|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 11 January 2006
doi: 10.1242/dev.02239
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Department of Genetics, University of Pennsylvania School of Medicine,
Clinical Research Building, Room 470, 415 Curie Boulevard, Philadelphia, PA
19104, USA.
2 Medical Genetics Branch, National Human Genome Research Institute, National
Institutes of Health, Department of Health and Human Services, Bethesda, MD
20892-3717, USA.
* Author for correspondence (e-mail: epsteind{at}mail.med.upenn.edu)
Accepted 6 December 2005
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Shh, Gene expression, Forebrain, Holoprosencephaly, Bac modification, Mouse
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). It is
from these sources that a morphogenic gradient of Shh is generated to promote
distinct neuronal progenitor subtypes in the ventral neural tube. In the
absence of Shh function, ventral CNS development is severely impaired because
of neuronal patterning and pathfinding defects
(Chiang et al., 1996;
Charron et al., 2003). In
humans, the ventral forebrain is particularly sensitive to the level of
Shh expression, given that a 50% reduction in its normal levels
causes holoprosencephaly (HPE), a structural brain malformation resulting in
the fusion of the cerebral hemispheres, optic vesicles and other craniofacial
anomalies (Roessler et al.,
1996). In mice, loss of both Shh alleles is required to
generate a similar HPE phenotype (Chiang et
al., 1996). Over recent years, significant progress has been made
in deciphering the molecular mechanisms used by Shh to pattern the ventral
neural tube (Jessell, 2000;
Patten and Placzek, 2000;
Briscoe and Ericson, 2001;
Ruiz i Altaba et al., 2003).
By contrast, much less is known of the transcription factors acting upstream
in the pathway that control Shh expression in key signaling centers,
including the floor plate and axial mesoderm.
The etiology of HPE is complex, with multiple genetic and environmental
factors contributing to the phenotype. To date, mutations in seven genes,
several of which disrupt Shh or Nodal signaling, have been shown to account
for nearly 20% of HPE cases in humans
(Ming and Muenke, 2002).
Additional causative loci have been identified in mice
(Hayhurst and McConnell, 2003;
Zoltewicz et al., 2004).
Surprisingly, these genetic studies have yet to uncover the transcription
factors directly responsible for regulating Shh expression in the
rostral forebrain and prechordal plate. Although Shh expression is
preferentially lost in the ventral forebrain of mouse embryos with attenuated
Nodal signaling, the phenotype appears to result from a failure in the
induction of prechordal plate mesoderm, rather than a direct effect on
Shh transcription per se (Lowe et
al., 2001; Dunn et al.,
2004). This contrasts with the situation in zebrafish, where Nodal
signaling is thought to directly regulate the expression of Shh in
the ventral midline of the neural tube
(Muller et al., 2000;
Rohr et al., 2001).
Our efforts to identify the genes regulating Shh transcription in
the ventral midline of the mouse CNS commenced with the analysis of 35 kb of
genomic DNA surrounding Shh for regulatory potential in a transgenic
mouse reporter assay (Epstein et al.,
1999). Multiple enhancers were shown to activate lacZ
expression at discrete positions along the AP axis of the neural tube
(Epstein et al., 1999). Two
Shh floor-plate enhancers, SFPE1 and SFPE2, were identified that
regulated reporter activity in a Shh-like pattern in the ventral
midline of the spinal cord and hindbrain. A third regulatory element,
Shh brain enhancer 1 (SBE1), directed reporter activity to the
ventral midbrain and caudal region of the diencephalon. The absence of
reporter activity in rostral regions of the ventral forebrain in the 35 kb
surveyed suggested that the enhancers controlling Shh expression in
this domain must operate over greater distance. Two additional hints suggested
that the Shh forebrain enhancers were long-range acting. First, a
translocation breakpoint mapping 250 kb upstream of Shh was
identified in an individual exhibiting a mild form of HPE, raising the
possibility that the translocation separated a forebrain enhancer from the
Shh transcription start site
(Belloni et al., 1996). Second,
Shh expression in the limb bud is regulated by sequences mapping
1 Mb upstream of Shh
(Lettice et al., 2003;
Sagai et al., 2005).
In this study, we describe a functional genomic approach to the analysis of Shh regulatory sequences. Using modified Bac clones as reporter constructs, we employed an enhancer trap assay to systematically screen 1 Mb of DNA surrounding the Shh locus for the ability to target reporter gene expression to sites of Shh transcription in transgenic mouse embryos. Highly specific patterns of reporter expression were identified in the axial mesoderm, gut endoderm, limb bud and CNS, all known sites of Shh transcription. By coupling the Bac reporter assay with comparative sequence analysis, we identified three novel enhancers located over 400 kb from the Shh transcription start site that directed Shh-like expression to the ventral forebrain. This brings the total number of Shh CNS enhancers to six, the combined activity of which covers all regions of Shh transcription along the AP axis of the mouse neural tube. We also assessed the relative contribution of each CNS enhancer to the overall pattern of Shh expression by deleting individual elements in the context of the Bac reporter assay. Redundant mechanisms regulated Shh-like expression in the ventral spinal cord, hindbrain and subventricular zone of the medial ganglionic eminence (mge) in the telencephalon. Whereas Shh reporter activity in the ventral midbrain, much of the ventral diencephalon and the ventricular zone of the mge was found to be dependent on unique elements. The multitude of enhancers controlling Shh transcription in the CNS is further indication of the complex mechanisms operating to regionalize the ventral midline of the neural tube along the AP axis.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). All
references to nucleotide positions for mouse Chromosome 5 listed below were
obtained from the mm5-May'04 assembly. Homology arms A and B flanking mouse
Shh exon 1 (corresponding to the following nucleotide positions Chr5:
26864937-26865255) were synthesized by PCR using the primer sets E169/E170 and
E239/E243, respectively (see Table S1 in the supplementary material for the
list of primer sequences described throughout this section). The fragments
were cloned into the AscI and PacI restriction sites of the
pLD53SCAEB shuttle vector (provided by N. Heintz, Rockefeller University). A
detailed description of the Bac modification protocol is available
(Jeong and Epstein, 2005). The
correct positioning of eGFP within the individual Bacs was verified by probing
XbaI-digested DNA with external probes on Southern blots.
