|Molecular Vision 2004;
Received 22 January 2004 | Accepted 23 March 2004 | Published 24 March 2004
Molecular profiling: Gene expression reveals discrete phases of lens induction and development in Xenopus laevis
Brian E. Walter, Yimin Tian, Amy K. Garlisch, Maria E.
Carinato, Matthew B. Elkins, Adam D. Wolfe, Jonathan J. Schaefer,
Kimberly J. Perry, Jonathan J. Henry
Department of Cell and Structural Biology, University of Illinois, Urbana, IL
Correspondence to: Jonathan J. Henry, Department of Cell and Structural Biology, University of Illinois, 601 South Goodwin Avenue, Urbana, IL, 61801; email: email@example.com
Purpose: Experimental tissue transplant studies reveal that lens development is directed by a series of early and late inductive interactions. These interactions impart a growing lens-forming bias within competent presumptive lens ectoderm that leads to specification and the commitment to lens fate. Relatively few genes are known which control these events. Identification of additional genes expressed during lens development may reveal key players in these processes and help to characterize these tissue properties.
Methods: A large suite of genes has been isolated that are expresssed during the process of cornea-lens transdifferentiation (lens regeneration) in Xenopus laevis. Many of these genes are also expressed during embryonic lens development. Genes were selected for expression analysis via in situ hybridization. This group consisted of clones with possible roles in cell determination and differentiation as well as novel clones without previous identities. The spatiotemporal expression of these genes in conjunction with previously described genes were correlated with key events during embryonic lens formation.
Results: Eighteen of the thirty clones analyzed via in situ hybridization demonstrated observable expression in the developing lens. These genes were initially expressed in the presumptive lens ectoderm at a variety of timepoints throughout development. Expression is restricted to discrete time intervals during lens development. However, in most cases, expression was maintained throughout lens development after being initially upregulated.
Conclusions: The expression of these genes suggests that a genetic hierarchy exists in which an increasing number of genes are upregulated and their expression is maintained throughout lens development. Suites of genes appear to be upregulated at specific timepoints during development, correlating with stages of lens induction, specification, commitment, lens placode formation, and lens differentiation, while suites at additional timepoints suggest that other, previously unreported stages exist as well. This analysis provides a genetic framework for characterizing these processes of lens development.
During development, the vertebrate lens forms from the ectodermal tissue that overlies the optic vesicles. Lens development is similar throughout the various vertebrate groups, and amphibians in particular have served as classic systems for studies of lens induction and development. Studies utilizing the anuran Xenopus laevis (X. laevis) in particular have culminated in a model whereby the lens development process is directed by a series of inductive tissue interactions [1-3]. Anterior head ectoderm first develops an autonomous window of competence to respond to lens-inductive signals between stages 10.5 and 12  (see  for staging criteria). At this time, this tissue responds to an "early phase" of lens induction, in which signals from neural plate ectoderm, together with signals from the underlying mesodermal and endodermal tissues, begin to impart this tissue with an increasing "lens-forming bias." This early phase of lens induction preceeds the time of optic vesicle formation and has been shown to be vital for lens development, as presumptive lens ectoderm tissues transplanted as to bypass early induction generally fail to form lenses in X. laevis [1,6]. The "late phase" of induction, which begins during neurulation (stage 19), involves signaling from the evaginating optic vesicle. This induction increases lens-forming bias and serves to pinpoint the location of lens placode formation as well as provide for the continued differentiation and growth of the lens epithelium and fiber cells. Overt differentiation is apparent with the formation of the lens placode (stage 26), observed as a morphological transition from squamous to columnar epithelium, coupled with the initial production of lens crystallin proteins [7-9]. Ultimately, the placode evaginates and forms a vesicle which separates itself from the surface epithelium and comes to rest within the optic cup. As differentiation proceeds, the lens vesicle becomes comprised of specialized, anucleate lens fiber cells and a distally-arranged, proliferative lens epithelium.
The extent of lens-forming bias can be determined by the amount of lens growth and differentiation achieved via transplantation studies . Bias increases through a series of stages as lens development proceeds, and these stages are related to the early and late phases of induction. During early induction, the continued signaling from the neural, mesodermal, and endodermal tissues causes an increase in lens-forming bias until stage 19, at which point the presumptive lens ectoderm is considered to be "specified," or able to form differentiated lens tissue without the requirement of any further inductive signal [10,11]. Additional signaling which occurs during the late phase of induction continues the growing lens-forming bias to the point where the presumptive lens ectoderm is "committed" to form lens tissue and undergoes overt differentiation (lens placode formation at stage 26).
