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First published online 2 April 2008
doi: 10.1242/dev.016576
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1 Department of Biological Sciences, Vanderbilt University, VU Station B, Box
35-1634, Nashville, TN 37235, USA.
2 Department of Embryology, Carnegie Institution of Washington, 3520 San Martin
Drive, Baltimore, MD 21218, USA.
* Author for correspondence (e-mail: josh.gamse{at}vanderbilt.edu)
Accepted 10 March 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Left-right asymmetry, Pineal complex, Diencephalon, Epithalamus, Zebrafish, from beyond mutant
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Borg et al., 1983). In adult
fish, the pineal organ is attached to the brain via a stalk, which emerges
just to the left of the midline (Liang et
al., 2000). The parapineal organ is situated within the brain to
the left of the pineal stalk (Gamse et
al., 2002). Adjacent to the pineal complex are the bilateral
habenular nuclei (habenulae). Along with the medial forebrain bundle, the
habenulae and their efferent connections are a principal pathway connecting
the limbic forebrain with the midbrain. Habenular axons travel via the
fasciculi retroflexus (FR) to the interpeduncular nucleus (IPN) of the
midbrain; this habenulo-interpeduncular connection is highly conserved
throughout the vertebrate lineage
(Sutherland, 1982). In
teleosts, including zebrafish, axons from the left versus right habenula
innervate different dorsoventral regions of the IPN, in contrast to other
vertebrates, in which the two habenulae exhibit similar connections to the IPN
(Kuan et al., 2007a).
In zebrafish, the asymmetric location of the parapineal organ imposes
laterality on the flanking habenulae, which exhibit L-R differences in their
neuropil density (Concha et al.,
2000) and in the size and expression of genes such as
leftover (lov; also known as kctd12.1 - ZFIN)
(Gamse et al., 2003). In
mutants in which the parapineal is on the right side of the brain, structural
and gene expression differences in the habenulae are also L-R reversed
(Gamse et al., 2003;
Concha et al., 2000).
Selective destruction of the parapineal by laser ablation reveals its
instructive role on the left habenula. In the absence of the parapineal, both
habenulae develop with equivalent size, patterns of gene expression, and
neuropil density characteristic of the right habenula
(Gamse et al., 2003;
Concha et al., 2003). This
abnormal development of the habenulae also affects efferent projections to the
IPN. In wild-type (WT) embryos, the left habenular neurons project to both the
dorsal and ventral regions of the IPN
(Gamse et al., 2005;
Aizawa et al., 2005). However,
in parapineal-ablated larvae, left habenular neurons no longer project
dorsally, but instead innervate the ventral IPN, characteristic of right
habenular neurons (Gamse et al.,
2005). Therefore, the development of the parapineal is important
for asymmetry of the dorsal diencephalon as well as for the formation of
diencephalic connectivity to other brain regions.
The parapineal organ is morphologically distinguishable between 28 and 32
hours post-fertilization (hpf) as a cluster of cells adjacent to the anterior
left edge of the pineal anlage (Gamse et
al., 2003; Concha et al.,
2003). Studies of fixed samples suggest that over the course of
the next 3 days, the parapineal becomes located more posteriorly and ventrally
relative to the pineal organ (Gamse et
al., 2002). A single parapineal organ is detected in 95% of WT
zebrafish larvae on the left side of the brain, and only very rarely
(<0.3%) are bilateral or no parapineal organs observed
(Gamse et al., 2003;
Concha et al., 2000).
Precursors of the parapineal and pineal organ are thought to derive from a
common pool of cells, as shown by lineage labeling of the pineal complex
anlage at 22-24 hpf (Concha et al.,
2003). The genes that regulate how parapineal cells are specified
from this precursor pool, and that are responsible for their ultimate
left-sided localization, are unknown.
