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First published online 24 January 2007
doi: 10.1242/dev.02780
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1 CNRS-UPR2197 DEPSN, Institut Fessard, Avenue de la Terrasse, 91198
Gif-sur-Yvette cedex, France.
2 MSNC INRA Group, Institut Fessard, Avenue de la Terrasse, 91198 Gif-sur-Yvette
cedex, France.
3 Department of Biology, University of Maryland, MD 20742, USA.
Author for correspondence (e-mail:
Sylvie.Retaux{at}iaf.cnrs-gif.fr)
Accepted 13 December 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Teleost, Telencephalon, Subpallium, Olfactory bulbs, LIM-homeodomain, Nkx, Dlx, GABA, Proliferation, Hypothalamus, Cavefish, Astyanax mexicanus
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Murakami et al., 2005;
Puelles et al., 2000;
Rubenstein et al., 1994;
Striedter, 1997;
Wullimann and Mueller, 2004),
even though the morphology and connectivity between forebrain areas vary
considerably among different vertebrate species
(Butler and Hodos, 1996).
Early in embryogenesis, the forebrain develops under the influence of
signalling centres, called `secondary organisers' in mammals, which secrete
morphogen molecules such as Wnt (Wingless-Int), Bmp (Bone morphogenetic
protein), Fgf (Fibroblast growth factor), or Shh (Sonic hedgehog)
(Marin and Rubenstein, 2001;
Ohkubo et al., 2002;
Rubenstein and Beachy, 1998;
Rubenstein et al., 1998;
Wilson and Rubenstein, 2000).
These signalling centres are located at the dorsal (Wnt, Bmp), anterior (Fgf8)
and ventral (Shh) midline of the telencephalon, respectively. Likewise,
diencephalon development is largely controlled by the floor or basal plate and
by the secondary organiser of the zona limitans intrathalamica (zli), which
both produce Shh (Kiecker and Lumsden,
2004; Vieira et al.,
2005; Zeltser,
2005). In amniotes, these midline signalling centres and
interactions between them (Ohkubo et al.,
2002; Shimogori et al.,
2004; Tole et al.,
2000) create a field of organisation in their zone of influence,
and regulate the growth, patterning and regionalisation of forebrain areas.
Thus, Shh midline signalling is well placed to have a major impact, not only
on the development but also on the evolution of the forebrain, through subtle
changes in signal intensity and/or position. For example, although the global
developmental organisation of the forebrain is highly conserved in lampreys
(jawless vertebrates) compared with gnathostomes
(Murakami et al., 2001;
Murakami et al., 2005), their
ventral telencephalon `lacks' a pallidum
(Weigle and Northcutt, 1999).
Strikingly, there is no Shh signalling at the lamprey telencephalic ventral
midline (Osorio et al., 2005),
suggesting that Shh expression in the anterior ventral midline was
responsible for the appearance of a novel forebrain subdivision, and further
that midline signalling may be a powerful motor for forebrain evolution in
jawed vertebrates.
The surface-dwelling and cave-living forms of Astyanax mexicanus
have been used as an advantageous model system in evolutionary developmental
biology (Jeffery, 2001). The
two forms of this single species split from a common ancestor about 10,000
years ago, a relatively short period of time during which the cave animals
have evolved both regressive and constructive features. These include the loss
of eyes, pigmentation and aggressive behaviour, and the increase in feeding
apparatus (jaws, teeth, taste buds) and body fat content. Among these
evolutionary changes, the loss of eyes in cavefish has drawn much attention.
Recently, Yamamoto et al. (Yamamoto et
al., 2004) have shown that eye regression is a consequence of
early overexpression of hedgehog genes (Hh, including Shh)
at the ventral midline of cavefish embryos. This expansion of midline Hh
signalling causes hyperactivation of downstream genes, lens apoptosis and eye
degeneration. Thus, a gain of function in Hh signalling is at the origin of
the regressive eye phenotype. In fact, eyes may have been lost by default as a
consequence of natural selection for constructive traits, such as feeding
structures, that are positively regulated by Hh signalling
(Jeffery, 2005).