Bac reporter constructs harboring deletions of SFPE1, SFPE2 and SBE1 in Bac
429M20eGFP were generated according to the same method described above.
Homology arms A and B flanking the sequences to be deleted corresponding to
mouse SFPE1 (Chr5: 26874573-26875234), SFPE2 (Chr5: 26857299-26858064) and
SBE1 (Chr5: 26858065-26858607) were generated by PCR using the following
primer sets: E260/E261, E262/E263 (SFPE1); E268/E269, E270/E271 (SFPE2); and
E264/E265, E266/E267 (SBE1), and cloned into the pLD53SCAEB shuttle vector
after removing the eGFP cassette. The construct carrying deletions of SFPE1
and SFPE2 was generated by deleting SFPE2 from Bac
429M20eGFP
SFPE1. Bac reporter constructs harboring deletions
of SBE2, SBE3 and SBE4 in Bac 447L17ßlacZ were generated by the same
approach. Homology arms A and B flanking the sequences to be deleted
corresponding to mouse SBE2 (Chr5: 27274453-27275587), SBE3 (Chr5:
27291667-27292041) and SBE4 (Chr5: 27184220-27185333) were generated by PCR
using the following primer sets: E289/E290, E291/E292 (SBE2); E295/E296,
E297/E298 (SBE3); and E360/E361, E362/E363 (SBE4), and cloned into the
pLD53SCAEB shuttle vector after removal of the eGFP cassette. The constructs
carrying combined deletions of SBE2 and SBE3, SBE2 and SBE4, SBE3 and SBE4,
and SBE2, SBE3 and SBE4 were generated sequentially.
Transposon-based Bac modification
The Tn7ßlacZ targeting vector (provided by D. Duboule, University of
Geneva) containing Tn7 transposable elements surrounding the ß-globin
promoter, lacZ gene and SV40 poly(A) signal was introduced into
overlapping Bacs obtained from the RPCI-23 (208K5, 447L17, 265M1, 214O17) and
RPCI-24 (265M10) libraries (CHORI, BacPac Resources) according to the method
of Spitz et al. (Spitz et al.,
2003). Tn7ßlacZ vector (40 ng) was mixed with 200 ng of Bac,
GPS buffer and TnsABC (NEB). After incubation at 37°C for 10 minutes,
Start Solution was added and the reaction was prolonged for 1 hour. After heat
inactivation at 75°C for 10 minutes, electrocompetent DH10B cells were
transformed with 1 µl of this incubate and grown overnight on LB plates
containing 20 µg/ml Kanamycin and 12.5 µg/ml Chl. Positive colonies were
identified by PCR using a primer pair corresponding to sequences from the
ß-globin promoter and lacZ gene. Modified Bac clones were
further analyzed by restriction enzyme digestion and pulsed-field gel
electrophoresis to determine the number and location of integration sites.
Only intact Bacs with a single Tn7ßlacZ integration were used in the
transgenic reporter assay.
Reporter constructs
All regulatory sequences were cloned into a reporter vector containing the
Shh minimal promoter, lacZ gene and SV40 poly(A) signal.
Each of the conserved regions (ECR1-ECR7) from the overlap between Bacs 447L17
and 265M1 was amplified by PCR (ECR1, Chr5: 27274453-27275587; ECR3, Chr5:
27291667-27292041; and data not shown) using primer sets E278/E279 (ECR1) and
E282/E283 (ECR3). SBE4 (Chr5: 27184220-27185333) was amplified by PCR using
primers E337 and E338. Conserved SBE2 sequences from human (Chr7:
155560539-155561314), chicken (Chr2: 7701492-7702259), frog (Scaffold57:
2747939-2748753) and tetraodon (Chr6: 4066565-4067332) were amplified from
genomic DNA by PCR using primer sets: E311/E313 (human); E309/E310 (chicken);
E319/E320 (frog); E321/E322 (tetraodon). Conserved SBE4 sequences from chicken
(Chr2: 7641242-7642395), frog (Scaffold57: 2689775-2690912) and tetraodon
(Chr6: 4063308-4064467) were amplified from genomic DNA by PCR using primer
sets: E372/E373 (chicken); E368/E369 (frog); E374/E375 (tetraodon). The frog
DNA derived from X. tropicalis was kindly provided by Dr Frank Conlin
(UNC, Chapel Hill, NC).
Electromobiliy shift assays (EMSA)
pCMV or pCMV-Nkx2.1 plasmids were transfected into NIH3T3 cells using
FuGENE 6 transfection reagent (Roche). After 48 hours, whole-cell lysates were
prepared in a buffer containing 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM DTT,
protease inhibitor cocktail and 25% glycerol. For EMSA, 10 µg of protein
from the cell lysates was incubated for 10 minutes at room temperature in a
DNA-binding buffer containing 10 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1 mM EDTA,
1 mM DTT, 5% glycerol, 200 ng poly (dI-dC), 1 µg BSA in the presence or
absence of competitor double stranded oligonucleotides. After 0.1 ng
(5x104 to 10x104 cpm) of probe was added to
the mixture, incubation was continued for an additional 20 minutes.
Protein-bound DNA complex was separated from free probe on a 6.5% acrylamide
gel run in 1xTBE (Tris-borate-EDTA) buffer. The sequence of probe and
competitors were as follows: probe (also used as wild-type competitor),
5'-ACAGAAATTGTTTTTTAAGTAGTTGCCCTCTGAAAATAT-3'; mutant
competitor,
5'-ACAGAAATTGTTTTTTGGAGCATTGCCCTCTGAAAATAT-3'.
