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Extralenticular Expression of Xenopus laevis a-, [}-, and y-Crystallin Genes Gerd A. Brunekreef,* Siebe T. van Genesen* Olivier H.]. Destree,-\ and Nicolette H. Lubsen* Purpose. Extralenticular expression of a- and /?-crystallin genes has been demonstrated in mammals and expression of y-crystallin genes has been shown in Xenopus laevis. To determine a possible correlation between lens determination and crystallin gene expression, the site of expression of (a member of) the a-, 0-, and y-crystallin gene families was observed before and during lens formation in X. laevis. Methods. The partial complementary DNAs (cDNAs) of aA- and /?A4-crystallin and a y-crystallin were cloned from an X. laevis lens cDNA library. The corresponding antisense RNAs were used to analyze the expression of these genes during X. laevis development by wholemount in situ hybridization. Results. Expression of the /?A4- and y-crystallin (but not a-crystallin) genes could first be detected in the animal cap of the X. laevis gastrula. The /5A4- and y-crystallin messengers were also found in the first stage of lens development, when the ectodermal tissue overlying the optic vesicle thickens to form the lens placode. aA-crystallin messenger RNAs were only detectable when the lens epithelial cells were formed. Conclusions. In contrast to observations in most vertebrates, expression of the /3A4- and ycrystallin genes was observed to precede that of the aA-crystallin gene during lens development of X. laevis, reflecting the determination that in amphibians, the (presumptive) fiber cells are formed before the epithelial cells, whereas in vertebrates, the order is reversed. Expression of /3A4- and y-crystallin genes in the ectodermal tissue of the X. laevis gastrula shows that these genes are expressed when this tissue gains competence for lens formation. Invest Ophthalmol Vis Sci. 1997;38:2764-2771. 1 he optical properties of the lens are determined by the high concentration and close-range order of abundant structural proteins, the so-called crystallins.1 On the basis of the distribution of these proteins in different species, the crystallins can be divided into two groups: ubiquitous and taxon-specific crystallins.2'3 The group of ubiquitous crystallins, which have been found in every vertebrate examined so far, consists of the a-, ft-, and y-crystallins. Until now, approximately 12 taxon-specific crystallins, each present in a restricted set of species, have been identified.4 From the "Department of Molecular Biology, University of Nijmegen; and the •fHubrecht Laboratory, Utrecht, The Netherlands. Supported in part by the EC-HCM grant CHRX-CT93-0175 (NHL). The research was earned out under the auspices of the Netherlands Foundation for Chemical Research. Submitted for publication March 12, 1997; revised July 30, 1997; accepted July 31, 1997. Proprietary interest category: N. Reprint requests: Nicolette H. Lubsen, Department of Molecular Biology, University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands. 2764 High-level expression of the crystallins is limited to the lens; in other tissues low-level expression of at least some crystallins has been detected, and it has become clear that crystallins are not merely structural proteins of the lens but may have other roles as well. The a-crystallins, for instance, which are an evolutionary relative of small heat-shock proteins,0 have been shown to act as molecular chaperons6'7 and are able to convey thermotolerance.8 Expression of aA-crystallin is found in spleen and thymus tissues9 and expression of aB-crystallin in heart, muscle, kidney, and brain tissues.10 The taxon-specific crystallins have a close resemblance or are identical to metabolic enzymes and often have retained their enzymatic activity in the lens.1112 Expression