As shown in various model species, the Hedgehog family of secreted proteins regulates pattern formation, proliferation and differentiation. Here we have taken advantage of enlarged Shh expression in the embryonic midline of cavefish relative to surface fish embryos to test the idea of the impact of the ventral midline on the development and evolution of forebrain patterning, neuronal organisation and wiring. Through the analysis of Shh downstream genes of the Nkx and LIM-hd (LIM-homeodomain) families, cell proliferation patterns and GABAergic neuron differentiation, we show that Shh overexpression has multiple and region-specific effects on neuronal development in the hypothalamus, subpallium and olfactory bulbs. Moreover, comparison between cave and surface embryos allows the identification of new developmental mechanisms for neuronal specification in the fish telencephalon.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
cDNA cloning
Total RNA from surface fish brains was reverse transcribed with random
primers using AMV reverse transcriptase (Promega). Partial cDNA sequences for
Nkx2.1b (278 bp, GenBank accession no. DQ431669), Lhx6 (408
bp, DQ431667), Lhx9 (1.2 kb, EF175738) and GAD65 (445 bp,
DQ431668) were amplified by PCR using degenerated primers (sequences on
request) designed after alignments of several teleost sequences including
zebrafish. PCR products were subcloned in TOPO-PCR II vector (Invitrogen) and
sequenced. Nkx2.1a (AY661435), Shh (AY661431) and
Pax6 (AY651762) cDNAs were previously isolated by the Jeffery
laboratory. The Lhx7 and Dlx2 plasmids were kindly provided
by David Stock.
Phylogenetic analysis
Sequences of Lhx6 and Lhx7 genes (accession numbers on
request) were aligned with Clustal W
(Thompson et al., 1994).
Phylogenetic tree (Neighbour-Joining) and bootstrap values were generated
using the MEGA3.1 program (Kumar et al.,
2004). During this work, an Astyanax Lhx6 sequence was
isolated by David Stock (DQ822512). The two Lhx6 sequences shared
100% identity in the overlapping fragment.
Whole-mount in situ hybridisation
cDNAs were amplified by PCR, and digoxygenin- or fluorescein-labeled
riboprobes were synthesised from PCR templates
(Nguyen et al., 2001). A
protocol for automated whole-mount in situ hybridisation (Intavis) was
performed (Deyts et al.,
2005). For probe penetration, embryos were treated with proteinase
K for 30 minutes at 37°C (10 µg/ml for 36 hpf, 20 µg/ml for 48 hpf,
30 µg/ml for 60 hpf, 40 µg/ml for 72 hpf). Embryos were
paraffinsectioned at 8 µm, or their brains were dissected out and mounted
in glycerol. Depending on the gene marker and the stage considered, five to 20
embryos from each population were analysed (except for cyclopamine treatments,
where n=4-5), and three to four were sectioned. Quantification of the
expansion of Nkx2.1 and Dlx2 domains was performed using ImageJ, on in toto
pictures of embryos at 24, 36 and 48 hpf coming from at least two independent
experiments.
PCNA and phosphohistone H3 immunohistochemistry
For Proliferating cell nuclear antigen (PCNA) immunohistochemistry embryos
were fixed in Clark's solution (3:1 ethanol 100: acetic acid) overnight at
4°C. They were processed as described
(Candal et al., 2005).
Quantification was performed on the same serial section in cavefish and
surface fish embryos. Classical morphometric measurement was done, using a
grid superimposed on the sections, and counting the arbitrary surface covered
by PCNA-positive cells.