To identify genes involved in asymmetric development of the epithalamus, we performed a genetic screen for mutations that disrupt L-R differences of lov expression in the habenulae. Here, we report the isolation and characterization of the from beyond (fby) mutation that produces homozygous embryos with reduced lov expression in the left habenula. We identify fby as a lesion in the T-box2b (tbx2b) gene that disrupts parapineal development. Time-lapse analysis reveals details of the morphogenetic process, showing that parapineal cells migrate leftward away from the pineal complex anlage as a cluster. By contrast, the parapineal cells of fby mutants are fewer in number and are scattered near the midline of the brain. We propose that Tbx2b assigns cells to the parapineal lineage and regulates their asymmetric migration.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and
WIK (Rauch et al., 1997), the
transgenic lines Tg(foxd3:GFP)fkg17 and
Tg(foxd3:GFP)fkg3
(Gilmour et al., 2002), the
ethylnitrosourea-induced mutations knypekm119 and
trilobitem747
(Solnica-Krezel et al., 1996),
and fbyc144 were used.
Mutagenesis and screening
ENU mutagenesis was performed by placing male zebrafish in 3 mM ENU for
three 1-hour treatments with 48 hours between treatments. F1 fish were
generated by crossing ENU-mutagenized males to AB females. F2 fish were
generated by intercrossing F1 males and females. F3 larvae were generated from
F2 intercrosses and at 4 dpf were fixed for in situ hybridization with the
lov probe. Putative mutations showing reversed or bilaterally
symmetric expression of lov in the habenular nuclei were
re-identified in F2 heterozygotes and lines established through outcrosses to
AB fish.
Cloning of fby
Heterozygous fish (AB background) carrying the fbyc144
mutation were crossed with wild-type WIK fish. F2 mutant larvae were used for
meiotic mapping with simple sequence length polymorphism (SSLP) markers, as
previously described (Bahary et al.,
2004). For identification of the lesion in the tbx2b
gene, cDNA was prepared using Superscript II reverse transcriptase
(Invitrogen) from mRNA isolated from 4-day-old AB and fby mutant
larvae using Trizol (Invitrogen). For sequencing, cDNA was amplified using
primers that flanked the tbx2b open reading frame and Pfu DNA
polymerase, and subcloned into the pCRII-Topo vector (Invitrogen). Clones were
sequenced using an ABI 3730xl sequencer and sequence data were analyzed with
Sequencher software (GeneTools).
Genotyping fby mutants
To genotype larvae, DNA was extracted by boiling larvae for 10 minutes in
25 µl of Embryo Lysis Buffer (Bahary et
al., 2004). Proteinase K (Roche Applied Bioscience) was added to a
final concentration of 0.5 mg/ml and samples were incubated at 55°C for 2
hours, then boiled for 10 minutes to deactivate the proteinase K. The
fby mutation introduces an MseI restriction site at bp 134
of exon 3. Primers flanking this site were used to amplify genomic DNA
(forward primer, 5'-TGTGACGAGCACTAATGTCTTCCTC-3'; reverse primer,
5'-GCAAAAAGCATCGCAGAACG-3'). The PCR product was digested with
MseI (NEB) at 37°C for 1 hour, then run on a 3% agarose gel.
Morpholino injections
Antisense morpholino (MO) sequences for tbx2b were derived from
the translation initiation site and the splice donor site of the third exon as
described [Tbx2b-ATG and Tbx2b-SP (Gross
and Dowling, 2005)]. The MO stock solution (10 mg/ml) was diluted
to 6 ng/nl in distilled H2O, and embryos (1- to 2-cell stage) were
pressure-injected with 
1 nl to deliver 6 ng of MO per embryo. Embryos
from the transgenic line Tg(foxd3:GFP)fkg17 were used so
that the location of the parapineal could be readily assessed by fluorescence
in live larvae at 3-4 dpf.
