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Vol. 13, No. 3, pp. 259-269, February 1, 1999
1 Division of Molecular Biology, Institute for Molecular and Cellular Biology, Osaka University, Suita, Osaka 565, Japan; 2 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST)
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Abstract |
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 Top
 Abstract
 Introduction
Results
Discussion
Materials and methods
References
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Both lefty-1 and lefty-2 genes are expressed on the left side of developing mouse embryos and are implicated in left-right (L-R) axis formation. With the use of transgenic analysis, the transcriptional regulatory regions of these genes responsible for their L-R asymmetric expression have now been investigated. The 9.5-kb upstream region of lefty-1 and the 5.5-kb upstream region of lefty-2 reproduced the expression pattern of the corresponding gene. Examination of deletion constructs revealed the presence of a left side-specific enhancer (ASE) that is essential and sufficient for lefty-2 asymmetric expression. In contrast, the asymmetric expression of lefty-1 was shown to be determined by a combination of bilateral enhancers and a right side-specific silencer (RSS). The 9.5-kb region of lefty-1 and the 5.5-kb region of lefty-2 responded to iv and inv, upstream genes of lefty-1 and lefty-2. The regulation of lefty-2 by iv and inv was mediated by ASE. These results suggest that, in spite of the similarities between lefty-1 and lefty-2, different regulatory mechanisms underlie their asymmetric expression.
[Key Words: Left-right asymmetry; lefty; transcriptional regulation]
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Introduction |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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Vertebrates exhibit numerous left-right (L-R) asymmetries, such
as the positions of the heart and spleen on the left side of the
body. In rodents, L-R asymmetry appears to be
determined at the presomite stage (Brown et al. 1991
; Fujinaga and
Baden 1991a,b). One of the earliest morphological indicators of
laterality is the looping of the cardiac tube toward the right side.
This event is accompanied by a clockwise axial rotation referred to as
embryonic turning, and is followed, much later, by the asymmetric orientation of various visceral organs.
The molecular mechanism that underlies L-R axis formation has remained
largely unknown until recently, when insights into this important
process have been gained (for review, see King and Brown 1997; Varlet
and Robertson 1997; Levin and Mercola 1998). The entire process by
which L-R asymmetry is established can be divided into three phases:
(1) the initial determination of L-R polarity, (2) L-R asymmetric
expression of signaling molecules, and (3) L-R asymmetric
morphogenesis induced by these signaling molecules. The mechanism of
the initial determination of L-R polarity remains unknown, although
several models have been proposed (Brown and Wolpert 1990; Yost 1992).
In mice, several mutations are known to affect L-R patterning (Hummel
and Chapman 1959; Yokoyama et al. 1991; Heymer et al. 1997; Melloy et
al. 1997), two of the more extensively studied of which are iv
and inv. L-R specification is randomized in
iv/iv mice, whereas it is reversed in
inv/inv mice. These phenotypes suggest that the
iv and inv genes likely contribute to the initial
(early) determination of L-R polarity. The iv and
inv genes have been shown to encode a protein related to
axonemal dynein (Supp et al. 1997), and a novel protein with ankyrin
repeats (Mochizuki et al. 1998), respectively. However, the precise
function of these proteins remains unclear.
With regard to the second phase of L-R axis formation, the L-R
asymmetric expression of signaling molecules, Levin et al. (1995) have
shown that the activin receptor typeIIA, sonic
hedgehog, and cNR-1 genes are expressed asymmetrically in
chick embryos, and they have proposed a hierarchy for these genes.
cNR-1 is a chick homolog of mouse nodal (Levin et al.
