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First published online May 16, 2007
doi: 10.1242/10.1242/dev.002980
Department of Organismal Biology and Anatomy, The University of Chicago, 1027 E. 57th Street R107, Chicago, IL 60637, USA.
* Author for correspondence (e-mail: iskromne{at}uchicago.edu)
Accepted 29 March 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cdx, Caudal, Hox, Retinoic acid, Segmentation, Rhombomeres, Hindbrain, Spinal cord, Central nervous system, Chordates, Vertebrates, Evolution
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Fraser et al., 1990;
Orr, 1887). The forebrain
neuromeres (or prosomeres) and the more familiar hindbrain rhombomeres are
important for proper neuronal organization and structural patterning of the
adult brain and brain stem (reviewed by
Borday et al., 2004;
Kiecker and Lumsden, 2005;
Puelles and Rubenstein, 2003).
In the hindbrain, extensive genetic and molecular data have shown the
existence of a regulatory network of transcription and secreted factors that
specify the identity and order of rhombomere formation
(Waskiewicz et al., 2002)
(reviewed by Moens and Prince,
2002). In sharp contrast, the spinal cord remains unsegmented,
although an anterior-posterior iterative arrangement of various neuronal
populations can be seen to different degrees in Amphioxus, zebrafish
and in certain tetrapods (Bone,
1960; Fetcho,
1987; Forehand and Farel,
1982; Myers, 1985;
Roberts and Clarke, 1982;
Stern et al., 1991). However,
these iterated arrangements are not the consequence of cryptic segmentation,
as demonstrated by the extensive mixing of cells in clonal analysis
experiments (Morin-Kensicki and Eisen,
1997; Stern et al.,
1991), but are the result of spinal cord neurons responding to
paraxial mesoderm-derived signals
(Detwiler, 1934;
Eisen and Pike, 1991;
Ensini et al., 1998;
Keynes and Stern, 1984;
Lewis and Eisen, 2004).
Despite the striking difference in segmentation, the hindbrain and spinal cord
share a number of characteristics: both derive from the caudal neural plate
(Brown and Storey, 2000;
Muhr et al., 1997;
Schoenwolf, 1992), are
patterned along their anterior-posterior axis by Hox transcription factors
(Deschamps and van Nes, 2005;
Krumlauf et al., 1993;
Lumsden and Krumlauf, 1996)
and have a common evolutionary origin
(Ghysen, 2003;
Hirth et al., 2003).
Therefore, we set out to investigate the mechanisms that direct the caudal
neural plate to develop as hindbrain or as spinal cord.
Cdx (Caudal) transcription factors have been implicated in the development
of trunk and tail structures across all major animal groups by controlling the
sequential addition and identity of body segments
(Copf et al., 2004). Within
vertebrates, the three family members Cdx1, Cdx2 and Cdx4
are expressed in nested domains in the trunk and tail of the embryo
(Davidson et al., 2003;
Frumkin et al., 1993;
Gamer and Wright, 1993;
Joly et al., 1992;
Lohnes, 2003;
Marom et al., 1997;
Meyer and Gruss, 1993;
Pillemer et al., 1998;
Reece-Hoyes et al., 2002).
Most of our understanding of Cdx function is restricted to their role in
paraxial mesoderm in mouse, where they have been shown to integrate FGF,
retinoic acid and Wnt signals into coherent Hox gene expression (reviewed by
Deschamps and van Nes, 2005;
Lohnes, 2003). This role seems
to be conserved in zebrafish and Xenopus
(Davidson et al., 2003;
Davidson and Zon, 2006;
Isaacs et al., 1998;
Pownall et al., 1998;
Pownall et al., 1996;
Shimizu et al., 2006;
Shimizu et al., 2005). The
function of Cdx genes in CNS development, however, is poorly understood,
despite the fact that expression of Cdx genes in the caudal neural plate is
highly conserved across vertebrates
(Ehrman and Yutzey, 2001;
Frumkin et al., 1993;
Joly et al., 1992;
Marom et al., 1997;
Meyer and Gruss, 1993;
Nordstrom et al., 2006;
Pillemer et al., 1998;
Reece-Hoyes et al., 2002).
Using a variety of morphological, cellular and molecular criteria we
present evidence that spinal cord specification in zebrafish is dependent on
the partially redundant functions of Cdx1a and Cdx4. In agreement with a
previous study (Shimizu et al.,
2006), we show that zebrafish embryos lacking full Cdx1a and Cdx4
functions develop an expanded hindbrain. In addition, we show that this
expanded hindbrain is organized into segmental units arranged in a
mirror-image duplicated pattern of ectopic rhombomeres within the trunk region
of the embryo. We also show that Cdx factors can induce the development of
spinal cord cell types and posterior Hox gene expression, when misexpressed in
rostral regions of the CNS. We propose that Cdx transcription factors normally
function to prevent rhombomere formation in the caudal neural plate and that
by preventing the posterior-most region of the neural plate from following a
segmented developmental program, Cdx transcription factors help determine the
size of the prospective hindbrain and spinal cord territories. We hypothesize
that this newly proposed function of Cdx transcription factors allowed the
development of the dorsal, hollow and unsegmented caudal neural tube that is
characteristic of the vertebrate lineage.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Embryos from wild-type AB stock,
kgghi2188A (Golling et
al., 2002), Tg[isl1:GFP]
(Higashijima et al., 2000) and
Tg[ßactin:GFP]
(Gillette-Ferguson et al.,
2003) were obtained from natural spawning, grown at 28°C and
staged as described (Kimmel et al.,
1995). Injections were carried out at the one-cell stage.
