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Keywords:

  • regeneration;
  • transdifferentiation;
  • development;
  • wound healing;
  • subtracted cDNA library;
  • lens;
  • cornea

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Few directed searches have been undertaken to identify the genes involved in vertebrate lens formation. In the frog Xenopus, the larval cornea can undergo a process of transdifferentiation to form a new lens once the original lens is removed. Based on preliminary evidence, we have shown that this process shares many elements of a common molecular/genetic pathway to that involved in embryonic lens development. A subtracted cDNA library, enriched for genes expressed during cornea-lens transdifferentiation, was prepared. The similarities/identities of specific clones isolated from the subtracted cDNA library define an expression profile of cells undergoing cornea-lens transdifferentiation (“lens regeneration”) and corneal wound healing (the latter representing a consequence of the surgery required to trigger transdifferentiation). Screens were undertaken to search for genes expressed during both transdifferentiation and embryonic lens development. Significantly, new genes were recovered that are also expressed during embryonic lens development. The expression of these genes, as well as others known to be expressed during embryonic development in Xenopus, can be correlated with different periods of embryonic lens induction and development, in an attempt to define these events in a molecular context. This information is considered in light of our current working model of embryonic lens induction, in which specific tissue properties and phases of induction have been previously defined in an experimental context. Expression data reveal the existence of further levels of complexity in this process and suggests that individual phases of lens induction and specific tissue properties are not strictly characterized or defined by expression of individual genes. © 2002 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

In vertebrate embryos, specific tissue interactions (inductive interactions) play key roles in the determination of cell fates during development. Such interactions lead to the formation of the germ layers, the central nervous system, and various organ systems (Grunz, 1997; Kimelman and Griffin, 1998; Baker and Bronner-Fraser, 2001). For over 100 years, the vertebrate lens has served as a model system for studying the nature of embryonic induction (Grainger, 1992, 1996). As a result of numerous tissue transplantation and explant culture experiments, we have developed a firm understanding of the interactions involved in this process. These studies have allowed us to develop a conceptual model defining certain properties inherent in both the inducing and responding tissues (Fig. 1). Embryonic lens formation is directed by a series of inductive interactions that take place during two principal phases (Henry and Grainger, 1987, 1990; Grainger, 1992, 1996). Experiments carried out with the frog Xenopus laevis indicate that an “early phase” of lens induction begins during gastrulation (approximately stage 11; all Xenopus stages follow those of Nieuwkoop and Faber, 1956) when the ectoderm is first competent to respond to these signals (see Fig. 1A; Henry and Grainger, 1987; Servetnick and Grainger, 1991). This early phase of lens induction involves signals emanating from the developing central nervous system (neural plate) that begin to specify fates in the adjacent, contiguous sheet of placodal ectoderm by means of planar induction (Henry and Grainger, 1990). The early phase also appears to involve transverse tissue interactions between the presumptive lens ectoderm and tissues underlying this region (e.g., mesoderm; see Henry and Grainger, 1990). The “late phase” of lens induction begins when the developing optic vesicles come into contact with the overlying head ectoderm (stage 19), and continues through later stages of development (Fig. 1A). This late phase of lens induction pinpoints the exact site of lens formation in a larger field of head ectoderm to ensure coordinated development of the lens and retinal tissues.

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Figure 1. Diagrams illustrating the model of embryonic lens induction and development in Xenopus laevis. The various graphs represent different tissue properties revealed by tissue transplantation and explant culture experiments (Henry and Grainger, 1987, 1989; Servetnick and Grainger, 1991). The X axes indicate developmental stages (according to Nieuwkoop and Faber, 1956). The Y axis represents the relative intensity of that property. A: Embryonic lens formation appears to be directed by two principal phases of induction, which include essential “early,” as well as “late” tissue interactions. Some slight overlap of these phases of induction occurs around stage 19 when the neural folds undergo fusion, which eventually separates the neural plate from the adjacent placodal ectoderm (terminating planar signals), and the optic vesicles begin to protrude from the developing forebrain and contact the presumptive lens ectoderm. B: Embryonic ectoderm gains an autonomous window of competence (A.C. in Fig. 4) to respond to lens inductive interactions, which is tied to its ability to respond to these interactions in the appropriate context. This property reaches a maximum at approximately stage 11.5, as indicated. Presumably competence to respond to these signals is sustained while inductive interactions continue to take place during later stages. C: When competent tissues receive these inductive signals, they gain an increasing lens-forming “bias” or propensity to form lens cells. After an initial period of “specification,” sufficient inductive signals reach the responding tissue such that when it can then be cultured in isolation to produce a lens, the tissue is then considered to be “specified” (stage 19). Ultimately, the tissue becomes “committed” to the lens cell fate, when it can no longer respond to other signals (definitions according to Slack, 1991; Grainger, 1992, 1996). Presumably this occurs by stage 26, when the lens undergoes cellular differentiation, after a phase of “commitment.” D: Hypothetical (temporal) patterns of gene expression that might be correlated with and establish each of the different properties described above. See text for further details.

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Embryonic ectoderm first develops an autonomous window of “competence” to respond to lens inductive interactions early during development (see Fig. 1B), which corresponds with initiation of lens induction (between stages 11 and 12; Henry and Grainger, 1987; Servetnick and Grainger, 1991). Presumably, competence is maintained once ectodermal tissues receive lens inductive signals (Fig. 1B). As further induction takes place, the responding tissues develop an increasing “lens-forming bias” or propensity to form lenses (Fig. 1C). Like the other properties, bias is measured experimentally, relative to the duration of the inductive interactions. The response is measured in terms of an increasing percentage of cases that will form lenses, as well the overall extent of lens epithelial and fiber cell differentiation that ultimately takes place in the responding tissues (Fig. 1C). Growing bias represents the process of “specification” and “commitment” that ultimately culminates in lens “differentiation.” By stage 19, the presumptive lens ectoderm is “specified,“ as this tissue will go on to differentiate some lens cells when cultured in isolation (Fig. 1C; Henry and Grainger, 1990). At some stage the lens is irreversibly “committed,” which presumably takes place by stage 26 when the lens placode is forming and lens crystallin proteins are being produced (Fig. 1C; general definitions according to Slack, 1991; Grainger, 1992, 1996). Although we have a firm understanding of the tissue interactions involved in vertebrate lens induction, much less is known about the genes controlling the processes of lens cell determination and differentiation. One presumes, however, that each of the concepts defined in this model of embryonic lens induction should be correlated with specific changes in gene expression. In other words, one may be able to identify specific phases of lens induction by examining spatial and temporal patterns of gene expression during lens development.