Underlined regions of the sequence indicate wild-type and mutant Nkx2-binding
sites, respectively.
Production and genotyping of transgenic mice
Conventional plasmid transgenes were prepared for microinjection as
described (Epstein et al.,
1999). Bac transgenes were linearized with the PI-SceI
(NEB) restriction enzyme prior to injection
(Jeong and Epstein, 2005).
Transient transgenic embryos or mouse lines were generated by pronuclear
injection into fertilized eggs derived from the (BL6xSJL) F1 mouse
strain (Jackson Labs). The primers listed in parentheses were used for PCR
genotyping of mice carrying: 429M20eGFP and 389P3eGFP (E128 and E129);
208K5ßlacZ, 447L17ßlacZ, 265M1ßlacZ, 214O17ßlacZ and
265M10ßlacZ (E276 and E277).
Whole-mount ß-galactosidase staining, in situ hybridization and immunohistochemistry
The assessment of ß-galactosidase activity was performed as described
(Epstein et al., 1999).
Whole-mount RNA in situ hybridization was performed using a
digoxigenin-UTP-labeled Shh riboprobe
(Echelard et al., 1993).
Stained embryos were photographed after dehydration in methanol and clearing
in benzyl alcohol:benzyl benzoate (1:1). Representative embryos were
rehydrated, immersed in 30% sucrose overnight, embedded and frozen in OCT and
sectioned at 20 µm on a cryostat. Primary antibodies used for
immunohistochemistry and dilutions were as follows: eGFP (Molecular Probes),
1:1000; Shh (5E1, Developmental Studies Hybridoma Bank), 1:100. Detection of
primary antibodies was achieved using Cy3- (Jackson ImmunoResearch
Laboratories) or Alexa 488- (Molecular Probes) conjugated goat anti-mouse
secondary antibodies.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) (see
Materials and methods for a description of the targeting strategy). Transgenic
embryos were then generated with the modified Bacs and assessed for reporter
activity. At 9.5 dpc, embryos carrying either of the 429M20eGFP or 389P3eGFP
transgenes displayed eGFP expression in the ventral midline of the CNS in a
similar pattern to that generated from the locally acting Shh
regulatory elements (Fig. 1A,B; Table 1)
(Epstein et al., 1999). In
addition, Shh-like reporter activity was detected in the hindgut of
embryos carrying 429M20eGFP but not 389P3eGFP, indicating that cis-acting
sequences controlling Shh expression in the gut endoderm are located
on Bac 429M20 (Fig. 1A,B).
|
SFPE1 or 429M20eGFPSFPE2 showed
no loss of eGFP expression in the floor plate of the hindbrain or spinal cord
(compare Fig. 1B,I with
1C,K and
1D,L). By contrast, embryos
carrying 429M20eGFPSFPE1, SFPE2, a Bac transgene in
which both floor plate enhancers were deleted, showed barely detectable levels
of eGFP staining in the ventral midline of the hindbrain and spinal cord,
despite normal expression in the ventral midbrain
(Fig. 1E,M). Conversely,
embryos carrying 429M20eGFPSBE1, a Bac with a deletion of
SBE1, showed an absence of eGFP expression in the ventral midbrain and caudal
diencephalon, despite normal staining in the hindbrain and spinal cord
(compare Fig. 1B,I,J with
F,N,O). These results suggest that Shh-like transcription
in the floor plate of the hindbrain and spinal cord is dependent on the
redundant activities of SFPE1 and SFPE2, and that Shh-like expression
in the ventral midbrain and caudal diencephalon is solely dependent on
SBE1.
Positioning long-range Shh regulatory sequences on Bacs using an enhancer trap assay
The absence of reporter activity in the rostral forebrain of embryos
carrying 429M20eGFP or 389P3eGFP suggested that the sequences regulating
Shh expression in this tissue must operate over considerable
distance. To further explore this possibility, we had to develop an efficient
strategy of screening large amounts of DNA for regulatory activity.
Comparative analysis of genomic DNA from divergent organisms is an effective
method of identifying evolutionary conserved regions (ECR) that may function
as regulatory elements (Nobrega et al.,
2003; Thomas et al.,
2003; Woolfe et al.,
2005). The problem with applying this approach over large genomic
intervals is that the number of ECRs is often too numerous to easily identify
the ones of interest. For example, in the 800 kb interval between Shh
and Rnf32, the next neighboring gene upstream, there are 170
ECRs (>70% identity over 100 bp) shared between human and mouse, of which
57 are also found in chicken (ECR browser:
http://ecrbrowser.dcode.org/).
Rather than testing each of the ECRs independently for Shh reporter
activity, we implemented an enhancer-trap assay similar to the one designed by
Spitz et al. (Spitz et al.,
2003) in their study of the HoxD global control region. In this
way, modified Bacs could be used as reporter constructs to first determine
whether a given genomic interval possessed Shh enhancer activity.
Five overlapping Bacs extending 1 Mb upstream of Shh were
isolated (CHORI) and modified to incorporate a single copy reporter cassette
containing a minimal ß-globin promoter and lacZ gene
(Fig. 2). The reporter cassette
was randomly inserted into each of the Bacs by Tn7 transposition in E.
coli (Spitz et al.,
2003). Two independent transposition events were generated for
each Bac and subsequently assayed for reporter activity in transgenic
embryos.
Remarkably, of the five Bacs tested, four directed highly specific and reproducible patterns of reporter expression to the axial mesoderm, ventral forebrain and posterior limb bud, all known sites of Shh transcription (Fig. 2). Of particular interest was the observation that X-gal staining in the ventral forebrain of embryos carrying either of two overlapping Bacs (447L17ßlacZ and 265M1ßlacZ) was similar to each other and highly reflective of the pattern of Shh expression in the ventral diencephalon and telencephalon (compare Fig. 2A with C,D).
