, Piscataway, NJ). These reactions were then electrophoresed and analyzed using an ALFexpress automated DNA sequencer (Pharmacia Biotech). DNA sequences were analyzed using DNASIS sequence analysis software for Windows (Hitachi Software, San Bruno, CA).
Results & Discussion
Normal developmental embryology of the mouse lens begins by days 9.5-10 (E9.5-10) of gestation, when the developing optic vesicles have approached the surface ectoderm, which begins to thicken, forming the lens placode [24,25]. At E10.5, the lens placode begins to invaginate to form the lens vesicle . At the same time, the optic vesicle invaginates to form the optic cup. At E11-13, the lens is characterized by elongation of the cells from the posterior wall of the lens vesicle, leading to obliteration of the vesicle cavity. Further enlargement of the lens continues by proliferation of lens epithelial cells at the lens equator to form lens fibers, a process that continues throughout adult life. Since both MP19 and MIP are found only in the lens fiber cells, we would anticipate that messenger transcripts coding for these proteins would only be found from approximately stage E11 on.
To identify the temporal expression of the genes of MIP, Cx50, and Lim2 in early mouse embryonic stages, we performed RT-PCR analysis using MIP-, Cx50-, and MP19-specific oligo primers, respectively.
We carried out several control reactions to determine whether we were amplifying only the correct sequence (ie, only MP19 mRNA and not some other product) and whether or not we were amplifying genomic DNA or only mRNA. As can be seen in Figure 1 and Figure 3, these RT-PCR reactions both cross at least one intron. If we were amplifying genomic DNA, we would observe more than one product, having a considerably greater size than the predicted sequence obtained from amplifying only mRNA. With cx50 (Figure 2), we were not able to cross an intronic space to obtain RT-PCR product, therefore the size of the product would be the same whether or not we had DNA contamination or not. We therefore carried out a negative control reaction with all three genes, whereby we destroyed the reverse transcriptase before carrying out the PCR reaction. This would result in a product only if there was contaminating DNA present in our preparations. In Figure 4 we observe a product only in samples which have not been pretreated at 95 °C for 10 min (Figure 4, lanes 2, 4, and 6). When identical samples were pretreated by heating to 95 °C for 10 min before carrying out the PCR reaction, there was observed no product (Figure 4, lanes 3, 5, and 7). This result indicated that we were indeed only amplifying mRNA from each of the genes in question.
In order to determine the nature of the RT-PCR product obtained, we cloned each RT-PCR product into a TA cloning vector and carried out double stranded DNA sequencing. This result indicated that all three of the RT-PCR products was pure, and sequence analysis indicated that the anticipated product was, indeed, sequence from each of the subject mRNA.
mRNA transcripts coding for Cx50, a major gap junction protein in the lens, were first observed at day E9 (8.5-9.75 dpc) of embryonic development (Figure 5, lane 3), and levels appeared to remain relatively constant throughout the developmental stages observed (Figure 1). At day 9 (typically a range of embryonic development in a litter of about plus or minus 1/2 day) there is very little in the way of eye development. The lens placode certainly is present, and the lens pit is just beginning to form. The presence of Cx50 transcripts at this early stage is rather surprising, however this protein may be important for further differentiation of lens epithelial cells to fiber cells. It is also very likely that a large portion of the Cx50 transcripts observed in the very early periods of development (E9-E11) would be originating from other areas of the mouse, since at these early stages of collection, the entire embryo was used as a source of RNA. It is well known that Cx50 is found in other areas of the body, including the ciliary body  and Muller cells and astrocytes of the retina .
MIP transcripts were clearly detected in E11.5 (11-12.25 dpc) embryos (Figure 6, lane 7). The primer set used was designed to amplify across an intron, thus eliminated the possibility of amplifying contaminating genomic DNA. The stage at which MIP transcripts were first detected is relatively early in lens development, about the time when the lens vesicle is completely separated from the surface epithelium (E11.5).
Embryos of the same gestational age may differ slightly in their stage of development. Different mouse strains develop at different rates and, in some cases show differences in the relative rates of development of different organs as discussed in the "Staging Criteria" for the Edinburgh Mouse Atlas. The possible range for developing mouse embryos at stage E11.5 would possibly be varied from 11 to 12 dpc. This range would include Theiler stage 19 (11-12.25 dpc), at which time the lens vesicle is completely separated from the surface epithelium. Also at Theiler stage 19, the anterior, equatorial, and posterior epithelium have formed, however, the lens vesicle cavity is still present. It is not until Theiler stage 21 (12.5-14 dpc) that lens fiber cells have formed and the lens vesicle has lost it's lumen. It is clear that MIP transcripts are present at the very beginning of lens fiber cell formation. The earliest stage that MIP protein has been detected has been at E12.5 [29-31]. It is interesting that the gene for MIP is activated at least a day before the protein is detected.
In comparison, MP19 mRNA was first detected in E12 embryos, shown in Figure 7, lane 10, which indicated that the Lim2 gene begins to be activated at about 11.5-13 dpc. The stage at which Lim2 transcripts were detected is about the period when primary lens fibers begin to elongate in the lens vesicle. Again, the expected range of E12 embryos is between 11.5 and 13 dpc. If the timing of mRNA transcripts until the presence of protein is the same as with MIP, it would be expected that MP19 protein would be first observed at about 13 to 15 dpc.
This study demonstrates that the mRNAs coding for MIP and MP19 in the mouse lens fiber cell is present quite early in mouse lens development, and considerably before their coded proteins are observed in the lens. It is important to point out that we are observing only the production of gene transcripts. It would not be surprising to find gene transcripts present in tissues some time before the presence of actual protein products.
Variation in the temporal expression between the genes of Cx50, MIP, and Lim2 over the course of development may suggest the gene-coordinated regulation during lens fiber cell development. The data reported here provide a basis for further studies on the temporal expression of the lens membrane protein genes and their relationship to protein translation.
This research was supported in part by National Institutes of Health grants R01 EY11516 and R01 EY12301 to RLC, F32 EY07022 to LZ, and T32 EY07092, P30 EY06360, C06 EY06307, the Knights Templar Educational Foundation of Georgia, Inc., and a Departmental Grant from Research to Prevent Blindness, Inc. RLC is presently a Research to Prevent Blindness Senior Scientific Investigator. Some of these data were presented in abstract form at the 2001 meeting of the Association for Research in Vision and Ophthalmology.
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