1995), which belongs to the TGF
superfamily. The pattern of
nodal expression is conserved among vertebrates; it is
expressed in the left half of mouse, chick and Xenopus embryos
(Collignon et al. 1996; Lowe et al. 1996). We have identified
previously two highly related TGF-related genes, lefty-1
and lefty-2, that are also expressed on the left side of
developing mouse embryos (Meno et al. 1996, 1997). Whereas lefty-1 is expressed predominantly in the prospective floor
plate (PFP), lefty-2 is expressed in lateral plate mesoderm
(LPM). Misexpression experiments in chick and Xenopus, as well
as analyses of knockout mice support the notion that these genes encode
signaling molecules for L-R positional information (Levin et al. 1997;
Sampath et al. 1997; Meno et al. 1998). For example, mice deficient in
lefty-1 show a variety of positional defects in visceral organs,
indicating that lefty-1 is required for the correct positioning of
these organs (Meno et al. 1998). In lefty-1
/ mice,
lefty-2 and nodal are ectopically expressed on the
right side, suggesting that Lefty-1 restricts the expression of
nodal and lefty-2 to the left side, perhaps by acting
as (or inducing) a midline barrier. The observation that expression of
lefty-1, lefty-2, and nodal is affected in
iv and inv mutant embryos suggests that
lefty-1, lefty-2, and nodal all act
downstream of the iv and inv genes.
With regard to the third phase of establishment of L-R asymmetry,
situs-specific morphogenesis in response to L-R asymmetric signals,
genetic evidence in mice (Oh and Li 1997) suggests that type II
activin receptors (ActRIIA and ActRIIB) serve as functional receptors for Nodal (and also for Lefty-2). Furthermore, recent studies
(Logan et al. 1998; Meno et al. 1998; Piedra et al. 1998; Ryan et al.
1998; Yoshioka et al. 1998) have indicated a role for Pitx2 (also known
as Rieg, Otlx2, and Brx1; Mucchielli et al. 1996; Semina et al. 1996;
Gage and Camper 1997; Kitamura et al. 1997), a bicoid-type homeobox
transcription factor, in mediating transmission of signals from Nodal
and Lefty-2. In addition, Snr-1, which encodes a zinc
finger-type transcription factor, is expressed on the right side of
chick embryos (Isaac et al. 1997) and may be negatively regulated by Nodal.
In spite of the recent progress, our knowledge of L-R determination remains limited. For example, the mechanism by which the initial determination of L-R polarity eventually results in the asymmetric expression of signaling molecules is not known. An understanding of the early events that take place before the asymmetric expression of lefty-1, lefty-2, and nodal will require elucidation of the mechanisms by which these genes are regulated. We have now identified the transcriptional regulatory elements of lefty-1 and lefty-2 that are responsible for the asymmetric expression of these genes. Our results indicate that lefty-2 is regulated by an asymmetric enhancer that is active exclusively on the left side. In contrast, lefty-1 is controlled by an asymmetric silencer that represses expression on the right side. These observations suggest that, although both lefty-1 and lefty-2 are expressed asymmetrically, different regulatory mechanisms underlies the L-R asymmetric transcription of the two genes.
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Results |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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The upstream regions of lefty-1 and lefty-2 determine the expression patterns of these genes
lefty-1 and lefty-2 are tightly linked on mouse
chromosome 1, being separated by a distance of only 30 kb (Fig. 1A;
Meno et al. 1997). Both genes exhibit similar
exon-intron structures and possess a putative TATA box at an expected
location. We first examined the transcriptional activity of upstream
regions of lefty-1 and lefty-2. For lefty-1,
the 9.5-kb upstream region containing the TATA box (from 
9.5 kb to
+80 bp relative to the TATA box) was linked to lacZ, yielding
L1-9.5 (Fig. 1A). For lefty-2, the 5.5-kb upstream
region including the TATA box (5.5 kb to +90 bp relative to the
TATA box) was fused to lacZ, yielding L2-5.5 (Fig.
1A). L1-9.5 and L2-5.5 were injected separately into
the pronuclei of fertilized embryos at the one-cell stage. The embryos were allowed to develop in utero until embryonic day 8.2 (E8.2), at
which time they were recovered and the expression of the lacZ transgene was examined by staining with X-gal
(5-bromo-4-chloro-3-indoyl--D-galactoside). Embryos
injected with L1-9.5 showed marked X-Gal staining in the PFP
(Fig. 1B,C). Transverse sections of the embryos indicated that X-gal
staining was localized predominantly in the left half of the PFP (Fig.