The establishment of competence within the presumptive lens epithelium and the various stages of lens bias (specification, commitment) represent experimentally defined, conceptual ideas, and so far their definition at the molecular level has been elusive. Only a small number of described factors are known to be expressed during lens development, despite the fact that a number of studies suggest many molecules are involved in lens development. For example, mouse lenses containing constituitively-active Pax6 revealed that at least 500 genes may lie downstream of Pax6 during lens development [12-14].
Of the factors that have been previously described, only a few have been implicated in the development of competence. Pax6 has been implicated as having a role in the establishment of competence in the chick and mouse [15,16]. Additionally, head ectoderm from mutant rats with phenotypes resembling Pax6-deficient Sey mice fail to respond to early lens-forming inductive signals when transplanted into wild-type rats . Sox3 also has also been claimed to have a role in establishing lens competence in medaka, as the ectopic expression of Sox3 leads to the formation of ectopic lenses within head ectoderm .
The expression of Pax6 and Otx2 in competent presumptive lens ectoderm (stages 11 to 11.5) transplanted to early neurula hosts (stage 14) provided evidence for the development of lens-forming bias within presumptive lens ectoderm prior to specification in X. laevis . Additionally, Sox3 expression was found to be an indicator of the later development of bias after the presumptive lens ectoderm had been specified. Other factors, such as XmafB, Xlmaf, Pitx3, and xSix3, have been shown to be upregulated just prior to placode formation [20-22]. Of these factors, Pitx3 has been demonstrated to play a role in lens commitment and placode formation, as mice lacking Pitx3 in the presumptive lens ectoderm fail to form lens placodes .
In order to identify additional genes involved in lens formation, we have examined the process of cornea-lens transdifferentiation (lens regeneration) in X. laevis, originally reported by Freeman . The process of cornea-lens transdifferentiation closely resembles that of lens development in regards to the source of the lens and morphological changes that lead to the regeneration of the lens . During regeneration, the lens arises from the corneal epithelium, which is derived from tissue that gave rise to the embryonic lens. Both processes produce a thickened placode that gives rise to a polarized lens vesicle containing primary fibers that later form secondary lens fibers through subsequent growth. We have produced a subtracted library consisting of genes that are upregulated during this process , and we have established that many genes expressed during the process of cornea-lens transdifferentiation are also expressed during lens development [25,26], suggesting that these processes share elements of a common molecular pathway leading to lens formation. Here, we describe the spatiotemporal expression of a large subset of these clones during embryonic development. This study comprises one of the largest sets of genes examined during the process of embryonic lens formation. The initial expression of these genes in the lens occurs over a range of time points during both the early and late phases of lens induction, and these observations suggest that groups of genes are upregulated in a coordinated fashion. These expression patterns serve to define different phases of lens development and provides additional insight into the model of lens induction.
Previously, a subtracted cDNA library, enriched for genes expressed during the process of cornea-lens transdifferentiation, was prepared . Corneas isolated over the first four days following lens removal were used for RNA extraction. This timeframe was chosen to limit the inclusion of terminal-differentiation transcripts (such as crystallin mRNAs), which are synthesized at a much greater level compared to other genes of interest. Clones were sequenced by the University of Illinois Biotechnology Center using the ABI Prism Dye Terminator Cycle Sequencing "Ready Reaction" kit (ABI Prism, Foster City, CA). As the cDNAs were produced by oligo-dT priming from the 3' end of the mRNAs, the resultant cDNAs were initially sequenced from the 5' ends to avoid the less conserved 3'UTRs. The National Center for Biotechnology Information website programs blastn, blastx, or blast2 were used for sequence comparisons. Partial sequences could be extended by comparisons with the expressed sequence tag (EST) database provided for comparisons with all other submitted Xenopus cDNAs. The inferred amino acid sequences were analyzed via the National Center for Biotechnology Information's conserved domain database (CDD) for known motifs and domains contained within the sequences.