In Xenopus, lenses can also arise through the process of cornea-lens transdifferentiation (also called “lens regeneration”). In cornea-lens transdifferentiation, a new lens forms from the outer corneal epithelium when the original lens is removed from the eye of a premetamorphosed tadpole (Freeman, 1963). This process is triggered by factors present in the eyecup, which appear to be manufactured by the neural retina (Freeman, 1963; Henry and Mittleman, 1995; Bosco et al., 1997). Normally, these factors are prevented from reaching the outer cornea due to the presence of the lens and the inner corneal endothelium, which is derived from neural crest (Filoni et al., 1997; Henry and Elkins, 2001). We have shown recently that certain genes expressed during embryonic lens development are re-expressed during cornea-lens transdifferentiation, including several transcription factors (i.e., Pax-6, Xotx2, xSOX3, Xprox1) and one of the γ-crystallin family members, γ6-cry (Schaefer et al., 1999; see also Mizuno et al., 1999a,b). These findings suggest that lenses forming from corneal tissue arise by means of a cellular and molecular pathway similar to that taken during embryogenesis. Thus cornea-lens transdifferentiation is a viable system with which to isolate additional genes involved in lens formation.

In this study, we have exploited the phenomenon of cornea-lens transdifferentiation as a convenient system to isolate genes expressed during lens formation. We have prepared a subtracted cDNA library enriched for clones representing gene activity associated with the process of cornea-lens transdifferentiation. Clones isolated from this library provide a profile of the genes expressed during cornea-lens transdifferentiation and corneal wound healing (the latter being a consequence of the surgery required to initiate transdifferentiation). Some of these genes are also expressed during embryonic lens development. These findings provide support for arguments that the processes of embryonic lens development and cornea-lens transdifferentiation share elements of a common molecular/genetic pathway. The expression patterns of these genes, as well as others previously identified, serve as markers that can be correlated with specific phases of lens induction. This information is used to evaluate our current model of embryonic lens induction and reveals the existence of further levels of complexity within this process.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Library Construction

Although there are several procedures that can be used to characterize novel patterns of gene expression (e.g., see Lisitsyn, 1995; Liang and Pardee, 1998), subtractive hybridization has proven to be a successful technique in the cloning of genes unique to various developmental processes (e.g., for Xenopus see Sive et al., 1989; Lemaire et al., 1993). We have argued that the process of cornea-lens transdifferentiation shares elements of the same molecular pathway as that involved in embryonic lens development (Schaefer et al., 1999; see also Mizuno et al., 1999b); therefore, we prepared a subtracted cDNA library enriched for genes expressed during cornea-lens transdifferentiation in Xenopus. To isolate genes expressed during the early phases of lens cell determination and differentiation, transdifferentiating corneas were only collected during the first 4 days after lens removal, to limit the representation of those genes expressed during more terminal phases of lens differentiation, such as the crystallins. For instance, crystallin protein expression is detected as early as 5 days after lens removal (Brahma and McDevitt, 1974; Henry and Mittleman, 1995), and γ-cry6 mRNA is not detected before 4 days after lens removal (Schaefer et al., 1999; see also Mizuno et al., 1999a).

The primary libraries contained 1.6 × 106 colony-forming units (pSport 1 plasmid transdifferentiating cornea library with 99% of these containing inserts as determined by Xgal/IPTG blue-white colony selection) and 2.2 × 106 colony-forming units (pSport 2 plasmid control cornea library with 99% of these containing inserts). The subtracted library contained approximately 1.4 × 105 cloned inserts (80% of the transformed cells possessed inserts, as determined by Xgal/IPTG blue-white colony selection). On the basis of transformation efficiencies measured by using known quantities of control DNA, we estimated that less than 1 ng of the starting transdifferentiating cornea ssDNA remained after subtraction. The rather large size of the subtracted cDNA library is attributed to the large amount of starting material used in the subtraction (600 ng of target ssDNA), rather than the overall complexity of this library. In fact, the overall complexity is much lower, as several the clones actually represent duplications of the same cDNAs (see below).

Library Screening and Gene Expression Profiles

Sive et al. (1989) prepared a Xenopus neurula stage (stage 23) subtracted cDNA library and calculated that the biotin-based subtraction procedure resulted in an approximately 50-fold enrichment in the representation of head-specific sequences. Wieland et al. (1990) estimate that these subtraction procedures can enrich target sequences up to 1,000-fold. Considering the degree of enrichment that can be achieved in the construction of subtracted cDNA libraries, a significant proportion of the cDNA clones present in our subtracted library should be transdifferentiation-specific (see evidence presented in the next section). To obtain a profile of the genes represented in the subtracted cDNA library, we sequenced several randomly selected clones. Only those clones containing inserts greater than or equal to 500 bp in length were sequenced. Although some interesting genes may be represented in the smaller size range, we chose not to examine them for the following reasons: (1) to ensure that more clones would be of full length, (2) to ensure that more sequence information could be obtained for reliable similarity searches, and (3) to ensure that the clones would be of ample size to generate labeled antisense RNA for in situ hybridization expression analyses (see below).

A total of 250 clones was ultimately selected for sequencing. Insert size ranged from 500 to 4,000 bp, with an average size of 975 bp. In each case, 400–600 bp of sequence was obtained from the 5′ end in an attempt to avoid the less conserved 3′ untranslated regions. The nucleotide and inferred amino acid sequences were compared with those contained in the GenBank database by using blastn and blastx search programs provided by the National Center for Biotechnology Information. An initial batch of 93 clones was sequenced, and it was found that three species were represented multiple times in this batch. One of the unidentified sequences (e.g., B46) was represented by seven identical clones in this group (nearly 8% of the initial batch of clones examined), and another unidentified sequence was found in four (4%) of the clones examined (e.g., B97). Finally, two other sequenced clones were also identical to one another (i.e., B127 and C8, representing an unidentified sequence). Because the subtracted library has not been amplified, each one of these clones passed through the subtraction process independently.

In an attempt to avoid resequencing these same clones, differential colony lift hybridization was employed by using a mixed probe to the three duplicated species mentioned above. An additional 157 clones were sequenced. Within these additional clones, seven unique species were each represented twice (e.g., C46, D93, H26, H73, J28, J93, J129). Therefore, a grand total of 233 unique clones were sequenced. In most cases (173 clones), the sequences examined did not exhibit significant degrees of similarity to characterized sequences present in the databases. Although these clones may represent novel genes, it is also likely that we do not have sufficient sequence data to ascertain their identities. In fact, some of the sequences do not contain good open reading frames, so one would ultimately need to screen for larger clones to obtain complete sequence information. However, many identities were readily assigned, and 45 of these clones exhibited significant degrees of sequence similarity to other known genes (Table 1). An additional 15 species displayed similarities to novel sequences contained in the databases that encode putative, uncharacterized proteins. Of the 233 unique sequences analyzed, 27 clones displayed exact matches to previously characterized Xenopus ESTs, and 64 other clones displayed some similarity to other ESTs represented in various databases (information not included in Table 1). All sequences of the 233 unique clones, along with information pertaining to sequence similarities, are available on-line at http://www.life.uiuc.edu/henry.