RNA in situ hybridization
Whole-mount RNA in situ hybridization was performed as described previously
(Gamse et al., 2003), using
reagents from Roche Applied Bioscience. RNA probes were labeled using
fluorescein-UTP or digoxygenin-UTP. To synthesize antisense RNA probes,
pBS-otx5 (Gamse et al.,
2002), znot (flh)
(Talbot et al., 1995), pBS II
SK-znr1 (cyc) (Sampath
et al., 1998), and pBK-CMV-leftover
(Gamse et al., 2003) were
linearized with EcoRI and transcribed with T7 RNA polymerase;
pCRII-tbx2b, zPitx2 (Tsukui et
al., 1999) and pBK-CMV-right on
(Gamse et al., 2005) with
BamHI and T7 RNA polymerase; pCRII-dexter
(Gamse et al., 2005) with
XhoI and SP6 RNA polymerase; pBS II SK-ATV (lft1)
(Thisse and Thisse, 1999) with
NotI and T7 RNA polymerase; pBSK+ southpaw
(Long et al., 2003) with
SpeI and T7 polymerase; pGEM-fzd7a
(El-Messaoudi and Renucci,
2001) with ApaI and SP6 polymerase; pBS II
SK-wnt11 (Makita et al.,
1998) with HindIII and T3 polymerase; and
pBS-gfi1 (Dufourcq et al.,
2004) with SacII and T3 RNA polymerase. Embryos were
incubated at 70°C with probe and hybridization solution containing 50%
formamide (60% for cyc). Hybridized probes were detected using
alkaline phosphatase-conjugated antibodies and visualized by 4-nitro blue
tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) staining for
single labeling, or NBT/BCIP followed by iodonitrotetrazolium (INT) and BCIP
staining for double labeling. For sagittal sections, larvae were embedded in
OCT (Sakura) and sectioned on a Leica cryostat. All in situ data were
collected on a Leica DM6000B microscope with a 5x or 20x
objective.
Immunofluorescence
For whole-mount immunohistochemistry with rabbit or mouse antibodies, 4-dpf
larvae were fixed overnight in 4% paraformaldehyde and stored at -20°C in
methanol. Samples were permeabilized by treatment with 10 µg/ml proteinase
K, refixed in 4% paraformaldehyde, and blocked in PBS with 0.1% Triton X-100,
10% sheep serum, 1% DMSO, 1% BSA (PBSTrS). For antibody labeling, rabbit
anti-Lov or anti-Ron (1:500) (Gamse et
al., 2005), rabbit anti-GFP (1:1000, Torrey Pines Biolabs), mouse
anti-opsin (1:500) (Adamus et al.,
1991) or mouse anti-Zpr1 (1:250, Zebrafish International Resource
Center, Eugene, OR) were used. Larvae were incubated overnight in primary
antibody diluted in PBSTrS. Primary antibody was detected using goat
anti-rabbit or goat anti-mouse secondary antibodies conjugated to the Alexa
568 or Alexa 488 fluorophores (1:350, Molecular Probes).
To simultaneously detect Lov and Ron, chicken anti-Lov polyclonal antibody
was generated. A bacterially expressed C-terminal fragment of Lov was
generated and purified as described previously
(Gamse et al., 2005), injected
into hens, and total IgY was isolated from egg yolks (Aves Labs). For Lov/Ron
double labeling, larvae were fixed in 100% Prefer fixative (Anatech) overnight
and stored in PBS containing 0.1% Tween 20 for up to 2 weeks. Samples were
permeabilized, refixed and blocked as above, then incubated overnight with
chicken anti-Lov (1:500 dilution of total IgY) and rabbit anti-Ron (1:500)
antibodies simultaneously in PBSTrS, and primary antibodies detected with goat
anti-chicken:Alexa 568 and goat anti-rabbit:Alexa 488 (1:350, Molecular
Probes). All immunofluorescence data were collected on a Zeiss/Perkin Elmer
spinning disk confocal microscope with a 40x oil-immersion objective and
analyzed with Volocity software (Improvision).
