Department of Genetics, University of Pennsylvania School of Medicine, Clinical Research Building, Room 470, 415 Curie Blvd, Philadelphia, PA 19104, USA
* Author for correspondence (e-mail: epsteind{at}mail.med.upenn.edu)
Accepted 12 May 2003
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SUMMARY |
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Key words: Shh, Floor plate, Notochord, Node, Gene regulation, Central nervous system
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INTRODUCTION |
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The origin of the floor plate has been the subject of ongoing controversy
(Placzek et al., 2000;
Le Douarin and Halpern, 2000
).
Clarification of where and when the floor plate derives bears significantly on
understanding the molecular mechanisms underlying its development. The
prevailing hypothesis stipulates that an instructive signaling event between
the axial mesoderm and the medial cells of the overlying neural plate results
in floor plate induction (Jessell,
2000
). A challenge to this model has been proposed which attests
that in chick, zebrafish and frog embryos, the ventral midline of the neural
tube is derived from a population of precursors shared with the notochord that
are situated in Henson's node (chick), the organiser (frog) and the embryonic
shield (zebrafish) (Le Douarin and
Halpern, 2000
; Lopez et al.,
2003
). Segregation of these precursors into either a floor plate
or notochord lineage is thought to depend, at least in zebrafish and frogs, on
delta-notch signaling (Appel et al.,
1999
; Lopez et al.,
2003
). In the chick, floor plate precursors come to occupy their
ultimate position in the CNS by intercalating into the medial aspect of the
neural plate (Catala et al.,
1996
; Teillet et al.,
1998
). Although these two models of floor plate development are
not mutually exclusive their compatibility in mammals remains to be
determined.
Much of our knowledge of floor plate development has emerged from the study
of the Sonic hedgehog (Shh) signaling pathway. With the observation that Shh,
a secreted protein, is expressed in the node and notochord prior to floor
plate differentiation it was deemed an ideal candidate to mediate the
induction of the floor plate and other ventral neuronal cell types
(Echelard et al., 1993;
Roelink et al., 1995
;
Marti et al., 1995
;
Ericson et al., 1996
). Several
studies have determined that Shh is indeed the notochord-derived signal that
promotes patterns of cellular growth and differentiation within the ventral
neural tube including the homeogenetic (like-by-like) induction of its own
expression in the floor plate (see
Jessell, 2000
). Misexpression
of Shh or downstream effectors in its signal transduction pathway is
sufficient to induce the formation of an ectopic floor plate
(Echelard et al., 1993
;
Sasaki and Hogan, 1994
;
Roelink et al., 1995
;
Ruiz i Altaba et al., 1995
;
Epstein et al., 1996
;
Hynes et al., 1995
;
Hynes et al., 1997
;
Lee et al., 1997
). Moreover,
loss of Shh function results in mouse embryos lacking a floor plate
(Chiang et al., 1996
).
The Shh-dependent pathway resulting in floor plate formation relies on
triggering a transcription factor cascade culminating in the stable expression
of Shh in the ventral midline of the neural tube. Shh signaling from
the notochord activates Gli2, a zinc-finger transcriptional regulator, in the
overlying neural plate (Matise et al.,
1998; Ding et al.,
1998
). Gli2, which is required for floor plate development, is
responsible for initiating the transcription of Foxa2 (formerly Hnf3b)
(Sasaki et al., 1997
;
Matise et al., 1998
;
Ding et al., 1998
). Although,
the misexpression of Foxa2 in the CNS can under certain conditions
result in the ectopic activation of Shh, it remains unclear whether
Foxa2 is required to regulate Shh transcription within sites of
endogenous expression including the floor plate
(Ruiz i Altaba et al., 1995
;
Hynes et al., 1997
). Attempts
at addressing this question through conventional loss-of-function studies is
confounded by the requirement for Foxa2 in node formation, resulting in
Foxa2-/- embryos that lack both the notochord and floor
plate (Ang and Rossant, 1994
;
Weinstein et al., 1994
).