Table 1. Similarities Exhibited by Clones Contained in the Subtracted cDNA Librarya
Clone IDPutative/known identity% SimilarityPutative/known functionsReferences
  • a

    Putative or known identities are based on sequence similarity. Putative/known functions inferred from characterized genes. References, if available, pertain to identity and/or putative functions. Similarity represents % identity based on either the nucleotide or inferred amino acid sequences, as indicated. See text for further details. All sequences, NCBI accession numbers, and specific information pertaining to similarities to other sequences are available at http://www.life.uiuc.edu/henry. Registered NCBI EST clone IDs begin with “XLTC” followed by the clone ID indicated above (e.g., clone B25 is recorded as “XLTCB025”).

Lens cell-related proteins
 B25Gamma cry I87/175aaLens crystallin proteinSmolich et al. (1993)
 H109Beta-Cry BI74/203aaLens crystallin proteinQuax-Jeuken et al. (1984)
Berbers et al. (1984)
 H156Gamma cry I93/39aaLens crystallin proteinSmolich et al. (1993)
DNA replication/transcription/translation
 B4RBP10100/67aaRNA polymerase II subunitMcKune et al. (1995)
 B81Coactivator of AF-274/81aaNuclear protein involved in receptor binding and activationSauve et al. (2001)
 B87CRSP3493/315aaTranscriptional cofactor complex subunitRyu et al. (1999)
 C46SNC1 Human SNRNA (SNAP 43)80/80aaProximal sequencing element-binding transcription factor gamma subunitHenry et al. (1995)
 D616s rRNA92/232ntXenopus mitochondrial 16s rRNARoe et al. (1985)
 D62CPEB90/376ntRNA binding protein promotes polyadenylation and translationHake et al. (1998)
 H59PHD zinc finger (C4HC3 Family, WSTF)93/194aaTranscriptional control, Williams syndromeLu et al. (1998)
 H84Metallothionine transcriptional activator99/38aaTranscriptional controlXu (1993)
 H97Zinc finger C2H2 family (ZNF215)60/185aaTranscriptional control Beckwith-Weidman syndromeAlders et al. (2000)
 H153Human mitochondrial ribosomal protein S2174/83aaTranslational machineryLai et al. (2000)
Koc et al. (2001)
 H16740 S. ribosomal S3A99/207aaTranslational MachineryAmaldi et al. (1982)
 H186Mat186/238aaInvolved in CDK-7-Cyclin H transcriptional regulation of p53Ko et al. (1997)
 J102B-IND66/63aaNovel component of Rac1-signaling pathways leading to the modulation of gene expressionCourilleau et al. (2000)
Wound response proteins
 B66MMP-971/671aaMatrix metalloproteinases, connective tissue destruction, remodeling, and regenerationCarinato et al. (2000)
 B89MMP-1396/153aaYang et al. (1997)
   Yang and Bryant (1994)
 D31ATPase90/114aaVarious functions, wound healingAmino et al. (1997)
 H112Heparin cofactor II93/203aaAnti-coagulant-possibly involved in wound responseAndersson (1987)
Extracellular/transmembrane proteins and cell signaling
 B118Nuclear receptor binding protein70/192aaUnknown functionHooper et al. (2000)
 C76Olfactomedin57/128aaSecreted polymeric glycoprotein, conserved domain in TIGR, implicated in open angle glaucomaYokoe and Anholt (1993), Adam et al. (1997)
 D31Plasma-membrane Ca++-ATPase96/115aaCalcium transportDumont et al. (2001)
 H34OL-(protocadherin)88/436ntHomophillic cell-cell adhesion moleculeHirano et al. (1999)
 H127G-coupled protein receptor92/41aaUnknown functionWittenberger et al. (2001)
 H141p24 delta putative cargo transport receptor96/250ntER to cargo transportKuiper et al. (2001)
 H151Xoom98/93aaInvolved in gastrulation of ectodermal cellsHasegawa and Kinoshita (2000), Hasegawa et al. (1999, 2001)
 J7QNR-7180/77aaEncodes membrane protein with similarity to melanosomal proteins regulated by v-mycTurque et al. (1996)
 J64CD2 binding protein64/154aaInvolved in IL-2 upregulationNishizawa et al. (1998)
 J80T-cell receptor49/151aaImmunogenic responseTakihara et al. (1988)
 J64CD2 binding protein64/154aaInvolved in IL-2 upregulationNishizawa et al. (1998)
 J80T-cell receptor49/151aaImmunogenic responseTakihara et al. (1988)
 J133Lectin precursor42/97aaInvolved in cell-cell adhesion processesTiffoche et al. (1993)
Metabolism
 B109Amannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase77/93aaSynthesis of N-glycansYoshida et al. (1998)
 H13Enoyl CoA/Acyl CoA hydratase type 180/176aaFatty acid metabolismHiltunen and Qin (2000)
 H26p4792/117aaNADPH oxidaseZhang et al. (2000)
 H29Vaculor ATPase subunit Ac4597/483ntInvolved in the mediation of acidification of intracellular compartmentsHolthuis et al. (1999)
 H169L13 mitochonrial ribosomal protein89/176aaprotein component of the large ribosomal subunitSuzuki et al. (2001)
Miscellaneous
 B10Ovarian tumor protein60/67aaGerm cell developmentSteinhauer et al. (1989)
Steinhauer and Kalfayan (1992)
 C9Bactenacin—anti-microbial domain52/81aaAntimicrobial peptidesScott et al. (1996)
 E7Thiopurine S-methyltransferase70/219aaInvolved in detoxificationFessing et al. (1998)
 H12Ypt/Rab GTPase activating protein77/136aaCatalyzes GTPase activity of Ypt/Rab-GTPase (has TBC domain)Götte et al. (2000)
 H89Glucocerebrosidase77/122aaDegradation of cerebrosidesO'Neill et al. (1989)
 H98Fbox92/140aaInvolved in ubiquinationCenciarelli et al. (1999)
 H101Transposase, Tc-1 family70/104aaInactive pseudogene?Leaver (2001)
 J55An1-like zinc finger protein77/184aaInvolved in ubiquinationLinnen et al. (1993)
 J47Skeletal muscle protein67/68aaUnknownLeachman et al. (1992)
Unknown/hypothetical proteins
 B63Hypothetical protein KIAA052364/190aaUnknownNagase et al. (1998)
 C24Hypothetical protein FLJ1456659/172aaUnknown
 C29Hypothetical protein67/51aaUnknownCarninci and Hayashizaki (1999)
 C70Hypothetical protein FLJ1097593/38aaUnknownPascual et al. (2000)
Aasland et al. (1995)
 H28Hypothetical protein KIAA068379/173aaUnknownIshikawa et al. (1998)
 H73Hypothetical Protein68/97aaUnknownCarninci and Hayashizaki (1999)
Carninci et al. (2000)
 H77Hypothetical protein53/230aaUnknown
 H163Hypothetical protein66/163aaUnknownAdams et al. (2000)
 J26Hypothetical protein mgC1428875/34aaUnknown
 H145Hypothetical protein92/185aaUnknownZhang et al. (2000)
 J98Hypothetical protein87/174aaUnknown
 J100Hypothetical protein76/100aaUnknownNagase et al. (2000)
 J136Hypothetical protein52/133aaUnknown
 J138Hypothetical protein54/133aaUnknown
 H144Hypothetical protein84/50aaUnknownShibata et al. (2000)
Carninci and Hayashizaki (1999), Carninci et al. (2000)
Unidentified clones
 173 additional unique clones with no significant sequence similarity, and unknown functions. See text for further information.