Antisense cdx1a (5 ng) (Shimizu
et al., 2005) and cdx4 (20 ng)
(Davidson et al., 2003)
morpholino oligonucleotides (Gene Tools LLC) were injected alone or in
combination. For mRNA overexpression, 25 pg of hoxc6a, hoxb8a or
hoxa9a (Prince et al.,
1998a), or 40 pg of gap43-RFP (cell membrane marker, gift
of E. Amaya, University of Cambridge, Cambridge, UK) capped sense mRNA (SP6
mMessage mMachine Kit, Ambion) was injected using a standard injection
protocol (Bruce et al., 2001).
Retinoic acid signaling was blocked by incubating embryos in 1 µM BMS493 in
the dark.
Cell transplantation was performed as previously described
(Ho and Kane, 1990). Donor
embryos were injected at the one-cell stage with 40-kDa lysine-fixable
fluorescein (Invitrogen). Donor cells were collected at sphere stage and
approximately 20 cells were transplanted 2- to 5 cell-diameters away from the
margin of a stage-matched unlabeled host.
Transgenic constructs, genotyping and heat shock
To generate a heat-inducible cdx4 expression construct,
full-length zebrafish cdx4 was PCR amplified using the following
primers: mCdx5'-forward,
5'-CGATTCCGGGATCCACCGGTCGCCACCATGTATGGATCGTGTTTGCTCGAAAAAGAGGCAAGCATGTATCACCAA-3'
(BamHI and AgeI sites underlined, translation initiation
site in bold); and 3'MCScdx-reverse,
5'-TTGTCTAGAAAGCTTGGTACCGATCGATAGTTTGTAATCCTTTTTGACCAC-3'
(XbaI site underlined). The forward primer changes the 5' end
of the gene to that of mouse Cdx4, rendering it unrecognizable to the
zebrafish cdx4 morpholino. BamHI/XbaI-digested PCR
product was cloned in-frame into pcDNA3.1(-)/Myc-His B (Invitrogen). The
modified mouse 5'-zebrafish cdx4-myc-his
(m5'zcdx4) gene was digested with SphI, blunt
ended, digested with AgeI and gel purified. Separately, the zebrafish
hsp70 promoter (Halloran et al.,
2000) and the pBS-ISce-II KS vector
(Thermes et al., 2002) were
digested with SacI/AgeI and SacI/EcoRV,
respectively, and purified. A double ligation was then set up using the
purified fragments to generate phsp70:m5'zcdx4-ISce.
All constructs were confirmed by sequencing.
Stable transgenic fish were generated by injecting 1 µg
phsp70:m5'zcdx4-ISce plasmid and 1 unit of ISce-I
meganuclease in 1x ISce-I buffer (New England Biolabs) into fertilized
eggs during the first 15 minutes of development
(Thermes et al., 2002).
Embryos were grown to adulthood, pairwise crossed and their embryos genotyped
using the 3phsp70-forward (5'-GTATTACTTTGTTAACGTGGC-3') and BGHrev
(5'-TGAAAGBCACAGTCGAGG-3') primers (IDT). As positive PCR
controls, Wnt5a-12 (5'-CAGTTCTCACGTCTGCTACTTGCA-3') and Wnt-21
(5'-ACTTCCGGCGTGTTGGAGAATTC-3') primers were included in all
reactions. Founder fish whose progeny produced diagnostic bands were
out-crossed to wild-type fish. F1 fish were genotyped and individuals carrying
the transgene were used as the Tg[phsp70:cdx4] line founders. A
standard heat-shock protocol was used to induce transient cdx4
expression (Halloran et al.,
2000).
Whole-mount in situ hybridization and immunocytochemistry
Detection of cdx1a (Shimizu et
al., 2005); cdx4
(Joly et al., 1992);
cyp26a1 (Kudoh et al.,
2002); epha4a (Xu et
al., 1994); foxb1.2 (mar)
(Odenthal and Nusslein-Volhard,
1988); islet1 (Inoue
et al., 1994); krx20
(Oxtoby and Jowett, 1993);
hoxa2b, hoxb1a, hoxb3a, hoxb8a, hoxc4a, hoxd3a and hoxd4a
(Prince et al., 1998a;
Prince et al., 1998b);
myod (Weinberg et al.,
1996); olig2 (Park et
al., 2002); radical fringe (rfng)
(Cheng et al., 2004);
raldh2 (also known as aldh1a2 - ZFIN)
(Begemann et al., 2001);
retinoic acid receptor alpha (RAR
; also known as
raraa - ZFIN) (Hale et al.,
2006); and valentino (val)
(Moens et al., 1998)
expression by in situ hybridization was carried out as previously described
(Bruce et al., 2001), using
NBT/BCIP or Fast Red as the enzyme substrate.