1D,E). Less marked X-gal staining was also detected in the anterior
region of the LPM on the left side (Fig. 1B-E) This pattern of X-gal
staining was indistinguishable from that of the distribution of
lefty-1 mRNA determined previously by whole mount in situ
hybridization (Meno et al. 1997).
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Embryos injected with L2-5.5 showed marked X-gal staining in
left LPM as well as weaker X-gal staining in left PFP (Fig. 1F-I). At
E7.0, X-gal staining was detected in the emerging mesoderm (data not
shown). These staining patterns were identical to the distribution
profiles of lefty-2 mRNA determined previously by whole mount
in situ hybridization (Meno et al. 1997). Thus, L1-9.5 and
L2-5.5 were able to recapitulate the expression patterns of lefty-1 and lefty-2, respectively. These results also
indicate that lefty-1 and lefty-2 are regulated
independently, even though they are tightly linked on the same chromosome.
L-R asymmetric expression of lefty-2 is controlled by a left side-specific enhancer
To locate cis elements responsible for the asymmetric
expression of lefty-2, we tested various restriction fragments
derived from the 5.5-kb upstream region for the ability to confer
asymmetric expression on lacZ (Fig. 2A).
Whereas the 5.5-kb upstream fragment fully retained this ability,
asymmetric expression was not apparent with L2-3.3 or
L2-1.6 (Fig. 2A), suggesting the presence of a left
side-specific enhancer in the 2.2-kb region between 5.5 and 3.3
kb. Although L2-3.3 embryos failed to show X-gal staining in
LPM, symmetrical staining was detected in the paraxial mesoderm. Because X-gal staining in the paraxial mesoderm was not apparent in
L2-1.6 embryos (Fig. 2A), a paraxial mesoderm-specific
enhancer (PME; Fig. 2) is likely located between 3.3 and 1.6 kb.
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To delineate further the cis elements responsible for
asymmetric expression, we prepared two sets of deletion mutants from L2-5.5, all of which contained the 3.3-kb proximal upstream
region (Fig. 2B). Among the 5'-deletion mutants, left-sided
lacZ expression in LPM and PFP was observed with
L2-5'
25 and
L2-5'75 but not with
L2-5'199 (Fig. 2B,C). Of the
3'-deletion mutants, L2-3'56 showed asymmetric expression, whereas
L2-3'37 did not (Fig. 2B,C). These
results indicated the presence of a left side-specific enhancer referred to as ASE (asymmetric enhancer), in a
380-bp region between 4.1 and 3.7 kb. The observation that
X-gal staining in the paraxial mesoderm was often apparent when ASE was
deleted (L2-5'199 and L2-3'37; Fig. 2B) suggest that ASE
may be required not only for left-sided expression but also for
suppressing the paraxial mesoderm-specific enhancer.
Internal deletion of the 380-bp region containing ASE from
L2-5.5, yielding L237-199 (Fig.
3A), abolished asymmetric expression (Fig. 3D,G),
indicating that ASE is absolutely required for such expression. The
ASE-lef2p construct (Fig. 3A), in which the 380-bp ASE
fragment is linked to the minimal promoter region of lefty-2 (from 300 to +90 bp), yielded left-sided X-gal staining in left LPM
and PFP (Fig. 3B,E). Finally, constructs in which the 380-bp fragment
was linked to the hsp68 promoter instead of the
lefty-2 promoter [ASE-hsp (R) and
ASE-hsp (S); Fig. 3A] gave rise to X-gal staining
in left LPM and left PFP (Fig. 3C,F). These results demonstrate that
ASE is essential and sufficient for the asymmetric expression of
lefty-2.
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The left side-specific enhancer of lefty-2 is composed of multiple subdomains
The 380-bp region containing ASE was further dissected by
directional and internal deletion analysis (Fig.
4A,B). Serial deletion of the 380-bp region from the
5' end resulted in the sequential loss of lacZ expression
in the PFP and LPM; expression disappeared first from the PFP, then
from the posterior LPM, and finally from the anterior LPM (Fig. 4A,C).