Analysis of Differential Expression

To determine the effectiveness of the subtraction process and select transdifferentiation-specific genes for further study, sets of polymerase chain reaction (PCR) primers were designed to amplify specific fragments of selected clones from the original control vs. transdifferentiating cornea libraries. In some cases, differential dot-blot hybridization was also used to verify if these clones were present only in the original transdifferentiating cornea cDNA library. Fifteen of the sequenced cDNA clones were randomly selected. Examples are shown in Figure 2, and the results are summarized in Table 2. It was found that 47% of these clones (seven cases) were present only in the transdifferentiating cornea library. One of these seven clones represents a γ-crystallin gene (B25). The other eight clones are present in both cDNA libraries. Although these latter genes do not appear to have been removed by the subtraction process, some may represent genes that were significantly up-regulated during transdifferentiation. Such genes could have been preserved through the subtraction process. This conclusion is supported by in situ hybridization expression analyses, as Xmmp-9 expression (clone B66) cannot be detected in control corneas by means of in situ hybridization analyses (see Carinato et al., 2000). Furthermore, colony lift hybridization revealed that this gene is represented in approximately 1:20,000 clones in the original transdifferentiating cornea cDNA library, whereas this clone is represented in 1:150 clones in the subtracted cDNA library (data not shown). Thus the subtraction enriched the representation of this gene by approximately 130-fold. Another clone, B105, is represented in approximately 1:250,000 clones in the original transdifferentiating cornea cDNA library, whereas it is represented in 1:250 clones in the subtracted cDNA library, which represents a 1,000-fold amplification of this species (data not shown). The gene profiles represented by the sequenced clones (e.g., the presence of clones encoding lens proteins, see Table 1), along with the fact that 47% of the clones examined are only present in the transdifferentiating cornea cDNA library, indicate that the subtraction process was successful in enriching for clones specific to the processes of cornea-lens transdifferentiation and corneal wound healing (see Discussion section).

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Figure 2. Polymerase chain reaction (PCR) -based screens to determine whether specific clones recovered from the subtracted cDNA library are present in the transdifferentiating and control cornea cDNA libraries used to construct the subtracted library. The presence of Pax-6 was also examined. One nanogram of each clone's plasmid DNA was used in the positive (+) control reactions, and 10 ng each of the transdifferentiating (T) and control (C) cornea library cDNA were included in the other PCR reactions shown here. The blank, negative control reactions are not shown to conserve space. See text for further details.

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Table 2. Presence of Selected Clones in the Two cDNA Libraries Used to Construct the Subtracted cDNA Librarya
Clone IDPutative/known identityPresence(+) vs. absence(−) in cDNA libraries
TransdifferentiatingControl
  • a

    Verification performed by noting the presence (+) or absense (−) of descrete bands of the expected size generated in separate PCR reactions using equivalent amounts of DNA templates (see Figure 2).

  • b

    Differential dot-blot hybridization was also used to verify the presence or absence of these clones in the two cDNA libraries (E7 and E20 were only assayed using the differential dot-blot hybridiazation assay). See text for further details.

B25bGamma crystallin+
B29bUnknown++
B37Unknown++
B46Unknown+
B66MMP-9++
B97Unknown+
B105Unknown+
B127Unknown++
C9Bactenacin++
C46Unknown+
C63Unknown++
D26Unknown++
D50Unknown++
E7bThiopurine S-methyltransferase+
E20bUnknown+

Expression Analyses of Clones Obtained From the Subtracted cDNA Library

The expression patterns of some of the genes recovered from the subtracted cDNA library were examined to determine whether they are expressed during early development. We examined the embryonic expression of clones that were found to be differentially expressed in the control and transdifferentiating cDNA libraries, except clones B25 (a γ-crystallin) and clone E20 (see Tables 1 and 2). Other clones were chosen at random. Their expression was examined over a range of embryonic stages from stage 12 (gastrulation) through stage 42 (early larval stages after eyes and lenses have formed). The data are represented in Table 3. A total of 15 clones was examined, and in 6 cases, embryonic expression was detected. One of these clones, B66 (corresponding to Xenopus matrix metalloproteinase, MMP-9), is not expressed in the embryonic eye (see Fig. 3A, see also Carinato et al., 2000); however, the other five clones (B87, B99, B105, D43, and E7) were found to be expressed in the developing eye and lens, as well as in other tissues (see Fig. 3B–L).

Table 3. Temporal Expression of Genes Isolated from the Subtracted cDNA Library, During Embryonic Stagesa
cDNA clonePutative/known identityDevelopmental stage examined
14–1519–2026–2730–3538–42
  • a

    4–12 embryos were examined at each time point, as indicated. − indicates that no embryonic expression was noted at those embryonic stages. + indicates that expression was detected in various embryonic tissues (see text and Fig. 3 for specific details). Blanks indicate that these time points were not examined. Expression was also assayed at earlier stages (stage 12), but no expression was detected. All developmental stages are according to Nieuwkoop and Faber (1956).

B10Unknown
B37Unknown
B44Unknown
B46Unknown
B66MMP-9+++
B70Unknown
B87CRSP34++++
B89MMP-13
B97Unknown
B99Unknown+++
B105Unknown++++
B111Unknown
C46Unknown
D43Unknown+++++
E7Thiopurine S-methyltransferase++++
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Figure 3. Expression patterns of genes isolated from the subtracted cDNA library. RNA whole-mount in situ hybridization revealing the localization of six different mRNAs in Xenopus embryos. All whole-mount early larval stages are shown as right lateral views, with the anterior ends located to the right of the figure. All whole-mount embryos are shown as left dorsolateral views, with the anterior end directed toward the left side of the figure. A: Expression of B66 (Xmmp-9) at stage 35. Notice that expression is detected in several isolated mesenchymal cells, which lie just beneath the ectoderm. Most of these cells are located along the trunk and in the tail, and very few are seen in the head. B: Expression of B87 (Xenopus CRSP34) at stage 30. Note intense expression in the eye and lens (ey), as well as the otic vesicle (ot), pharyngeal arches (pa), spinal cord (sc), pronephros (pn), and axial musculature. The black arrowhead points to expression in the pineal gland. C: Expression of B99 (identity unknown) at stage 27. Note intense expression in the eye and lens, as well as one of the pharyngeal arches. Some weaker expression may also be seen in the otic vesicle. D: Expression of B105 (identity unknown) at stage 14. Expression is seen to the placodal ectoderm surrounding the anterior neural plate (np). The most intense expression is localized in the presumptive lens ectoderm (white arrowhead), whereas some expression is also detected in the area of the presumptive otic region (black arrowhead). Much weaker expression extends anteriorly into the region of the presumptive olfactory ectoderm. Thin lines of expression are also seen in the anterior neural folds (nf). E: Transverse section through the anterior neural plate showing expression in the presumptive lens ectoderm (ple) and cells in the neural folds. Relative plane of section is indicated by dotted white line shown in D. F: Expression of B105 (identity unknown) at stage 19. G: Expression of B105 at an even later stage of development (stage 34). Note intense expression in the eye and lens, as well as the otic vesicle. Asterisks mark the locations of segmental expression in the brain. Black arrowhead points to expression in the pineal gland. H: Cross-section through the eye and lens of a stage 30 larva showing expression in the lens (ln). rt, retina. I: Representative negative control showing no hybridization of sense D43 probe (D43s) to a stage 32 embryo. J: Expression of D43 at stage 15. Expression is seen in regions similar to those expressing B105. K: Expression of D43 at stage 32. Expression is detected in the head in structures including the eye, lens, and otic vesicle. Other expression is also seen in the spinal cord, the pronephros and pronephric duct, and in cells of the cloaca (cl). L: Expression of E7. Specimens shown in B and C have been cleared, whereas the others have not. Scale bars = 25 μm in E, 30 μm in H, 250 μm in J (applies to D,F,J), 500 μm in L (applies to A–C,G,I,K,L).