For in situ hybridization, 30 clones from this group were selected for analysis based upon a number of criteria. Our interest lies primarily in studying those clones that are most likely to be involved in controlling lens cell determination and differentiation. Thus, clones with homology to known transcription factors and/or containing domains known to be involved in DNA binding or transcription, as well as clones having presumed roles in tissue differentiation, were selected. These criteria would eliminate a large number of the clones that have no known homology or those with strong homology to hypothetical proteins, and clones within both of these categories may have interesting and informative expression patterns. Therefore, a number of these uncharacterized clones were included in the analysis as well.
Embryos were collected and prepared according to Henry and Grainger  and reared in 1/20x NAM  at 16 °C, while larvae were grown at room temperature in dechlorinated water. Embryos at appropriate stages were mechanically demembranated in 3/4x NAM, fixed in MEMFA for 90 min, and stored at -20 °C in 100% methanol. The in situ hybridizations were conducted as detailed by Harland  using embryos over an extensive range of stages through 35-37  to encompass both the early and late phases of lens induction. BM Purple (Roche, Indianapolis, IN) was used as the colorimetric substrate. After the colorimetric reaction, embryos were washed in alkaline phosphatase and phosphate-buffered saline (PBS) and were then fixed in 4% paraformaldehyde (in PBS) at 4 °C. In all cases, both sense and antisense probes were compared to control for any background signal in the reactions. No appreciable background stain was observed in the sense controls (data not shown). Results were recorded using a Nikon Coolpix digital camera and an Olympus SZX12 dissecting microscope.
Table 1 and Table 2 detail accession numbers, sequence lengths, and identities based upon similarities to known ESTs or proteins by blastn and blastx. Figure 1 and Figure 2 show the expression patterns of the clones from Table 1 over a range of developmental stages. Further discussion of these clones and their potential relevance to lens development are detailed below. Figure 3 provides a summary of the temporal expression of each clone within the lens.
Clones exhibiting expression during development
No significant hits resulted from blastn (EST) analysis. However, blastx resulted in a hit with 93% similarity over 162 amino acids to a hypothetical human protein currently without any known function. B63 was found to exhibit an expression pattern in the X. laevis embryo, particularly within the presumptive lens ectoderm, beginning by stage 24 (Figure 1 and Figure 3). Lens expression persisted through stage 35. Additional expression was observed in the otic vesicle and the pharyngeal arches.
Blastx analysis of B81 revealed no strong similarity to any known protein. However, a relatively low level of similarity was found to nuclear receptor coactivator CIA (coactivator independent of AF-2), in human (75% similarity) as well as CIA orthologs in Xenopus and mouse (67% and 75% similarity, respectively) over an 81 amino acid region (Table 1). CIA interacts with estrogen receptors within the nucleus, dependent upon overlapping LxxLL and ΦxxΦΦ motifs (where Φ is a hydrophobic residue) in the CIA protein . The inferred B81 protein sequence appears to lack these motifs, and overlaps with CIA over a region that does not contain any known functional domains, despite the fact that this region was found to have some transcriptional-activating activity . From these results, it appears that B81 represents a novel gene uncharacterized in any species that may provide a transcriptional regulatory function. B81 expression begins by stage 23, where expression can be observed in the anterior embryo, including the presumptive lens ectoderm (Figure 1 and Figure 3). Additional expression can be seen along the dorsum of the embryo within the CNS. At stage 31, B81 was observed throughout the anterior regions, including the retina and lens. Also, note that B81 is expressed within the somitic musculature, with a more intense staining in the ventral somitic regions. Expression continued in the cephalic region and somites through at least stage 35, and by this time B81 has been greatly upregulated in the lens.
Blastx of B87 resulted in a hit with 92% similarity (86% identity) to human CRSP34/p37 (CRSP subunit 8) over 188 amino acids, and B87 most likely codes for the Xenopus ortholog of this protein (Table 1). CRSP34 was originally identified as a 34 kDa member of a protein complex CRSP (cofactor required for Sp1 transcription activation, ) but was also identified as p37, a component of the TRAP/SMCC/PC2 complex . Both of these complexes are actually subunits within a larger complex (USA) known to regulate the activity of Sp1 . Due to the fact that a number of components, including CRSP34/p37, are known to participate in a variety of transcriptional complexes, Ryu et al.  suggest a combinatorial model of protein association that may potentially allow for a diverse array of transcriptional regulation. Examples of the embryonic expression of B87 between stages 26 and 33 are shown in Figure 1, and this represents an extension of previously-reported results . B87 was observed in the presumptive lens epithelium at stage 19 and remained expressed in the developing lens through stage 37. Expression was also observed in other tissues, including the otic vesicle, somites, pronephros, and pharyngeal arches.
