Download Extralenticular expression of Xenopus laevis alpha-, beta

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
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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
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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.
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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
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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
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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.
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