In addition to the ventral forebrain, X-gal staining was observed in the notochord in embryos carrying any of three overlapping Bacs (208K5, 447L17, 265M1), suggesting the existence of at least two long-range notochord enhancers (Fig. 2B,C,D). The early expression of Shh in the node, notochord, prechordal plate and ventral forebrain was also recapitulated in embryos carrying 447L17ßlacZ (see Fig. S1 in the supplementary material). However, in contrast to the normal expression of Shh, X-gal staining was not maintained at rostral levels of the notochord in older embryos (Fig. 2B-D).
The most distant acting enhancer identified in our study mapped to Bac
265M10ßlacZ and directed Shh-like reporter activity to the zone
of polarizing activity (ZPA) in the posterior region of the fore- and hindlimb
buds between 10.5 and 12.5 dpc (Fig.
2F; data not shown). This Shh ZPA enhancer is probably
the same as the one reported by Lettice et al.
(Lettice et al., 2003),
located 850 kb upstream of Shh.
Identifying long range Shh enhancers by comparative sequence analysis
The similar pattern of X-gal staining in the forebrain of embryos carrying
the 447L17ßlacZ and 265M1ßlacZ transgenes suggested that the
cis-acting sequences responsible for this expression localized to the 70 kb
region of overlap between the two clones. The pair-wise alignment (ECR
browser:
http://ecrbrowser.dcode.org/)
of genomic sequence from this interval between mouse and human identified
seven highly conserved regions of DNA showing at least 75% nucleotide identity
that ranged in size from 0.37 to 1.4 kb
(Fig. 3). To address their
regulatory potential, each of the ECRs in this interval was amplified by PCR
from mouse genomic DNA, cloned into a reporter cassette containing a minimal
Shh promoter and lacZ gene and assayed for forebrain
enhancer activity in transgenic embryos. Of the seven ECRs (ECR1-7) tested,
embryos carrying ECR1 and ECR3 showed consistent X-gal staining in the ventral
forebrain. The 1.1 kb ECR1 fragment, located 410 kb distal to Shh,
directed lacZ expression to the rostral region of the ventral
diencephalon in two lateral stripes that converged in the ventral midline at
the level of the optic vesicles in a manner consistent with Shh and
447L17ßlacZ reporter activity (compare Fig.
3D,J,P,V with
3A,G,M,S and
3B,H,N,T). Embryos carrying the
370 bp ECR3 element, which mapped 426 kb distal to Shh, directed
reporter activity to only part of the Shh expression domain in the
ventral telencephalon, encompassing the subventricular zone (svz) of the
medial ganglionic eminence (mge) (Fig.
3E,K,Q,W). As the expression of Shh and 447L17ßlacZ
reporter activity was also detected in the ventricular zone (vz) of the mge,
we presumed that a second telencephalic enhancer must regulate Shh
transcription at this site (Fig.
3S,T). None of the other ECR constructs directed reporter activity
to Shh-expressing tissues, including the mge and axial mesoderm,
suggesting that these enhancers must form on other sequences contained in Bac
447L17 (data not shown). In keeping with the nomenclature of previously
identified Shh regulatory elements, we refer to the rostral
diencephalic enhancer as Shh brain enhancer-2 (SBE2) and the telencephalic svz
enhancer as SBE3 (Table 1).
|
|
319 kb upstream of the Shh promoter
(Fig. 3). A 1.1 kb fragment
overlapping this ECR was amplified from the mouse genome and tested for
reporter activity. Transgenic embryos carrying the 1.1 kb fragment showed
X-gal staining in both the vz and svz of the mge in a manner comparable with
447L17ßlacZ and Shh expression
(Fig. 3F,X). X-gal staining was
also detected in the ventral diencephalon in the vicinity of prosomere 3
(Fig. 3F,L). This enhancer was
designated SBE4 (Table 1).
Requirement of long range Shh forebrain enhancers
We next addressed whether the sequences mediating SBE2, SBE3 and SBE4 were
required for the Shh-like pattern of expression in the ventral
forebrain of embryos carrying 447L17ßlacZ. We reasoned that if SBE2-4
were necessary then deleting their sequences from 447L17ßlacZ would
abrogate reporter activity in the ventral forebrain. However, if ventral
forebrain expression persisted, in whole or in part, in the absence of SBE2-4,
then additional regulatory elements would have to be implicated.
|
SBE2 were
devoid of reporter activity throughout most of the rostral-ventral
diencephalon with the exception of a small region corresponding to prosomere 3
(p3) that showed weak bilateral patches of X-gal staining (compare
Fig. 4A-C with
Fig. 3B,H,N). Reporter activity
in the ventral telencephalon and notochord were unaffected by the deletion of
SBE2 (Fig. 4A,D).
Interestingly, embryos carrying 447L17ßlacZSBE2,
SBE3 showed a similar pattern of X-gal staining to those carrying
447L17ßlacZSBE2
(Fig. 4E-H). This finding
indicates that SBE3 is not required for svz expression in the ventral
telencephalon and that another forebrain element in Bac 447L17 must compensate
in its absence (Fig. 4D,H). As
SBE3 and SBE4 were each capable of directing reporter expression to the svz of
the ventral telencephalon, it is likely that the two enhancers function in a
partially redundant manner. To test this hypothesis, we engineered deletions
in Bac 447L17ßlacZ of either SBE4 on its own or in combination with SBE3
and tested the respective transgenes (447L17ßlacZSBE4,
447L17ßlacZSBE3, SBE4) for reporter activity.
Embryos carrying 447L17ßlacZSBE4 showed no X-gal
staining in the vz of the ventral telencephalon, but did retain staining in
the svz (compare Fig. 4L with
Fig. 3T). Only when SBE3 and
SBE4 were both deleted (447L17ßlacZSBE3, SBE4)
was X-gal staining completely eliminated from the ventral telencephalon
(Fig. 4M,P). These results
confirm that the Shh-like expression derived from Bac
447L17ßlacZ in the svz of the ventral telencephalon is dependent on both
SBE3 and SBE4, and that the vz expression in the mge is solely dependent on
SBE4.
|
SBE2, SBE4) was reporter activity
abrogated from the p3 domain (Fig.