Antibody labeling was performed as previously described
(Svoboda et al., 2001).
Monoclonal mouse anti-acetylated Tubulin (Sigma-Aldrich), mouse anti-myosin HC
(A4.1025, Developmental Studies Hybridoma Bank, IA, USA), mouse
anti-neurofilament 160k (RMO44, Zymed, CA, USA) and polyclonal rabbit anti-GFP
conjugated to FITC (Invitrogen) were used at 1:500, 1:100, 1:5000 and 1:1000,
respectively. Goat anti-mouse [Alexa Fluor 647 (Invitrogen) and/or FITC
(Jackson ImmunoResearch)] or goat anti-rabbit (Alexa Fluor 488, Invitrogen)
secondary antibodies were used at 1:2000.
Image processing
Deyolked embryos were manually sectioned using a scalpel. Specimens were
photographed with a Nikon D1 digital camera mounted on a Leica MZFL III or
Zeiss Axioskop microscope. For confocal microscopy, single optical sections
and image stacks were obtained using a Zeiss laser-scanning confocal imaging
system (LSM 510). Three-dimensional reconstructions were produced with the
Zeiss LSM 510 software and ImageJ 1.32 (NIH). Figure panels were constructed
using Photoshop 7.0 (Adobe).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Davidson and Zon,
2006; Joly et al.,
1992; Shimizu et al.,
2005). We tested the function of Cdx4 in zebrafish CNS development
by examining the distribution of hindbrain and spinal neuronal populations in
wild-type and cdx4 morpholino-injected embryos
(Fig. 1A-J). These embryos,
referred to hereafter as cdx4-deficient embryos, are phenotypically
indistinguishable from cdx4 loss-of-function mutants
kgghi22188A and
kggtv205 (Davidson
et al., 2003; Golling et al.,
2002). We used the vagal motor neurons and the T reticular
interneurons as landmarks for the posterior hindbrain. These neurons are
located in the spinobulbar junction, and they have a characteristic
organization in vertebrates that is evolutionarily conserved
(Fetcho, 1987;
Kimmel et al., 1985;
Wake, 1993). We found that in
cdx4-deficient embryos, the size of the vagus expanded posteriorly by
two somites as compared with wild-type siblings
(Fig. 1A,F). Similarly, the
RMO44-immunopositive T reticular interneurons also expanded posteriorly in
these embryos (Fig. 1B,G).
Other hindbrain-specific populations, such as the branchiomotor and
reticulospinal neurons of rhombomeres (r) 1-6, appeared unaffected
(Fig. 1B,G and data not shown).
We also examined three distinct spinal cord populations in
cdx4-deficient embryos: motor neurons and their exit roots,
oligodendrocytes and Rohon-Beard sensory neurons. In wild-type embryos, the
axons of the first spinal motor neurons exit at the level of somite 2
(Fig. 1C). By contrast, these
axons exited at the level of somite 4 in cdx4-deficient embryos
(Fig. 1H), correlating with the
two-somite posterior expansion of the hindbrain neuronal populations
(Fig. 1F,G). The distribution
of olig2-expressing primary motor neurons and oligodendrocytes and
isl1-positive Rohon-Beard sensory neurons also shifted posteriorly in
cdx4-deficient embryos (Fig.
1D,E,I,J), indicating a generalized posterior shift of the spinal
cord territory. Together, these data suggest that Cdx4 is necessary in
zebrafish for the proper determination of the axial location of the transition
from hindbrain to spinal cord.
|
;
Lohnes, 2003), raising the
possibility that cdx1a and cdx4 might cooperate in
determining the hindbrain-spinal cord transition in zebrafish. To test this
hypothesis, we injected wild-type and cdx4-deficient embryos with a
cdx1a morpholino (Shimizu et al.,
2005) and examined the distribution of cell populations described
above (Fig. 1K-T). Whereas
wild-type embryos injected with a cdx1a morpholino showed no CNS
defects (Fig. 1A-E,K-O),
reduction of cdx1a function in a cdx4-deficient background
led to an almost complete loss of spinal cord identities: spinal motor neurons
and oligodendrocytes were absent, and the number of isl1-positive
Rohon-Beard sensory neurons was greatly reduced
(Fig. 1R-T). Conversely, there
was a posterior expansion of r7 and r8 in these embryos, as shown by the trunk
and tail distribution of isl1-GFP-positive branchiomotor neurons
(Fig. 1P). Most of these
branchiomotor neurons had axonal projections and morphologies characteristic
of vagal cells, although separate and distinct neuronal clusters were also
observed near the tail of these embryos
(Fig. 1P). A posterior
expansion of T reticular interneurons was also observed in these embryos
(Fig. 1Q). Together, these data
suggest that Cdx1a and Cdx4 function redundantly in the specification of
spinal cord-specific neuronal identities, and in their absence most of the
caudal neural plate takes on an expanded hindbrain fate.