For phospho-H3 immunohistochemistry, embryos were fixed in 4% PFA. A primary rabbit polyclonal anti-phosphoH3 antibody (1/100e, Euromedex) was used. Quantification in the hypothalamus was performed by counting the total number of phospho-H3 positive cells in the hypothalamus of each embryo through all serial transverse sections obtained. For control quantification in the tectum, the first five sections at tectal level were counted. Results were expressed as phospho-H3 positive cell density after measurement of the surface area of the hypothalamus or the tectum for each section using ImageJ. Statistical analysis was carried out using Student's t-test.
Cyclopamine treatment
At 15 hpf embryos were treated for 9 hours in 200 µmol/l or 20 µmol/l
cyclopamine (Sigma) in water. Control embryos were exposed to 0.1% ethanol, as
the cyclopamine stock solution was diluted in 100% ethanol. After treatment,
they were washed twice, maintained in fresh water until 48 hpf and fixed.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) have recently shown that midline Shh expression is
expanded at early embryonic stages (neural plate though 1-4 somites, or 10
hpf), and indirectly causes eye degeneration in cavefish. Here, we have
investigated the impact of Shh modification later in forebrain
embryogenesis, from 24 hpf to 1 week of development.
Shh expansion is maintained during cavefish development
First, we asked whether enlarged Shh expression in cavefish was
maintained throughout development. At 20, 36 and 48 hpf, Shh was
still more largely expressed in cavefish compared with surface fish
(Fig. 1). This expanded
expression included all anterior Shh expression domains, including
the floor plate, the zli, the hypothalamus
(Fig. 1A-H) and the subpallium
(Fig. 1G,H). These data
indicate that Shh expansion was not transient in cavefish, but rather
was maintained and even spread to new locations during late embryogenesis,
opening the possibility of multiple consequences on forebrain development.
The expression of Nkx family factors is enlarged in cavefish in a region-specific manner
As a next step, we investigated the expression of patterning and
regionalisation genes of the Nkx family. Nkx2.1 and Nkx2.2 homeodomain factors
are downstream in the Shh signalling pathway
(Briscoe et al., 1999;
Rallu et al., 2002;
Xu et al., 2005). In other
well-studied teleost species (zebrafish, medaka), there are two Nkx2.1 members
(Alunni et al., 2004;
Rohr et al., 2001). In
Astyanax, we also found that Nkx2.1a
(Yamamoto et al., 2004) is
accompanied by a duplicated gene, which we named Nkx2.1b. Consistent
with expression patterns in other fishes, Nkx2.1a was found
exclusively in the presumptive hypothalamus, encompassing almost the entire
structure (Fig. 2I). By
contrast, Nkx2.1b was expressed both in restricted nuclei of the
hypothalamus, in the subpallial telencephalon (SP, subpallium), including the
future basal ganglia and septum (see
Wullimann and Mueller, 2004;
Alunni et al., 2004), and in
the preoptic region (po; Fig.
2E-G). Finally, Nkx2.2 showed a typical `alar-basal
boundary' pattern, adjacent to and following the Shh pattern
(Fig. 2C).
There was no significant difference in Nkx2.2 or Dlx2 (subpallial marker) expression between surface and cave embryos at any stage (Fig. 2A-D). Quantification showed a non-significant +4.8% increase in Dlx2 territory in cavefish, suggesting that global pallial and subpallial patterning was identical in both populations. Nkx2.1b expression was similar in the presumptive hypothalamus of cavefish and surface fish embryos (Fig. 2E-H), but was significantly expanded in the cavefish SP and preoptic region relative to surface fish embryos (Fig. 2E-H; +35% after quantification). This result was found reproducibly from 24 to 72 hpf. To investigate whether the Nkx2.1b expansion was at the expense of the rest of the subpallium, double labelling for Dlx2 and Nkx2.1b were performed. They showed that the domain expressing only Dlx2 was unchanged, and that Nkx2.1b territory enlargement was due to an anteroventral expansion of the Nkx2.1b domain (see Fig. S1A,B in the supplementary material). Finally, between 24 and 72 hpf, Nkx2.1a expression was enlarged in the developing hypothalamus of cavefish compared with their surface counterparts (Fig. 2I,J; +38%), possibly defining a larger hypothalamic territory.