Efforts to determine whether Foxa2 and/or other genes regulate Shh
transcription in the floor plate have thus turned to the analysis of
cis-acting sequences controlling Shh expression
(Epstein et al., 1999;
Müller et al., 1999
;
Müller et al., 2000
;
Goode et al., 2003
). Multiple
enhancers were shown to regulate the expression of Shh at discrete
positions along the anteroposterior axis of the mouse neural tube
(Epstein et al., 1999
).
Regulatory sequences mediating the activity of two of these enhancers,
Shh brain enhancer 1 (Sbe1) and Shh floor plate enhancer 2
(Sfpe2), were found to reside on contiguous DNA fragments within the second
intron of the Shh gene. Sbe1 activity alone was sufficient to direct
reporter expression to rostral regions of the CNS including the ventral
midline of the midbrain and portions of the diencephalon. Importantly, the
combination of Sbe1 and Sfpe2 activities were required to direct reporter
expression to more caudal regions of the neural tube including the floor plate
of the hindbrain and spinal cord, as neither enhancer on its own was
sufficient for this expression (Epstein et
al., 1999
). Further indication of the cooperative nature of these
two enhancers came from mutational studies showing that the deletion of two
Foxa2 binding sites in sequences mediating Sbe1 activity compromised the
ability of Sfpe2 to consistently drive reporter expression to the floor plate
(Epstein et al., 1999
). These
data suggest that Foxa2 must be acting with additional transcription factors
to regulate Shh expression in the floor plate. To ascertain the
identity of the cooperating transcription factors we sought to uncover the
critical regulatory sequences mediating Sfpe2 activity.
We report here that cross-species comparison of intron 2 sequences from mouse, human, chicken and zebrafish have narrowed Sfpe2 activity to an 88-bp interval. A key feature of this floor plate element is its responsiveness to Shh signaling, probably reflecting that the transcription factors promoting Shh expression in the floor plate are activated by notochord-derived Shh. Further inspection of the 88-bp sequence identified three highly conserved binding sites matching the consensus for homeodomain, T-box related (Tbx) and Foxa transcription factors. Our studies reveal that the combined action of homeodomain and Foxa2 proteins is required to positively regulate transcription from the Shh floor plate enhancer, whereas the Tbx factor has a role in repressing transcription from areas of the CNS where Shh is not normally expressed. Because Sfpe2 activity was also observed in the node of early somite-stage embryos it provided the opportunity to trace the lineage of Shh-expressing cells from this structure. Interestingly, both Sfpe2 reporter activity and Shh expression were restricted to the ventral (mesodermal) layer of the node and notochord plate with no apparent mixing of X-gal-positive ventral cells with X-gal-negative dorsomedial cells (prospective floor plate). Thus, in contrast to the chick, floor plate cells in the mouse embryo do not derive from precursors shared with the notochord that subsequently insert into the neural plate, but are generated by inductive Shh signaling that initiates in the node.
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MATERIALS AND METHODS |
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To generate the mouse HR-c trimer (Rc9), excess amounts of the 88 bp HR-c fragment, derived from Rc7 upon NotI digestion, were used for ligation into the NotI site of the same reporter construct yielding multi-copy clones. Rc9 comprises a total of 3 copies of the HR-c fragment in the reverse orientation with respect to the lacZ cDNA. HR-c sequences from chicken and zebrafish were generated by PCR amplification of their respective genomic DNA using the following primers: Rc10, (E133) 5'-ATTAGCGGCCGCGGATTTTAATTAGAGAACCCAAA-3' and (E134) 5'-ATTAGCGGCCGCTAGTAGTGTAGCAGCTCTACAAA-3'; Rc11, (E138) 5'-ATTAGCGGCCGCGGTTTTTAATTAGAGCAGTCCAGG-3' and (E139) 5'-ATTAGCGGCCGCTAGGCTGTAAAGCTTCTCTAAAAAAC-3'. The orientation of the trimeric HR-c fragments cloned in Rc10 and Rc11 is the same as that in Rc9. The E number in parentheses refers to the lab stock code for each primer.