The inferred amino acid sequence has a strong identity (96%) to X. laevis MMP-13 (collagenase 3), a matrix metalloproteinase family member which functions to degrade a variety of collagen species during the remodeling of the ECM (Table 1). MMP-13 has been shown to be involved in rat corneal wound healing, as its transcription is initiated within six h following corneal perturbation . Additionally, the transcription of MMP-13 was shown to be regulated by proinflammatory factors IL-1β and TNF-α in human corneal cell cultures . Our screen has revealed a number of MMPs, including MMP-18 and MMP-9. Interestingly, MMP-9 is known to be a substrate for MMP-13 . MMP-9 was determined to be upregulated specifically as a wound-response factor , and although the involvement of B89/MMP-13 in lens formation is currently unknown, the embryonic expression pattern of B89 (Figure 1 and Figure 3) certainly suggests a role in lens development. B89/MMP-13 expression was first detected in the anterior CNS and presumptive lens epithelium at stage 24, and this expression continues at least through stage 35.
B105 analysis resulted with no significant hits with blastn or blastx. We had originally reported B105 expression within a number of embryonic stages , and the images presented in Figure 1 expand on this study. Expression was observed within the developing lens beginning within the stage 14 presumptive lens ectoderm , through at least stage 35 (Figure 1). Additionally, expression was seen in the somitic mesoderm.
No identity for B118 was found using blastn(EST), and the best match found using blastx was a hypothetical human protein with 70% similarity over 192 amino acids to the inferred B118 protein. However, the inferred B118 protein also showed 62% similarity to the human and mouse nuclear binding receptor protein NRBP [37,38] (Table 1). NRBP contains a variety of domains, including a kinase-like domain, a SH2 domain, and a pair of nuclear receptor binding motifs LxxLL, and is predicted to be an adapter protein involved in signal transduction and protein trafficking [37,38]. B118 does not contain any LxxLL motifs over the corresponding region, and, due to the fact that the 5' end of the B118 clone falls over 1.0 kb short of the human NRBP, no further domain characterization could be made. The overall blastx similarities may be too low for B118 to be considered the Xenopus ortholog of NRBP, although high similarity to NRBP was seen in a number of discrete regions which could possibly be important for B118 function. B118 was found to be expressed faintly in the presumptive lens ectoderm beginning at stage 26 (Figure 1 andFigure 3). By stage 35, expression was more intense in the lens, and some expression was also observed in the otic vesicle.
Blastx analysis revealed that C76 codes for Xenopus tiarin, a secreted factor which functions to specify dorsal neural tissue and retinal tissue . The expression of tiarin was characterized by Tsuda et al.  through stage 19, where it was observed in a region bordering the anterior neural plate which includes the presumptive lens ectoderm. Our studies continued the examination of tiarin expression through stage 35 (Figure 1 and Figure 3). At stage 24, tiarin is expressed in the anterior epidermis and the proctodeum, but, interestingly, no expression was seen in the presumptive lens ectoderm after stage 19. Continued expression was observed in the proctodeum as well as anteriorly in the region of the pineal gland through stage 35 and, by stage 31, additional expression appeared within the pronephric duct. Based upon the embryonic expression pattern, it seems that C76/tiarin is expressed during a narrow window of time within the presumptive lens ectoderm, suggesting a possible transient role in lens development.
Both blastn(EST) and blastx analysis revealed no significant hits. This analysis expands on the previously reported expression of D43, in which a low intensity of expression was observed by stage 14 in the presumptive lens ectoderm . This expression was maintained through stage 36, at which point strong expression was seen principally in the lens (Figure 1 and Figure 3).
Blastx analysis revealed that E7 has 70% similarity (52% identity) to Rattus norvegicus thiopurine S-methyltransferase over 219 amino acids (Table 1). Generally found in the heart, erythrocytes, kidney, liver, pancreas, and intestine , thiopurine S-methyltransferase has been shown to play a role in the in vivo transition of mercaptopurine to 6-methylmercaptopurine, a reaction important in anticancer treatments. The expression of E7, however, does not appear to be consistent with those of rat, mouse, and human, as E7 is expressed almost exclusively within the developing lens, beginning at stage 26 (Figure 1 and Figure 3, also see ). This observation, together with the low percentage of homology with the various known thiopurine S-methyltransferase orthologs, suggests that this may be a related protein with an altogether different role.