of/3-crystallins outside the lens has also been reported, namely in chicken and mouse retinas, whereas extralenticular expression of y-crystallins was found in Xenopus laeuis.l3~l° For these proteins no function other than a structural one has been established. During lens development, the crystallins are dif- Investigative Ophthalmology & Visual Science, December 1997, Vol. 38, No. 13 Copyright © Association for Research in Vision and Ophthalmology Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933421/ on 05/03/2017 Crystallin Gene Expression During Xenopus laevis Development ferendally expressed. In mammals, in the embryonic stage a-crystallin is found in the lens placode, whereas ft- and y-crystallin expression is not detectable until primary fiber cell differentiation occurs.16"18 In chicken, <5-crystallin is predominant in the embryonic lens, whereas the /?-crystallins become the major proteins in the lens after hatching.19 In the duck, e-crystallin expression is preceded by the expression of aBand r-crystallin.12'20 In X. laevis, y-crystallin messenger RNAs (mRNAs) are detectable at the early stages of lens development.15'21 In this report, we describe the temporal and spatial expression of a member of each of the three classes of ubiquitous crystallin genes during X. laevis development, using wholemount in situ hybridization. The /3A4- and y-crystallin mRNAs are detectable from the early stages of lens development onward. a?A-crystallin messengers could not be detected until between stages 33 and 34, when in the young tadpole the lens ectoderm invaginates, and the first cells start to differentiate. We also detected expression of the y- and the /?A4-crystallin genes in gastrulas (at stage 10). This demonstrates that members of these classes of crystallins are present at a time that part of the ectoderm of the gastrula has gained competence to form lens tissue.22"24 2765 and sequenced according to standard procedures.2'1 Computer alignments and comparisons of the sequences were performed, using the GCG package of the CAOS-CAMM Center at the University of Nijmegen. Wholemount In Situ Hybridization Wholemount in situ hybridization was essentially performed, as described by Harland.28 Sense and antisense RNA probes were made, using the DIG RNA labeling kit according to the manufacturer's protocol (Boehringer Mannheim). To enhance the sensitivity of the assay, 10% polyvinyl alcohol (13 to 23 kD; Aidrich, Milwaukee, WI) was included in the color-reaction mixture.2930 After the color reaction, the embryos were refixed in 4% formaldehyde for 4 hours. The embryos were stored in methanol at —20°C until they were embedded in paraffin. RESULTS Cloning of Xenopus laevis a-, (i-, and y-Crystallin Complementary DNA Low-stringency screening of a Xgt22 cDNA library with a calf /3A3-crystallin probe resulted in the isolation of a /?-crystallin cDNA (Fig. 1A). The cDNA insert is 706 bp long (excluding the polyA tail), METHODS which correlates well with the length of the corresponding mRNA, determined by Northern blot analEmbryos ysis (data not shown). It codes for a protein of 196 Xenopus laevis embryos were obtained from the Depart- amino acids. Sequence comparison revealed that ment of Zoology of the University of Nijmegen or the this protein is the X. laevis homologue of /3A4-crysHubrecht Laboratory in Utrecht and were maintained tallin. The deduced amino acid sequence shows a in one-third Ringer's solution until the desired stage similarity of 78.4% and 73% with the calf31 and was reached. Embryos were staged according to Nieuwchicken /3A4 proteins, 32 respectively (Fig. IB). Be25 koop and Faber. The investigation adhered to the cause we did not map the 5' end of the /3A4 mRNA, ARVO Statement for the Use of Animals in Ophthalmic we cannot exclude that this mRNA possesses a more and Vision Research. upstream