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The expression patterns of these five genes are reminiscent of various transcription factors, such as Pax-6, and other genes that are expressed broadly in the eye, lens, and central nervous system. One of these clones, B87 (Xenopus CRSP34), is expressed in the eye, lens, and central nervous system beginning at stage 19. Expression at stage 30 is shown in Figure 3B, when this gene continues to be expressed in the lens as well. B99 (unknown identity) is first expressed at stage 19 in anterior neural tissue and the placodal region. It continues to be expressed in the derivatives of these tissues, including the lens at later stages. Expression at stage 27 is shown in Figure 3C. B105 (unknown identity) is first expressed at stage 14 (see Fig. 3D). Figure 3E shows a transverse section through the neural plate at this stage showing expression in the outer ectoderm of the neural folds and in the inner sensorial ectoderm of the placodal, presumptive lens, ectoderm. Expression at stages 19 and 36 are shown in Figures 3F and 3G, respectively. A cross-section through the eye shows expression in the lens (Fig. 3H). Significantly, B105 is only found in the transdifferentiating cDNA library (see Table 2). D43 (unknown identity) is expressed early beginning at stage 14 in the placodal ectoderm and anterior neural folds (Fig. 3J). Expression is highest in the presumptive lens ectoderm. Later expression at stage 32 is shown in Figure 3K, where this gene is expressed in the derivatives of these tissues, which includes the lens. Its expression is similar to that of B105 (e.g., compare Figs. 3D and 3J). Clone E7 (similar to thiopurine S-methyltransferase) is expressed between stages 14 and 32, initially in the neural plate and the presumptive neural crest, and later in the spinal cord, anterior CNS, the eye, lens, and other tissues (see Fig. 3L). Lens expression is first detected at stage 26. No significant hybridization was detected by using sense control probes generated from any of the clones examined (e.g., see Fig. 3I). Temporal windows of embryonic expression in presumptive lens ectoderm, lens placode, and differentiating lens are summarized in Figure 4.

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Figure 4. Summary of temporal patterns of gene expression within embryonic lens ectoderm in Xenopus laevis. Windows of expression are correlated with specific stages of development and to different phases of lens induction, tissue competence, and lens forming-bias as defined in the text and Figure 1. A: Different embryonic stages are represented on a time line of early development. B: Temporal windows of lens forming bias, competence, and induction are mapped onto the time line of early development. C: Temporal windows of gene expression within the developing lens ectoderm are mapped onto the time line of early development. Data are based on the results of this study, as well as that of previous studies (Pannese et al., 1995; Kablar et al., 1996; Hirsch and Harris, 1997; Penzel et al., 1997; Zygar et al., 1998; Schaefer et al., 1999; Kenyon et al., 1999; Hollemann and Pieler, 1999; Zhou et al., 2000; Ishibashi and Yasuda, 2001; Pommereit et al., 2001).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

A Profile of Gene Expression in Transdifferentiating Cornea Tissue

The putative identities of many of the clones present in the subtracted cDNA library are consistent with known expression profiles characteristic of cells undergoing lens formation and corneal wound healing. For instance, three of the clones (B25, H109, H156) encoded lens crystallin proteins, which represent new members of the Xenopus β- and γ-crystallin families. Although some cystallins are normally expressed in other tissues at much lower levels (Smolich et al., 1994), we have not detected expression in control larval corneas by means of in situ hybridization (Schaefer et al., 1999), and clone B25 is not present in the original control cornea cDNA library (see Fig. 2). The presence of these different crystallin clones in the subtracted library is consistent with the timing of crystallin expression during cornea-lens transdifferentiation, as mRNA used to construct the transdifferentiating cDNA library was obtained from corneas collected 1–4 days after lens removal. γ-cry mRNA is first detected in transdifferentiating corneas on the fourth day after lens removal (Schaefer et al., 1999; see also Mizuno et al., 1999a).

Several other clones represent components of the transcriptional and translational machinery (see Table 1). The presence of these clones is not surprising, as the phenomenon of cornea-lens transdifferentiation involves changes in cell proliferation and gene activity, and such genes are likely to be activated or up-regulated during this process. Of greater interest is the fact that some clones represent genes that may be involved in tissue-specific and developmental regulation of transcription and translation. For example, one clone, B87, encodes the Xenopus homologue of CRSP34, a newly identified transcriptional cofactor complex subunit (Ryu et al., 1999). The CRSP complex is a coactivator required for Sp1 activation, and it is possible that specific combinations of these coactivator complex subunits confer specific patterns of gene expression. Other clones also appear to encode proteins involved in transcriptional control and activation, including three that are similar to known zinc finger DNA binding proteins (H59, H84, H97). Clone H186 represents Mat1, a regulator of p53, that could have potential roles in wound healing (Ko et al., 1997). MAT1 is one of three proteins that form Xenopus CAK (cdk-activating kinase [Devault et al., 1995]). CAK has been shown to play a role in the activation of cdks as well as in the function of TFIIH and RNA polymerase II. Retroviral transduction of antisense Mat1 constructs into fetal human lens epithelial cells in vitro has demonstrated that reduced expression of MAT1 protein leads to a decrease in lens epithelial cell proliferation (Kampmeier et al., 2000). Clone J109 is similar to B-IND, a novel component of the Rac1-signaling cascade involved in gene activation (Courilleau et al., 2000). Although its structure and exact function remain largely unknown, the human homologue of B-ind1 has been found to potentiate Rac1 induction of NF-κB, a transcription factor known to play a role in inflammation, differentiation, cell proliferation, and apoptosis (Courilleau et al., 2000). Clone H59 is similar to a PHD zinc finger transcription factor (WSTF of the C4HC3 family) implicated in Williams syndrome, a complex developmental disorder (Lu et al., 1998). Clone H97 appears to represent a C2H2 zinc finger protein with similarity to ZNF215, a gene implicated in Beckwith-Wiedemann syndrome (Alders et al., 2000). C2H2 zinc finger proteins play a variety of roles in cell determination and differentiation. Clones representing transcription factors, and their coactivators may represent key players in controlling cornea-lens transdifferentiation. Although we have not examined all the clones represented in the subtracted cDNA library, we have not yet found any of the transcription factors known to play roles in lens formation, such as Pax-6 (e.g., Altmann et al., 1997; Chow et al., 1999). PCR analyses indicate that Pax-6 clones are present in both the original control and transdifferentiating cDNA libraries (see Fig. 2); therefore, this gene, and others, could have been removed by the subtraction process.

