1 Developmental Genetics Program and the Department of Cell Biology, The
Skirball Institute of Biomolecular Medicine, New York University Medical
Center, 540 First Avenue, New York, NY 10016, USA
2 Harvard University, Department of Molecular and Cellular Biology, Cambridge,
MA 02138, USA
* Author for correspondence (e-mail: fishell{at}saturn.med.nyu.edu)
Accepted 13 July 2004
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SUMMARY |
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Key words: Telencephalon, Sonic hedgehog, Patterning, Oligodendrocyte, Interneuron
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Introduction |
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The requirement of Shh ligand for ventral patterning is strikingly similar
in the ventral telencephalon and all other levels of the ventral neuraxis
ventral cell fates are absolutely dependent on Shh, whereas more
lateral fates can still be achieved in the absence of this ligand
(Litingtung and Chiang, 2000;
Pierani et al., 1999
;
Rallu et al., 2002
).
Shh null mice have morphologically abnormal cortical midlines
observable as early as embryonic day 10.5 (E10.5)
(Rallu et al., 2002
). More
detailed analysis, using both gain-of-function and conditional
loss-of-function techniques, has also implicated Shh in specification
of oligodendrocyte precursors as well as maintenance of postnatal
telencephalic progenitor populations
(Machold et al., 2003
;
Nery et al., 2001
;
Tekki-Kessaris et al., 2001
).
The means by which Shh is able to effect these disparate
developmental events is unclear.
A logical place to examine the mechanistic differences that control these
processes lies in the only known mediators of mammalian Hh signaling, the
three Gli transcription factors (Bai et
al., 2004). With regards to dorsoventral patterning and
telencephalic size, only the repressor form of Gli3 has been shown as
yet to play a decisive role, suggesting that these different actions of Hh
signaling are not mediated by different Gli transcription factors
(Park et al., 2000
;
Rallu et al., 2002
). An
alternative possibility is that the functional consequences of Hh signaling
vary over time (Gunhaga et al.,
2000
; Kohtz et al.,
1998
). The complex, rapidly changing patterns of Shh
expression in and around responsive telencephalic tissue also suggests a model
of temporally distinct signaling events.
We have recently shown using a conditional Smoothened (Smo) allele
that at least some aspects of Hh signaling can be attributed to distinct
development time frames (Machold et al.,
2003). Smoothened is an obligate cell-autonomous mediator of all
Hh-signaling, hence all Hh-signaling is lost in Smo mutants
(Zhang et al., 2001
).
Experiments that removed Smo gene function in the CNS by E12 left
forebrain patterning unperturbed, while to differing degrees affecting
oligodendrogenesis and postnatal progenitor survival to differing degrees
(Machold et al., 2003
). These
data argue that Hh's control of intrinsic patterning molecules is complete and
set by this point in the developing telencephalon. The exploration of earlier
windows of Hh signaling in the telencephalon offers the potential to further
reconstruct the spatial and temporal requirement for Hh in the rapidly
developing neuroepithelium.
In these experiments, we have ablated Smo function by E9 in the mouse telencephalon. We see gross ventral patterning defects accompanied by complete dorsalization of the telencephalon. Consequently, we also see near complete loss of two telencephalic populations that are largely ventrally derived, interneurons and oligodendrocytes. Our data highlights a window of telencephalic Hh signaling that is crucial for the establishment of ventral patterning.
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Materials and methods |
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Tissue preparation, histology and in situ hybridization
E9.5, E10.5 and E12.5 embryos were dissected in cold PBS and fixed in 4%
paraformaldehyde (PFA) for 2-3 hours at 4°C. E18.5 embryos were
transcardially perfused and post-fixed for 2-3 hours. Embryos were then washed
in phosphate buffered saline (PBS) and cryoprotected in 30% sucrose in PBS.
Tissues were embedded in TissueTek, frozen on dry ice, and sectioned serially
at 18 µm for in situ hybridization and immunohistochemical analysis.
Section in situ hybridizations were performed as previously described using
nonradioactive DIG-labeled probes
(Wilkinson and Nieto, 1993).