To test the requirement of the homeodomain binding-site in HR-c (Rc12), the following pair of PCR primers were used to create a small deletion overlapping the TAATTA core motif at the 5' end of HR-c: (E59) 5'-ATTAGCGGCCGCCCACACAAGTCTCGGGCTTTCACACC-3' and (E34) 5'-ATTAGCGGCCGCTAGTCGTTGTAGCAACTGGACAAACATTCCAGAGGTTTGC-3'. Point mutations designed to disrupt DNA binding at the recognition sequences for FoxH1, T-box protein and Foxa2 were constructed by PCR with the following primer pairs. The mutated residues are underlined. FoxH1, (E33) 5'-ATTAGCGGCCGCGGATTTTAATTAGAAAATCCGAGCAAGTCTCGGGCTTTCA-3', and (E34); T-box (Rc14), (E147) 5'-ATTAGCGGCCGCGGATTTTAATTAGAAAATCCACACAAGTCTCGGGCTTTGGGGGCTTGGGCAAACCTCTGG-3' and (E34); T-box (Rc14.1), (E243) 5'-ATTAGCGGCCGCGGATTTTAATTAGAAAATCCACACAAGTCTCGGGCTTTCATTTCTTGGGCAAACCTCTGG-3' and (E34); Foxa2, (E32) and (E86) 5'-ATTAGCGGCCGCTAGTCGTTGTAGCAACTGGCGGGGGCTTCCAGAGGTTTGCCCAAGGTG-3'. Each of the PCR products was cloned into the Shh reporter cassette as a trimer to generate Rc13-15. The integrity and orientation of all constructs containing PCR-generated fragments was confirmed by DNA sequencing.
Production and genotyping of transgenic mice
Transient transgenic embryos or mouse lines were generated by pronuclear
injection into fertilized eggs derived from either (BL6xSJL)F1
(Jackson Labs) or FVBN (Charles River) strains essentially as described
(Hogan et al., 1994).
Transgenes were prepared for microinjection as described
(Epstein et al., 1996
). The
genotyping of embryos or adult mice carrying reporter constructs was performed
by PCR using Proteinase K-digested yolk sacs or tail biopsies as DNA
templates. An upstream primer directed against the Shh promoter (E56)
5'-GACAGCGCGGGGACAGCTCAC-3' and a downstream primer directed
against lacZ (E68) 5'-AAGGGCGATCGGTGCGGGCC-3' were used
as described (Epstein et al.,
1999
). The Shh+/- animals were kindly provided
by H. Westphal (NIH) (Chiang et al.,
1996
) and maintained on a CD-1 background (Charles River).
Ptclacz/+ animals were procured from the Jackson
Labs (Bar Harbor, ME, USA).
Whole-mount ß-galactosidase and in situ hybridization
The assessment of ß-galactosidase activity was performed by
histochemical staining using either X-gal (GibcoBRL) or Salmon-gal (Biosynth)
as substrates (Epstein et al.,
2000). Transgenic embryos were stained from 30 minutes to
overnight depending on the strength of transgene expression. Whole-mount RNA
in situ hybridization was performed essentially as described
(Matise et al., 1998
) using
digoxygenin-UTP-labeled Shh, Foxa2 (B. Hogan), and lacZ
riboprobes. After whole-mount staining some embryos were fixed in 4%
paraformaldehyde, embedded in 4% agarose and sectioned on a vibratome at 50-75
µm.