Blastn of the EST database revealed Xenopus cDNAs 94% identical to the entire H59 clone. Blastx results indicate that H59 is the Xenopus homolog of human PHD finger protein 10 , a protein previously unreported in Xenopus. It bears 94% similarity to the 410 amino acid human PHD finger 10 and 93% similarity to the mouse ortholog (BAB25323). PHD finger 10 has two isoforms and contains two zinc finger domains (C4HC3) in what is known as the "plant homeodomain region." Although the exact function of this zinc finger-containing domain in PHD finger protein 10 is unknown, these domains in other PHD-type proteins have been reported to have functions in transcription, the alteration of chromatin structure, and apoptosis [42-44]. Low levels of H59 expression are first seen at stage 32 and are maintained through stage 37 (Figure 2 and Figure 3). Expression was seen initially at low levels in the lens, and expression became more intense as lens development continued. Expression was also observed in the developing cornea, primarily in a ring surrounding the point where the lens had been separated. Low levels of H59 expression were also seen in the anterior CNS and the otic vesicles as well.
Blastx of H77 revealed a match with 98% similarity over 219 amino acids to a Xenopus protein in which the function and domains remain uncharacterized. At stage 19, expression of H77 was seen in the presumptive lens ectoderm, and this expression continued in the developing lens through stage 35 (Figure 2 and Figure 3). H77 expression was also observed in the neural crest, pharyngeal arches and head mesenchyme.
Blastx analysis revealed a 100% similarity to mouse MafY/Mt1a  and human RNA polymerase polypeptide K  over a 38 amino acid sequence (Table 1). These proteins are composed of a 58 amino acid sequence that contains a C4 zinc finger and are known to play a role in the transcriptional activation of metallothionein-1 . The expression pattern for H84 is quite striking (Figure 2). H84 transcripts were detected by stage 19 in the presumptive lens ectoderm and developing CNS. This expression continued through stage 24, at which time additional expression was seen in the somites. By stage 32, strong expression is seen primarily in the lens and the somites and remains until at least through stage 35. As no in situ expression data exists for any H84 homolog, this is the first documented expression for MafY/Mta1/polypeptide K.
The only significant homologies found to the H97 sequence using blastn of the EST database were that of a number of X. tropicalis clones, which had up to 88% identity to H97. Blastx reveals that the transcript codes for a protein with at least five C2H2 zinc finger motifs, with this number of consecutive motifs placing it in the multiple adjacent C2H2 (maC2H2) zinc finger subgroup . These zinc finger proteins are typically involved in DNA binding, usually at multiple sites, but can also bind RNA and other proteins . Blastx further revealed that the 170 amino acid zinc finger region of H97 has from 61% to 56% similarity to a number of hypothetical human and mouse zinc finger proteins (Table 1). The human and mouse proteins, however, have at least 90 amino acids of sequence N-terminal to the H97 inferred sequence. Based upon this fact, it is unlikely that H97 represents a full-length clone, and further zinc finger motifs may have yet to be uncovered. Also, due to the fact that H97 has relatively low similarities with the human and mouse proteins, it may represent a novel zinc finger protein. H97 was first observed to be expressed around stage 32. Strong expression levels were observed in both the differentiating lens and the CNS, and remained until at least stage 35 (Figure 2 and Figure 3). H97 transcripts were also apparent in the otic vesicle at stage 35.
The inferred H98 protein was found to have 92% similarity to the Xenopus early mitotic inhibitor protein (Emi1), an F-box protein which functions as a mitotic regulatory factor (Table 1). Although this was the strongest hit with blastx, a number of other Xenopus ESTs actually exhibit perfect matches to the Emi1 nucleotide sequence, while H98 has only an 98% identity. H98 is therefore more likely to be a non-Emi1 member of the F-box protein family. F-box proteins perform a variety of roles, including ubiquitination, transcriptional elongation, translational regulation, and nuclear localization . Expression was seen in embryos between stages 24 and 35 (Figure 2). At stage 24, a signal of low intensity was seen anteriorly in the presumptive lens ectoderm, and stronger expression was detected as the lens developed.