start codon. However, we feel that this is unlikely, because the predicted protein is of the Construction and Screening of Complementary same length as that of the calf and chicken /3A4DNA Libraries crystallin. a- and y-crystallin cDNAs were obtained by screening a Xgtll library with a polyclonal antiTotal RNA was isolated from lenses obtained from 10body directed against total calf crystallins. Two parto 12-day-old X laevis tadpoles, using the guanidinium tial cDNAs, one encoding the 56 C-terminal amino isothiocyanate procedure, described by Sambrook et 2<> acids of aA-crystallin (Fig. 2A), the other encoding al. Complementary DNA (cDNA) was prepared from the 106 C-terminal amino acids of a y-crystallin, the RNA according to the manufacturer's protocol were obtained (Fig. 3A). The similarity between the (Boehringer-Mannheim, Almere, the Netherlands), deduced amino acid sequence of the X laevis aAusing oligo(dT) primers, and were cloned into \gt22 crystallin and the previously identified aA-crystallins or Xgtll using EcoRI linkers. The \gt22 cDNA library ranges from 62.5% to 73.1% (Fig. 2B), with the highwas screened with a calf /?A3-crystallin cDNA probe M est score that of the North American opossum. The under low-stringency conditions. Expression screencDNA encoding the y-crystallin represents a new ing of the Xgtll cDNA library was performed ac27 member of the y-crystallin gene family, because it cording to standard procedures, using a rabbit polydiffers from the y-crystallin sequences identified by clonal antibody directed against calf crystallins. EcoRI Smolich et al38 (Fig. 3B). At the nucleotide level, inserts of positive phages were cloned into pBluescript Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933421/ on 05/03/2017 Investigative Ophthalmology & Visual Science, December 1997, Vol. 38, No. 13 2766 CCCCCAGCTCGGCTGCTGGTTCAAGGAAAAAGACAAAATGACTCAACATTGTACTAAATTTTCTGGACAC M T Q H C T K F S G H TGGAAGATAATTGTATGGGATGAAGAATGTTTCCAGGGACGGAGACATGAGTTTACAGCTGAATGCTACA W K I I V W D E E C F Q G R R H E F T A E C Y ATATCATGGAATGTGGATTTGAAACTGTTCGCTCATTTAAAATAGAGAGTGGAGCATGGGTTGGCTATGA N I M E C G F E T V R S F K I E S G A W V G Y E GCACTTGGGATTCCAAGGACAGCAATTTATATTGGAGAGAGGAGAGTACCCCCGCTGGGAGGCCTGGAGC H L G F Q G Q Q F I L E R G E Y P R W E A W S GGCGGCAATGCTTATCATGTTGAGAGAATGACTTCGTTCCGACCAATCGCTTGTGCTAACCATCGTGATT G G N A Y H V E R M T S F R P I A C A N H R D GTAAGATGTCGATATTTGAGAAGGAAAACTTCCTGGGAAGGAAAGGAGAGCTTGGTGAAGACTATCCCTC C K M S I F E K E N F L G R K G E L G E D Y P S CTTGCAAGCTATGGGATGGTGCAACAACGAAGTGGGCTCATTCCGTGTTCATTCTGGCGCTTGGGTGTGC L Q A M G W C N N E V G S F R V H S G A W V C TACCAGTATCCTGGTTACCGTGGATTCCAGTACATCATGGAATGTGACCGCCACTCTGGAGACTACAAGC Y Q Y P G Y R G P Q Y I M E C D R H S G D Y K ACTGGAGAGAATGGGGATCCCATGCACAAACCTTTCAGATTCAGTCCATCCGCAGAATTCAGCAGTAAAG H W R E W G S H A Q T F Q I Q S I R R I Q Q AGAATATTATACCCATTGCCGTTCTTTGTATTTTAACCTGAAGCAAATAAAGCATAAAATTATATTACAT TTATCAAAA 707 Motif 1 N-te Xenopue &A4 bovine &A4 MTQHCTKFSG HWKIIVWDEE CFQGRRHEFT AECYNIMECG FETVRSFKIE SLQ SA V G PSVL L L VL chicken [SA4 human &A4 I RRR P S L V PF K T STP R S Motif 2 51 Xenopue &A4 bovine (JA4 chicken 0A4 SGAWVGYEHL GFQGQQFILE RGEYPRWEAW SGGNAYHVER MTSFRPIACA F A YV S D NTS PA L V F C V C S D S T human &A4 Xenopue bovine chicken human Motif 3 C.P. 101. (5A4 NHRDCKMSIF EKENFLGRKG ELGEDYPSLQ AMGWCNNEVG SFRVHSGAWV BA4 SRLT 0 SD DG H &A4 V GRSQLLL Q Q R SD C P L GGSA L C ()A4 Xenopus bovine chicken human Motif 4 151 . &A4 CYQYPGYRGF QYIMECDRHS GDYKHWREWG SHAQTFQIQS IRRIQQ &A4 S F VL H F V (A4 S LL S T A E V G V V 0A4 S F VL H F P V l. (A) Nucleotide and deduced amino acid sequence of the complementary DNA encoding /3A4-crystallin of Xenopus laevis. The amino acids are shown below their respective codons. (B) Alignment of the /?A4-crystallin sequences of Xenopus laevis, calf,31 