Neural explant cultures
Embryos generated from matings between
Rc9+;Shh+/- males and
Shh+/- females were dissected in ice-cold L15 medium
(GibcoBRL) between 8.0 and 8.5 dpc (3-6 somites), and dissociated in 1 mg/ml
dispase (Boehringer Mannheim) essentially as described
(Alder et al., 1999). Neural
tissue isolated from the presumptive anterior spinal cord was bissected along
the midline and cultured on Transwell filters (0.4 µm, Costar) floating on
47.5% Dulbecco's minimum essential medium (Specialty Media), 47.5% F-12 Ham's
nutrient mixture (GibcoBRL), 2 mM glutamine (GibcoBRL), 100 U/ml
penicillin-streptomycin (GibcoBRL), and 5% rat serum (Harlan) in the presence
or absence of 1 µM recombinant Shh-N protein. After 30 hours of culture in
a CO2 incubator at 37°C, explants were fixed and stained with
X-gal. The genotype of each neural explant was established by PCR using DNA
from Proteinase K digested yolk sacs as template with the following primers
directed against neo (to detect the mutant Shh allele) (E131)
5'-GAACAAGATGGATTGCACGCAG-3' and (E132)
5'-TTCAGTGACAACGTCGAGCACA-3'); Shh intron2 (to detect the
wild-type allele) (E133) 5'-TGAGCAGCGGTAATCCAGCC-3' and (E134)
5'-CTCCAGGATCATGCTTTTGGC-3'; and the Rc9 transgene (E56 and
E68).
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RESULTS |
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|
HR-c constitutes a functionally conserved floor plate and notochord
enhancer
If one copy of HR-c is sufficient to interact with Sbe1 for floor plate
expression, albeit at weaker levels compared to the full-length fragment, we
reasoned that a construct containing multimers of HR-c might recapitulate the
full extent of Sfpe2 activity. Three copies of HR-c were cloned into the
reporter cassette in head to tail configuration and tested in transgenic
embryos (Rc9). In all 12 embryos carrying Rc9, X-gal staining was detected in
the floor plate in a pattern indistinguishable from those carrying the entire
746-bp fragment (Rc2) as assessed by the intensity of X-gal staining as well
as the minimal time required to initiate staining (30 minutes)
(Fig. 2A). Upon sectioning the
embryos, strong staining was detected in both the floor plate and notochord
(Fig. 2D), indicating that the
multimerized HR-c construct faithfully recapitulates these domains of
endogenous Shh expression
(Epstein et al., 1999).
|
The Shh floor plate enhancer is responsive to Shh
signaling
The induction of Shh expression in the floor plate is dependent on
Shh signaling from the underlying axial mesoderm (reviewed by
Jessell, 2000). If the
sequences mediating Sfpe2 activity contain the requisite complement of
transcription factor-binding sites we reasoned that they should be responsive
to Shh signaling. However, if sequences outside of these sequences are also
required, then the reporter should not be activated by Shh. Sfpe2
responsiveness to Shh signaling was assayed in neural explants derived from
wild-type and Shh-/- embryos. The use of explants from
Shh-/- embryos offers the advantage of performing the
experiments in an environment free of prior exposure to Shh and extraneous
floor plate signals. Stable transgenic mouse lines carrying three copies of
HR-c (Rc9) were crossed onto a Shh+/- background and
subsequently intercrossed to generate wild-type and
Shh-/-; Rc9+ embryos. Spinal cord explants were
isolated from 3-6 somite stage embryos, which precede the onset of
Shh expression in the CNS, and cultured in the presence or absence of
recombinant N-Shh for 30 hours. X-gal staining could not be detected in the
majority of wild-type; Rc9+ or any of the
Shh-/-; Rc9+ neural explants cultured in the
absence of N-Shh (Fig. 3A,B).
The few cells that stained positive in the wild-type culture probably reflect
their exposure to Shh signaling from the notochord prior to being explanted.