The hindbrain patterning and segmentation program is improperly induced in the CNS of cdx1a/cdx4-deficient embryos
We next examined the expression of Hox patterning genes in the CNS of
cdx1a/cdx4-deficient embryos (i.e. in embryos deficient for both
genes). In these embryos, the r7 and anterior spinal cord marker
hoxd4a was found to be expressed throughout the posterior CNS,
whereas expression of the spinal cord markers hoxb6a, hoxb8a and
hoxb10a was notably absent (Fig.
2A,B,G,H and data not shown). Within the hindbrain,
cdx1a/cdx4-deficient embryos showed no changes in the anterior limit
of hoxa2b (r2), hoxb1a (r4), hoxb3a (r5) and
hoxd4a expression as compared with wild-type embryos
(Fig. 2B,C,H,I and data not
shown) suggesting that only spinal cord fates fail to be specified in these
embryos. Together with our morphological data, these results show that the
native hindbrain region in embryos lacking Cdx1a and Cdx4 activities has a
normally ordered and nested set of rhombomeric identities and that the
increase in hindbrain size seen in these embryos is mostly owing to an
expansion of r7 and r8.
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), was also found to be expressed in the posterior CNS of
these embryos (Fig.
2D,D',J,J'). Similarly, krx20 (egr2b
- ZFIN) expression, normally confined to r3 and r5
(Oxtoby and Jowett, 1993), was
now present in the posterior CNS (Fig.
2G-I,K,K'). This shows that in cdx1a/cdx4-deficient
embryos, several members of the regulatory network controlling rhombomere
patterning are ectopically expressed outside of their native hindbrain
region.
The regulatory network controlling hindbrain patterning also controls its
segmentation into rhombomeres (Moens and
Prince, 2002; Waskiewicz et
al., 2002). Therefore, we examined the expression of epha4a,
radical fringe (rfng) and foxb1.2 (also known as
mar) in cdx1a/cdx4-deficient embryos, as these genes have
been shown to be involved in rhombomere cell-sorting and boundary formation
(Cheng et al., 2004;
Cooke et al., 2001;
Cooke et al., 2005;
Odenthal and Nusslein-Volhard,
1988). In wild-type embryos, epha4a is expressed within
odd-numbered rhombomeres as well as in the forebrain, midbrain and tail
notochord, whereas expression of rfng and foxb1.2 in the CNS
is restricted to the boundaries between rhombomeres
(Fig. 2E-F'). In
cdx1a/cdx4-deficient embryos, these genes were ectopically expressed
in small discontinuous domains in the posterior CNS
(Fig. 2K-L'), showing the
induction of hindbrain segmentation genes beyond their normal domain of
expression. Together, these data suggest that in the absence of Cdx1a and Cdx4
activities, the caudal neural plate not only fails to acquire spinal cord
characteristics, but it also becomes competent to initiate the molecular
program leading to the formation of supernumerary hindbrain segments and
boundaries.
Development and mirror-image patterning of supernumerary rhombomeres in the CNS of cdx1a/cdx4-deficient embryos
Despite the fact that several hindbrain- and boundary-specific genes were
ectopically expressed in the posterior CNS of cdx1a/cdx4-deficient
embryos, we noted the absence of definitively sized additional rhombomeres in
this region. We hypothesized that the expansion of the native r7/8 region in
cdx1a/cdx4-deficient embryos could be inhibiting the formation of
correctly sized rhombomeres within the transformed caudal neural plate. In
order to experimentally reduce the size of the r7/8 region, we took advantage
of the observation that retinoic acid (RA) signaling inhibition results in the
loss of r6-8 markers in wild-type (Begemann
et al., 2004; Maves and
Kimmel, 2005) and cdx1a/cdx4-deficient embryos
(Shimizu et al., 2006). We
treated embryos with the pan-RA receptor inhibitor BMS493 at mid-gastrulation
to reduce the size of r7/8 without affecting the development of more-rostral
rhombomeres (Begemann et al.,
2004; Maves and Kimmel,
2005). As predicted, these treatments led to the loss of the r8
marker hoxc4a and an altered expression limit of the r7/8 marker
hoxd4a (Fig.
3A,B,I,J). We then examined the expression of more-anterior Hox
genes including hoxa2b (r2/r3), hoxb1a (r4), hoxb3a
(r5/r6) and hoxd3a (r6) in comparison with the r3 and r5 marker
krx20 (Fig. 3C-F,K-N).
Excluding hoxa2b, whose expression domain was confined to r2/3 as in
wild-type embryos (Fig. 3N)
(Prince et al., 1998b),
ectopic expression of these markers was observed in rhombomere-sized domains
in the posterior CNS (Fig.
3K-M). These embryos expressed krx20 in three definitive
stripes (Fig. 3I-N),
contrasting with the more loosely organized expression seen in the posterior
of cdx1a/cdx4-deficient embryos without BMS493 treatment
(Fig. 2). Within the second and
third krx20 stripes, we observed broad hoxb3a expression
(r5/r6 marker, Fig. 3L) and,
nested within it, smaller hoxd3a (r6/7 marker,
Fig. 3K) and hoxd4a
(r7 marker, Fig. 3J) expression
domains. The r4 marker hoxb1a was expressed in two rhombomere-like
domains, between the first and second stripe of krx20 expression and
posterior to the third krx20-positive domain
(Fig. 3M). In addition, we
examined the expression of the rhombomere boundary markers rfng and
foxb1.2 (Cheng et al.,
2004; Odenthal and
Nusslein-Volhard, 1988) and found that
cdx1a/cdx4/RA-deficient embryos had nine evenly spaced, boundary-like
stripes (Fig. 3O,P), instead of
the six seen in wild-type embryos (Fig.