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Downstream of Shh and Nkx: Lhx6 and Lhx7 LIM-homeodomain factors
We next sought to estimate the impact of enlarged Nkx2.1a and
Nkx2.1b expression on neuronal specification processes in the
hypothalamus and basal telencephalon. In other vertebrates, Nkx2.1
governs the expression of Lhx6 and Lhx7, neuronal
specification factors of the LIM-hd family expressed in the basal forebrain
(Sussel et al., 1999;
Grigoriou et al., 1998). In
mouse, Lhx6 governs the tangential migration of GABAergic neurons
generated in the subpallium towards the pallium
(Alifragis et al., 2004;
Lavdas et al., 1999), whereas
Lhx7 governs the specification of cholinergic neurons
(Fragkouli et al., 2005;
Mori et al., 2004;
Zhao et al., 2003). The
Astyanax Lhx6 and Lhx7 genes were identified as orthologs of
Lhx6 and Lhx7 in other vertebrates, including zebrafish
(Fig. 4A), and their expression
was localised with respect to Shh expression using double-colour in
situ hybridisation (Fig. 4B,C).
In Astyanax, Lhx6 was expressed in the SP and preoptic region, and in
a complex hypothalamic pattern partly similar to that of Nkx2.1b
(Fig. 4B, see also Figs
6 and
9). Lhx7 transcripts
were similarly detected in the SP and po
(Fig. 4C, see also Figs
5 and
9), and also in the pituitary
(see Fig. 4C-E and
Fig. 5A-D). This pituitary
expression has never been reported for Lhx7 in other species
(Alunni et al., 2004;
Bachy et al., 2001;
Grigoriou et al., 1998), and
might be Astyanax-specific. Double-colour in situ hybridisation
further showed that Lhx6 and Lhx7 were expressed in the same
subpallial domain but had different expression territories in the preoptic
region, Lhx6 expression being restricted to the anterior po and
included in a larger Lhx7 domain
(Fig. 4D,E).
Lhx7: heterochrony and broader expression in the po of cavefish embryos
Lhx7 spatiotemporal expression patterns were compared between
surface fish and cavefish embryos (Fig.
5). First, we observed a shift in the onset of Lhx7
expression in the SP: whereas the cavefish basal telencephalon already
contained numerous Lhx7-expressing cells at 24 hpf, only two or three
cells were reproducibly detected in their surface fish counterparts at this
stage (Fig. 5A,B). This
heterochrony in the onset of Lhx7 in the telencephalon was rapidly
compensated, as we could not detect significant differences in SP expression
of Lhx7 in later embryos, ranging from stage 36 to 60 hpf
(Fig. 5C-H). Of note, and with
respect to the role of Lhx7 in cholinergic specification in mammals,
the Astyanax ortholog was found in a few cells of the lateral
subpallium at 48 and 60 hpf (Fig.
5G,H and inset). This region is called Vl in fish (for area
Ventralis lateralis) and contains cholinergic neurons
(Mueller et al., 2004).
Lhx7 expression at this level also appears identical in cavefish and
surface fish embryos (Fig.
5G,H). By contrast, Lhx7 was continuously expressed as a
larger domain in the po region of cavefish relative to surface fish embryos
(Fig. 5C-H). Finally,
hypothalamic and pituitary expression were identical in both populations
(Fig. 5C,D).
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Lhx6: expanded expression in the cavefish telencephalon and hypothalamus
At all studied stages from 24 to 72 hpf, Lhx6 expression domains
were significantly broader in cavefish embryos than in their surface fish
counterparts. This included the SP and po regions (shown in
Fig. 6A,B at 24 hpf), and the
hypothalamus (shown in Fig.
6C,D at 36 hpf). Moreover, starting at 48 hpf, an additional area
of Lhx6 expression was detected in the telencephalon of both
Astyanax populations (Fig.