In the presence of N-Shh, a significant portion of each explant stained
positive for lacZ expression in both wild-type; Rc9+
(n=16) and Shh-/-; Rc9+ (n=3)
genotypes (Fig. 3C,D). These
data show that Shh signaling is both necessary and sufficient to activate the
Sfpe2 enhancer, confirming that the combination of Sbe1 and HR-c possess the
appropriate array of transcription factor-binding sites needed to respond to
Shh signaling.
|
Two homeodomain binding-sites were identified within an AT-rich segment of DNA spanning approximately 20 bp including sequence proximal to HR-c (Fig. 4 and data not shown). Remarkably, this 20-bp stretch was virtually 100% conserved across phyla with only a single nucleotide substitution identified in zebrafish. The ATTA cores of the two homeodomain recognition sequences were found in opposite orientation separated by a two base-pair overlap. Sequences outside the core ATTA motif did not match recognition sequences for any of the transcription factors known to partner with Hox proteins, including Pbx and Meis.
|
|
A highly conserved Tbx binding site showing a perfect match with the
consensus was also identified in HR-c
(Smith, 1998)
(Fig. 4). Given that the
founding member of the Tbx family, brachyury, is expressed in the
axial mesoderm and that other members of this large transcription factor
family have been described in the ventral midline, the Tbx site seemed to be a
good candidate for regulating Sfpe2 activity. Nevertheless, no embryos
expressing the Rc14 transgene, which contains mutations in the Tbx
binding-site, showed any alteration to the pattern of X-gal staining in the
floor plate or notochord as compared to embryos carrying the wild-type
transgene (compare Fig. 4A with
4D). Unexpectedly, loss of the Tbx binding-site did result in
alterations to the pattern of X-gal staining in the diencephalon. Normally,
the rostral boundary of X-gal staining derived from Sbe1 coincides with
prosomere 3 (Fig. 5A). However,
in all 13 transgenic embryos expressing the mutated Tbx binding-site construct
(Rc14), the rostral boundary of X-gal staining extended into prosomere 5
(Fig. 5B). Moreover, X-gal
staining was detected in the ventral midline in contrast to where Shh
is normally expressed in the rostral diencephalon within two stripes
adjacent to the midline (Fig.
5C,D). To confirm that the ectopic Shh reporter activity
indeed resulted from the loss of the Tbx binding site rather than the
inadvertent creation of a new binding site supporting Shh reporter
activation, a second construct was generated containing a different set of
point mutations (Rc14.1; see Materials and Methods). Transgenic embryos
carrying this construct showed precisely the same ectopic expression of
Shh reporter activity in the ventral midline of the rostral
diencephalon (n=4, data not shown). These results suggest that
sequences encoding a consensus binding site for Tbx proteins are required to
recruit a transcription factor, probably a member of the Tbx family, which
functions to repress Sfpe2 activity and by extension, Shh
transcription, from the ventral midline of the diencephalon.
|
Homeodomain and Foxa2 binding sites are required at the initial
stages of Shh transcription in the floor plate
In the CNS of the developing mouse embryo, the expression of Shh
initiates at the 7-8 somite stage within the ventral midline of the
prospective midbrain and then extends rostrally to the forebrain and caudally
to the hindbrain and spinal cord (Echelard
et al., 1993). By the 13-somite stage, the ventral midline
expression intensifies and is continuous along the extent of the
anteroposterior neuraxis. We verified that transgenic lines carrying a
wild-type enhancer construct (Rc9) recapitulated this early progression of
Shh transcription (Fig.
6). With exception to the domain of Shh expression in the
rostral forebrain, embryos expressing the wild-type enhancer construct (Rc9)
simulated the ontogeny of Shh transcription in the ventral midline of
the CNS. With this information in hand, we next sought to determine whether
the consequences of mutating the homeodomain and Foxa recognition sequences on
X-gal staining in the floor plate and notochord reflected alterations in the
initiation and/or maintenance of lacZ expression.
