1 Department of Molecular Biology, Genentech Inc., 1 DNA Way, South San
Francisco, California 94080, USA
2 Department of Cell and Developmental Biology, University of Michigan, Ann
Arbor, Michigan 48109, USA
3 Rinat Neuroscience, 3155 Porter Drive, Palo Alto, California 94304, USA
* Author for correspondence (e-mail: craven{at}gene.com)
Accepted 9 December 2003
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SUMMARY |
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Key words: Gata, Serotonergic neurons, Sonic hedgehog
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Introduction |
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The coordinated specification of multiple neuronal cell types is best
understood in the ventral spinal cord. Sonic hedgehog (Shh), secreted from the
notochord and floor plate, forms a gradient within the ventral neural tube
that results in the concentration dependent expression of homeodomain (HD)
proteins by neural progenitors. These HD proteins are subdivided into two
groups, termed class I and II. Class I HD proteins are constitutively
expressed by neural progenitor cells and are repressed by Shh, whereas class
II HD proteins require Shh for expression
(Briscoe et al., 2000;
Jessell, 2001
). This mechanism
establishes a HD code that, through mutual repression of adjacent class I and
II HD proteins, defines five distinct ventral progenitor domains designated
from ventral to dorsal as p3, pMN, p2, p1 and p0
(Briscoe et al., 2000
;
Muhr et al., 2001
). Downstream
of the HD code, neural progenitors express subtype specific determinants that
direct the differentiation of distinct neuronal cell fates
(Briscoe and Ericson,
2001
).
Vertebrate serotonergic (5-HT) neurons constitute multiple diverse
populations along the rostrocaudal axis of the hindbrain that differ in the
timing and positioning of their induction, in their requirements for inductive
factors, and in their innervation targets and function
(Aitken and Tork, 1988;
Hynes and Rosenthal, 1999
).
The earliest 5-HT neurons to arise develop adjacent to the MHB at the ventral
midline in rhombomere 1 (r1), and send ascending projections that modulate the
function of multiple forebrain regions
(Hynes and Rosenthal, 1999
).
Subsequent 5-HT populations develop in more caudal positions, with many
sending descending projections to modulate spinal sensory and motoneurons
(Hynes and Rosenthal, 1999
). A
number of inductive signals have been implicated in the development of 5-HT
neurons, including Shh secreted from the floor plate, a Fgf activity present
in the hindbrain, and, for rostral populations, Fgf8 secreted from the MHB
(Ye et al., 1998
). Gene
ablation experiments further reveal that the HD transcription factor Nkx2.2,
the ETS transcription factor Pet1, and the LIM-HD transcription factor Lmx1b
are all involved in the genesis of 5-HT neurons, with the loss of each
resulting in a loss of 5-HT neurons early in development
(Briscoe et al., 1999
;
Hendricks et al., 2003
;
Ding et al., 2003
;
Cheng et al., 2003
).
Gata transcription factors, of which there are six in vertebrates, are
characterized by conserved C4-type zinc-finger domains and are
widely but distinctly expressed (Patient
and McGhee, 2002). Diverse developmental functions have been
ascribed to the Gata proteins, including cell fate specification, a role best
characterized in the hematopoietic system
(Patient and McGhee, 2002
).
Gata2 and Gata3 are expressed in largely overlapping domains in the central
nervous system (CNS), where relatively little is known about their function
(Nardelli et al., 1999
).
Expression data and evidence from gene targeting suggest an involvement in
neurogenesis, neuronal migration and axon projection
(Pandolfi et al., 1995
;
Nardelli et al., 1999
;
Pata et al., 1999
;
Karis et al., 2001
); however,
a role in specifying neuronal subtypes within the context of neural tube
patterning is only beginning to be understood
(Zhou et al., 2000
;
Karunaratne et al., 2002
).
Furthermore, the level of redundancy between Gata2 and Gata3 during CNS
development remains unclear.
Using both gain- and loss-of-function experiments in chick and mouse embryos, we describe a specific and essential role for Gata2 within a Shh-initiated transcriptional cascade for the development of 5-HT neurons in r1 of the hindbrain. Furthermore, we ascertain the interaction between Gata2 and the transcription factors Nkx2.2, Nkx6.1, Gata3, Lmx1b and Pet1 to translate the Shh signal to a rostral 5-HT cell fate. Specifically, we find that the Shh-activated HD proteins, Nkx2.2 and high levels of Nkx6.1 act in combination to induce expression of Gata2 in ventral r1. In turn, Gata2 is sufficient to activate the transcription factors Gata3, Lmx1b and Pet1, and to induce 5-HT neurons at the expense of other neuronal subtypes along the dorsoventral axis of the rostral hindbrain. Conversely, whereas the early development of rostral 5-HT neurons is unaltered in Gata3 null mice, Gata2 null mice fail to specify 5-HT progenitors or support 5-HT development, even in the presence of Gata3. Thus a combinatorial transcriptional cascade, in which individual Gata family members play distinct roles, translates the broad inductive signal of Shh into the development of a specific subpopulation of 5-HT neurons.
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Materials and methods |
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Morpholinos
The sequence of morpholino-modified antisense oligonucleotide with
lissamine (Gene Tools, LLC) targeted against a splice junction of chick Nkx6.1
was 5'-ATCGAGCCTTGATCTGCCGGAGGG-3'. A morpholino with the sequence
inverted was used as a control. The splice junction was identified by
comparison with the mouse Nkx6.1 genomic sequence, and the intron was
amplified by PCR of chick genomic DNA with primers based on the chick cDNA
Nkx6.1 sequence (5' primer sequence,
5'-CCGCCCTGGAGGGACGCCCG-3'; and 3' primer sequence,
5'-GAAAATCTGCTGCCCGGAGAACGTG-3').