4Q,R). Interestingly, transgenic embryos carrying
447L17ßlacZSBE2, SBE3, SBE4, in which all
three forebrain enhancers were deleted, showed no X-gal staining anywhere in
the ventral forebrain, despite persistent notochord staining, indicating that
all Shh-like expression generated from Bac 447L17ßlacZ is
regulated by SBE2-4 (Fig.
4U-X).
Conservation of Shh forebrain enhancer activity
The sequences mediating the activities of SBE2-SBE4 were identified based
on their high degree of conservation between mouse and human (80% for SBE2 and
SBE4 and 72% for SBE3). To determine the extent to which the conservation of
sequence reflected the conservation of enhancer function, we performed blat
and blast searches using the UCSC
(http://www.genome.ucsc.edu/)
and Ensemble
(http://www.ensembl.org/)
genome browsers, respectively, and identified enhancer sequences from a number
of vertebrate species. SBE2 sequences were identified in most vertebrate phyla
at various distances from the Shh promoter
(Fig. 5A,B). However, SBE3
sequences were only found in closely related organisms, including chimp and
rat, raising the possibility that the SBE3 element arose relatively recently
in the clade containing rodents and primates (data not shown). Sequences
mediating SBE4 were identified in most organisms surveyed; however, the degree
of conservation was reduced in comparison with SBE2
(Fig. 6A,B).
The SBE2 sequences from human, chicken and frog were each sufficient to drive lacZ expression into the ventral diencephalon of transgenic mice, in keeping with the significant preservation of nucleotide identity across much of this element (Fig. 5C-E). By contrast, the SBE2 sequence from puffer fish, which differed significantly from the other vertebrates, was incapable of regulating transcription in the ventral diencephalon of mouse embryos (Fig. 5F). Similarly, the SBE4 element from chicken showed conserved reporter activity in mice, but the more divergent frog and puffer fish SBE4 sequences were not active (Fig. 6C-E). Whether these findings indicate that puffer fish and frog use other cis-acting sequences to regulate distinct aspects of Shh expression in the forebrain, or that the sequences tested can only accommodate enhancer function in the puffer fish and frog brains remains to be determined.
Dependency of SBE3 reporter activity on an Nkx2 binding site
To begin to understand the molecular mechanisms governing Shh
forebrain enhancer activity, we surveyed the sequence of one of the enhancers,
SBE3, for known transcription factor-binding sites and considered only those
sites showing a correlation between the expression of their cognate
DNA-binding protein and that of Shh. A binding site matching the
consensus for Nkx2 proteins (T(T/C)AAGT(A/G)(G/C)TT)
(Watada et al., 2000) was
identified in the sequence of the SBE3 enhancer in a region that was 100%
conserved between human and mouse (Fig.
7A). The expression of Nkx2.1 overlaps with that of
Shh in the ventral forebrain including the svz of the mge
(Shimamura et al., 1995).
Moreover, the telencephalic expression of Shh is downregulated in
Nkx2.1-/- embryos
(Sussel et al., 1999).
However, as the mge fails to form in these mutants, it is unclear whether the
regulation of Shh by Nkx2.1 is direct. To address this issue, we
performed electromobility shift assays, the results of which suggested that
Nkx2.1 protein was able to bind to its target recognition sequence in the SBE3
enhancer (Fig. 7B). We next
determined the requirement of the Nkx2-binding site in the context of the SBE3
reporter assay. Transgenic embryos carrying an SBE3 reporter construct that
lacked the core Nkx2 binding site (AAGTAG) failed to activate lacZ
expression in the svz of the mge (Fig.
7D,F). Based on these findings, we conclude that Nkx2.1 is a
direct regulator of SBE3 activity.
|
|
|
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Woolfe et al.,
2005). This is particularly problematic when the enhancers are
spread out across a large genomic landscape, as with Shh, because the
number of highly conserved sequences can be large. Using our methodology, we
were able to rapidly identify several novel enhancers distributed over 400 kb
from the Shh promoter that regulate Shh transcription in the
embryonic forebrain. Together with our previous analysis of locally acting
Shh regulatory sequences, our studies have uncovered a total of six
enhancers that are capable of directing reporter activity to all sites of
Shh transcription in the ventral neural tube of the mouse embryo
(Fig. 8;
Table 1).
One limitation for all approaches that use multi-species sequence
comparison to uncover cis-acting regulatory elements is the potential to
overlook enhancers whose sequence or position in the genome has not been
conserved across phyla (Frazer et al.,
2004). This may explain why we were unable to identify the precise
location of the Shh notochord element on Bac 447L17. In such
instances, deletion mapping may be a more suitable strategy to narrow down the
location of the enhancer of interest.
|
).
According to our results, this homeogenetic activation of Shh
transcription within the floor plate of the spinal cord and hindbrain is
dependent on the redundant activities of SFPE1 and SFPE2. Both floor plate
enhancers are reliant on Gli2, as Shh expression and consequently
floor-plate formation are absent in Gli2-/- embryos
(Ding et al., 1998;
Matise et al., 1998). However,
Gli2 is unlikely to be regulating SFPE1 and SFPE2 directly as Gli-binding
sites were not identified in sequences mediating these enhancers
(Epstein et al., 1999;
Jeong and Epstein, 2003).
Instead, Foxa2 and Nkx6 proteins are probably acting as transcriptional
intermediaries downstream of Gli2 as conserved binding sites matching their
consensus are required for SFPE2 activity
(Jeong and Epstein, 2003). To
determine whether SFPE1 and SFPE2 are under the influence of the same
transcription factors, we screened the 300 bp segment mediating SFPE1 for
Nkx6- and Foxa2-binding sites. However, no sequences even remotely matching
their consensus binding sites were identified (data not shown). Taken
together, these findings suggest that although SFPE1 and SFPE2 possess
equivalent functions, the transcription factors responsible for their
activation appear to be different.