Blastx revealed a match to Xenopus oocyte membrane protein (Xoom) over 93 amino acids. The Xoom protein contains 380 amino acids, and the 5' extent of the H151 clone codes for the 93 C-terminal amino acid region of Xoom only. Xoom exists as both a cytosolic protein and as a membrane glycoprotein, and has been shown to have a role in tissue movement during gastrulation [49,50]. In those studies, the expression of the protein prior to stage 20 was examined and found to be localized primarily in the cortex of the animal blastomeres before and during gastrulation. At stage 19, Xoom was observed ubiquitously, although at higher levels in the neural tube and neural crest cells . Xenopus Xoom is known as adhesion regulating molecule-1 (ARM-1) in human and chick, and has been shown to be upregulated in metastatic tumor cells . In situ hybridization has shown that H151 is expressed between stage 14 through 39, and its was expressed in presumptive lens and differentiating lens tissues in all stages examined. It was observed in the developing CNS at stage 19, as previously described , although the reported low level of ubiquitous expression was not observed here. The in situ data further shows an increased intensity of expression in the developing lens during stage 32 through stage 39 (Figure 2 and Figure 3). This could represent a possible role for H151/Xoom in the growth and invagination of the lens vesicle.
Blastx of H163 revealed an 86% similarity over 137 amino acids to an uncharacterized human protein. Faint expression was seen in the presumptive lens ectoderm between stages 23 and 32, and expression was stronger in the lens by stage 35 (Figure 2 and Figure 3). Additional expression was seen in the CNS by stage 35 as well.
This clone has an open reading frame that codes for a 237 amino acid sequence that matches the Xenopus Mat1 ring finger protein using blastx . Mat1 is a protein that contains a C3HC4 RING finger domain and a hydrophobic domain that has been shown to interact with cyclinH and cdk7 to stabilize their binding in the CAK subunit, part of the TFIIH transcription and DNA repair complex [52,53]. The sequenced portion of the H186 clone codes for the amino-terminal end of the Mat1 protein as well as a portion of the 5'-UTR. Although Mat1 has been described in a variety of organisms, including human, mouse, and starfish [52,54,55], its expression pattern has not been previously reported. In X. laevis embryos, Mat1 is expressed by stage 14 in the anterior of the embryo, primarily within the CNS, including the developing optic vesicles. By stage 32, Mat1 was found to be expressed in the lens and otic vesicles, and this expression continues through at least stage 39 (Figure 2 and Figure 3). Interestingly, Mat1 therefore appears to be expressed in a tissue-specific fashion, and presumably genes involved in the lens development pathway modulate its expression.
Initially, Blastx revealed human and mouse homologs of J55 known as testis expressed sequence 27 (Tex27, [56,57]) that have 78% and 77% respective similarities. Blastn revealed a number of Xenopus cDNAs which are 95%-98% identical to J55. J55, with several of these clones, were compared using ClustalW, and they were found to fall into two groups, which we termed Tex27A/J55 and Tex27B. From the various Tex27A ESTs, a full open reading frame coding for a 226 amino acid protein was determined for Tex27A/J55. The J55/Tex27A nucleic acid sequence is 81% identical to human Tex27 while the inferred amino acid sequence is 92% similar (85% identical). Only a partial open reading frame exists for Tex27B; however, the 440 bp of the known coding region has 95% identity to J55/Tex27A at the nucleotide level and 96% identity at the inferred amino acid level. Thus, J55/Tex27A and Tex27B may represent paralogous genes resulting from the allotetraploid genotype of X. laevis . Although the protein sequences have been documented, no functional characterization or expression of Tex27 has been established. Using the NCBI Conserved Domain Database, J55 was found to contain a C4H2C2 zinc finger domain similar to that found in Xenopus AN1, a ubiquitin-like protein. No similarity was found to ubiquitin, however. J55 was expressed during all of the stages examined (Figure 2 and Figure 3). From stage 14 through stage 39, J55 was expressed in the CNS. At stage 26, it becomes expressed in the eye region, and by stage 32, J55 is expressed in the lens. Also at this time, the otic vesicle expresses J55.