Stable mouse lines expressing the mutated homeodomain binding-site transgene (Rc12) were generated to test the role of the homeodomain site on ventral midline expression at early stages of CNS development. At the 6-somite stage, X-gal staining was detected in the notochord of embryos from 4 independent lines in a pattern similar to embryos carrying the wild-type transgene (Fig. 6A,B). By the 9-somite stage, X-gal staining was detected in the region of the presumptive midbrain in embryos from all 4 lines expressing Rc12. However, unlike embryos expressing the wild-type enhancer construct, staining was excluded from the ventral hindbrain, thus indicating that the caudal progression of Sfpe2 activity was impaired by the mutation in the homeodomain binding-site (Fig. 6C,D). By the 13-15 somite stage, X-gal staining was completely absent from the floor plate in 3 of 4 transgenic lines carrying the homeodomain binding-site mutation (Rc12), with the fourth showing patchy expression in the rostral portion of the spinal cord (Fig. 6F,H). The patchy X-gal staining could result from limited activity of the enhancer in the absence of the homeodomain binding site or perdurance of ß-gal from an earlier progenitor cell. To distinguish between these two possibilities whole-mount in situ hybridization was performed to detect lacZ mRNA transcripts. Transgenic embryos carrying the wild-type enhancer construct (Rc9) showed high levels of lacZ mRNA in the floor plate and notochord at the 13-somite stage (Fig. 6I). Embryos derived from the reporter line showing patchy X-gal staining in the floor plate showed weak and patchy expression of lacZ mRNA consistent with the pattern of X-gal staining (Fig. 6J,K). Because the lacZ expression is only detected at the time that Shh is normally transcribed in the floor plate (after the 8-somite stage) it argues against the probability that the X-gal-positive cells were derived from a precursor that expressed lacZ at earlier stages. These studies confirm that the homeodomain binding-sites in HR-c are required for the initiation of Sfpe2 activity in the floor plate between 9 and 13 somites.
With respect to the Foxa binding site, at no stage of development analyzed did embryos expressing a reporter construct containing a mutated Foxa binding-site (Rc15) display X-gal staining in the floor plate or notochord, suggesting that Foxa2 is also required at the initiation of Sfpe2 activity. Because of the failure to activate reporter expression at early stages in embryos carrying binding-site mutations, we cannot exclude the possibility that homeodomain and Foxa proteins also function to maintain Sfpe2 activity at later stages.
Tracing the lineage of Shh-expressing cells in the node
In addition to the floor plate and notochord we observed Sfpe2 activity in
the node of the mouse embryo as early as the onesomite stage
(Fig. 7A and data not shown).
In comparing the distribution of X-gal staining in the node of 5-6 somite
stage embryos to that of endogenous Shh we observed that both are
restricted to the ventral (mesodermal) layer
(Fig. 7A-D). Examination of
thin sections (6 µm) generated through X-gal-stained embryos at the 5-6
somite stage (n=7) from the level of the posterior node to the
hindbrain confirmed the absence of any X-gal-positive cells in the dorsal
layer of the node or ventral midline of the neural plate
(Fig. 7A). Only the ventral
layer of the node and its derivatives the notochord progenitors
showed X-gal staining at the 5-somite or any other stage examined
prior to the onset of Shh transcription in the CNS (n=37
embryos examined between 0 and 7 somite stages, data not shown). This
contrasts with the expression of Shh in the chick, which shows a
uniform distribution at low levels in both the ventral and dorsal (ectodermal)
layers of Henson's node (Teillet et al.,
1998). Lineage tracing studies of Henson's node using chick-quail
chimeras suggested that the floor plate and notochord are derived from a
common precursor (Catala et al.,
1996
; Teillet et al.,
1998
). The same is unlikely to be true in the mouse because X-gal
staining is not detected in the dorsal layer of the node, from where the floor
plate is derived. Furthermore, the clear demarcation between mesoderm and
ectoderm as evidenced by the segregation of X-gal-positive and X-gal-negative
cells in and around the node (Fig.
7A), argues against the probability that the mouse floor plate
arises from a common precursor with the notochord that subsequently inserts
into the medial portion of the neural plate as was previously reported for the
chick (Catala et al., 1996
;
Teillet et al., 1998
). The
expression of Ptc and Foxa2 in the dorsal layer of the node
and medial neural plate at more rostral axial levels in response to Shh
signaling is consistent with the model that floor plate development initiates
in the node via inductive signaling (Fig.
7F,G).