Time-lapse imaging
For time-lapse imaging, wild-type or fby mutant embryos carrying
the transgene (foxd3:GFP)fkg3 were either uninjected or
injected with a 6 ng/nl solution of tbx2b splice MO. Embryos were
mounted in a 0.8% solution of low-melting-point agarose containing 0.003%
1-phenyl-2-thiourea (PTU) and anesthetized with 0.04% Tricaine. Images were
collected on a Zeiss/Perkin Elmer spinning disk confocal microscope with a
40x oil-immersion objective every 15 minutes from 24-48 hpf and analyzed
using Volocity software.
Cell ablation
Ablation of a subset of parapineal cells was performed using
Tg(flh:GFP)c161 or Tg(foxd3:GFP)fkg3
31-hpf embryos, mounted dorsal-side-up in 0.8% agarose in 35 mm Petri dishes.
Parapineal cells were visualized by GFP fluorescence and 5 cells were
ablated by 5-10 pulses/cell from a 440 nm laser beam (Photonic Instruments)
focused through a 40x water-immersion objective mounted on a Leica
DM6000B microscope. An equivalent number of cells contralateral to the
parapineal were ablated in the controls.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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)
(Fig. 1A). The from
beyond (fby) mutation reproducibly caused reduced expression of
lov in the left habenula (Fig.
1B). In WT larvae, the left habenula is larger and contains more
neuropil than the right (Gamse et al.,
2003; Concha et al.,
2000), whereas in fby mutants the size and neuropil
content of the left habenula was similar to the right (data not shown).
Intercrosses between carriers from the original F2 family revealed that
fby segregates as a simple mendelian recessive mutation (104/438,
24%).
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2 weeks
of age. Rarely, mutants survived to adulthood (4/101, 4%). A morphological
phenotype of reduced otic vesicle size was also detected, with 30% of mutant
larvae showing fused otoliths in one or both otic vesicles at 4 dpf, and 100%
by 7 dpf (data not shown).
Reduced lov expression in the left habenula was associated with
other alterations in the usual L-R asymmetric pattern of the habenulae. In WT
larvae, a smaller area of the left than of the right habenula expresses the
KCTD genes right on (ron; also known as kctd12.2 -
ZFIN) and dexter (dex; kctd8 - ZFIN)
(Gamse et al., 2005)
(Fig. 1C,E). By contrast, the
left habenula of fby mutant larvae had an expanded region of
ron- and dex-expressing cells similar to the right habenula
(Fig. 1D,F).
In addition to asymmetric gene expression, the left and right habenulae
differ in their axonal projections to the IPN
(Kuan et al., 2007b;
Gamse et al., 2005;
Aizawa et al., 2005). In WT
larvae, Lov+ axons project to the dorsal and ventral regions of the
IPN, whereas Ron+ axons project to the ventral IPN only
(Gamse et al., 2005)
(Fig. 1G,I-K). In fby
mutant larvae, the dorsal/ventral (D/V) pattern of L-R habenula projections
was altered. Fewer Lov+ axons projected dorsally and more projected
ventrally (Fig. 1H,L), and an
increased number of Ron+ axons were detected ventrally
(Fig. 1M,N). These data are
consistent with left habenular efferents adopting a projection pattern
characteristic of the right habenula.
The abnormal laterality in the brains of fby mutants prompted an
examination of asymmetric Nodal signaling, a key early determinant of L-R axis
formation (Shen, 2007). In WT
embryos, two Nodal genes, southpaw (spaw) and
cyclops (cyc; also known as ndr2 - ZFIN), are
expressed in the zebrafish left lateral plate mesoderm (LPM), as are the
Nodal-induced genes lefty1 (lft1) and pitx2
(Sampath et al., 1998;
Rebagliati et al., 1998b;
Rebagliati et al., 1998a;
Long et al., 2003;
Liang et al., 2000;
Concha et al., 2000;
Bisgrove et al., 2000) (see
Fig. 2A,B). cyc, lft1
and pitx2 were also expressed in the left side of the epithalamus
(Fig. 2C-E). In fby
mutants, expression of spaw, cyc, lft1 and pitx2 was
indistinguishable from that in WT siblings in both the left LPM and left
epithalamus (Fig. 2F-J). The
right-sided position of the pancreas and rightward looping of the heart tube,
which are influenced by Nodal signaling in the left LPM
(Yan et al., 1999), were also
unaffected in fby mutants (data not shown).