6E-H). At 48, 60 and 72 hpf, two bilateral columns of
Lhx6-expressing cells extended from the dorsal aspect of the
Lhx6-positive SP domain (dSP, Fig.
7A,B), ending as a pack of strong Lhx6-expressing cells
located in the anterolateral part of the pallium
(Fig. 6E-H and
Fig. 7A,B). After analysis in
sections, this Lhx6 expression pattern is suggestive of cells
migrating out of the SP towards pallial areas, and may therefore represent the
fish counterpart of the Lhx6-positive,
ganglionic-eminence-originated, tangentially migrating GABAergic neurons in
mammals. Importantly, Lhx6 expression was much more intense in the
columns of cells emanating from the dorsal SP in cavefish than in surface fish
embryos (Fig. 6E-H). Moreover,
at 72 hpf, Lhx6 expression in the SP and associated bilateral columns
of putative migratory cells continued to be stronger in cavefish embryos
(Fig. 6G,H).
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). We could confirm that Lhx6 cells cross the
pallio-subpallial border, represented by the Pax6-expressing band in
the telencephalon (Fig. 7J,K),
and reach their target in a pallial region that also expresses weak levels of
Pax6 (Fig. 7J-M). To
further explore the characteristics of the columns of Lhx6-expressing
cells, we showed that at 60 hpf, when the columns are well developed, they
also expressed Nkx2.1b (Fig.
7E,F, see also Fig.
2G,H at 48 hpf). Moreover, the columns of Lhx6-positive
cells expressed Astyanax GAD65 (Glutamic acid decarboxylase),
demonstrating that they are GABAergic (Fig.
7C,D). Finally, we found that the columns ended in a pallial area
corresponding to the developing olfactory bulbs, as demonstrated both by the
positions of their termini in the forebrain and by fluorescent dye (DiI)
labelling of the olfactory bulbs through the primary olfactory projection
(Fig. 7G-I). As the
Lhx6-positive columns are more important in cavefish than in surface
fish, these data suggest that the expansion of the `ventralising' Shh
morphogen has an impact on the establishment of neuronal circuits in various
parts of the brain, including regions that are not directly in the range of
Shh signalling activity.
Forebrain patterning modifications in cavefish are due to Shh expansion
Finally, we sought to demonstrate that the modifications of forebrain
neuronal patterning in cavefish were indeed Shh-dependent. To this end, we
performed cyclopamine treatments on cavefish embryos, trying to phenocopy the
surface-type patterns by reduction of Shh signalling. Because we are studying
late events in neurodevelopment, cavefish embryos were treated with
cyclopamine between 15 and 24 hpf, and fixed at 48 hpf
(Fig. 8). After cyclopamine
treatment, Dlx2 expression was affected in the SP and thalamic
prosomere p3 but not in the hypothalamus
(Fig. 8A-D), and
Nkx2.2 expression was strongly affected
(Fig. 8E-H). The
Nkx2.1a expression domain in the hypothalamus was also reduced by
cyclopamine, phenocopying the difference between surface fish and cavefish
embryos (Fig. 8I-L). Although
Nkx2.1b expression was not strongly modified in the hypothalamus, it
was reduced in size in the po and SP of treated cavefish
(Fig. 8J-L). Finally,
Lhx6 and Lhx7 expression were affected in the telencephalon
(SP and po) of cyclopamine-treated embryos
(Fig. 8Q-X), but not in the
pituitary (Lhx7) nor in the hypothalamus (Lhx6). Of note,
the columns of Lhx6-positive cells were affected similarly to the
difference observed between cavefish and surface fish
(Fig. 8U-X). In sum,
cyclopamine treatments showed dose-dependent, gene-specific and
region-specific effects, which demonstrate the Shh-dependence of the above
described modifications in regional and neuronal patterning in cavefish
(Fig. 9).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), in the
transcriptional regulation of Shh has been changed in cavefish. Alternatively,
a global increase of an upstream regulator that would quantitatively control
Shh expression independently of spatial regulatory elements is
possible. In any case, as increased Shh signalling has been found in several
independently evolved Astyanax cavefish populations
(Yamamoto et al., 2004), it
will be extremely interesting to identify this viable regulatory mutation.