Ross et al. (1995) and Yu et al. (1995) have defined suites of differential gene expression in healing rat corneas. Because cornea-lens transdifferentiation was elicited by surgical removal of the original lens through the corneal margin, we anticipated that genes expressed during the process of corneal wound healing would also be present in the subtracted cDNA library (see Table 1). We found that two of the clones (B66 and B89) represent different matrix metalloproteinases (MMP-9 and MMP-13, respectively). MMPs play major physiological roles in degrading and remodeling the ECM. The presence of a clone encoding gelatinase-B (B66) is significant, because MMP-9 has been shown to be expressed in wounded corneal epithelium and is implicated in corneal ulceration and graft rejection (Berman, 1989; Fini et al., 1995, 1996, 1998; Barro et al., 1998; Ye and Azar, 1998; Carinato et al., 2000). MMPs have also been implicated in regeneration (Yang and Bryant, 1994; Yang et al., 1997). Several other clones could also be involved in wound healing, such as C9, D31, E7, H29, H89, H98, J55, and H29 (see Table 1).

Several clones encode extracellular or transmembrane proteins, and a few appear to be involved in cell signaling (see Table 1). For instance, clone H127 may represent a G-coupled protein receptor of unknown function (see Wittenberger et al., 2001). Such clones may be of potential significance, as the process of cornea-lens transdifferentiation is initiated by soluble signals provided by the neural retina, and these must be transduced by means of specific receptors and their intracellular pathways. Other clones encode a variety of proteins, which are involved in other processes, including various metabolic pathways, cell–cell adhesion, and others listed in Table 1. One of these (H101) appears to encode a transposase, which like many others, may represent an inactive pseudogene (Leaver, 2001). Finally, the majority of clones did not exhibit any significant degree of similarity to genes of known functions in the databases. Expression data for some of these unidentified genes indicates that they are expressed in the developing eye and lens and are likely to represent important players in the process of lens formation (e.g., B99, B105, D43; see Fig. 3C–K).

Relationships Between the Process of Embryonic Lens Development and Cornea-Lens Transdifferentiation

In choosing this approach to isolate additional genes expressed in the process of lens formation, we made one key assumption, which appears to have been valid. We assumed that similar molecular genetic pathways would be operating in both the processes of embryonic lens development and cornea-lens transdifferentiation. Although this assumption may have at first seemed obvious, as the ultimate end point (lens formation) appears to be identical in both cases, there was no a priori reason to believe it was true. There has been considerable debate about the relationships between various developmental and regenerative processes (Lopashov et al., 1986; Stocum, 1991; Gardiner et al., 1995; Schaefer et al., 1999; Mizuno et al., 1999a,b). Some studies have demonstrated conserved elements of gene expression between these processes. For instance, Del Rio-Tsonis et al. (1995a, 1999) have demonstrated that both pax-6 and Prox1 are re-expressed during Wolffian lens regeneration in the newt Notophthalmus viridescens. Schaefer et al. (1999) and Mizuno et al. (1999b) demonstrated conserved patterns of gene expression between these processes in both Xenopus and Cynops pyrrhogaster. As previously mentioned, there has been some speculation as to whether the inductive interaction required for transdifferentiation may be related to that occurring during the late phase of embryonic lens induction (Bosco et al., 1979, 1986; Filoni et al., 1982; Schaefer et al., 1999). The expression patterns of genes such as those represented by clones B105 and D43, however, indicate that the library contains genes expressed during very early phases of embryonic lens development. Together with the observation that other early genes, such as Xotx2 and Pax-6, are also expressed during transdifferentiation (Schaefer et al., 1999; Mizuno et al., 1999b), the data suggest that such early genes also play a role in cornea-lens transdifferentiation. Of the 15 clones chosen to examine embryonic expression in the present study, 6 cases exhibited some form of embryonic expression, with 5 (33%) of these displaying expression in the lens. These findings clearly point to the close molecular/genetic relationships between this process and that of embryonic lens development.

Although there is obviously a close relationship between embryonic lens development and cornea-lens transdifferentiation, we may, in fact, find molecular/genetic differences specific to each of these processes. That several of the genes examined do not appear to be expressed during embryonic development (Table 3) indicates that some gene expression may be specific to the process of cornea-lens transdifferentiation. Some of these genes may represent genes involved in wound healing, such as Xmmp-9 (Carinato et al., 2000); others may represent corneal genes that inadvertently passed through the subtraction process, which have nothing to do with lens formation. A key difference between embryonic development and transdifferentiation, or regeneration, involves the initial generation of progenitor cells by means of the localized de-differentiation of cell fates (Eguchi and Kodama, 1993; Del Rio-Tsonis et al., 1995b; Kodama and Eguchi, 1995; Stocum, 1995; Kosaka et al., 1998). It is likely that significant patterns of gene expression will be associated with these early phases of transdifferentiation, which may not take place during development. On the other hand, it is also possible that some patterns of gene expression will be specific to the process of embryonic lens development. Ultimately, we will be able to decipher these differences through extensive comparative studies.

Some authorities estimate that hundreds of genes may be involved in lens and eye development (Halder et al., 1995). For instance, more than 100 spontaneous mouse eye mutants have been reported (Graw, 1996). The results presented here demonstrate that the system of cornea-lens transdifferentiation is a viable one for isolating additional genes expressed during lens formation. What roles these genes play in the processes of lens cell determination and differentiation remain to be addressed by functional analyses. Directed screens will ultimately uncover all the molecular players involved in lens formation, and this information will lead us to fully understand the connections between related lens developmental, regenerative, and transdifferentiative processes, and the reasons why some organisms are able to undergo lens regeneration or transdifferentiation.