fby mutants have fewer and mispositioned parapineal cells
Previous studies have shown that the left-sided parapineal organ is
required for the asymmetric identity of the habenulae, and therefore for L-R
habenular neurons to adopt D/V differences in their projection to the IPN
(Gamse et al., 2003;
Gamse et al., 2005;
Concha et al., 2003;
Aizawa et al., 2005). To
determine if the altered laterality of the habenulae in fby mutants
was due to disrupted development of the parapineal, we examined pineal complex
formation.
The pineal complex adopted an abnormal morphology in fby mutants.
In 24-hpf WT larvae, the otx5 gene is expressed throughout the pineal
complex anlage (Gamse et al.,
2002) (Fig. 3A). By
34 hpf, the otx5-expressing parapineal is revealed as a
morphologically distinct group of cells emerging from the left anterior border
of the pineal complex anlage (Gamse et
al., 2002) (Fig.
3C). By 4 dpf, parapineal cells are found in a more posterior and
ventral position adjacent to the left habenula
(Gamse et al., 2003;
Concha et al., 2003)
(Fig. 3E,G). In fby
mutants, the pineal complex anlage appeared similar to WT at 24 hpf
(Fig. 3B), but by 34 hpf a
distinct parapineal organ was not found
(Fig. 3D). At 4 dpf, individual
otx5-expressing cells were ectopically located below the pineal organ
(Fig. 3F,H).
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) (see Fig.
4A,C and Table 1)
and are never detected in the pineal. We also examined pineal-specific
markers, in the context of the transgenic line
Tg(foxd3:GFP)fkg17 that labels both pineal and parapineal
cells with GFP (Gilmour et al.,
2002; Concha et al.,
2003) (see Fig. S1 in the supplementary material). The entire
pineal complex of WT larvae consisted of 60±10 GFP-positive cells
(n=13, Table 1).
Red-green cone cells labeled by the Zpr1 antibody were found in the pineal but
were absent from the parapineal (Fig.
4E). Almost half of the GFP-positive pineal cells were identified
in confocal sections as red-green cone cells (44%, n=13,
Table 1). Rod cells in the
dorsal midline of the pineal organ were labeled by the anti-rhodopsin antibody
(Concha et al., 2000).
Parapineal cells were negative for rhodopsin expression
(Fig. 4G,I,
Table 1).
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Migration of a group of cells, such as the lateral line primordium,
requires polarization of the cell cluster (reviewed by
Ghysen and Dambly-Chaudiere,
2007). The failure of parapineal migration in fby mutants
could therefore be a secondary consequence of specifying too few parapineal
cells to form a polarized group. To test whether parapineal migration and
cohesion depend on cell number, 5 cells were ablated at 31 hpf, prior to
their movement away from the pineal complex anlage, leaving a number of cells
comparable to fby mutants (Fig.
5A-F). The placement of the remaining parapineal cells was assayed
at 55 hpf, when they have moved leftward, as well as posteriorly and
ventrally, and begin to express gfi1. We found that the remaining
parapineal cells formed a coherent and correctly positioned, albeit smaller,
parapineal organ, similar to controls (Fig.
5G-L).
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) revealed two genes, tbx2b and tbx4, in
this region. The tbx2b gene was a likely candidate based on previous
reports of expression in the pineal anlage
(Ruvinsky et al., 2000;
Dheen et al., 1999) and the
observation that antisense morpholino (MO)-treated embryos developed abnormal
otic vesicles similar to those seen in fby mutants
(Gross and Dowling, 2005).