Continuous expanded Shh signalling would be expected to induce
major perturbations in brain development. However, the global patterning of
forebrain development is not modified in cavefish relative to surface fish
embryos, and only some of the specific gene cascades downstream of Shh
signalling are increased in these embryos (summarised in
Fig. 9). Hence, the expression
of Nkx2.2 is identical in surface and cavefish, and so is the
expression of Dlx2 (see also Xu
et al., 2005), reinforcing the specificity of the effects
discussed below, and suggesting that at least some natural compensatory
mechanisms must be at work in cavefish to avoid a massive perturbation in
brain development after Shh expansion. In fact, when cavefish are
treated with cyclopamine, Dlx2 and Nkx2.2 expressions are
diminished, demonstrating the functional regulatory link between Shh and Dlx2
or Nkx2.2, and unmasking a probable compensatory mechanism that has evolved in
cavefish. Moreover and globally, the cyclopamine treatments also show that the
observed effects on regionalisation and neuronal specification genes are
indeed due to increased Shh signalling.
As schematised in Fig. 9,
Nkx2.1a and Nkx2.1b expressions are enlarged in the cavefish
hypothalamus and SP or po, respectively. However, Nkx2.1b is
unchanged in the hypothalamus. This differential regulation can be viewed in
terms of subfunctionalisation of the two Nkx2.1 genes, which may constitute a
feature characteristic of teleosts. Concerning LIM-hd genes, the enlarged
expression of Lhx6 in the hypothalamus can be interpreted as a
consequence of Nkx2.1a expansion, and the enlarged expression of
Lhx7 and Lhx6 in the SP or po as a consequence of
Nkx2.1b expansion. Thus, we uncover specificity of the increased
`cascades', where each Nkx2.1 paralog has an effect on the expression
of one or the two LIM-hd paralogs, and where Shh signalling eventually
regulates the neuronal composition of the forebrain. These results fit nicely
with recent findings in mouse showing that Shh signalling during neurogenesis
maintains cortical interneuron identity through regulation of Nkx2.1
(Xu et al., 2005).
Shh and cell proliferation: a bigger hypothalamus in cavefish
Our findings suggest that the expanded Nkx2.1a and Nkx2.1b,
Lhx6 and Lhx7 expression domains in the hypothalamus and po of
cavefish embryos reflects a higher level of cell proliferation in these
regions relative to surface fish embryos, eventually resulting in enlargement
of this part of the basal forebrain. Consistent with this possibility, the
mature hypothalamus is larger in cavefish than in surface fish adults (D.
Soares and W. R. Jeffery, unpublished). The implication of Shh in the control
of proliferation in the brain (and other organs) is documented
(Britto et al., 2000;
Fu et al., 2004;
Ishibashi and McMahon, 2002;
Lewis et al., 2004;
Moshiri et al., 2005;
Wechsler-Reya and Scott,
1999). However, despite the fact that Shh controls the
proliferation of both ventral and dorsal brain structures
(Dahmane et al., 2001), we
observed a specific effect only in the hypothalamus and preoptic region. By
contrast to the case of the telencephalon, where other organising centres
secreting Bmps or Fgfs might naturally compensate for Shh
overexpression (Ohkubo et al.,
2002; Shimogori et al.,
2004; Tole et al.,
2000), the hypothalamus develops under the direct influence of the
ventral midline and alar-basal boundary and in the vicinity of the zli
diencephalic organiser, all of which secrete Shh. Thus, there is probably no
possible compensation by neighbouring centres in this part of the forebrain,
and this might explain why Shh expansion results in a bigger
hypothalamus in cavefish. Does this have any physiological and/or adaptive
consequences? Cave animals are fat and have lost aggressive behaviours
compared with surface fish (Jeffery,
2001). These traits might be related to the neuroendocrine
controls exerted by the hypothalamus. However, several nuclei of the
hypothalamus and preoptic region serve as tertiary gustatory centres
(Folgueira et al., 2003;
Lamb and Finger, 1996), and
the increase in these nuclei might correspond to the increase in taste bud
number in cavefish (Jeffery et al.,
2000). Further investigations are clearly needed to functionally
interpret this hypothalamic difference between cavefish and surface fish.