2F and data not shown). Together, these results show that upon
RA-pathway inhibition, cdx1a/cdx4-deficient embryos can develop three
supernumerary rhombomeres in the posterior CNS in addition to the normal
seven. These supernumerary rhombomeres express Hox identity genes in a reverse
anterior-posterior orientation in what seems a mirror-image duplication of the
hindbrain, as follows: r2, r3, r4, r5, r6, r7, r6, r5, r4
(Fig. 3I-L, summarized in
Fig. 4).
|
;
Begemann and Meyer, 2001;
Begemann et al., 2001). In
support of this, close re-examination of hindbrain-specific gene expression in
cdx1a/cdx4-deficient embryos not treated with BMS493 showed that
these embryos had signs of incipient mirror-image duplication in the CNS,
despite having normal expression levels of genes involved in the synthesis,
reception and degradation of RA (see Fig. S2 in the supplementary material).
For example, the expanded hoxd4a-positive r7/8 territory was flanked
by the r5/6 marker val, which in turn was bordered by the r4-marker
hoxb1a (Fig. 2H-J,
summarized in Fig. 4).
Together, these results suggest that the absence of Cdx1a and Cdx4 functions
allows the posterior CNS to adopt a segmented pattern of development with the
potential to develop supernumerary, hindbrain rhombomere-like fates.
|
; Muhr et al.,
1999; Muhr et al.,
1997; Nordstrom et al.,
2002; Nordstrom et al.,
2006), raising the question of whether Cdx function is required in
the CNS, paraxial mesoderm, or in both tissues, to specify spinal cord. We
transplanted cdx1a/cdx4-deficient, fluorescein-labeled cells into the
CNS of wild-type host embryos and analyzed the expression of the r5/6 marker
val (n=8; Fig.
5A-E), as this gene is ectopically expressed in the posterior CNS
of cdx1a/cdx4-deficient but not wild-type embryos
(Fig. 2D,J). As shown in
Fig. 5, transplanted
cdx1a/cdx4-deficient cells were typically found populating the entire
length of the CNS by the 20-somite stage. In rostral regions, transplanted
cdx1a/cdx4-deficient as well as wild-type host cells located in the
native r5/6 territory expressed val
(Fig. 5C). In the posterior
CNS, only the transplanted cdx1a/cdx4-deficient cells, but not
surrounding wild-type cells, expressed val, even when in isolation
(n=8; Fig. 5D,E,
arrowheads). In reciprocal experiments, transplantation of wild-type cells
into the paraxial mesoderm of a cdx1a/cdx4-deficient host failed to
prevent val expression within the posterior CNS (n=2;
Fig. 5F-H). Taken together,
these transplant experiments indicate that Cdx function is required
autonomously within neural ectoderm for correct spinal cord fate
specification. We also transplanted wild-type cells into the CNS of cdx1a/cdx4-deficient embryos (n=5; Fig. 5G,I,J). Wild-type cells were able to contribute to the entire length of the CNS. In rostral regions, wild-type and cdx1a/cdx4-deficient cells were evenly distributed within the tissue, with transplanted cells expressing val only when located in the native r5/6 territory (Fig. 5I). Interestingly, in posterior regions, wild-type cells were found segregating from host cdx1a/cdx4-deficient cells and did not express val (black arrowhead). However, in the posterior CNS, isolated wild-type cells surrounded by cdx1a/cdx4-deficient cells were occasionally seen expressing val (Fig. 5J, white arrowheads). Together, these results suggest that the expression of the hindbrain-specific val gene can be controlled by the level of Cdx function within the CNS.
cdx4 overexpression in the hindbrain induces spinal cord development
Our experiments indicate that Cdx activity is required for the caudal
neural plate to develop as spinal cord instead of as segmented hindbrain. This
hypothesis predicts that cdx4 overexpression in the hindbrain should:
(1) interfere with the segmentation of this region; (2) change hindbrain
neuronal identities; and (3) induce spinal cord neuronal markers.
cdx4 overexpression by mRNA injection at the one-cell stage causes
severe gastrulation defects (Davidson et
al., 2003) (data not shown). To overcome this limitation, we
generated a transgenic fish line, Tg[hsp70:cdx4], carrying a
5'-end modified zebrafish cdx4 gene under the control of the
heat-inducible hsp70 promoter, which enables the rapid and ubiquitous
induction of transgene expression at any point during development by
incubating the embryos for 1 hour at 37°C
(Halloran et al., 2000) (see
Fig. S3A,B in the supplementary material and data not shown).