chicken,32 and human.33 The amino acid sequence of the Xenopus laevis /?A4-crystallin is shown completely; for the other sequences, only differences are specified. Dots indicate sequence not determined. Above the sequence, the predicted limits of the structural motifs are indicated. FIGURE sequence identity between the cloned region of newly identified 7-crystallin, which we call xcryf, and xcrya is 86.4%, whereas the predicted amino acid sequence identity with xcrya is 91.5%. The predicted amino acid sequence identity of xcryf with xcryb to xcrye is less, approximately 80%. Although the genome of X. laevis is tetraploid, xcryf probably does not represent the duplicated gene of xcrya, in that most duplicated genes share 96% to 98% sequence identity in their coding regions.15 Crystallin Expression During Lens Development The expression of these a-, (5-, and 7-crystallin genes during lens development of X laevis was analyzed by wholemount in situ hybridization. The first crystallin messengers to be detected during X. laevis lens development are the 7-crystallin mRNAs, which are already present at stage 28 (Fig. 4). At this stage, the internal layer of the prospective lens ectoderm, underlaid by the optic vesicle, thickens to form the lens placode. At stage 28, 7-crystallin gene expression is limited to these internal layers of the ectoderm (Fig. 4). In wholemount in situ hybridization, /?A4-crystallin mRNAs were detectable somewhat later, at stage 29, when the lens ectoderm thickens further (data not shown). At this stage, /?A4-crystallin mRNA could not be visualized in sections of lenses, presumably because of the low level of expression. At stages 30 and 33/34, the /0A4-crystallin as well as the 7-crystallin messengers are located in the central cells, which probably correspond to the prospective primary lens fiber cells (Figs. 4, 5). The posteriorly located cells do not show expression of the /?A4- or the 7-crystallin gene. These cells are thought to be precursors of the lens epithelial cells.21 The fact that 7-crystallin gene expression is detectable before /3A4-crystallin gene expression does not necessarily mean that transcription of the latter gene is induced later during lens development: The signal obtained with the 7-crystallin probe is the sum CGCCGATATCGCCTTCCTTCCAATATGGATCAGAACTCTGTGAGCTGCACTCTGTCTGCG R R Y R L P S N M D Q N S V S C T L S A 60 GACGGGATCCTCACTTTCTTCGGTCCCAAACTGCAATTCAACATGGACTCCAGCCACAGC D G I L T F F G P K L Q F N M D S S H S 120 GATAGGACCATTCCTGTGTCCAAGGAGGAGAAATCAGGCTCATCCTCCTAAAGGCCTGCC D R T I P V S K E E K S G S S S 180 CCTTGGCCCAATCTCTCTGTGCTGCCCCCGGTGCTTCCTCTGGAGCCCCCTGAGATACAT 24 0 ACTCCTGTGTGTGGGAAGGTGGGCGTAGCGCTTAATAAAGAGTTCTGACTATC 294 B. Xenopus Opossum Frog Rat Human aA aA aA aA aA 116 173 RRYRLPSNMDQNSVSCTLSADGILTFFGPKLQFNMDSSHSDRTIPVSKEEKS--GSSS V SAI S M S IH NM D S R PTLAPSS L S I S S MMS LV E P R PTSAPSS V SAL S M S V SGL AG E A R PSSAPSS V SAL S M C I TGLDA- TE A R PTSAPSS FIGURE 2. (A) The complementary DNA and the deduced amino acid sequence of the 3' part of aA-crystallin of Xenopus laevis. The amino acids are shown below their respective codons. (B) Sequence comparison of the deduced amino acid sequence of the C-terminal part of aA-crystallin from X. laevis with the aA-crystallin sequences of the North American opossum,34 the frog (Rana temporaria) ,35 the rat,36 and human.37 The deduced amino acid sequence of the X. laevis a-crystallin complementary DNA is shown completely; for the other sequences, only differences are specified. A dash indicates a gap in the corresponding sequence. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933421/ on 05/03/2017 Crystallin Gene Expression During Xenopus laevis Development ATGGGCTTCAATGACAACATCAGGTCCTGTCGCTTTATTCCTCAAGAGAATGGCCAATAC M G F N D N I R S C R F I P Q E N G Q Y 60 AAAATGAGAATCTATGAAAAAGGAG ACTATC AAGGGCAGATGATGGAGTTCTTTGATGAC K M R I Y E K G D Y Q G Q M M E F F D D 120 TGCCCCAATACTT ATGATCGATTCCGTTTCCATGAC ATTCACTCCTGCAATGTGTTTGAT C P N T Y D R F R F H D I H S C N V F D 18 0 GGCCACTGGATGTTCTACGAGGAACCCAACTACAGGGGGCGGCAATACTACCTGAGACCT G H W M F Y E E P N Y R G R Q Y Y L R P 24 0 GGCGAATACAGGAAATACAGTGACTGGGGAGCCTCAAGCCCCAGAATTGGATCATTCAGA G E Y R K Y S D W G A S S P R I G S F R 300 AGAGTTTATC ACAAGTTTTAAATCAACTC AGACATTTACTAATACATTTCTATTAGTTCA R V Y H K F 36 0 ATAAGTTCAATTAAAGCAACATGCTCAAAAAAAAAAAAA ryf rya rye ryb ryd rye MGFNDNIRSCRFIP--QENGQYKMRIYEKGDYQGQMMEFFDDCPNTYERFNFHDIHSCNV L --NYQ S MS YVSHQ S R R --NHH EY K S R --HPHS Y K MS Y--HQ N S ryf rya rye ryb ryd rye 130 177 FDGHWMFYEEPNYRGRQYYLRPGEYRKYSDWGASSPRIGSFRRVYHKF R N R N R A H I SE F TA H M S K RF A H MV N R A H LV 2767 ment. Smolich et al15 observed that the y-crystallin genes are already expressed in the gastrula stages, using RNase protection and Northern blot analyses. During neurulation (stages 12 to 18), the expression levels decreased but were still detectable at the Northern blot level. To examine the site of crystallin expression in early X. laevis development and to determine whether other crystallin genes are also active at this stage, we analyzed gastrula stages of X. laevis by wholemount in situ hybridization, using the aA-, /3A4-, and y-crystallin probes. The weak and nonreproducible signal obtained with the aA-crystallin probe (Fig. 6) did not differ from the signal obtained with the sense probe (data not shown) and therefore probably represents nonspecific hybridization. The strong and reproducible signals obtained with the y- and /3-crystallin probes show that not only are y-crystallin mRNAs present during gastrulation, but /?A4-crystallin messengers as well (Fig. 6). Expression is localized to the animal cap and marginal zone, extending to the dorsal lip (Fig. 6; note however, that the vegetal part of the gastrula is a poor substrate for in situ hybridization). DISCUSSION FIGURE 3. (A) The (partial) complementary DNA and deduced amino acid sequence of y-crystallin xcryf. The amino acids are shown below their respective codons. (B) Sequence comparison of the deduced amino acid sequence of y-crystallin xcryf and the deduced amino acid sequences of previously identified Xenopus laevis y-crystallin coding sequences.38 The amino acid sequence of xcryf is shown completely; for the other sequences, only differences are specified. Dashes denote gaps in the relevant sequence. In this study, the spatial and temporal expression of a member of each class of the ubiquitous crystallin genes, a-, ft-, and y-crystallins, in X. laevis is described. The (partial) cDNAs encoding these crystallins were obtained from Xenopus lens cDNA libraries. Alignment of the deduced amino acid sequences identified these crystallin as the cxA- and /?A4-crystallins and as a novel member of the y-crystallin family. The /3A4- and y-crystallin transcripts are detectof expression of several of the y-crystallin genes, in able at the first stage of lens development, characterthat the members of this gene family are highly homolized by a thickening of the presumptive lens ectoderm, ogous. 38 The /3A4-crystallin probe is likely to detect which subsequently develops into the lens placode only its corresponding messenger, because in other (stages 26/27 to 30). During further lens developspecies, the /?