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;
Zhao et al., 2003), and mice
lacking Lhx7 show impaired spatial learning and memory
(Fragkouli et al., 2005). More
recently, Lhx7 was also involved in the control of GABAergic fate in
the basal forebrain (Manabe et al.,
2005; Bachy and Rétaux, 2006). There, Lhx7
inhibits GABAergic differentiation at early stages of embryogenesis to
preserve a pool of future cholinergic neurons, the number of which is tightly
controlled and which will undergo terminal differentiation much later in
development (Bachy and Rétaux, 2006). Thus, our observation of earlier
Lhx7 expression in cavefish embryos might simply reflect the fact
that upstream Shh and Nkx2.1b are increased and reach a
`threshold' level earlier than in surface fish to induce Lhx7
expression. Then, when the correct pool of cholinergic neurons is obtained,
progenitors stop dividing, and the expression of Lhx7 in surface fish
and cavefish becomes similar after 36 hpf. In this case, and contrary to the
case of Lhx6 regulation, the (unknown) mechanisms that control the
number of cholinergic neurons in the forebrain would naturally compensate for
the primary effects of Shh overexpression in cavefish, and would not be
expected to have any obvious consequence on SP cholinergic neuronal
patterning.
|
) and
other nonmammalian vertebrates (Marin et
al., 1997; Pombal et al.,
2001; Rodriguez-Moldes et al.,
2002). Moreover, in fish, the po projects to the olfactory bulbs
(Folgueira et al., 2004), and
these projections might represent the fish counterpart of the pathway from
basal forebrain cholinergic nuclei of the diagonal band onto the olfactory
bulbs of mammals (Shipley,
1985). Importantly, this cholinergic innervation of the bulbs
actively participates in olfactory functions, such as early olfactory learning
and discrimination (Fletcher and Wilson,
2002; Linster and Cleland,
2002; Wilson et al.,
2004). Further analysis will be needed to investigate whether
cavefish indeed have enhanced cholinergic projections to the olfactory bulbs,
and whether this has functional implications (see also below).
Lhx6: a marker for a subpallium to pallium migratory stream in fish?
We interpret the Lhx6 expression domain in Astyanax
telencephalon as a stream of migrating cells originating from the SP and
populating the pallial region forming the olfactory bulbs. These cells fulfil
criteria for migrating cells: (1) they express Nkx2.1b, a subpallial
marker, expressed in the ventricular zone of the SP; (2) their pallial region
of destination never expresses Nkx2.1 or Lhx6 in the
ventricular or subventricular zone, suggesting that they are not derived from
the pallial progenitor zone; (3) they follow a tangential trajectory in the
neuroepithelium, where they end up in a subpial, marginal zone location; and
(4) they cross the pallio-subpallial border defined by Pax6 domain. These
cells are GABAergic, as they express GAD65, and thus probably
constitute a population of interneurons. We therefore propose that
Lhx6 labels the equivalent of the mammalian rostral migratory stream
(RMS), which contributes granule and periglomerular GABAergic interneurons to
the olfactory bulbs (Lois and
Alvarez-Buylla, 1994; Luskin,
1993; Wichterle et al.,
2001). In mammals, the RMS begins to migrate during the late
embryonic and perinatal periods and continues throughout adulthood
(Wichterle et al., 2001). The
timecourse observed here in fish is similar, as the peak of Lhx6
expression in the putative migratory cells is observed in 48 hpf embryos and
continues at later stages. We did not observe at any stage
Lhx6-positive cells that could constitute the fish counterpart of
tangentially migrating GABAergic cortical interneurons
(Anderson et al., 1999;
Anderson et al., 1997;
Anderson et al., 2001;
Marin and Rubenstein, 2001;
Marin and Rubenstein, 2003).