The cDNA probes used included Gli1, Foxg1, Nkx2.1, Gsh2, Pax6, Emx2, Ngn2,
Mash1, Dlx2, Lhx6, GAD67, Olig2 and Pdgfra.
Immunohistochemistry
The following antibodies were used: mouse anti-TuJ1 (1:1000, Covance),
rabbit anti-Caspase3 (1:200, Cell Signaling), mouse anti-BrdU (1:100, Becton
Dickinson), rabbit anti-Calbindin (1:5000, Swant), rabbit anti-GABA (1:1000,
Sigma), mouse anti-GalC (1:10, Chemicon), mouse anti-O4 (1:10, Chemicon).
Triton X-100 was not used when O4 was the primary antibody. Secondary
antibodies were obtained from Jackson ImmunoResearch Laboratories and were
raised in both donkey and goat.
Oligodendrocyte culturing
E16.5 cortical culturing experiments were performed as previously
described, except that the culturing media had no mitoC added
(Nery et al., 2001).
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Results |
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Removal of telencephalic Hh responsiveness
To more precisely visualize the timing and extent of Smo
recombination in telencephalic progenitors, we crossed the
Foxg1Cre mice with the R26R ROSA reporter strain, in which
a floxed Neo cassette interrupts the ubiquitous expression of the
lacZ gene (Soriano,
1999). Foxg1Cre-mediated recombination is
present in the E7.5 pre-somitic embryo in the rostral part of the neural plate
(Fig. 1A). Recombination at
this stage appeared mosaic, with ß-gal-positive cells dispersed
throughout the medio-lateral extent of the anterior neural plate. During the
stages of embryonic turning (Fig.
1B), dynamic changes of Cre recombinase expression were observed.
At 10 somites (
E8.25) recombination was complete in the anterior neural
ridge and had initiated in the ventral telencephalon. By the 16-somite stage,
recombination appeared complete throughout the telencephalon as judged by ROSA
reporter analysis. All progenitors along the entire A-P extent of the
telencephalon displayed Cre-mediated recombination, as evidenced by
ß-gal staining extending from the telencephalic-diencephalic border to
the floor of the ventral diencephalon (Fig.
1C). In addition to the telencephalon expression, mosaic Cre was
found at the mid-hindbrain junction and the branchial arches, two domains of
known Foxg1 expression (Hatini et
al., 1999
; Hebert and
McConnell, 2000
). A low level of mosaic Cre expression was
detected throughout the rest of the embryo and is attributable to mouse
strain-specific ectopic Cre expression from the Foxg1Cre
transgene (data not shown) (Hebert and
McConnell, 2000
). To functionally assess the degree of
Smo removal, we examined the expression of Gli1 transcripts,
a sensitive indicator of Hh signaling events
(Bai et al., 2002
). At both E9
and E10.5 in the conditional knockout there was no detectable Gli1
expression in the telencephalon, a clear indication that all of Hh signaling
had been abrogated (Bai et al.,
2004
) (see Fig. S1A,B in supplementary material,
Fig. 1D,E). Genetic removal of
Hh-responsiveness prior to the onset of neurogenesis in
Smoc/;Foxg1Cre mice generated a severe
telencephalic phenotype (Fig.
1F). At E18.5,
Smoc/;Foxg1Cre mice had smaller
telencephalic vesicles than wild-type controls and a large hole in the ventral
telencephalon (Fig. 1G and data
not shown). Smoc/;Foxg1Cre olfactory
bulbs were also reduced in size. The appearance of holoprosencephalic facial
defects, including a rudimentary proboscis and centrally-displaced eyes, was
most probably because of a disruption of Hh signaling in the facial ectoderm
and branchial arches, two other sites of Foxg1Cre-mediated
Smo excision (Fig.
1F).