Multiple enhancers regulate Shh expression in the ventral forebrain
Ventral midline cells of the forebrain exhibit distinct properties from
floor-plate cells in more posterior regions of the CNS and are thought to
emerge from a discrete pool of progenitors through slightly different
inductive signaling events (Placzek and
Briscoe, 2005). Lineage tracing studies performed in the chick
indicate that ventral midline cells of the forebrain derive from a small
domain, termed `area a', situated immediately rostral to Hensen's node
(Garcia-Martinez et al., 2004; Patten et
al., 2003). The prechordal plate that underlies `area a' cells is
the source of Shh and Nodal, the combination of which is thought to convert
`area a' progenitors into ventral midline cells
(Patten et al., 2003). This
contrasts with the origin of floor-plate precursors in more posterior regions
of the embryo, which derive from the node and are dependent on
notochord-derived Shh signaling for their formation
(Placzek and Briscoe, 2005).
Studies in zebrafish suggest that in addition to Nodal and Shh, suppression of
Wnt signaling in the ventral midline of the rostral diencephalon plays a role
in distinguishing the hypothalamic infundibulum from floor-plate cells that
occupy more posterior regions of the neural tube
(Kapsimali et al., 2004). It
remains to be confirmed if `area a' cells and attenuated Wnt signaling are
involved in ventral midline development in the mouse forebrain. Nevertheless,
the distinct signaling properties of the prechordal plate versus the notochord
are likely to play a significant part in distinguishing Shh
expression in the ventral forebrain from other regions of the CNS as evidenced
by the separate enhancers controlling Shh transcription in rostral
versus caudal region of the neural tube.
The finding that multiple enhancers control Shh expression in the
forebrain argues that multiple transcription factors must be involved in this
regulation. One likely candidate, the Nkx2.1 homeodomain protein, is expressed
in the basal telencephalon and rostral region of the ventral diencephalon in a
pattern that overlaps with Shh
(Shimamura et al., 1995).
Importantly, Shh transcripts were significantly reduced from these
domains in Nkx2.1-/- embryos
(Sussel et al., 1999).
Indication that at least one aspect of Shh regulation by Nkx2.1 is
direct comes from our identification of a functional Nkx2.1-binding site in
sequences mediating SBE3. In the absence of this binding site, SBE3 reporter
activity was abrogated from the svz of the mge
(Fig. 7). SBE2 and SBE4 also
direct Shh-like reporter activity in a manner that overlaps with
Nkx2.1, raising the possibility that Nkx2.1 binds to these regulatory
sequences as well. However, as SBE2 and SBE4 control different aspects of
Shh expression in the forebrain, Nkx2.1 would have to be acting in
concert with other transcription factors.
Although the identities of the transcription factors that cooperate with
Nkx2.1 to positively regulate Shh expression in the forebrain remain
unclear, at least one negative regulator, Tbx2, has been proposed.
Shh is not expressed in the ventral midline of the diencephalon
corresponding to the hypothalamic infundibulum, despite the presence of Nkx2.1
in this domain. Instead, Shh transcripts are found in two bilateral
stripes adjacent to the ventral midline. Recent data in the chick suggest that
Tbx2 is activated specifically in the region of the hypothalamic infundibulum
in response to Bmp signaling from the prechordal plate and is responsible for
the repression of Shh in this domain (E. Manning and M. Placzek,
personal communication). There is some evidence to suggest that the repression
of Shh by Tbx2 is direct as the removal of a Tbx-binding site in
sequences mediating SFPE2 caused ectopic lacZ expression in the
ventral midline of the diencephalon (Jeong
and Epstein, 2003). Analysis of the crucial sequences and cognate
transcription factors required for the activities of SBE2 and SBE4 should
provide additional insight to the mechanisms regulating Shh
transcription in the mouse forebrain.
Blocking access of Shh enhancers to their promoter: a potential cause of HPE
HPE is a genetically heterogeneous condition caused predominantly by
mutations in Shh (Ming and
Muenke, 2002). The initial indication for Shh as an HPE
candidate gene was based on the analysis of two individuals with HPE who were
carrying cytogenetically detectable rearrangements involving chromosome 7q36,
where Shh was shown to map
(Belloni et al., 1996).
Interestingly, the translocation breakpoints in these individuals did not
disrupt Shh-coding regions but instead mapped 15 kb and 250 kb distal
to the Shh gene. It is unlikely that these translocations interrupted
other genes in the area as the interval between Shh and
Rnf32, the next distal gene over, is separated by an 840 kb gene
desert. Rather, our findings suggest that the cause of HPE in these
translocation cases was due to the displacement of Shh enhancers from
its promoter (Fig. 8). A
similar explanation may also account for the HPE phenotype exhibited by the
short digits (Dsh) mouse mutant, which contains an 11.7 Mb inversion
on mouse chromosome 5 with a distal breakpoint 13 kb upstream of Shh
(Niedermaier et al.,
2005).
The positioning of each of the forebrain and axial mesoderm enhancers on
the distal side of the translocation breakpoints in the individuals with HPE
does little to resolve which elements in particular are required for proper
forebrain development. In principle, a reduction in Shh transcription
from any of the non-redundant enhancers could cause specific phenotypes
associated with HPE. The expression of Shh in the prechordal plate
and ventral forebrain is responsible for establishing patterns of growth and
differentiation of neuronal and glial precursors in the rostral CNS, including
bifurcation of the telencephalic vesicles and craniofacial structures
(Chiang et al., 1996;
Rallu et al., 2002; Fucillo et
al., 2004). Gain- and loss-of-function studies in several organisms have
implicated the source of Shh in the prechordal plate as requisite for ventral
forebrain development (reviewed by
Hayhurst and McConnell, 2003;
Wilson and Houart, 2004).