The L2-5'501 construct was
expressed in LPM but showed virtually no expression in the PFP. The
mutants L2-5'98,
L2-5'505, and
L2-5'502 showed asymmetric
expression in the anterior portion of LPM, but they exhibited a gradual
loss of expression in the posterior LPM as the extent of the deletion increased; the graded pattern of X-gal staining along the
anteroposterior (A-P) axis was most evident with
L2-5'505 and
L2-5'502 (Fig. 4C). Finally, with
L2-5'503 and
L2-5'504, expression in the anterior LPM was virtually abolished (Fig. 4A).
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Serial deletion of the 380-bp region from the 3' end resulted in
the sequential loss of expression in the anterior LPM
(L2-3'12 and
L2-3'54) followed by that in the
posterior LPM and PFP (L2-3'52). These results suggest that ASE is composed of multiple (at least three)
subdomains; in the 5' to the 3' direction, a subdomain that
controls expression in the PFP, one for the posterior portion of LPM,
and one for the anterior portion of LPM. Consistent with the results
obtained with the directional deletion mutants, internal deletion
mutants lacking the 3' portion of the 380-bp region
(L2-54-504 and
L2-54-199) showed X-gal staining in the
posterior portion of LPM but lost that in the anterior portion of LPM
(Fig. 4C).
Regulation of lefty-1 by bilateral enhancers and a right side-specific silencer
To localize the transcriptional regulatory elements of
lefty-1, we tested various restriction fragments derived from
the 9.5-kb upstream region for the ability to confer asymmetric
expression (Fig. 5A). The 6.0-kb upstream fragment
(L1-6.0) fully retained the ability to mediate lacZ
expression in the PFP and left LPM (Fig. 5B,E). Whereas L1-3.0
and L1-1.3 retained asymmetric transcriptional activity in the
PFP [transverse section indicated that X-gal staining in PFP was left
sided (data not shown)], no expression was apparent in LPM (Fig.
5C,D,F,G). These results suggested the presence of an LPM-specific
enhancer (LPE) in the 3.0-kb region between 6.0 and 3.0 kb.
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The asymmetric expression in the PFP was maintained in L1-3.0
and L1-1.3 (Fig. 5), suggesting the presence of a PFP-specific enhancer(s) within the 1.3-kb upstream region. To delineate further the
position of the PFP-specific enhancer, we constructed a series of
5'-deletion mutants from L1-1.3. Because the asymmetric
expression of lacZ in the PFP was not apparent with
L1-5'9 or
L1-5'8, the PFP-specific enhancer
referred to as NPE (neural plate
enhancer) was localized to a 200-bp region between
1.3 and 1.1 kb.
To examine whether LPE and NPE possess L-R specificity, LPE (the
1.5-kb fragment from 4.5 to 3.0 kb) and NPE (the 0.2-kb fragment from 1.3 to 1.1 kb) were linked to the hsp68
promoter, yielding LPE-hsp and NPE-hsp,
respectively (Fig. 6A). Unexpectedly, LPE-hsp gave rise to X-gal staining on both sides of LPM
(Fig. 6B). As with L1-9.5 (Fig. 1D,E), the anterior portion,
but not the posterior portion, of LPM was positive for X-gal staining. NPE-hsp also lacked L-R specificity, with bilateral X-gal
staining apparent in a broad region of the neural plate along the A-P
axis (Fig. 6C,F,I,M). Therefore, unlike ASE of lefty-2,
neither LPE nor NPE alone confers L-R specificity. It was most likely
that LPE and NPE are bilateral enhancers, and that L-R specificity was
determined by some other cis element.
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Because L1-1.3 showed left-sided X-gal staining in the PFP
(Fig. 5A,D,G), a cis element that controls asymmetric
expression appeared to reside in the proximal promoter region (between
1.3 and +80 bp). When LPE was linked to an 0.9-kb proximal upstream region (0.9 kb to +80 bp) of lefty-1
(LPE8), left-sided X-gal staining was apparent
in the anterior portion of LPM (Fig. 6B). However, when the proximal
promoter region was further deleted toward the TATA box
(LPE10 containing LPE and an upstream fragment from 70 to +80 bp), X-gal staining in LPM was bilateral (Fig. 6B).