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In marked contrast to the absence of ventral telencephalic patterning, the
dorsal cortical midline of
Smoc/;Foxg1Cre mutants appears well
preserved. This is quite surprising in light of the Shh null
phenotype, in which both ventral and dorsal midline structures are affected
(Chiang et al., 1996). Marker
analysis of this region confirms the presence of all normal dorsal midline
signaling centers (data not shown), confirming the morphological prediction
that this structure has formed normally in
Smoc/;Foxg1Cre mutants. This implies
that Hh signaling is required before E9 in patterning the dorsal cortical
midline.
The absence of cortical interneurons
The profound early ventral patterning defects present in
Smoc/;Foxg1Cre mutants enabled us to
examine the importance of Hh signaling in populations that have significant
ventral contributions, namely cortical interneurons and oligodendrocytes. A
large body of work including genetic analysis, fate-mapping and
transplantation studies indicates that most, if not all cortical interneurons
are derived from the ventral ganglionic eminences
(Anderson et al., 1997;
Nery et al., 2002
;
Pleasure et al., 2000a
;
Wichterle et al., 2001
). In
light of the complete absence of ventral patterning in the
Smoc/;Foxg1Cre mutants, we decided to
examine whether cortical interneurons could still be generated prenatally by
some Hh-independent process. The perinatal lethality of our mutants precludes
analysis of specific mature interneuron phenotypes in an in vivo context.
However, a great deal can be learned by correlating early expression of genes
important in acquisition of interneuron character with more general perinatal
markers of the GABAergic inhibitory interneurons class. To this end, we first
analyzed the telencephalic expression of several genes essential for the
proper maturation of interneurons. Dlx2 is part of an evolutionarily
conserved gene cluster that is widely expressed throughout the basal
telencephalon and has been implicated in the tangential migration of ventrally
derived interneurons (Anderson et al.,
1997
). In E12.5 wild-type brains, Dlx2 expression is
observed in all three ventral eminences
(Fig. 5A). As holds true for
all observed ventral patterning genes, Dlx2 expression is completely
absent from the Smoc/;Foxg1Cre mutant
telencephalon (Fig. 5B).
Lhx6 is a LIM homeodomain transcription factor that is expressed in
cells migrating tangentially from the MGE to the cortex, a population that is
thought to consist largely of interneurons
(Fig. 5C)
(Lavdas et al., 1999
). Like
Dlx2, Lhx6 is also absent from all ventral domains of
Smoc/;Foxg1Cre mutants
(Fig. 5D). The loss of
Lhx6 and Dlx2 in
Smoc/;Foxg1Cre mutants does not preclude
the existence of normal interneuron progenitor pools but rather implies that
any interneurons that could still be generated would probably have defects in
tangential migration. We therefore examined GAD67 in situ
hybridization staining to better assess the state of interneuron progenitor
development. GAD67 (Gad1 Mouse Genome Informatics)
is a gene that codes for an isoform of glutamic acid decarboxylase, a
rate-limiting enzyme in the synthesis of the inhibitory neurotransmitter,
GABA. Wild-type GAD67 staining is found in the mantle of all three
eminences as well as in the midline septum, whereas in
Smoc/;Foxg1Cre mutants, only ventral
diencephalic GAD67 staining can be observed (compare
Fig. 5E with 5F).
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Oligodendrocyte formation is severely perturbed in Smoc/;Foxg1Cre mutants
The ventral telencephalon has also been shown to be an important source of
oligodendrocyte precursors (OPCs) that undergo tangential migration into the
cortex. The association of OPC markers with regions of Shh expression
and in vivo Shh gain and loss of function studies have intimately
linked the Hh pathway and oligodendrogenesis
(Nery et al., 2001;
Olivier et al., 2001
;
Tekki-Kessaris et al., 2001
).
The Smoc/;Foxg1Cre mutants provide a
genetic approach to assessing OPC formation in progenitor cells that are
insensitive to all Hh ligands. Analysis of the expression of embryonic OPC
markers was combined with in vitro primary culture assays to determine the
potential of OPC in Smoc/;Foxg1Cre
mutants. The bHLH transcription factor Olig2 is proposed to have a
role in telencephalic OPC generation, based both on its ventral expression
pattern as well as its known function as a motor neuron to oligodendrocyte
switch in the spinal cord (Fig.