To study the effects of cdx4 overexpression in rhombomere
formation, one-cell stage Tg[hsp70:cdx4] embryos were injected with
gap43-RFP mRNA, a membrane-tagged red fluorescent protein, to follow
the formation of rhombomere boundaries after heat shocking the embryos at the
three-somite stage. This labeling method has been used to reveal the
rhombomere boundaries before they become morphologically distinct
(Moens et al., 1998). In
transgenic heat-shocked and control embryos, boundary formation initiated at
the six-somite stage (data not shown) and visible boundaries were apparent by
the 14-somite stage, although less well defined in embryos carrying the
cdx4 transgene (Fig.
6A,B). By the 20-somite stage, however, the characteristic
rhombomere bulges seen in wild-type embryos were not present in their
transgenic siblings (Fig.
6C,D). At this stage, heat-shocked embryos also showed loss of
rfng and foxb1.2 expression at the rhombomere boundaries,
and downregulation of the cell adhesion molecule-encoding epha4a gene
in r1, r3 and r5 (Fig. 6E-H).
Less severe defects were obtained when the transgene was induced at other
developmental stages [from 75% epiboly to the ten-somite stage, 8-14 hours
post-fertilization (hpf), data not shown]. These results show that Cdx4
interferes with rhombomere cell sorting and boundary formation.
We then examined the effects of cdx4 overexpression on hindbrain neuronal populations and patterning. cdx4 overexpression caused the anterior expansion of r7/8 neuronal populations such as the vagal motor neurons (nX) and the T reticular interneurons (Fig. 6I-L). Furthermore, rostral neuronal populations, including the trigeminal (nV in r2) and facial (nVII in r4) motor neurons as well as the MiD2 (r5), MiD3 (r6) and in some cases Mauthner (r4) reticulospinal neurons, were lost in these embryos (Fig. 6I-L and data not shown). The posteriorization of hindbrain neuronal populations was also reflected at the level of Hox gene expression. Although cdx4 overexpression had no effect on Hox genes normally expressed in r2-4 (hoxa2b in r2/r3, hoxb1a in r4, hoxb2a in r3/r4; Fig. 6M,N and data not shown), it induced generalized hoxb3a, hoxb5a, hoxb8a and hoxb10a hindbrain expression (Fig. 6O,P and data not shown). Despite the variability in response of different rhombomeres to cdx4 overexpression, these results corroborate that Cdx4 has the ability to posteriorize the hindbrain.
|
), after cdx4 overexpression. We found that this gene
was ectopically expressed in the hindbrain of heat-shocked transgenic but not
wild-type embryos (Fig. 6Q,R),
suggesting that Cdx4 can divert the development of hindbrain cells to a spinal
cord fate. Taken together, these results suggest that Cdx factors normally
promote spinal cord development by inducing and later patterning neuronal cell
types specific to this region, and by interfering with molecular pathways
leading to hindbrain patterning and segmentation.
Hox-independent specification of spinal cord fates by Cdx factors
Hindbrain expansion in cdx1a/cdx4-deficient embryos was
accompanied by the loss of posterior Hox gene expression
(Fig. 2). Since posterior Hox
genes are under direct Cdx regulation
(Charite et al., 1998) and
their activity is required to override anterior Hox function in caudal regions
of the embryo (Duboule, 1991),
it was important to know whether this hindbrain expansion was due to the lack
of posterior Hox activity or more directly to the loss of Cdx function itself.
We overexpressed the posterior Hox genes hoxc6a, hoxb8a and
hoxa9a by injecting 25 pg of the respective mRNAs into
cdx1a/cdx4-deficient isl1-GFP transgenic embryos, and
examined the distribution of GFP-positive branchiomotor neurons at 50 hpf
(Fig. 7A-C). Previously, it was
shown that posterior Hox gene overexpression rescues the loss of the red blood
cells observed in cdx1a/cdx4-deficient embryos
(Davidson and Zon, 2006).
Although we were able to observe the rescue of the red blood cell marker
gata1 in our injected embryos (see Fig. S4 in the supplementary
material), we never observed the rescue of the CNS defects seen in the
cdx1a/cdx4-deficient embryos. For example, branchiomotor
neurons were still present along the trunk of cdx1a/cdx4-deficient
embryos overexpressing these posterior Hox genes
(Fig. 7A-C). This suggests that
Cdx factors have a Hox-independent role in spinal cord specification.
We also examined the effect that posterior Hox gene overexpression had on
the mirror-image patterning of the CNS of cdx1a/cdx4-deficient
embryos (Fig. 7D-I). In the
native hindbrain, r3 and r5 krx20 expression was reduced or absent,
whereas the r5/r6 val expression domain was mostly unaffected
(Fig. 4D-I), consistent with
the ability of middle and posterior Hox genes to transform anterior
rhombomeres into posterior ones (Bruce et
al., 2001). We also found, as previously reported
(Shimizu et al., 2006), that
posterior Hox gene overexpression prevents ectopic krx20 expression
in the posterior CNS of cdx1a/cdx4-deficient embryos
(Fig. 7G-I, arrowhead).