In fish, these GABAergic pallial interneurons constitute a relatively scarce
neuronal population (Anglade et al.,
1999), and the absence of pallial Lhx6 expression,
together with the absence of Dlx gene expression in the fish pallium
(Akimenko et al., 1994;
Alunni et al., 2004;
Zerucha and Ekker, 2000)
(present data), suggest that these interneurons do not share common genetic
specification mechanisms with their mammalian counterparts. In fact, an Lhx6.1
isoform of Lhx6 in mouse was reported to be expressed both in the
cortex and the olfactory bulbs (Kimura et
al., 1999). Altogether, this may suggest that an
Lhx6-positive migratory pathway from the SP to the olfactory bulbs is
ancestral in vertebrates, and that the dorsal pallium-directed Lhx6
migrations are an innovation of mammals.
Strikingly, lampreys do not express Shh and Nkx2.1 in
their embryonic ventral forebrain
(Murakami et al., 2001;
Osorio et al., 2005), they do
not have a pallidal division in their adult subpallium
(Weigle and Northcutt, 1999),
and they do not possess GABAergic interneurons in their pallium
(Melendez-Ferro et al., 2002).
Thus, the presence of regulatory cascades controlling cell migrations and
generating diversity in neuronal patterning in the forebrain seems to be a
novelty that emerged at the transition from agnathans to fish, and for which
the impact of Shh midline signalling is crucial.
Cavefish have increased Lhx6-positive migrations to the olfactory bulbs
We have observed an increased Lhx6-positive migratory stream in
the forebrain of cavefish relative to surface fish. From an adaptive point of
view, what kind of advantage does having more olfactory bulb interneurons
confer to cavefish for their life in perpetual darkness? In the mammalian
bulbs, periglomerular GABA interneurons increase the contrast between
activation levels in different glomeruli, and augment odour discrimination
(Aungst et al., 2003;
Lledo et al., 2004), whereas
granular cells increase contrast and specificity of olfactory information by
synchronising the discharges of mitral projection neurons
(Lagier et al., 2004;
Laurent, 2002;
MacLeod and Laurent, 1996;
Saghatelyan et al., 2005). In
sum, bulbar interneurons are crucial in olfactory circuitry, and a positive
correlation between the abundance of newly generated bulbar interneurons and
olfactory performances was reported
(Scotto-Lomassese et al.,
2003). An increase in olfactory modality through an augmentation
or more rapid turnover of the RMS might therefore be advantageous for
cavefish. Indeed, Shh was shown to be required for the production of new
olfactory neurons from the stem cell niche of the forebrain subventricular
zone in vivo (Palma et al.,
2005).
Conclusions
Using the two forms of Astyanax as an evolutionary developmental
model, we have shown that changes in Shh midline signalling can have various
effects on brain development and evolution. Because Shh expansion in
cavefish is physiological and probably adaptive, it could be compensated by
other signalling pathways to prevent deleterious effects on brain development.
Thus, Shh expansion in cavefish has highly specific consequences on
the expression of downstream genes of the Nkx2.1 and LIM-hd families,
resulting either in expression heterochrony of Lhx7, which is rapidly
compensated and has no consequences on telencephalic patterning, or in an
increase of Lhx6 expression, which might confer a selective advantage
to the cavefish, or in an increase of the size of the hypothalamus, which is
more difficult to interpret in terms of adaptation. The finding that cavefish
have enhanced olfactory and neuroendocrine structures when compared to surface
fish illustrates how midline signalling centres can have a major impact on
forebrain development, evolution and diversification.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/5/845/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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