These results suggested the presence of a right side-specific silencer
referred to as RSS (right side-specific
silencer), in the proximal promoter region (between 0.9
kb and 70 bp).
Similar experiments were performed with a set of constructs containing
NPE. In contrast to the L-R specificity of L1-1.3 (Fig. 5),
the deletion of the proximal promoter region between 0.9 kb and
70 bp (NPE10) resulted in bilateral
lacZ expression throughout the neural plate (Fig. 6A,D,G,J,N),
with the exception that left-sided X-gal staining was maintained in the
most anterior region. Furthermore, the addition of the 0.8-kb region
(from 0.9 kb to 70 bp) to NPE-hsp, yielding
NPE-RSS-hsp, rescued left-sided expression in the PFP (Fig.
6E,H,K,O); when compared with NPE-hsp, NPE-RSS-hsp gave rise to left-sided X-gal staining in the PFP with the exception of
the middle portion of the PFP. These results again suggested the
presence of an RSS element in the 0.8-kb region between 0.9 kb and
70 bp. We therefore concluded that the asymmetric expression of
lefty-1 in the PFP and LPM is determined not by enhancers but by RSS, a silencer that can repress transcription on the right side
when combined with the bilateral enhancers.
Transcriptional regulatory elements of lefty-1 and lefty-2 respond to iv and inv mutations
Expression of lefty-1 and lefty-2 is affected in
situs mutants such as iv/iv and
inv/inv mice; the pattern of expression is randomized (left-sided, right-sided, or bilateral) in
iv/iv mutants, and reversed in
inv/inv mutants (Collignon et al. 1996; Lowe et al. 1996; Meno et al. 1996, 1997). Therefore, lefty-1 and
lefty-2 both act downstream of the iv and
inv genes. We examined whether expression of the
lefty-1-lacZ and lefty-2-lacZ transgenes was affected by the iv and inv mutations, by establishing
permanent transgenic lines containing L1-9.5 or
L2-5.5 (lines 1-1 and 2-1, respectively). Embryos derived from
these permanent lines showed X-gal staining patterns identical to those
observed in the transient transgenic embryos; line 1-1 embryos showed
marked X-gal staining in left PFP as well as weaker staining in left
LPM, whereas line 2-1 embryos exhibited marked X-gal staining in left
LPM and weaker staining in left PFP (data not shown).
Line 2-1 mice were mated with iv/iv mice and the
lacZ transgene was transferred to the
iv/iv background. The
iv/iv embryos harboring the L2-5.5
transgene showed three different patterns of X-gal staining in LPM;
left-sided, right-sided, or bilateral (Fig. 7A-C).
The L2-5.5 transgene was also transferred to the inv/inv background by mating line 2-1 animals
with inv/+ mice (inv/inv homozygotes are lethal). Among the
embryos obtained by crossing inv/+, L2-5.5 mice with inv/+
mice, ~25 % of lacZ+ embryos (14/46), which were
inv/inv homozygotes, showed abberant X-gal staining pattern in LPM;
the staining pattern in such embryos was either right-sided (11/14 embryos;
Fig. 7F) or bilateral (3/14 embryos; Fig. 7E). These X-gal staining patterns
observed in the iv/iv and
inv/inv mice were identical to the patterns of
lefty-2 mRNA distribution in these mutants (Meno et al. 1997).
Therefore, we concluded that the 5.5-kb upstream region of
lefty-2 can reproduce the response to the iv and
inv mutations.
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To examine whether ASE alone can respond to the iv mutation, we also established a permanent line containing ASE-hsp (line 2-2). Line 2-2 embryos expressed lacZ in left LPM and PFP (data not shown), as observed with the transient transgenic embryos (Fig. 3C,F). Line 2-2 mice were then crossed with iv/iv mice and inv/+ mice. The iv/iv embryos harboring an ASE-hsp transgene showed either left-sided, bilateral, or right-sided X-gal staining both in LPM and PFP (Fig. 7G-I). The inv/inv embryos harboring the ASE-hsp transgene showed either right-sided or bilateral X-gal staining in LPM and PFP (Fig. 7K,L). These results indicate that the regulation of lefty-2 by iv and inv genes is mediated through ASE.