However, when we additionally examined markers of posterior hindbrain
identities, such as val, we observed that the expression of these
genes in cdx1a/cdx4-deficient embryos was still present in both the
native hindbrain and posterior CNS after Hox gene overexpression
(Fig. 7D-F, asterisks). This
result shows that hindbrain patterning genes can be differentially affected by
Hox gene overexpression in the absence of Cdx activity.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cdx promotes spinal cord development
In zebrafish, at the beginning of gastrulation, the hindbrain and spinal
cord precursor cells are broadly distributed along the margin of the epiblast
and are not yet committed to their fate
(Woo and Fraser, 1995;
Woo and Fraser, 1998).
Commitment occurs towards the end of the gastrulation period, when hindbrain
and spinal cord cells occupy the anterior and posterior halves of the caudal
neural plate, respectively (Woo and
Fraser, 1995). This segregation and commitment of prospective
hindbrain and spinal cord cells correlates with the restriction of
cdx4 transcripts to the posterior third of the neuroectoderm from an
initial broad, ventral-to-dorsal gradient of expression at the margin of the
epiblast (see Fig. S1 in the supplementary material)
(Davidson et al., 2003;
Davidson and Zon, 2006;
Shimizu et al., 2005). In
addition to the posterior CNS expression domain, cdx4 and
cdx1a are expressed in a lateral-posterior domain of the tailbud that
contains the spinal cord precursor cells, among other lineages
(Kanki and Ho, 1997). This
nested expression explains the partially redundant function of cdx1a
and cdx4 in spinal cord specification and patterning; only the loss
of both genes causes severe tail truncations, absence of spinal cord and, as
previously shown, lack of hematopoietic stem cells
(Davidson and Zon, 2006). This
partially redundant function of Cdx1a and Cdx4 in the development of the
spinal cord is not unlike the situation described in the paraxial mesoderm of
mouse (Chawengsaksophak et al.,
2004; van den Akker et al.,
2002; van Nes et al.,
2006).
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The loss of Cdx functions in the developing nervous system leads to the formation of a larger than normal hindbrain region in which both expanded and ectopic rhombomeric identities can be found within the former spinal cord territory. We have also observed that in cdx1a/cdx4-deficient embryos, the hindbrain boundary markers rfng and foxb1.2 are ectopically expressed within the CNS in the tailbud region, and that this incipient segmentation resolves into recognizable rhombomere-like structures upon partial inhibition of RA signaling. The involvement of Cdx factors in not only spinal cord specification and anterior-posterior patterning, but perhaps also inhibition of segment formation, places these factors at an important regulatory crossroad.
Cdx repression of hindbrain development
A formal possibility is that Cdx factors might allow spinal cord
specification by repressing hindbrain-specific characteristics within the
posterior CNS; such a function would be consistent with the expression pattern
of cdx4 within the spinal cord region of the zebrafish. The creation
of a heat-inducible cdx4 transgenic line has enabled the
overexpression of a Cdx factor at the end of the hindbrain determination
period. We have shown that cdx4 overexpression affects correct
hindbrain formation. For example, the loss of rfng and
foxb1.2 expression within the hindbrain suggests that a relatively
late step in the hindbrain segmentation cascade, namely the formation of
segmentation boundaries, can be disrupted by cdx4 overexpression.
Therefore, this type of experiment suggests that by interfering with the
hindbrain segmentation program, Cdx factors might be able to direct the caudal
neural plate cells to a spinal cord fate.
By contrast, the analysis of more-upstream hindbrain segmentation pathway
components such as krx20, epha4a and hoxb3a, gave variable
results in our overexpression assays, depending on which region of the
hindbrain was analyzed. This variation can be attributed to the heterochrony
of the region, as different rhombomeres form at different times during
development and express different sets of genes
(Moens and Prince, 2002). In
these experiments, we confined our analyses to cases in which cdx4
overexpression was accomplished by heat-shock treatment administered at the
three-somite stage, the developmental stage at which cdx4
overexpression caused the most severe hindbrain abnormalities. At this time,
genes involved in hindbrain patterning and rhombomere boundary formation such
as hoxb1a, val and krx20, are already expressed in the
hindbrain (Moens et al., 1998;
Prince et al., 1998b;
Waskiewicz et al., 2002).
Under these experimental conditions, the hindbrain region expresses what
appears to be a mixed hindbrain/spinal cord identity with some aspects of
hindbrain fate, such as the formation of vagal motor neurons, now overlapping
with aspects of spinal cord fate, such as the ectopic expression of the spinal
motor neuron marker olig2. It is likely that cdx4
overexpression at different times of development will give different outcomes,
a possibility we are currently testing. Despite this caveat, we have shown
that the overexpression of cdx4 is able to interfere with both the
segmentation and specification of individual rhombomeric identities in the
zebrafish hindbrain.
Specification and patterning of the spinal cord territory by Cdx
Although our work, like that of many others (reviewed by
Deschamps and van Nes, 2005;
Lohnes, 2003), shows that Cdx
genes have roles in the establishment of Hox gene expression limits, we
further propose that the initial function of Cdx in establishing the spinal
cord field might be independent of a role in Hox gene regulation. We suggest
that Cdx factors initially function to establish the prospective spinal cord
territory by preventing the posterior-most region of the caudal neural plate
from adopting a segmental developmental program (hindbrain fate) and by
inducing or promoting the expression of spinal cord-specific gene expression.