Clones exhibiting no expression during development
The clones listed in Table 2 are those which did not produce an observable embryonic pattern with in situ hybridization, and a few are highlighted here. C46 has a strong match to other Xenopus ESTs as well as similarity to human small nuclear RNA-activating protein complex (SNAPc) polypeptide SNAP43/PTFγ . SNAPc functions as a non-TATA box basal promoter necessary for transcription of short-nuclear RNA genes by RNA polymerases II and III . J98 has strong blastn and blastx hits, matching the rat hydin protein, a factor implicated in the hydrocephalus 3 mutation . Both C46 and J98, respectively, represent partial characterizations of novel SNAP43/PTFγ and hydin orthologs in Xenopus. H112 has strong similarity to the Xenopus heparin cofactor II protein. Heparin cofactor II is a thrombin inhibitor involved in hemostasis, and it is most likely upregulated as a wound response factor triggered by the surgical removal of the original lens.
We have cloned a number of genes expressed during cornea-lens transdifferentiation (lens regeneration) in X. laevis. Thirty clones were selected and examined for potential identities, and whole mount in situ hybridization was used to characterize the spatiotemporal expression of these clones during lens development. Results from this survey have uncovered new genes relevant to lens development and further demonstrates close relationships as well as certain differences between two different processes of lens formation; embryonic lens development and cornea-lens transdifferentiation. Additionally, the expression profiles of these clones and other previously characterized genes provide insight into the conceptual model of lens induction [2,25].
Of the thirty clones examined, eighteen (listed in Table 1) had observable embryonic expression patterns via in situ hybridization, representing one of the largest reported assemblies of genes with characterized expression throughout embryonic lens development. Seventeen of these eighteen clones ultimately exhibited expression within the differentiated lens; the only gene without late expression in the lens was C76/tiarin, and this gene had transient expression in the presumptive lens ectoderm between stages 14 and 19.
Table 2 lists the twelve clones for which in situ hybridization analysis did not result in any observable embryonic expression patterns. The fact that these clones did not produce an observable expression pattern could possibly be due to limitations in sensitivity of the in situ hybridization process. On the other hand, these clones were retrieved from a cornea-lens transdifferentiation library, a phenomenon involving wound healing and cell state changes (dedifferentiation) from larval corneal tissue. Expression of a number of these genes may therefore be limited to one of these specific processes (e.g., H112/heparin cofactor II, as previously discussed).
Some of the clones that exhibited embryonic expression appeared to be highly restricted to the developing lens itself, while other genes exhibited additional expression in the developing otic vesicle and/or the CNS. The otic vesicle and lens are both derived from ectodermal placodes, and many ectodermal placodes have been shown to express a number of characterized genes in common (see review , as well as [63,64]). Furthermore, many previously described genes, including Sox3, xSix3, Pax6, and XmafB [21,65-67], have been shown to be expressed in both the lens and CNS as well. The coincident occurrence of gene expression between the lens and the CNS and otic vesicle reinforces the developmental and evolutionary relationship between these tissues .
Gene expression during lens development
Figure 3 depicts the temporal expression of genes with observed patterns in the developing lens. The clones from this study with expression in the lens (bolded blue) were observed to be upregulated at the particular timepoints shown. The clones are grouped at the earliest examined timepoint in which they show expression in the lens. Other genes (black), previously characterized in different studies [21,25,26,65-67,69-73], are placed according to their recorded timepoints of expression in the developing lens. Although this represents a relatively small subset of genes that are undoubtedly expressed in the developing lens, a number of observations can be made regarding the sequence of temporal expression of these markers. First, no gene expression is observed in the presumptive lens ectoderm prior to stage 14, during the time when competence is initiated and lens-forming bias is first established. Second, when a particular gene initially becomes upregulated, its expression in the lens is usually maintained throughout later stages of development, at least as long as through stage 35, without being fully downregulated. The only apparent deviation from this trend involves the downregulation of C76/tiarin in the presumptive lens ectoderm by stage 19 (Figure 2 and Figure 3). Although shown to play a role in dorsal-ventral specification of the CNS and optic cup , no role for tiarin in the developing lens has yet been established. Third, these genes (or groups of genes) are expressed in a staggered fashion over the course of lens development. From this, it appears that lens development involves the sequential upregulation and addition of genes in an overlapping and seemingly hierarchical fashion.