-crystallin transcripts do not cross-hybridment, when the primary fiber cells are formed (stages 39 ize. 35 to 41), the expression of the /?A4- and the y-crysaA-crystallin mRNA is not detectable until later tallin genes is greatly enhanced. At stage 33/34, when stages of lens development (from stage 33/34 on, Figs. the final shape of the lens, a central mass of differenti4, 5). This coincides with the formation of the singleating fiber cells surrounded by a single layer of epithelayered epithelium surrounding the fiber mass.21 Allial cells, becomes recognizable,21 aA-crystallin gene though aA-crystallin gene expression was clearly deexpression also becomes detectable. The results obtectable in the wholemount embryos (Fig. 5), the low tained in this study correspond well with the observalevel of expression could not be seen in sections of tions made by others at the protein level.21'40 Using these embryos (Fig. 4). Sections of lenses of stage 40 antibodies directed against total lens protein, crysor 42 embryos (Fig. 4) show that aA-crystallin gene tallin proteins could be detected from stage 29/30 on expression is highest in the posteriorly located cells, in the area were fiber cells are forming. Later during whereas the /?A4- and y-crystallin messengers are lolens development, at stage 41, fluorescence was also cated in the differentiated fiber cell zone. seen in the epithelial layer. Crystallin Expression During Early The developmental order of expression of the Development ubiquitous crystallins observed in X. laevis differs from that of crystallins in other vertebrates. In most verteRecently, it has been reported that the y-crystallin genes are also expressed during early X. laevis develop- brates—for instance, in the rat—/?- and y-crystallin Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933421/ on 05/03/2017 Investigative Ophthalmology & Visual Science, December 1997, Vol. 38, No. 13 2768 P30/ a40 FIGURE 4. Sections (10 pm) of albino embryos of different stages, as indicated, hybridized with aA-, /3A4-, and y-crystallin antisense probes. Magnification, X25. of presumptive fiber cells is formed first. In X. laevis, a lens vesicle cannot be detected until stage 36/37, after which the typical single-layered epithelium is formed. This process coincides with the onset of ocAcrystallin gene expression. Smolich et al15 showed that y-crystallin genes are also expressed during gastrulation and neurulation, long before lens formation. In the neurula stage y-crystallin mRNAs could be detected in the anterior, middle, and posterior parts of the embryo, although most of the transcripts seemed to be localized anteriorly. Our wholemount in situ hybridization experiments confirm and extend these 30 observations. We find that not only y-crystallin genes but also a /3-crystallin gene, the /?A4 gene, is expressed in stage 10 embryos. However, in contrast to the findings of Smolich et al, we did not detect hybridization in embryos of the neural tube 34 stage (data not shown). This is most likely because of the insensitivity of the in situ hybridization in FIGURE 5. Expression of the aA-, /JA4-, and y-crystallin genes comparison with RNase protection, because the during lens development in Xenopus laevis. Wholemoimt in level of the y-crystallin mRNAs in the neurula was situ hybridizations with aA-, /3A4-, and y-crystallin antisense 13 probes to stage 29/30 and stage 33/34 albino embryos. Em- reported to be lower than that in gastrula stages. Alternatively, it is possible that expression of these bryos were photographed in methanol. Magnification, X4. gene expression is preceded by a-crystallin gene expression.'6"'8'41 This order reflects the differences in the pattern of lens development between X. laevis21 and the other vertebrates."2 Whereas in most vertebrates a lens vesicle whose cellular arrangement is similar to that of the final lens is formed after the lens placodal stage, in X. laevis, an irregular flattened mass a y Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933421/ on 05/03/2017 2769 Crystallin Gene Expression During Xenopus laevis Development ylO otlO FIGURE 6. Expression of /3A4-, and •y-crystallin in stage 10 albino embryos. (A) Wholemount in situ hybridizations with oA-, /3A4-, and y-crystallin antisense probes to stage 10 albino embryos. Embryos