However, as the ventral forebrain is missing in mutants lacking a prechordal
plate, the specific contribution of Shh signaling from the ventral forebrain
itself remains unclear. Interestingly, in chick embryos, the severity of
craniofacial defects resulting from a block in Shh signaling correlated with
where and when the signal was disrupted
(Marcucio et al., 2005). These
studies showed that the expression of Shh in the ventral diencephalon
and telencephalon had a significant impact on craniofacial morphogenesis. The
inactivation of specific enhancers in the mouse that direct Shh
expression to the ventral diencephalon (SBE2) and ventral telencephalon (SBE4)
will provide a unique opportunity to investigate more thoroughly the
contribution of Shh from a given source with respect to its role in forebrain
and craniofacial development.
The objective of the current study was to identify enhancers controlling
Shh transcription in the mouse CNS. Coincidently, reporter activity
from Bac transgenic embryos was also detected in the gut, axial mesoderm and
posterior limb bud, all known sites of Shh expression. Previous
studies have shown that the Shh limb enhancer is located 1 Mb
away from Shh-coding sequences and is mutated in cases of preaxial
polydactyly in humans, mice and chickens
(Lettice et al., 2003; Sagai
et al., 2004; Maas and Fallon,
2005). The types of defects caused by alterations in Shh
limb enhancer activity are representative of the growing number of
developmental anomalies that result from the disruption of cis-acting
regulatory sequences, either by mutation or position effect resulting from
chromosomal rearrangements (Kleinjan and
van Heyningen, 2005; Lettice
and Hill, 2005). Undoubtedly, this number will continue to rise
with improved methods of identifying functional regulatory elements.
Approaches such as ours will be useful in identifying other long-range
enhancers that regulate tissue-specific expression of genes during
development.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/3/761/DC1
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Belloni, E., Muenke, M., Roessler, E., Traverso, G., Siegel-Bartelt, J., Frumkin, A., Mitchell, H. F., Donis-Keller, H., Helms, C., Hing, A. V. et al. (1996). Identification of Sonic hedgehog as a candidate gene responsible for holoprosencephaly. Nat. Genet. 14,353 -356.[CrossRef][Medline]
Briscoe, J. and Ericson, J. (2001). Specification of neuronal fates in the ventral neural tube. Curr. Opin. Neurobiol. 11,43 -49.[CrossRef][Medline]
Charron, F., Stein, E., Jeong, J., McMahon, A. P. and Tessier-Lavigne, M. (2003). The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance. Cell 113,11 -23.[CrossRef][Medline]
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H. and Beachy, P. A. (1996). Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383,407 -413.[CrossRef][Medline]
Ding, Q., Motoyama, J., Gasca, S., Mo, R., Sasaki, H., Rossant, J. and Hui, C.- C. (1998). Diminished Sonic Hedgehog signaling and lack of floor plate differentiation in Gli2 mutant mice. Development 125,2533 -2543.[Abstract]
Dunn, N. R., Vincent, S. D., Oxburgh, L., Robertson, E. J. and
Bikoff, E. K. (2004). Combinatorial activities of Smad2 and
Smad3 regulate mesoderm formation and patterning in the mouse embryo.
Development 131,1717
-1728.
Echelard, Y., Epstein, D. J., St-Jacques, B., Shen, L., Mohler, J., McMahon, J. A. and McMahon, A. P. (1993). Sonic hedgehog, a member of a family of putative signaling molecules is implicated in the regulation of CNS and limb polarity. Cell 75,1417 -1430.[CrossRef][Medline]
Epstein, D. J., McMahon, A. P. and Joyner, A. L. (1999). Regionalization of Sonic hedgehog transcription along the anteroposterior axis of the mouse central nervous system is regulated by Hnf3-dependent and -independent mechanisms. Development 126,281 -292.[Abstract]
Frazer, K. A., Tao, H., Osoegawa, K., de Jong, P. J., Chen, X.,
Doherty, M. F. and Cox, D. R. (2004). Noncoding sequences
conserved in a limited number of mammals in the SIM2 interval are frequently
functional. Genome Res.
14,367
-372.
Fuccillo, M., Rallu, M., McMahon, A. P. and Fishell, G.
(2004). Temporal requirement for hedgehog signaling in ventral
telencephalic patterning. Development
131,5031
-5040.
Garcia-Lopez, R., Vieira, C., Echevarria, D. and Martinez, S. (2004). Fate map of the diencephalon and the zona limitans at the 10-somites stage in chick embryos. Dev. Biol. 268,514 -530.[CrossRef][Medline]
Gong, S., Yang, X. W., Li, C. and Heintz, N.
(2002). Highly efficient modification of bacterial artificial
chromosomes (BACs) using novel shuttle vectors containing the R6Kgamma origin
of replication. Genome Res.
12,1992
-1998.
Hayhurst, M. and McConnell, S. K. (2003). Mouse models of holoprosencephaly. Curr. Opin. Neurol. 16,135 -141.[CrossRef][Medline]
Jeong, Y. and Epstein, D. J. (2003). Distinct
regulators of Shh transcription in the floor plate and notochord
indicate separate origins for these tissues in the mouse node.
Development 130,3891
-3902.
Jeong, Y. and Epstein, D. J. (2005). Modification and Production of Bac transgenes. Chapter 23, Unit 11. Current Protocols in Molecular Biology. Hoboken (NJ): John Wiley.
Jessell, T. M. (2000). Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat. Rev. Genet. 1,20 -29.[CrossRef][Medline]
Kapsimali, M., Caneparo, L., Houart, C. and Wilson, S. W.
(2004). Inhibition of Wnt/Axin/beta-catenin pathway activity
promotes ventral CNS midline tissue to adopt hypothalamic rather than
floorplate identity. Development
131,5923
-5933.