What are the implications of the presence of such a genetic hierarchy? Tissue transplant experiments [1,6,10] have identified early and late phases of lens induction and led to the definition of a number of conceptual states through which presumptive lens tissue proceeds during the course of development (e.g., specification, commitment). Although no genes are expressed coincidently with the initiation of lens induction, a number are expressed at stage 19, when the presumptive lens ectoderm is specified and the late phase of induction is initiated. A number of other genes (XProx1, Sox2, B118, and E7) are expressed at stage 26, when the presumptive lens ectoderm is committed and the lens placode forms. Finally, suites of genes appear to be expressed at stage 32 when the lens vesicle separates from the overlying epithelium . Many clones have been found to begin expression at other time points as well, and they may define new stages of lens development. For example, the suite of genes upregulated at stage 14 may correspond to a neural plate "intermediate" stage such as that proposed by Grainger et al. . This may represent the initiation of planar induction once the neural plate and neural folds have formed. Another suite of genes also appears to be upregulated at stage 24, potentially representing yet another stage which occurs just prior to placode formation. As the expression of additional clones is determined, further stages may be resolved. However, the expressions of some key regulators (e.g., Xpitx-1 and XmafB) do not appear to cluster at the onset of defined stages and rather are interspersed at various time points. The activity of these genes could contribute to the phenomenon of increasing lens-forming bias that has been observed in the presumptive lens ectoderm throughout lens development. For instance, a large number of genes are upregulated sequentially between stages 21 and 26 when growing lens-forming bias drives the presumptive lens ectoderm toward commitment. Therefore, lens development appears to involve an increasing complexity of gene activity interspersed with the coordinated upregulation of suites of genes at discrete timepoints that define developmental milestones (such as, specification or commitment, Figure 3).
The progression of this hierarchy results in numerous genes being expressed toward the end of eye development rather than at the onset. The phenomenon of this hierarchical gene upregulation may be due to the fact that an increasing number of genes are required to take what is initially a broad, flat, and generalized tissue and produce a spherical and complex structure composed of a variety of specialized cell types. Gene activity is necessary to coordinate the movement, communication, compartmentalization, and differentiation of tissues as the lens develops. Although this observation may appear to be intuitively obvious, it is hardly trivial, and it serves to exemplify the basic molecular concepts underlying such developmental processes.
A number of reviews have described genes expressed during eye development [9,74], and expression profiling has provided insight into hierarchies involved in retinal development and growth as well. For example, Zuber et al.  examined the expression and interactions of a number of eye field expression factors in Xenopus, including ET, Rx1, Pax6, and Optx2, and their role in eye field specification. They determined that these genes are expressed in a sequential fashion between stages 10 and 12.5 and many became strongly expressed over a narrow window of time (between stages 12 and 12.5), at a point when the retina field has become specified . Furthermore, Perron et al.  provide evidence of an additional genetic hierarchy related to proliferation within the ciliary marginal zone following initial retinal morphogenesis. Only a handful of genes are expressed within the cells of the distal ciliary marginal zone, but as proliferation continues and displaces these cells proximally, additional groups of genes are sequentially expressed.
In this study, the coincident expression of subsets of genes over a narrow window of time suggests that they function as part of a genetic network(s) which function(s) toward the realization of a common goal within the developing lens (e.g., specification, differentiation). For instance, genes such as H59, H97, H186/Mat1, and J55/Tex27A, which are all expressed at stage 32, are most likely involved in the promotion of the final morphology of the lens. It will undoubtedly be useful to define the position of these genes within molecular networks to gain further understanding of lens-development processes.
Cornea-lens transdifferentiation versus embryonic lens induction
All of the genes analyzed here were recovered from a subtracted cDNA library enriched for genes expressed during cornea-lens transdifferentiation , and the examination of the temporal periods of gene expression provides further insight into the process of transdifferentiation. A number of genes shown to be expressed during transdifferentiation are expressed in the eye during the early phase of lens induction [25-26, and shown here] and we argued that, to a large extent, cornea-lens transdifferentiation recapitulates the process of embryonic lens development. However, the formation of a new lens from the cornea might simply represent an extension of the late phase of embryonic lens induction in which the cornea maintains competence to respond to the lens-inductive interactions provided by the optic cup. On the other hand, the fact that genes that are upregulated during the early phase of induction remain expressed throughout the late phase of lens induction may indicate that their continued expression is required or subject to new pathways and feedback loops as development proceeds (e.g., Pax6 ). The observed expression of these particular genes during transdifferentiation may therefore be due to transcriptional control from genes normally expressed during the late stages of lens development and induction.
This research was supported by NIH/NEI research grant EY09844.
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