Consistent with this hypothesis are our data showing that the overexpression
of 5' Hox genes fails to rescue the loss of the spinal cord markers seen
in cdx1a/cdx4-deficient zebrafish embryos and only causes the
posterioization of the expanded hindbrain. If Cdx functioned solely through
the control of Hox gene expression, then the general overexpression of a
posterior Hox gene would be predicted to prevent the expansion seen in
cdx1a/cdx4-deficient embryos. Since this was not the case, we propose
that separate hindbrain and spinal cord territories must be established prior
to becoming receptive to Hox gene functions. In cdx1a/cdx4-deficient
embryos, posterior hindbrain identities are still present when 5' Hox
genes are overexpressed. Our conclusion differs from that of Shimizu et al.
(Shimizu et al., 2006), who
interpreted their overexpression studies as showing that Hox genes could
prevent the ectopic expression of posterior hindbrain fates in
cdx1a/cdx4-deficient embryos. We note that Shimizu et al.
(Shimizu et al., 2006) only
utilized the r5 marker krx20; however, when we additionally evaluated
the r5/6 marker val it was clear that ectopic hindbrain fates were
still present in the Hox-overexpressing embryos
(Fig. 7). Therefore, our data
suggest that in vertebrates, Cdx might have homeotic functions independent of
those of Hox factors, similar to the function of the caudal gene in
the Drosophila adult (Moreno and
Morata, 1999). This homeotic function may act both prior to and
independent of any downstream control of Hox genes, similar to the ability of
Drosophila Caudal to repress Abd-B transcription and induce
Distal-less, brachyenteron and even skipped gene expression
during analia development (Moreno and
Morata, 1999). Further work will be required to characterize the
Hox-independent function of Cdx during spinal cord development.
Another function of Cdx factors within the nervous system is to allow the
hindbrain and spinal cord regions to differentially respond to gradients of
FGF, RA and Wnt signals in the embryo. This is illustrated by the striking
mirror image expression of ectopic hindbrain patterning genes in
cdx1a/cdx4-deficient embryos and by the failure of 5' Hox gene
overexpression to prevent this phenotype. As the caudal neural plate fails to
be specified as spinal cord in cdx1a/cdx4-deficient embryos, it
retains hindbrain characteristics and remains responsive to surrounding
signals, particularly FGF and RA, in a manner similar to the native hindbrain
region. For example, in wild-type embryos, r4-derived FGF signals are
responsible for inducing and patterning the r5 and r6 regions
(Maves et al., 2002). During
normal development, the native hindbrain territory is located far removed from
the tailbud region, which is another source of FGF signals
(Draper et al., 2003;
Griffin et al., 1995).
However, in cdx1a/cdx4-deficient embryos, the expanded hindbrain now
comes into close contact with the tailbud. As shown by Shimizu et al.
(Shimizu et al., 2006),
tailbud-derived FGF signals are able to mimic the FGF-dependent, r5- and
r6-inducing activity of r4. This signaling activity, coupled with paraxial
mesoderm-derived RA signals, is responsible for the induction of ectopic r4,
r5, r6 and r7/8 identities in the trunk region of the embryo (summarized in
Fig. 4). Because the gradients
of FGF from the tailbud region of the embryo are reversed relative to the FGF
gradients found in the native hindbrain region, the pattern of ectopic
rhombomeric identities is likewise reversed within the trunk region.
Therefore, the mirror-image pattern of rhombomeric identities seen in the
trunk region of cdx1a/cdx4-deficient embryos could be produced by the
normal responses of hindbrain tissues to the same types of signals that they
would be exposed to in the hindbrain's native location. This is further
supported by transplantation experiments, in which individual
cdx1a/cdx4-deficient cells in the spinal cord region of a wild-type
host responded to FGF and RA factors as if they were located in the hindbrain.
Therefore, we propose that the functions of the cdx1a and
cdx4 genes in the nervous system of the zebrafish are to inhibit the
hindbrain developmental program by preventing the tissue from inappropriately
segmenting and taking on inappropriate anterior-posterior identities.
Evolutionary implications of Cdx function in spinal cord development
Based on patterns of expression and functional similarities across species,
we propose that the control of hindbrain and spinal cord development by Cdx
transcription factors might be common to all vertebrates. Remarkably, the
rostral limit of the most anteriorly expressed Cdx gene coincides with the
position of the hindbrain-spinal cord transition in zebrafish,
Xenopus, chick and mouse
(Cambronero and Puelles, 2000;
Frumkin et al., 1993;
Lohnes, 2003;
Marom et al., 1997;
Meyer and Gruss, 1993;
Pillemer et al., 1998;
Reece-Hoyes et al., 2002)
(this work); Cdx factors thus define the prospective spinal cord territory in
the caudal neural plate and, by exclusion, the region that will give rise to
hindbrain. Careful re-examination of Cdx function in chordate neural tube
patterning might prove useful in addressing the underlying developmental
mechanisms and evolutionary origin of the vertebrate spinal cord.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/11/2147/DC1
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ACKNOWLEDGMENTS |
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