were photographed in Murray's. Magnification, X50. The blastocoele has partially collapsed during the in situ hybridization procedure. Note that the signal obtained with the a-crystallin probe is weak and nonreproducible and therefore is likely to represent background hybridization. (B) Sagittal section (10 //) of stage 10 albino embryo hybridized with antisense y-crystallin probe showing staining of die animal cap. Magnification, X50. a small area of the head ectoderm. Later, at the neural tube stage of development, when this region is underlaid by the optic vesicle, lens formation is induced. Finally, the primary and secondary differentiations of lens cells take place. In view of this model, the animal cap cells of a X. laevis gastrula, which express /?A4- and "y-crystallin mRNA, might be envisaged as the earliest precursors of lens cells. The acquisition of competence for lens formation of these cells could be coupled with a low level of expression of the /3A4- and •y-crystallin genes. This would imply that the first steps toward lens formation are part of a cascade of signals that determine the ectodermal phenotype. This might also explain the relative ease with which a number of tissues of neuroectodermal origin—for instance, the retina46"'18 or cornea49—transdifferentiate into lens cells, given the right (culture) conditions. Alternatively, the relatively uniform expression of these crystallin genes in the ectodermal tissue of the X. laevis gastrula embryo could be independent of lens induction. The presence of these crystallin mRNAs at the time when tissue is committed to lens formation may be the explanation for recruitment of these genes as crystallin genes. This would suggest an additional function of these proteins during embryogenesis. Key words crystallins, gastrula, lens development, Xenopus lazvis Acknowledgments genes during neurulation is more diffuse, and therefore below our detection limit. In the model of Grainger22 for early tissue interactions leading to lens formation, four stages are distinguished. In the first stage, during early gastrulation, the head ectoderm acquires competence for lens formation. Transplantation of these tissues to, for instance, a lens-ablated eye cavity, results in the formation of lens cells.23'24'43"'13 At the neural plate stage, the capacity of the ectoderm to form lens cells is limited to The authors thank A. Koster, K. Yzema, and P. Landser for their help with the in situ hybridizations. We also would like to thank J. Derksen and J. Peterson for their advice and help with the photography. References 1. Delaye M, Tardieu A. Short-range order of crystallin proteins accounts for eye lens transparency. Nature. 1983;302:415-417. 2. Wistow G, PiatigorskyJ. Lens crystallins: The evolution and expression of proteins for a highly specialized tissue. Annu Rev Biochem. 1988;57:479-504. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933421/ on 05/03/2017 2770 Investigative Ophthalmology & Visual Science, December 1997, Vol. 38, No. 13 3. Bloemendal H, De Jong WW. Lens proteins and their genes. ProgNucl Acid Res Molec Biol. 1991;41:259-281. 4. PiatigorskyJ, Wistow G. The recruitment of crystallins: New functions precede gene duplication. Science. 1991;25:1078-1079. 5. Ignolia TD, Graig EA. Four small heat shock proteins are related to each other and to mammalian a-crystallin. Proc Natl Acad Sci USA. 1982; 79:2360-2364. 6. Horwitz J. Alpha-crystallin can function as a molecular chaperon. Proc Natl Acad Sci USA. 1992; 89:1044910453. 7. Jakob U, Gaestel M, Engek K, Buchner J. Small heat shock proteins are molecular chaperones./ifco/ Chem. 1993;268:1517-1520. 8. Klementz R, Frohli E, Steiger RH, Schafer R, Aoyama A. aB-crystallin is a small heat shock protein. Proc Natl Acad Sci USA. 1991;88:9292-9296. 9. 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