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DISCUSSION |
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Functional conservation of Shh regulatory sequences
Our approach towards identifying regulators of Shh expression in
the notochord and floor plate relied on comparing candidate regulatory
sequences from multiple organisms to narrow down the critical elements based
on homology. The assumption that conservation of sequence underscores
conservation of function was validated with the identification of a highly
conserved 88-bp fragment from intron 2 of the Shh gene (HR-c) that
cooperates with Sbe1 to direct lacZ reporter expression to the floor
plate and notochord in transgenic mice. Further confirmation of this principle
was obtained when the corresponding chicken DNA was found sufficient to direct
transgene expression to the notochord and floor plate in mouse embryos.
A survey of the 88-bp fragment for transcription factor binding sites identified those matching the consensus for homeodomain, Tbx and winged-helix family members. In determining the requirement of each binding site on transgene expression we uncovered a novel interaction between homeodomain and Foxa2 proteins as binding sites for each were required for the initiation of Shh floor plate enhancer activity. In addition to the positive elements mediating Sfpe2 function, we found that a well-conserved Tbx binding site is required for the repression of transgene expression in a region of the ventral diencephalon where Shh is not normally detected. The identity of the Tbx protein that represses Sfpe2 activity is presently unknown.
Interestingly, the more divergent zebrafish HR-c sequence which was
incapable of directing ventral midline expression in transgenic embryos showed
significant sequence homology in and around the area of the homeodomain
recognition sequences but not in the vicinity of the Foxa binding site.
Shh floor plate enhancer activity has been mapped in zebrafish to
sequences inclusive of the conserved homeodomain binding-site and extending
160 bp upstream of HR-c (Müller et
al., 1999). Upon surveying these sequences we identified a Foxa
binding site located in the reverse orientation 25 bp downstream of a
previously reported Tbx site. This suggests that the transcriptional control
mechanisms responsible for regulating Shh expression in the floor
plate of zebrafish, although slightly displaced in position, may remain
conserved in function.
Cooperative interactions regulate Shh floor plate enhancer
activity
Our studies suggest that Foxa2 is not sufficient to mediate Sfpe2 function
and that cooperative interactions with a homeodomain transcription factor are
required to direct reporter expression to the floor plate in a
Shh-like manner. These results are seemingly inconsistent with
previous reports documenting that forced expression of Foxa2 is sufficient to
activate Shh transcription (Hynes
et al., 1995; Ruiz i Altaba et
al., 1995
). However, additional observations are supportive of our
conclusion. First, Foxa2 is expressed along the length of the floor plate yet
the activities of the enhancers regulating Shh are regionalized along
the anteroposterior axis of the neural tube
(Epstein et al., 1999
).
Moreover, the sequences mediating Sbe1 and Sfpe2 activity, although both
possessing Foxa binding sites, cannot independently direct reporter expression
to the floor plate even when multimerized. Therefore, additional transcription
factors must be acting in concert with Foxa2 to regulate Shh
expression in the floor plate. To reconcile differences between our results
and the Foxa2 gain-of-function studies, we speculate that: (1) Foxa2 may be
inducing the expression of the cooperating transcription factor(s) (see
Fig. 8B); (2) Foxa2 may only be
capable of activating Shh transcription where the cooperating
transcription factor(s) is/are expressed. Restrictions in where Foxa2 can
activate Shh within the neural tube have been described
(Ruiz i Altaba et al., 1995
);
and 3) ectopic expression of Foxa2 may be activating Shh
transcription through enhancers other than Sfpe2.
|
Notwithstanding the agreement of our data with a role for Nkx6 family
members in regulating Shh expression in the floor plate, genetic
studies supporting the requirement of Nkx6 genes in this process have
not been forthcoming (Sander et al.,
2000; Vallstedt et al.,
2001
; Cai et al.,
2001
). Thus, we cannot rule out the possibility that homeodomain
proteins other than Nkx6.1 or Nkx6.2 regulate Sfpe2. Furthermore, detecting a
down-regulation in Shh transcription in Nkx6 mutants may be
confounded by the presence of another floor plate enhancer (Sfpe1), located
upstream of the Shh gene which may compensate in the absence of
Sfpe2. It is interesting to note that the regulation of Shh
expression in more rostral regions of the CNS is also dependent on an
Nkx gene. In embryos lacking Nkx2.1, Shh expression in the
ventral telencephalon is completely absent. Not surprisingly Nkx2.1
is the only Nkx family member expressed in this region of the CNS
(Sussel et al., 1999
).