Similarly, the L1-9.5 transgene was transferred to the inv/inv and iv/iv backgrounds. The iv/iv embryos containing the L1-9.5 transgene showed either left-sided (Fig. 7M,P), right-sided (Fig. 7O,R), or bilateral (Fig. 7N,Q) expression in the PFP and LPM. When inv/+, L1-9.5 mice were mated with inv/+ mice, most of the transgene-positive inv/inv embryos showed right-sided X-gal staining in the PFP (Fig. 7U,X), whereas the remainders exhibited bilateral expression in the PFP (Fig. 7T,W). These results indicate that the regulation of lefty-1 by iv and inv genes is mediated by the 9.5-kb upstream region.
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Discussion |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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Regulation of lefty-1 and lefty-2 by distinct mechanisms
lefty-1 and lefty-2 are tightly linked on mouse chromosome 1. However, we have now demonstrated that the two genes are regulated independently and by distinct mechanisms (Fig. 8). The asymmetric expression of lefty-2 appears to be determined solely by a left side-specific enhancer (ASE), given that ASE alone is necessary and sufficient for L-R asymmetric expression. In contrast, L-R asymmetric expression of lefty-1 is determined not by the enhancers (LPE and NPE) but by an RSS.
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Despite the similarities in structure and expression patterns between
lefty-1 and lefty-2, several differences are apparent between the two genes. First, their major expression domains differ; whereas lefty-1 is expressed predominantly in the PFP,
lefty-2 is expressed predominantly in LPM. In addition,
lefty-1 expression slightly precedes that of lefty-2;
lefty-1 expression begins in the PFP at the two-somite pair
stage, whereas the asymmetric expression of lefty-2 begins in
left LPM at the three-somite pair stage (Meno et al. 1997; Heymer et
al. 1997). Our data suggested that, although both are expressed in left
LPM, lefty-1 and lefty-2 are regulated differently.
This difference may reflect distinct cell lineages for
lefty-1-expressing and lefty-2-expressing mesoderm cells; lefty-2 expression in LPM begins at the left side of the node, whereas lefty-1 expression in LPM begins at the left foregut. Expression of lefty-1 and that of lefty-2 are
affected similarly in iv and inv mutants (Meno et al.
1997) but differently in other mutants such as nt and
ft. In nt mutant mice, for instance, lefty-1 is not expressed, whereas lefty-2 expression is bilateral
(Melloy et al. 1998). A second difference between lefty-1 and
lefty-2 is that they likely play distinct roles in L-R
determination. Recent analysis of
lefty-1/ mutant mice has
suggested that the role of lefty-1 is to restrict the
expression of lefty-2 and nodal to the left side of
the embryos, whereas lefty-2 and nodal may encode
signals for leftness (Meno et al. 1998). Thus, lefty-1 appears
to act upstream of lefty-2 and nodal. These
differences between the two genes may explain, at least in part, the
existence of distinct mechanisms for the regulation of
lefty-1 and lefty-2.
The expression patterns of nodal and lefty-2 are
highly similar; the timing and sites of their expression are
indistinguishable. Furthermore, these two genes may play similar roles
in L-R determination; as mentioned above, both Lefty-2 and Nodal may
act as determinants for leftness (Meno et al. 1998). In addition, both
Lefty-2 and Nodal can induce Pitx2 expression in chick embryos
(Logan et al. 1998; Piedra et al. 1998; Yoshioka et al. 1998).
Therefore, the asymmetric expression of nodal and
lefty-2 may be regulated by similar mechanisms. We and others
have recently identified a left side-specific enhancer in the
nodal gene (H. Adachi, Y. Saijoh, S. Ohishi, K. Mochida, and
H. Hamada unpubl.; E.J. Robertson, pers. comm.).
Our data suggest that ASE is composed of multiple subdomains. Thus, it
is likely that different sets of cis-regulatory sequences within the 380-bp region contribute to the regulation of
lefty-2 in the PFP, the anterior LPM, and the posterior LPM.
This notion is supported by our observations with lefty-1
mutant mice (Meno et al. 1998). In
lefty-1/ embryos, both
lefty-2 and nodal are bilaterally expressed in LPM.