6A) (Zhou et al.,
2001
). The normal ventral Olig2 expression, found in a
high medial to low lateral pattern, is completely absent from
Smoc/;Foxg1Cre mutants at E12.5
(Fig. 6B). Pdgfra,
which seems to mark OPC before their migration in the developing spinal cord,
is normally expressed in a punctate array in the anterior entopeduncular area
of the telencephalon and later in the ventral eminences
(Fig. 6C)
(Nery et al., 2001
;
Tekki-Kessaris et al., 2001
).
As judged by Pdgfra in situ staining, there were no OPC remaining in
the E12.5 mutant ventral telencephalon
(Fig. 6D). A more direct in
vivo investigation of mature oligodendrocyte populations was impossible,
because of the perinatal lethality of the
Smoc/;Foxg1Cre mutants. Consequently, an
in vitro cortical culture system was employed to examine whether the lack of
early OPC markers correlated with a decrease in the formation of more mature
oligodendrocyte populations. Cortical cultures were made from E16.5
disassociated cortex of wild-type and mutant mice and were subsequently grown
for four days before being fixed and stained with various markers of
oligodendrocyte maturation. Immunoreactivity to O4 antibody, which labels
O-antigens on differentiated oligodendrocytes, was normalized to the number of
total cells in culture, as ascertained by DAPI and TuJ1 staining
(Fig. 6E,F). Wild-type cortical
cultures produced 25-fold more oligodendrocytes than
Smoc/;Foxg1Cre mutant cultures, which
yielded virtually no O4-positive cells
(Fig. 6G).
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Discussion |
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Ventral patterning is never established in Smoc/;Foxg1Cre mice
The most striking phenotype seen in
Smoc/;Foxg1Cre mice is the complete
absence of ventral telencephalic markers
(Fig. 3), accompanied by an
expansion of dorsal markers (Fig.
4). Moreover, rather than being lost after transient expression,
the earliest markers of ventral telencephalic fate are never established in
Smoc/;Foxg1Cre mutants. This dramatic
phenotype could be attributed either to decreases in ventral progenitor cell
proliferation, localized increases in ventral cell death, regional fate
transformation or a lack of proper specification. Cell proliferation levels
are largely unaltered in E10.5
Smoc/;Foxg1Cre mutants, suggesting that
Hh signals at this early time period may not greatly alter progenitor
proliferation rates (Fig. 2).
Analysis of Smoc/;Foxg1Cre mice at E9.5
and at E10.5 show marked increase in cell death compared with wild-type
controls. A fairly widespread pattern of cell death at E9.5 gave way to a more
dorsally localized focus of apoptosis by E10.5. However, at no time
investigated is there evidence of a massive, ventral-specific apoptosis that
could have ablated the entire population of early ventral telencephalic
progenitors. These observations suggest that either a failure in the
specification of ventral cell types or a dorsal to ventral fate transformation
has occurred in the telencephalon of
Smoc/;Foxg1Cre mice. In support of the
latter, the ventral regions of the mutant telencephalon, although expressing
cortical markers maintain a pattern of neurogenesis that resembles that seen
in the wild-type LGE (Fig.
2E,F). Regardless, it seems likely that these two alternatives
represent different sides of the same patterning process
(Ericson et al., 1996). As has
been demonstrated in the spinal cord, telencephalic Hh signaling is probably
first required to create a region where dorsal patterning genes are excluded.
Within this domain, Hh signaling can specify progenitor domains by a
derepression mechanism, most likely through the modulation of levels of the
Gli3 repressor protein (Litingtung and
Chiang, 2000
).
To investigate whether ventral telencephalic patterning requires direct Hh signaling or acts through some relay mechanism of non-telencephalic Hh, we analyzed Smo null chimeras. We observe that in Smo null chimeras, null cells are largely excluded from the ventral-most territory (Supp. 2A). When Smo null cells are present in this territory, they fail to express pan-ventral telencephalic markers (Supp. 2A-D). These results are consistent with the loss of ventral patterning resulting from failure of direct Hh specification within ventral progenitors.