To explain how Foxa2 and the cooperating homeodomain factor may be
interacting to regulate Shh transcription we draw from previous
studies which have shown that the binding of Foxa proteins to their
recognition sites on active enhancers can result in the stabilization of
nucleosome position, thus facilitating the binding of additional transcription
factors to the enhancer complex (Cirillo et al., 1999;
Chaya et al., 2001). Foxa2 may
be functioning in a similar capacity on Sfpe2 by promoting the stable binding
of homeodomain proteins such as Nkx6 family members. This model is
particularly attractive because it can also explain why Foxa2 sites in Sbe1
are unable to compensate when a similar site in Sfpe2 is mutated. Foxa2 may
only be able to act locally in generating an environment permissive for
transcription, concordant with the close proximity of Foxa2 binding sites to
those for factor X in Sbe1 and the homeodomain site in Sfpe2
(Fig. 8).
The floor plate and notochord do not share a common precursor in the
node
That the mutation in the homeodomain binding-sites in HR-c only affected
reporter expression in the floor plate stipulates that the cooperating
homeodomain factor is not involved in regulating Sfpe2 activity in the
notochord. This result bears significance on the timing of floor plate
specification and argues that although initial steps may be occurring in the
node, the final step in the process happens well after floor plate progenitors
have emerged from the node.
Using Sfpe2 reporter activity to trace the lineage of
Shh-expressing cells in the node we showed that X-gal staining was
restricted to the ventral layer, as was endogenous Shh. Consequently,
only the notochordal plate, a mesodermal derivative emerging from the ventral
layer of the node was positive for X-gal staining. Because the floor plate
precursors residing in the dorsal layer of the node showed no X-gal staining,
we conclude that floor plate and notochord progenitors in the mouse node do
not derive from a common origin (Fig.
8A). These results are consistent with previous dye I labeling
studies of the mouse node (Beddington,
1994; Sulik et al.,
1994
) but are in disagreement with data from the chick which
supports a common origin for floor plate and notochord precursors
(Catala et al., 1996
;
Teillet et al., 1998
). In the
chick, floor plate precursors in the node segregate from a common population
of progenitors and subsequently insert into the medial position of the
overlying neural plate (Catala et al.,
1996
; Teillet et al.,
1998
). Because mixing between X-gal-positive ventral cells and
X-gal-negative dorsal cells in or around the mouse node was not observed, we
conclude that floor plate precursors in the mouse are not generated by the
same mechanism as in chick. Instead, we concur with the prevailing model that
the mouse floor plate forms by inductive Shh signaling.
Our observation that the Shh target genes Ptc and Foxa2 are expressed in the dorsal layer of the node (Fig. 8A) offers further support that the process of floor plate induction begins in the mouse node at early somite stages and doesn't terminate until Shh transcription is activated in the ventral midline of the CNS between 8 to 12 somite stages. In this homeogenetic model of floor plate induction, Shh secreted from the axial mesoderm signals to the overlying neural plate to activate effectors of the Shh signal transduction cascade (Fig. 8B). A consequence of this vertical signaling step is the initiation of Shh transcription, through the direct binding of Foxa2 and a homeodomain protein to sequences in HR-c. Given that sequences mediating Sbe1 activity are also required for floor plate expression, we speculate that additional transcriptional activators are participating in the regulation of Shh expression (Fig. 8B). Identifying the critical sequences mediating Sbe1 activity and the factors binding to these sites should further elucidate how Shh expression is activated in the floor plate of the mouse spinal cord.
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ACKNOWLEDGMENTS |
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