However, the ectopic expression of lefty-2 in right LPM was always confined to the anterior portion, suggesting that
lefty-2 is regulated differently in the anterior and posterior LPM.
Regulation of lefty-1 and lefty-2 by signals downstream of iv and inv
Given that the expression of lefty-1, lefty-2, and
nodal is affected in iv/iv and
inv/inv mutant embryos (Collignon et al. 1996;
Lowe et al. 1996; Meno et al. 1996, 1997), these three genes all likely
act downstream of the iv and inv genes. Thus, signals downstream of the iv and inv genes must reach the
transcriptional regulatory regions of lefty-1 and
lefty-2. The 5.5-kb upstream region of lefty-2 was
able to respond to the iv and inv mutations. Furthermore, ASE-hsp lacZ also responded to these mutations.
Therefore, signals downstream from the iv and inv
genes likely target the 380-bp region of ASE. The 9.5-kb upstream
region of lefty-1 was also able to respond to the iv
and inv mutations.
Regulation of lefty genes by the iv and inv
genes must be indirect because these latter two genes appears to act at
an earlier stage of L-R determination. Furthermore, the iv
gene encodes a protein related to axonemal dynein, not a transcription
factor (Supp et al. 1997). Clarification of the mechanism by which
signals downstream of iv and inv regulate
lefty genes will require identification of transcription
factors that interact with ASE of lefty-2 and those that bind
to RSS of lefty-1. The 380-bp region of ASE and the 0.7-kb
region of RSS each possess a number of potential binding sequences for
known transcription factors. To identify transcription factors that
bind to ASE and RSS, detailed mutational analysis of ASE and RSS as
well as the sequence information on other asymmetric enhancers (such as
the ones found in nodal gene and human lefty-2) are needed.
Positive signals on the left side or negative signals on the right side?
Theoretically, L-R asymmetric transcription of left side-expressed genes can be achieved by one of the two regulatory mechanisms; transcriptional activation on the left side or transcriptional silencing on the right side. Although our data may appear to support the former mechanism for lefty-2 and the latter mechanism for lefty-1, it is premature to reach such a conclusion. In the case of lefty-2, for instance, the L-R specificity of ASE could be conferred either by the presence of an ASE-activating signal on the left side and its absence on the right side, or by the presence of an ASE-repressing signal on the right and its absence on the left. The same logic applies to RSS of lefty-1. Distinguishing between the two possible mechanisms for the asymmetric expression of lefty-1 and lefty-2 will require identification of the transcription factors that bind to ASE and RSS.
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Materials and methods |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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lacZ constructs
Chromosomal lefty-1 and lefty-2 genes were
isolated from a genomic library derived from 129/Sv mice.
The 3' end of the lefty-1-upstream fragment
(SalI-HindIII 9.5 kb) is located in the
5'-untranslated region (+80 bp relative to the putative TATA box,
20 bp relative to the translation initiation codon). The 3' end
of the lefty-2 upstream fragment
(SalI-HindIII 6 kb) is also located in the
5'-untranslated region (+90 bp relative to the putative TATA box,
10 bp relative to the initiation codon). These upstream fragments
were linked to the lacZ fragment
(HindIII-BamHI 4 kb) derived from pCH110 (Pharmacia)
and were then subcloned in Bluescript or pGEM, yielding L1-9.5
and L2-5.5. In the 5' set of L2-5.5 deletion
mutants, deletion proceeded from the SalI site located at the
5' end of L2-5.5 toward the 3' end. In the 3'
set of L2-5.5 deletion mutants, deletion proceeded from the
internal BamHI site (located at 3.3 kb) toward the 5'
end (3'-deletion mutants were constructed by adding each of
3'-deleted fragments to L2-3.3 in a correct orientation). For generation of lacZ constructs containing the
hsp68 promoter, various DNA fragments were rendered blunt
ended and subcloned at the SmaI site of hsp68 lacZpA
[Kothary et al. 1989; Logan et al. 1998; a generous gift from A. Joyner (New York University Medical Center) and J. Rossant (Samuel
Lunenfeld Research Institute, Mount Sinai Hospital)]. For all the
constructs, lacZ fragments free of vector sequences were
isolated by gel electrophoresis and used for microinjection.