Sequencing of tbx2b cDNA from fby mutants at 4 dpf revealed
a T-to-A transversion introducing a premature stop codon
(Fig. 6B). This lesion lies
within the T-box sequence and is predicted to prevent translation of 9 of the
20 DNA-contacting residues (Sinha et al.,
2000). Two different MOs that blocked either tbx2b
translation or removal of the third intron
(Gross and Dowling, 2005)
effectively phenocopied the fby mutation
(Fig. 6C,D). Microinjection of
tbx2b MOs into fby mutants did not cause a further reduction
in the number of gfi1-expressing cells (data not shown), supporting
the premise that the fby mutation eliminates tbx2b
function.
tbx2b is expressed early in the pineal complex anlage
Expression of tbx2b in the cells that give rise to the medially
located pineal complex anlage was present at the six-somite stage, in two
clusters of cells at each lateral edge of the neural plate
(Fig. 7A). By the ten-somite
stage, when the lateral edges of the neural plate meet to form the neural rod,
a single dorsal midline domain of tbx2b expression was detected
(Fig. 7B)
(Ruvinsky et al., 2000). At 24
hpf, expression of tbx2b was found in many cells of the pineal
complex anlage, and was most intense in the midline
(Ruvinsky et al., 2000;
Dheen et al., 1999;
Cau and Wilson, 2003)
(Fig. 7C). At 4 dpf,
tbx2b expression was maintained in the parapineal organ and the
pineal stalk (Fig. 7D). In
fby mutants, tbx2b RNA expression was still detected in the
pineal complex anlage at a similar level to that in WT
(Fig. 7E,F), indicating that
the fby mutation did not cause nonsense-mediated decay.
The tbx2b expression pattern resembled that of flh, a
homeobox transcription factor also expressed in the pineal complex anlage
(Masai et al., 1997), although
the two expression patterns did not completely overlap. During somitogenesis,
many flh-positive cells expressed tbx2b
(Fig. 7G). However, some medial
cells of the pineal complex anlage expressed tbx2b only
(Fig. 7H). By 24 hpf, the
pineal complex anlage expressed both flh and tbx2b, except
at its most lateral edges, where only flh expression was detected
(Fig. 7I,J).
|
), and that in the developing mouse hindbrain, Tbx2
regulates expression of Wnt planar cell polarity (PCP) genes including
Fzd7 (Song et al.,
2006). However, we find that disrupting the Wnt PCP signaling
pathway, by mutation of the proteoglycan knypek (glypican)
(Topczewski et al., 2001) or
van gogh-like 2 (strabismus; also known as
trilobite in zebrafish) (Jessen
et al., 2002), has no effect on tbx2b expression,
parapineal specification or migration (see Fig. S2A-I in the supplementary
material). In addition, neither fzd7a nor other PCP genes (wnt11,
wnt5, fzd7b, trilobite) are expressed in the epithalamus (see Fig. S2J-Q
in the supplementary material; data not shown).
Migration of parapineal cells is disrupted in fby mutants
In fby/tbx2b mutants, the absence of a distinct left-sided
parapineal organ and the development of parapineal cells in an ectopic ventral
location suggested that parapineal cells failed to move to the left side of
the brain. Although studies have inferred that parapineal precursors migrate
out from the anlage to the left side of the brain
(Gamse et al., 2003;
Concha et al., 2003), these
cellular movements have not been directly documented in live embryos. We
performed time-lapse analysis using foxd3:gfp transgenic embryos,
recording cell locations between 24 and 48 hpf (see Movie 1 in the
supplementary material). One or two highly protrusive cells extending multiple
short and wide filipodia were detected at the anterior left edge of the pineal
complex anlage at 31 hpf (Fig.
8A). Over the next 8 hours, these cells migrated in a leftward and
slightly caudal direction (Fig.
8D,G). They were very closely followed by more cells that
originated from the same anterior left region of the pineal complex anlage
(Fig. 8J). By 48 hpf, the cells
stopped moving, they coalesced into a tightly packed cluster and extended
highly dynamic, thin processes (Fig.