Examining the Model of Embryonic Lens Induction

As described in the Introduction section, a conceptual model has been proposed to explain the process of embryonic lens induction (see Grainger 1992, 1996, and Fig. 1). By examining the expression of several different genes during embryonic lens formation in Xenopus, one can evaluate the present model of lens induction and attempt to define tissue properties in a molecular context. Some experimental data indicate that changes in Pax-6 expression can be correlated with developmental changes in lens-forming competence in embryonic head ectoderm in the chicken (Li et al., 1994). In a recent study, Zygar et al. (1998) demonstrated that certain changes in gene expression are triggered by specific phases of lens development in Xenopus. Some of the genes isolated in the present study also exhibit expression patterns, which appear to be coordinated with certain phases of lens development. Those genes represented by clones B105, and D43 are initially expressed during the early phase of lens induction beginning at stage 14, whereas B87 and B99 are first expressed at the beginning of the late phase of lens induction (stage 19), and E7 is first expressed in the differentiating lens placode (stage 26). These temporal windows of gene expression are shown in Figure 4, and are correlated with specific stages of development and different phases of lens induction, tissue competence, and lens-forming bias, as defined in the model outlined in the Introduction section and illustrated in Figure 1.

Can the temporal expression patterns of different genes be correlated with specific tissue properties defined in the model? Does their expression reveal additional phases of lens development not identified in the current model? None of the genes examined appear to be correlated with the development of the initial autonomous phase of competence, which reaches a maximum around stage 11.5 (i.e., none like example I shown in Fig. 1D). Likewise, there are no examples that might be correlated with the development of sustained competence (i.e., examples III or IV shown in Fig. 1D). The two genes known to have the earliest zygotic expression in the presumptive lens ectoderm are Xotx2 and Xlens1, which are first expressed around stage 13 (Fig. 4; Kablar et al., 1996; Penzel et al., 1997; Kenyon et al., 1999; note that maternal Xotx2 mRNA is also present in the ectoderm at much earlier stages; Pannese et al., 1995, Zygar et al., 1998). Their expression could be triggered by the early phase of induction; however, both competence and the early phase of induction are initiated before this time at approximately stage 11 (i.e., there are no genes like examples II or III shown in Fig. 1D). Many examples such as Xotx2, Xlens1, and Xpitx-1, which is first expressed at stage 16 (Hollemann and Pieler, 1999), do not correlate directly with specific events known to take place during lens development (Fig. 4), but their continued expression through later stages of development could be involved in maintaining competence to respond to further lens inductive interactions (i.e., similar to example IV shown in Fig. 1D). There are no genes with expression limited solely to the early phase of lens induction (i.e., none like those depicted as example II in Fig. 1D). On the other hand, B87 and B99 are first expressed at stage 19 when the late phase of lens induction is initiated (see Fig. 4, like example VI shown in Fig. 1D), and their expression could be in direct response to signals provided by the optic vesicle (see also Zygar et al., 1998). Although no examples were found with expression limited solely to the period of specification or commitment (i.e, examples II or V in Fig. 1D), several genes are first expressed in the presumptive lens ectoderm at different points during the period of commitment (i.e., XSix3, stage 20; xSOX3, stage 21; XmafB, stage 22; XL-maf and Pitx3, stage 24, see, Pennzel et al., 1997; Zhou et al., 2000; Ishibashi and Yasuda, 2001; Pommereit et al., 2001, and Fig. 4). In some cases, such as the Sox and maf genes, their expression is known to be involved in the regulation of crystallin expression (Kamachi et al., 1998; Ishibashi and Yasuda, 2001). The staggered expression of these genes may represent elements of the sequential chain of events that prepares the embryonic ectoderm for a final stage of commitment before it can undergo tissue differentiation. Once differentiation takes place (stage 26) several terminal differentiation products, such as the crystallin genes (e.g., α-cry, β-cry, and γ-cry), are finally expressed (Fig. 4; like example VII in Fig. 1D; Schaefer et al., 1999; Mizuno et al., 1999a). Understandably, XProx1, thought to be involved in the differentiation of mature lens fiber cells, is also first expressed at this time (Schaefer et al., 1999; Mizuno et al., 1999b; Wigle et al., 1999).

It may be of significance that several genes (Xotx2, Xlens1, Pax-6, B105, D43) appear to first be expressed at stages 13–14 (Fig. 4). Stage 14 (the open neural plate stage) has been used as a convenient time point for examining the effects of the early period of lens induction in many transplantation experiments, even though the early phase of induction begins before this stage of development (approximately stage 11). These observations may indicate that key cellular and molecular events are initiated during this time interval (stage 13–14). Perhaps this represents a difference in the action of certain early lens inductors. Experiments suggest that trans-germ layer inductive interactions from underlying tissues (e.g., the foregut endoderm and anterolateral mesoderm) serve as early lens inductors, as well as planar interactions involving the anterior neural plate (Henry and Grainger, 1990). Perhaps there is a key distinction in the timing of these different interactions, and planar lens induction does not begin until stage 13–14. In fact, regionalization of anterior neural structures is a progressive process (Gamse and Sive, 2000, 2001), and complete separation of the embryonic eye primordia does not occur until stage 16 in Xenopus (Eagleson et al., 2001).

It may be hard to define the properties of competence and lens-forming bias, as they are not distinguished in a temporal context. On the other hand, one may eventually identify genes expressed very early that initiate the autonomous window of competence. Whether or not the expression of particular genes serves as an indicator of tissue competence or increasing lens-forming bias within the placodal ectoderm would have to be tested in tissue transplantation and explant culture experiments (see Zygar et al., 1998).

In all cases examined, once a particular gene is detected, it appears to be expressed continuously through later stages of lens development and cellular differentiation (stage 32). With the exception of the late phase of lens induction, and the time that the lens undergoes cellular differentiation, there are no genes with expression correlated strictly with the other experimentally defined tissue properties in this system. Furthermore, none of the genes examined is restricted solely to the presumptive lens ectoderm. This finding suggests that a higher level of complexity is involved in defining the different properties associated with embryonic lens development, and the specification of particular cell fates probably involves the combinatorial inputs of several key regulatory genes (Davidson, 2001; Carroll et al., 2001). To date, we have only examined a relatively small number of molecular markers known to be expressed during Xenopus lens development, and there are clearly other genes involved in this process, which are yet to be identified.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Subtracted cDNA Library Construction