The early lack of ventral specification seen in
Smoc/;Foxg1Cre mutants has profound
consequences for the remainder of telencephalic development. There is no
recovery of later ventral progenitor fates through a Smo-independent
patterning mechanism. By all markers examined,
Smoc/;Foxg1Cre mutants are devoid of all
aspects of ventral telencephalic character at E12.5
(Fig. 4). Thus, it seems that
telencephalic progenitors require Hh signaling around E9 to specify ventral
telencephalic fates. In conjunction with previous studies from our lab that
show grossly normal ventral patterning in
Smoc/;NestinCre mutants, it would seem
that the time period between E9 and E12 is critical for specifying ventral
character. It is important to clarify that these timepoints are conservative
estimates of when complete removal of Hh signaling is achieved. The
Foxg1Cre driver is active in prospective ventral
telencephalon as early as E7 and thus creates a gradual loss of Hh
responsiveness beginning at this timepoint. Likewise,
NestinCre-mediated recombination begins ventrally at E9.5
and completes removal of Hh signaling by E12. In addition, both Cre drivers
have a ventral to dorsal sequence of activation that mimics the neurogenic
gradient (Fig. 1). Therefore,
this E9-E12 window could in reality be shifted at least a day earlier. It also
remains possible that initial aspects of Hh-dependent ventral patterning could
take place at gastrulation, as has been suggested by in vitro explant work in
the chick telencephalon (Gunhaga et al.,
2000). Although in principle this could be stringently addressed
using Cre-driver lines with earlier patterns of telencephalic expression, such
reagents are not yet available.
Cortical interneurons are virtually absent in Smoc/;Foxg1Cre mice
The complete absence of ventral patterning throughout the development of
Smoc/;Foxg1Cre mutants provides an
interesting context for investigating the importance of Hh signaling in the
ventral progenitors that contribute to mature dorsal telencephalic cell types.
A large body of evidence in mice has shown that the majority of interneurons
are derived from the ventral telencephalon
(Anderson et al., 1997;
Wichterle et al., 2001
;
Nery et al., 2002
). However,
work in human embryonic cortical slices, in addition to early dorsal cortical
culturing experiments hints at possible dorsal sources of interneurons
(Letinic et al., 2002
).
Irrespective of their origin, it is presently unclear whether Hh is required
by all interneuron precursors or if a Hh-independent subset of interneurons
exists. Our work demonstrates that the vast majority of embryonically born
interneuron precursors are Hh dependent. A small population of Hh-independent
neurons having immunological profiles consistent with interneuron fate, seems
to persist in the paleocortical region of the
Smoc/;Foxg1Cre mutant telencephalon
(Fig. 5). Because of the
perinatal lethality of these mutants, the mature class of these potential
Hh-independent interneuron precursors remains unknown. It seems likely,
however, that as all telencephalic Dlx expression is absent in
Smoc/;Foxg1Cre mutants, this population
of Hh-independent interneurons would probably represent a
Dlx-independent class of interneuron precursors.
Oligodendrogenesis requires hedgehog signaling between E9 and E12
Previous work from many labs has shown that Shh signaling is intimately
linked with telencephalic oligodendrogenesis. The expression of early
oligodendrocyte markers, such as Pdgfra and plp/dm20, is
localized to the Shh-expressing region of the anterior hypothalamic
neuroepithelium and anterior entopeduncular area. Loss of function data has
argued both for and against Hh-independent oligodendrocyte formation
(Nery et al., 2001;
Tekki-Kessaris et al., 2001
).
In vivo analyses of Nkx2.1 mutant mice, which lose telencephalic
Shh expression, show a strong correlation between the loss of early
OPC markers and the loss of Hh ligand. Strangely however, in vitro cortical
cultures from Shh null mice are capable of generating
oligodendrocytes (Nery et al.,
2001
; Tekki-Kessaris et al.,
2001
). Our results suggest that most, if not all oligodendrocyte
precursors are Hh-dependent (Fig.