Kleinjan, D. A. and van Heyningen, V. (2005). Long-range control of gene expression: emerging mechanisms and disruption in disease. Am. J. Hum. Genet. 76, 8-32.[CrossRef][Medline]
Lettice, L. A. and Hill, R. E. (2005). Preaxial polydactyly: a model for defective long-range regulation in congenital abnormalities. Curr. Opin. Genet. Dev. 15,294 -300.[CrossRef][Medline]
Lettice, L. A., Heaney, S. J., Purdie, L. A., Li, L., de Beer,
P., Oostra, B. A., Goode, D., Elgar, G., Hill, R. E. and de Graaff, E.
(2003). A long-range Shh enhancer regulates expression in the
developing limb and fin and is associated with preaxial polydactyly.
Hum. Mol. Genet. 12,1725
-1735.
Lowe, L. A., Yamada, S. and Kuehn, M. R. (2001). Genetic dissection of nodal function in patterning the mouse embryo. Development 128,1831 -1843.[Abstract]
Maas, S. A. and Fallon, J. F. (2005). Single base pair change in the long-range Sonic hedgehog limb-specific enhancer is a genetic basis for preaxial polydactyly. Dev. Dyn. 232,345 -348.[CrossRef][Medline]
Marcucio, R. S., Cordero, D. R., Hu, D. and Helms, J. A. (2005). Molecular interactions coordinating the development of the forebrain and face. Dev. Biol. 284, 48-61.[CrossRef][Medline]
Matise, M. P., Epstein, D. J., Park, H. L., Platt, K. P. and Joyner, A. L. (1998). Gli2 is required for induction of floor plate but not most ventral neurons in the mouse central nervous system. Development 125,2759 -2770.[Abstract]
Ming, J. E. and Muenke, M. (2002). Multiple hits during early embryonic development: digenic diseases and holoprosencephaly. Am J. Hum. Genet. 71,1017 -1032.[CrossRef][Medline]
Müller, F., Albert, S., Blader, P., Fischer, N., Hallonet, M. and Strahle, U. (2000). Direct action of the nodal-related signal cyclops in induction of sonic hedgehog in the ventral midline of the CNS. Development 127,3889 -3897.[Abstract]
Niedermaier, M., Schwabe, G. C., Fees, S., Helmrich, A., Brieske, N., Seemann, P., Hecht, J., Seitz, V., Stricker, S., Leschik, G. et al. (2005). An inversion involving the mouse Shh locus results in brachydactyly through dysregulation of Shh expression. J. Clin. Invest. 115,900 -909.[CrossRef][Medline]
Nobrega, M. A., Ovcharenko, I., Afzal, V. and Rubin, E. M.
(2003). Scanning human gene deserts for long-range enhancers.
Science 302,413
.
Patten, I. and Placzek, M. (2000). The role of Sonic hedgehog in neural tube patterning. Cell Mol. Life Sci. 57,1695 -1708.[CrossRef][Medline]
Patten, I., Kulesa, P., Shen, M. M., Fraser, S. and Placzek,
M. (2003). Distinct modes of floor plate induction in the
chick embryo. Development
130,4809
-4821.
Placzek, M. and Briscoe, J. (2005). The floor plate: multiple cells, multiple signals. Nat. Rev. Neurosci. 6,230 -240.[CrossRef][Medline]
Rallu, M., Machold, R., Gaiano, N., Corbin, J. G., McMahon, A.
P. and Fishell, G. (2002). Dorsoventral patterning is
established in the telencephalon of mutants lacking both Gli3 and Hedgehog
signaling. Development
129,4963
-4974.
Roessler, E., Belloni, E., Gaudenz, K., Jay, P., Berta, P., Scherer, S. W., Tsui, L. C. and Muenke, M. (1996). Mutations in the human Sonic Hedgehog gene cause holoprosencephaly. Nat. Genet. 14,357 -360.[CrossRef][Medline]
Roessler, E., Ward, D.E., Gaudenz, K., Belloni, E., Scherer, S. W., Donnai, D., Siegel-Bartelt, J., Tsui, L. and Muenke, M. (1997). Cytogenetic rearrangements involving the loss of the Sonic Hedgehog gene at 7q36 cause holoprosencephaly. Hum. Genet. 100,172 -181.[CrossRef][Medline]
Rohr, K. B., Barth, K. A., Varga, Z. M. and Wilson, S. W. (2001). The nodal pathway acts upstream of hedgehog signaling to specify ventral telencephalic identity. Neuron 29,341 -351.[CrossRef][Medline]
Ruiz i Altaba, A., Nguyen, V. and Palma, V. (2003). The emergent design of the neural tube: prepattern, SHH morphogen and GLI code. Curr. Opin. Genet. Dev. 13,513 -521.[CrossRef][Medline]
Sagai, T., Hosoya, M., Mizushina, Y., Tamura, M. and Shiroishi,
T. (2005). Elimination of a long-range cis-regulatory module
causes complete loss of limb-specific Shh expression and truncation of the
mouse limb. Development
132,797
-803.
Shimamura, K., Hartigan, D. J., Martinez, S., Puelles, L. and Rubenstein, J. L. (1995). Longitudinal organization of the anterior neural plate and neural tube. Development 121,3923 -3933.[Abstract]
Spitz, F., Gonzalez, F. and Duboule, D. (2003). A global control region defines a chromosomal regulatory landscape containing the HoxD cluster. Cell 113,405 -417.[CrossRef][Medline]
Sussel, L., Marin, O., Kimura, S. and Rubenstein, J. L. (1999). Loss of Nkx2.1 homeobox gene function results in a ventral to dorsal molecular respecification within the basal telencephalon: evidence for a transformation of the pallidum into the striatum. Development 126,3359 -3370.
GGAGCA) did not alter the shifted complex (lanes 5, 6).
(C-F) X-gal staining of transgenic embryos carrying a wild-type (C,E)
or mutant SBE3 reporter construct in which the Nkx2 core recognition sequence
(AAGTAG) was deleted (D,F). Embryos carrying the wild-type SBE3 reporter
construct show consistent X-gal staining in the svz of the mge (C,E). By
contrast, embryos carrying an SBE3 reporter construct lacking the Nkx2 site
(