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Dev Growth Differ
2005 Apr 01;473:131-40. doi: 10.1111/j.1440-169X.2005.00789.x.
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Requirement for betaB1-crystallin promoter of Xenopus laevis in embryonic lens development and lens regeneration.
Mizuno N
,
Ueda Y
,
Kondoh H
.
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Regulation of the lens-specific betaB1-crystallin promoter in Xenopus laevis was investigated using transgenic larvae and tadpoles. Comparison of the promoter sequence with that of chicken betaB1-crystallin gene indicates significant sequence similarity over a span of several hundred base pairs starting from the transcriptional start site. Remarkably, PL-1 and PL-2 sequences identified in the chicken promoter as essential binding sites of MAF, Pax6 and Prox1 transcription factors were conserved. Mutations of X (Xenopus) PL-1 and XPL-2 sequences eliminated the promoter activity, indicating a conserved mechanism regulating betaB1-crystallin promoter among vertebrate species. A stepwise deletion of the promoter sequence starting from 2800 bp indicated that the proximal 260 bp directly upstream of the transcription initiation site is sufficient for eliciting lens-specific expression, but the 150 bp promoter sequence is inactive despite it containing the XPL-1 and XPL-2 sequences, suggesting the presence of an additional and essential regulatory sequence located between -150 and -260 bp. Activity of the betaB1-crystallin promoter during lens regeneration from cornea was examined using transgenic tadpoles and found to have the same dependence on promoter regions as in embryonic lens development, indicating that gene regulation is largely shared by the two lens-generating processes.
Fig. 1. Surgical removal of the lens from right eye of Xenopus laevis tadpole. (A) An incision was made through cornea with a fine tungsten
needle, while the body is held by a smooth glass rod support against wet cotton wool. (B) The lens was squeezed out of the eye by
pressing the lateral wall of the eye against the glass rod support. The lens (indicated by the arrow) is now held on the glass rod.
Fig. 2. Comparison of βB1-crystallin promoter sequences of X. laevis and other vertebrates previously determined for activity in the
lens. Nucleotides indicated by green are conserved among all four species, those in red are common among three of four species,
and those in yellow or purple are shared by two species. (–), gap introduced to align the sequences, and position 1 is the
transcriptional start site determined for chicken βB1-crystallin promoter (Duncan et al. 1995). The TATA box (asterisks), PL-1 and PL-
2 sequences are also indicated. Newt, chicken and mouse sequences are from DDBJ/EMBL/GenBank databases with accession
numbers AB113881, U09951 and AF106854, respectively.
Fig. 3. Generation of X. laevis
tadpoles with 370-EGFP transgene
constructs. (A) Scheme of
linearized transgene DNA carrying
cytomegalovirus (CMV) promoterdriven
DsRed2 and βB1-crystallin
promoter-driven EGFP genes.
(B) Comparison of chicken (C)
and X. laevis (X) PL-1 and PL-2
elements of the βB1-crystallin
promoter with consensus binding
sequence of MAF dimer (T-MARE)
(Kataoka et al. 1994). Nucleotides
matching the consensus are indicated
by green letters, and those
deviating from the consensus but
identical between chicken and
X. laevis sequences are indicated
by orange letters. Mutated XPL-1
and XPL-2 sequences are shown
below where altered nucleotides
are indicated by blue letters. (C)
Summary of transgene expression
in the lens of larvae at stage 51.
EGFP expression in the lens was
not affected when mutation was
introduced into only either XPL-1
or XPL-2 sequence, but was lost
when both XPL-1 and XPL-2 were
mutated. Frequency of EGFP expression
among transgenic larvae
are also indicated. (D) Examples
of transgene expression. (a–c)
Transgenic larva at stage 51 carrying
EGFP transgene with wild-type
βB1-crystallin promoter. (a) Brightfield
image. (b) EGFP expression in
the lens. (d) Widespread DsRed2
fluorescence. (d–f) Transgenic
larva at stage 51 carrying the βB1-
crystallin promoter with mutated
XPL-1 and XPL-2 sequences.
(d) Bright-field image. (e) EGFP
expression in the lens (arrow)
is missing, while (f) DsRed2 expression confirms success of transgenesis. (g) In situ hybridization of βB1-crystallin transcript in a
section of normal larval eye. Hybridization was detected only in the lens fiber. (h) In situ hybridization of EGFP transcript in transgenic
larval eye carrying wild-type 370-EGFP construct. Hybridization signal was detected only in the lens fibers. co, cornea; l, lens fibers;
re, retina. Pigmented epithelial cell layer appears as a black lining surrounding the eye chamber.
Fig. 4. Analysis of βB1-crystallin
promoter activity in the lens of transgenic
X. laevis. (A–F) Transgenic
X. laevis larva carrying 370-EGFP/
DsRed2 construct, showing lensspecific
EGFP expression. (A)
Transgenic larva at stage 34,
bright-field view. (B) EGFP expression
evident in the lens. (C) Widespread
DsRed2 expression. (D,E,F)
Enlargement of the eye area of
(A), (B) and (C), respectively.
(G–L) Transgenic X. laevis larva
carrying 150-EGFP/DsRed2 construct,
with no EGFP expression
in lens. (G) Transgenic larva at
stage 34, bright-field view. (H) EGFP
fluorescence not detectable in
the lens. (I) Widespread DsRed2
expression, indicating successful
transgenesis. (J,K,L) Enlargement
of the eye area of (G), (H) and
(I), respectively. Arrowheads in
(K) and (L) indicate the lens. (O)
Summary of transgene expression
after introduction of constructs
carrying various lengths of the
promoter sequence. Left column
indicates the scheme of EGFP
transgene. Blue boxes indicate the
promoter and intron 1 sequences,
red boxes, exons 1 and 2, and
yellow box, the region spanning
XPL-1, XPL-2 and TATA box.
Fig. 5. Analysis of βB1-crystallin
promoter activity during lens regeneration.
(A,B) Removal of the lens.
(A) Lens was removed from the eye
of a transgenic tadpole, carrying
3.5 K-EGFP construct and placed
on the embryo surface. The original
eye is shown on the right of the
panel, and the isolated lens on the
left. (B) Fluorescence micrograph
of the same field as (A), showing
bright EGFP fluorescence of the
lens (arrow) and complete absence
of fluorescence in the eye chamber,
confirming clean and complete removal
of the lenstissue. (C–E) EGFP
expression in regenerating lens
observed 6 days after removal of
lens from an analogous tadpole.
(C) Bright-field view. (D) EGFP
fluorescence of the same larva,
showing strong EGFP expression in
regenerating lens. (E) In situ hybridization
of regenerating lens detecting
EGFP mRNA, indicating lens
fiber-specific expression. co, cornea;
ir, iris; lf, lens fiber. (F–H) Absence of
EGFP expression in regenerating
lens of transgenic tadpole carrying
150-EGFP/DsRed2 construct. Bright
field view of lens-regenerating tadpoleeye (F), without expression of
EGFP in regenerating lens (G), but
regenerating lens expresses Ds
Red2 (H). (I) Summary of transgene
expression in regenerating lenses
of transgenic tadpoles carrying
EGFP constructs with various lengths
of the promoter sequence.
Left column indicates scheme of
EGFP transgenes. Symbols are
identical to those used in Fig. 4.