6). Marker analysis of oligodendrocyte precursors at E12.5 showed
that these early populations were entirely absent from mutant forebrains.
Smoc/;Foxg1Cre cortical cultures, taken
from E16.5 mice and grown four days in culture, give rise to few if any
oligodendrocytes in comparison with wild-type controls. The apparent failure
of OPC specification in Smoc/;Foxg1Cre
mutants is in contrast with the oligodendrocyte phenotype seen in Shh
nulls, suggesting that the in vitro production of OPCs reflects the
upregulation of other Hh ligands. Perhaps the greater severity of
Smoc/;Foxg1Cre as compared with
Shh/ mutants with regard to ventral
patterning reflects a similar upregulation of Desert or Indian hedgehog in the
latter.
With regard to oligodendrocyte and interneuron formation, a direct
comparison of the phenotypes resulting from the
NestinCre-versus Foxg1Cre-mediated
removal of the conditional Smo is informative. Although both
oligodendrocytes and interneurons were lost in the
Smoc/;Foxg1Cre, both were clearly
present in the Smoc/;NestinCre, albeit
with some reduction in the oligodendrocyte population. This demonstrates that
the same critical window needed for the establishment of ventral identity is
the period required for the specification of these two cell types. It is
tempting to suspect that the loss of cell specification is a direct result of
the loss of patterning, implying that all interneuron and oligodendrocytes
come from ventral origin. However, some caution should be taken with this
interpretation. As Smoc/;Foxg1Cre die at
birth, one possibility is that there is a Hh-independent postnatal phase of
oligodendrogenesis and perhaps interneuron production. The observation that
Cre directed from the Emx1 locus, a gene confined to dorsal domains
throughout development, fate maps oligodendrocytes in the corpus callosum is
consistent with this possibility (Gorski
et al., 2002). Nonetheless, the exclusion of GABAergic cells from
this same fate-mapping argues against a postnatal phase of dorsal interneuron
production.
In striking contrast to the severe ventral phenotype observed, the dorsal cortical midline of Smoc/;Foxg1Cre mutants appears to be unaffected. Consistent with morphological data, gene expression studies indicate that this region is unchanged in mutants (M.F. and G.F., unpublished). These results suggest that Hh-dependent patterning of the dorsal midline occurs earlier than E9, a prospect that we are currently exploring.
Our results have further elucidated temporal changes in the requirement for
telencephalic Hh signaling. Specifically, we demonstrate that it is required
at some time prior to E9 for dorsal patterning, then from E9-E12 for ventral
telencephalic patterning. The
Smoc/;NestinCre mutants uncovered a
third role for Hh signaling in maintenance of postnatal neural progenitors
(Machold et al., 2003). As a
consequence of removal of Hh signaling beginning at E9, interneurons and
oligodendrocytes, two populations thought to be derived from ventral
progenitor domains, are missing. In conjunction with data from the
NestinCre-mediated removal of Hh-responsiveness, we show
that temporal division of Hh action is an important mechanism of Hh functional
diversification in the mammalian telencephalon. Of the potential effectors of
Hh, to date only Gli3 gene function has been shown to have relevance
to telencephalic patterning (Park et al.,
2000
; Rallu et al.,
2002
). Furthermore, unlike the spinal cord, where both activator
and repressor forms of the Gli proteins are employed in generating diverse
cell types, only the repressor form of Gli3 seems to be required in the
forebrain (Bai et al., 2004
;
Rallu et al., 2002
). With Hh
signaling only serving to orchestrate the levels of this negative
transcriptional regulator, the question of positive activators of ventral
telencephalic patterning remains unanswered. Recent work has shown that in the
spinal cord, Fgf and retinoid signaling may be direct activators of ventral
patterning genes (Diez del Corral et al.,
2003
; Novitch et al.,
2003
). Both signaling pathways have been implicated in forebrain
development, although neither has been directly associated with establishment
of ventral patterning. Our data highlight an important window of ventral
telencephalic development in which to look for the candidate signaling
pathways that actively specify early ventral patterning, and the cell types
later derived from these regions.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/20/5031/DC1
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