Adult X. laevis were obtained from Xenopus I (Ann Arbor, MI). Fertilized eggs and embryos were prepared as previously described (Henry and Grainger, 1987; Schaefer et al., 1999). Xenopus tadpole larvae, stages 50–52 were anesthetized in 1× NAM (normal amphibian media, Slack, 1984) containing a 1:3,000 dilution of MS222 (Sigma, St. Louis, MO). By using fine iridectomy scissors, 2,148 control corneas and 1,910 transdifferentiating corneas were collected over the first 4 days after lens removal (the latter represented in the following proportions: 1 day = 26%; 2 days = 25%; 3 days = 28%; 4 days = 21%). Nucleic acids were extracted in a buffer consisting of 7 M urea, 2% sodium dodecyl sulfate, 0.35 M NaCl, 1 mM EDTA, and 10 mM Tris, pH 8.0. After phenol/chloroform extraction and ethanol precipitation, genomic DNA was subsequently removed by treating the samples with RQ1 DNase following the manufacturer's instructions (Promega, Madison, WI). These tissues yielded 119.7 μg of total control cornea RNA and 97.6 μg of total transdifferentiating cornea RNA. The total RNA yielded approximately 1 μg each of poly(A+) RNA after oligo(dT) selection (Invitrogen, Carlsbad, CA). Subsequently, two separate cDNA libraries were prepared from these mRNA samples based on standard procedures (Sambrook et al., 1989). First-strand cDNA synthesis was carried out by using oligo(dT)-NotI primer adapters, and Superscript reverse transcriptase (Invitrogen). dsDNA was then prepared by Gubler-Hoffman replacement synthesis. SalI adapters were blunt-end-ligated to the double-stranded cDNA and digested with NotI, which, after size selection (Cizdziel and Wang, 1993), allowed for the forced directional cloning of the cDNAs into plasmid expression vectors pSport 1 (transdifferentiating cornea clones) and pSport 2 (control cornea clones) (Invitrogen). The plasmid DNA was then electroporated into ElectroMAX DH12S Escherichia coli to generate the functional cDNA libraries (Invitrogen).

By using a biotin-based subtraction method originally developed by Sive and St. John (1988), the two plasmid cDNA libraries were used to generate a subtracted cDNA library enriched for clones unique to the process of cornea-lens transdifferentiation, following the protocol of Gruber et al. (1993). A total of 600 ng of the ssDNA prepared from transdifferentiating corneas was then hybridized for 48 hr at 42°C to an excess of biotin-labeled cRNA (70.4 μg) prepared from the control cornea cDNA library. Common sequences (hybrids) were removed by addition of streptavidin followed by phenol/chloroform extraction (3 times) according to the method of Sive and St. John (1988). A complete second round of subtractive hybridization was then carried out by using the remaining ssDNA and 32.5μg of biotinylated cRNA, followed by streptavidin addition, and three rounds of phenol/chloroform extraction. This approach yielded subtracted, transdifferentiating cornea-specific ssDNA that was subsequently converted to dsDNA by using the NotI primers and Taq polymerase, and electroporated into ElectroMAX DH12S E. coli to generate the final subtracted library (Invitrogen).

DNA Sequencing

Sequencing was completed by the University of Illinois Biotechnology Center. The universal primer T7 was used to obtain sequence from the 5′ end of each clone. “Sequencher” (ABI Prism, Foster City, CA) was used to edit and analyze the sequence. Comparison of the sequence against other sequences in the Genbank database was accomplished by using the blastn and blastx search programs found on the National Center for Biotechnology Information web site (www.ncbi.nlm.nih.gov).

Colony Lift Hybridization

After sequencing, the first 98 clones, it was found that three species were represented as multiple copies (see Results section). To avoid re-picking these more abundant clones, a set of 32P random-prime labeled probes was generated from these clones (e.g., B46, B97, and B127) by using the RTS RadPrime DNA Labeling System (Invitrogen). The mixed probe was used to screen replicate colony lifts (MagnaGraph, Osmonics, Inc., Minnetonka, MN; Church and Gilbert, 1984), and only those colonies that did not hybridize were selected for additional sequence analyses.

Differential PCR Analysis

Based on the sequence analysis of individual clones, specific PCR primers were designed to amplify known DNA stretches to check for the presence or absence of these sequences in the two original cDNA libraries used to prepare the subtracted cDNA library (the control and transdifferentiating cornea libraries described above). For primer sequences see http://www.life.uiuc. edu/henry. In addition, gene specific primers were also designed to amplify a region of the Xenopus Pax-6 gene (5′-CAAGATTGCCCACTATAAGC-3′; 5′-CCTGGGTAAAAGATGTTCTG-3′; Ta = 50°C). The reactions each contained 2.5 mM MgCl2, 0.2 mM dNTPs (Roche, Indianapolis, IN), 0.2 μM each of the forward and reverse primers, and one unit of Taq polymerase in a final volume of 50 μl of 1× PCR buffer (Invitrogen). Ten, 20, and 100 ng of input library cDNA was used in separate PCR reactions. MgCl2 concentrations and annealing temperatures (Ta) were adjusted for optimal results starting with the recommended temperatures for each specific set of primers (see http://www.life.uiuc.edu/henry). Reactions were run through 30 cycles of the following: 45 sec at 94°C, 30 sec at Ta, and 1 min at 72°C, followed by a terminal 5-min extension at 72°C. Negative and positive controls by using 1 ng of the appropriate cloned cDNAs were run with every set of reactions. PCR products were resolved by using 1% agarose TBE gels (0.09 M Tris-borate, 0.002 M EDTA).

Dot Blot Hybridization

Representation of some clones was also checked by differential dot-blot hybridization. A total of 0.1, 1, 5, and 10 ng of plasmid DNA prepared from the control and regenerating cDNA libraries were spotted onto nylon membranes (Hybond N+, Amersham Pharmacia Biotech, England). pSport1 plasmid DNA was also blotted to the membranes as a negative control. Plasmid DNA was isolated from individual selected clones, and the inserts were removed by NotI and SalI digestion. The individual gel-purified inserts were used to prepare 32P random-prime labeled probes by using the RTS RadPrime DNA labeling System (Invitrogen) and these probes were hybridized to the various dot-blots using standard procedures (Church and Gilbert, 1984).

In Situ Hybridization

Digoxigenin (DIG) -labeled antisense and sense RNAs were obtained by digesting selected clones with NotI or SalI and transcribed with T7 and SP6 (Invitrogen), respectively, following the manufacturer's instructions (Roche, Indianapolis, IN). In situ hybridization was performed as previously described (Harland, 1991). Hybridized probe was detected by using anti-DIG-alkaline phosphatase followed by either of the alkaline phosphatase substrates, 4-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate, or BM-purple (Roche). Embryos (stage 12) through larval stages (stage 32) were examined for expression during normal development. In some cases expression at later stages (up to stage 42) was also examined. Some specimens were cleared in a 1:2 mixture of benzyl alcohol and benzyl benzoate. Some specimens were prepared for serial sectioning. These latter cases were post-fixed in MEMFA and washed with PBS (136 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 17.6 mM KH2PO4). They were then dehydrated through a graded series of ethanol, incubated in xylene, and infiltrated with paraffin at 60°C under vacuum. The specimens were embedded and sectioned at a thickness of 10 μm. The sections were dewaxed and cleared in xylene and mounted in Permount (Fisher Scientific, Pittsburgh, PA). Whole-mount specimens and sections were photographed by using either a dissecting microscope or light microscope equipped with a Spot digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Color images were processed by using Adobe Photoshop (Mountain View, CA).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The authors thank Ron Blackman for sharing his technical advice and assistance in the construction of the cDNA libraries. The authors also thank Becky Bergstrom and Paul Laski for their technical assistance. This research was supported by NIH-NEI research grant EY09844, and the Fight for Sight research division of Prevent Blindness America.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
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