8M,P).
In contrast to the robust migration of parapineal cells to the left side of the brain, leftward migrating cells were not observed in tbx2b MO-treated or fby homozygous mutant embryos (see Movies 2, 3 in the supplementary material). At 33 hpf, one or two highly protrusive cells were seen at the anterior end of the pineal complex anlage (Fig. 8B,C). They were joined by an additional two or three highly protrusive cells over the next 9 hours (Fig. 8E,F,H,I,K,L). However, these cells did not migrate to the left of the pineal, but remained close to the midline near the anterior end of the pineal complex anlage. Although in an ectopic location, these cells still extended long thin processes by 48 hours (Fig. 8N,O,Q,R). The time-lapse analyses indicated that tbx2b is required for the leftward migration of parapineal cells and their morphogenesis into a coherent cluster.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Expression of tbx2b in the pineal complex specifies parapineal cells
The tbx2b gene is expressed in many cells of the pineal complex
anlage, but only a dozen cells contribute to the parapineal organ. How does a
widely expressed transcription factor play a role in the development of a
limited number of parapineal cells? One likely model is that different cell
types in the pineal complex anlage are specified by different repertoires of
transcription factors that interact with tbx2b. Modulation of
enhancer-binding activity by interaction with other transcription factors
(particularly Lim, Gata and homeodomain proteins) is a common feature of Tbx
family members (Showell et al.,
2004; Naiche et al.,
2005). The tbx2b gene is co-expressed with the homeobox
gene flh in most of the pineal complex anlage at eight somites.
However, a small symmetric domain in the anterior medial region of the anlage
expresses tbx2b but not flh. We propose that cells from this
anterior medial domain will be specified as parapineal, whereas cells
expressing both flh and tbx2b will generate the pineal
organ. This model predicts that the fby mutation causes parapineal
precursors to be respecified to a pineal fate. In support of this model, we
find that in addition to reduced numbers of gfi1-expressing
parapineal cells, red-green cone cells typically restricted to the pineal are
instead situated in an ectopic position ventral to the pineal organ.
The absence of a tbx2b-exclusive domain in the pineal complex anlage of fby mutants affects parapineal specification; however, a few cells still adopt parapineal identity as assessed by gfi1 expression. The action of tbx2b in combination with additional transcription factors during parapineal and pineal specification will be directly tested by conditional misexpression in the epithalamus using tissue-specific promoters.
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;
Dheen et al., 1999).
tbx2b specifies parapineal cells without affecting Nodal signaling
In addition to expression in the pineal complex, tbx2b is
expressed in the chordoneural hinge (CNH), a structure in the embryonic
tailbud (Ruvinsky et al.,
2000; Dheen et al.,
1999). Expression of the Nodal gene southpaw in the CNH
is required for its subsequent asymmetric expression in the left LPM, where it
regulates left-sided expression of another Nodal gene, cyc, in the
LPM and epithalamus (Gourronc et al.,
2007). Loss of spaw activity results in the absence of
cyc expression in the left epithalamus
(Long et al., 2003) and in L-R
randomization of parapineal location
(Gamse et al., 2005). However,
fby mutant embryos develop with normal left-sided expression of Nodal
genes, indicating that tbx2b activity in the CNH is not required for
initiation of asymmetric spaw expression. This suggests that
tbx2b acts in the pineal complex anlage to regulate specification of
the parapineal organ in a pathway that is independent of Nodal signaling.