Transient transgenic assay
Transgenic mice were generated by pronuclear injection of
lacZ fragments (4 ng/µl) into fertilized
eggs obtained from intercrosses between (C57BL/6×
C3H)F1 mice (Hogan et al. 1994; Sasaki and Hogan 1994). The injected
embryos were transferred into pseudopregnant recipients, and allowed to
develop in utero until E8.2. Because of the narrow window of
lefty expression, it was necessary to recover the embryos at a
stage between three and eight somite pairs, for which the recipient
mice were anesthetized with Nenbutal and the stage of embryos was
estimated by a size of the decudia. The E8.2 embryos were examined for
the presence of the transgene (by PCR) and for lacZ expression
(by X-gal staining). The activity of -galactosidase in dissected
embryos was detected by a standard protocol (Hogan et al. 1994). For
each construct, >120 embryos were microinjected and transferred, and
10 transgene-positive embryos were examined by X-gal staining. The
amount of -galactosidase activity was estimated from the extent of
X-gal staining after incubating in the staining buffer for 2, 8, and 24 hr. Primers used to amplify the lacZ sequence were
5'-TTGCCGTCTGAATTTGACCTG-3' and 5'
TCTGCTTCAATCAGCGTGCC-3' (Sasaki and Hogan 1996).
Permanent transgenic lines
For some lacZ fragments, the transgenic embryos were
allowed to develop to term and permanent transgenic lines were
established. The presence of the transgene was examined by Southern
blot analysis with the lacZ fragment as probe. The
lacZ transgenes were introduced into the background of
iv/iv or inv/+, by first
mating transgenic mice with iv/iv and
inv/+ mice, respectively. The genotype of the
resulting offsprings was determined by PCR or Southern blotting analysis. iv/iv, lacZ or
inv/+, lacZ mice thus obtained were
mated, and the expression of the transgenes at E 8.2 was examined. The iv alleles were genotyped by PCR with the primers,
5'-GCCAGCAATGAACGAGTGGCCCTCAAACCT-3' and
5'AGCTCTGGAAACCGTGGCTGGTGTGGCTGT-3', followed by TaqI
digestion. The wild-type iv allele is sensitive to
TaqI, yielding 50-bp fragments, whereas the mutant allele is
resistant to TaqI, yielding a 100-bp fragment (Supp et al.
1997). The inv alleles were also genotyped by PCR. A pair of
primers, 5'-GTACACACCCCTTGATTATGCA-3' and
5'-TTGGCTGCAGCGTCTTTCCTCA-3' were used to detect the wild-type
inv allele. These primers would amplify a 212-bp fragment that
is located in exon 11 and is deleted in the mutant allele. The primers
used to detect the mutant inv allele are
5'-TCAGAAGAGTATAATAGCCATC-3' and
5'-TGTCCACAAAAGCATGGTGAAGAAG-3'. These primers would amplify a
348-bp fragment derived from the tyrosinase transgene.
| |
Acknowledgments |
|---|
We thank Elizabeth Robertson and Christopher Wright for comments on
the manuscript; Hiroshi Sasaki for advice on transient transgenic
assay; Alexandra Joyner and Janet Rossant for hsp68lacZpA; Takahiko Yokoyama and Paul Overbeek for
inv+/ mice; Hiroaki Yamamoto for
advice on inv genotyping; and Martina Brueckner for
information on iv genotyping. This work was supported by CREST
of the JST and Uehara Memorial Foundation.
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked `advertisement' in accordance with 18 USC section 1734 solely to indicate this fact.
| |
Footnotes |
|---|
Received November 13, 1998; revised version accepted December 22, 1998.
3 Corresponding author.
E-MAIL hamada{at}imcb.osaka-u.ac.jp; FAX 81-6-878-9846.
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References |
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Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
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at the right time.
Curr. Biol.
7:
212-215[CrossRef].-family member lefty in mouse embryos.
Nature
381:
151-155[CrossRef][Medline].
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