Parapineal cells require tbx2b activity to migrate away from the pineal complex anlage
In WT embryos, migration of parapineal precursor cells from the pineal
complex anlage begins with the emergence of a single cell from the anterior
left region, followed by other closely apposed cells. This sequence suggests a
leader-follower mechanism for directional migration, in which cells at the
leading edge coordinate the behavior of the group. Such cellular behavior has
been best described in zebrafish for the migration of the lateral line
primordium, where leader cells expressing cxcr4b at the anterior end
of the primordium organize the migration of the rest of the cells
(Haas and Gilmour, 2006). The
first cells to emerge from the pineal complex anlage may be leader cells that
direct the left-sided movement of the parapineal organ. In fby
mutants, a reduced number of cells develop with parapineal characteristics,
including gfi1 expression and anteriorward axonal projections, and
they do not migrate to the left side of the epithalamus; rather, at 48 hpf
they are found in the midline, at the anterior edge of the pineal complex. The
failure of parapineal cells to migrate in fby mutants could be due to
the reduced number of parapineal precursor cells that are specified, the
inability of mutant cells to move, or a loss of leader cell function. We
believe that the latter hypothesis is correct. First, reducing the total
number of parapineal precursor cells by laser ablation to a number similar to
that of the scattered cells in the fby mutant, still allows a
cohesive organ to form on the left side of the brain. Second, fby
mutant parapineal cells still move posteriorly and ventrally, and thus are not
immobile. Therefore, we speculate that tbx2b is required for the
specification or maintenance of leader cells. Testing this hypothesis will
require the development of methods to identify the leader cells and
specifically destroy them as they emerge from the pineal complex.
One mechanism invoked to mediate tbx2b regulation of migration is
the Wnt-PCP pathway. Although tbx2b plays a role in PCP in other
contexts (Fong et al., 2005;
Song et al., 2006), we find no
role for PCP in parapineal migration.
Delayed parapineal migration away from the midline until after 48 hpf has
been observed in masterblind (mbl; also known as
axin1) and RNA-rescued one-eyed pinhead (Roep)
mutants in a significant percentage of larvae
(Gamse et al., 2002;
Carl et al., 2007). However, by
96 hpf, the parapineal cells of mbl and Roep mutants have
achieved their correct left-sided position, whereas the parapineal cells of
fby mutants remain scattered close to the midline. The data suggest
that mbl and oep regulate the timing or speed of migration,
but are not required for leader cell formation.
An evolutionarily conserved role for Tbx genes in parapineal formation?
Fish, frog, lizard and bird embryos all develop a pineal and a pineal
accessory organ (although in birds the parapineal does not persist in the
adult), whereas mammals seem to form only the pineal gland
(Hill, 1892;
Butler and Hodos, 1996;
Braitenberg and Kemali, 1970;
Borg et al., 1983). The
development of a pineal complex that includes both a pineal and an accessory
organ is phylogenetically correlated with the expression of Tbx homeobox genes
in the pineal complex anlage. Such expression is observed for tbx2b
in zebrafish (Ruvinsky et al.,
2000; Dheen et al.,
1999) (this study), and for the closely related Tbx3 gene
in chick (Gibson-Brown et al.,
1998) and Xenopus
(Takabatake et al., 2000).
However, in the mouse, neither Tbx2 nor Tbx3 is expressed in
the developing diencephalon (Chapman et
al., 1996). We propose that the pineal accessory organ of
non-mammalian vertebrates is specified by Tbx2/3 function.
In lizards and frogs, the pineal accessory organ (known as the parietal eye
or frontal organ, respectively) is photoreceptive, expresses certain opsin
family members and is thought to transmit diurnal information
(Engbretson et al., 1981;
Eldred et al., 1980;
Su et al., 2006;
Blackshaw and Snyder, 1997). In
mammals, which do not receive direct light input to the pineal, the major
source of diurnal information is stimulation of the melanopsin-expressing
retinal ganglion cells (Hattar et al.,
2002). The acquisition of photic input to the pineal via the eyes
in mammals rendered a photoreceptive pineal accessory organ unnecessary. Loss
of Tbx2/3 expression in the pineal complex anlage might be the
mechanism that prevents formation of a pineal accessory organ in mammals, by
altered specification and disrupted migration of precursors. How a
transcription factor regulates the number and behavior of parapineal cells
will be revealed by identification of its downstream target genes.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/9/1693/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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