In ovo electroporation
Chick embryos were unilaterally electroporated at HH stage 8-10, as
previously described (Hynes et al.,
2000). DNA at 3-6 mg/ml or morpholinos at 1 mM were microinjected
into the central canal of the neural tube, and platinum electrodes flanking
the neural tube delivered 6 square pulses of 28V with a duration of 40 ms and
an interpulse interval of 45 milliseconds. Chicks were harvested 2-4 days
later.
Explant culture
The ventral mid- and hindbrains of E8 embryos from Gata2
heterozygous matings were dissected and cultured in collagen as described
(Hynes et al., 1995;
Ye et al., 1998
). Explants
were cultured for 5 days prior to fixation with 4% paraformaldehyde, and then
processed for immunofluorescence or in situ hybridization as described below.
Yolk sac tissue was collected for subsequent genotyping.
In situ hybridization and immunofluorescence
Immunofluorescence on sections and wholemounts was performed as described
(Hynes et al., 1995;
Hynes et al., 2000
), using
rabbit anti-5-HT (Serotec), rat anti-5-HT (Chemicon), mouse anti-3C2
(Developmental Studies Hybridoma Bank, University of Iowa), mouse anti-islet1
(Developmental Studies Hybridoma Bank, University of Iowa), mouse anti-Gata2
(Santa Cruz Biotech), mouse anti-Gata3 (Santa Cruz Biotech), mouse anti-Nkx2.2
(Developmental Studies Hybridoma Bank, University of Iowa) and rabbit
anti-Nkx6.1 (Jensen et al.,
1996
). Single and double in situ hybridization were carried out as
described (Ye et al.,
1998
).
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Results |
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To examine the requirement of Gata2 and Gata3 in specifying endogenous 5-HT
neurons, we analyzed the development of these neurons in knock-out mice. Mice
homozygous for a targeted deletion in Gata3 die between E11 and E12
with massive internal bleeding, gross abnormalities in the nervous system and
disruption of fetal hematopoiesis
(Pandolfi et al., 1995).
Despite this, Gata3 null embryos possess similar numbers of 5-HT
positive neurons in r1 at E11 as their heterozygous littermates
(Fig. 4A,B), indicating that
Gata3 is not required for the proper specification of these 5-HT neurons.
Gata2 null mice die in utero between E9.5 and E10.5 from
pan-hematopoietic failure (Tsai et al.,
1994
). As 5-HT immunoreactivity is not yet detectable at E10.5, we
examined the expression of the early 5-HT neuron specific transcription factor
Pet1 (Hendricks et al., 1999
).
In Gata2-/+ embryos, Pet1 is present bordering the ventral
midline of the rostral hindbrain (Fig.
4C). By contrast, Gata2-/- embryos lack
expression of Pet1, even though Nkx2.2, and thus p3, neural progenitors are
present (Nardelli et al.,
1999
) (Fig. 4D,
left). Furthermore, expression of Gata3 is still found throughout much of the
hindbrain, including r1, as previously reported
[(Pata et al., 1999
) but
contrary to the findings of Nardelli et al.
(Nardelli et al., 1999
)]
(Fig. 4D, right), and partially
overlaps with Nkx2.2 (Fig. 4D,
bottom right), suggesting that both hindbrain 5-HT and interneuron progenitors
initiate expression of Gata3 in the absence of Gata2.
|
Shh induces 5-HT neurons in r1 through Gata2
The morphogen Shh patterns the neural tube along the dorsoventral axis and
is required for the development of 5-HT neurons
(Ye et al., 1998;
Hynes et al., 2000
),
suggesting that Gata2 may function as its downstream mediator. To investigate
this, we used a constitutively active form of the Shh co-receptor smoothened
(Smo-M2) that mimics Shh signaling (Xie et
al., 1998
; Hynes et al.,
2000
). Similar to Gata2, electroporation of Smo-M2 in the
hindbrain results in the cell-autonomous generation of 5-HT neurons throughout
r1 at HH24 (Fig. 5A-D).
Importantly, the induction of ectopic 5-HT neurons by Smo-M2 is preceded by
approximately 12-15 hours by the activation of both Gata2 and Gata3
(Fig. 5E and data not shown),
followed by the activation of Lmx1b and Pet1
(Fig. 5F and data not shown).
The induction of Gata2 occurs along the entire dorsoventral axis of r1
(Fig. 5G), and only cells that
express Smo-M2 ectopically express Gata2
(Fig. 5H). Furthermore only
cells that ectopically express Gata2 become 5-HT positive
(Fig. 5I), indicating that
Gata2 is correctly expressed to serve as a mediator between Shh and 5-HT
development.
|
Nkx2.2 and Nkx6.1 mediate induction of Gata by Shh in r1
To place Gata2 within a Shh-dependent pathway for the specification of
rostral 5-HT neurons, we undertook to identify the HD protein code that
controls Gata2 expression within r1. 5-HT neurons develop within the ventral
Nkx2.2-positive p3 domain in r1 (Fig.
1), and are largely absent in Nkx2.2 knock-out mice
(Briscoe et al., 1999). Thus we
first tested whether Nkx2.2 is sufficient to induce Gata2 and 5-HT neurons.
When Nkx2.2 is misexpressed in the chick hindbrain, ectopic Gata2, Gata3, and
5-HT neurons are induced in r1, but only within a narrow ventral domain
immediately lateral to endogenous 5-HT neurons
(Fig. 6A) and endogenous Gata
expression (Fig. 6B, top). This
suggests that Nkx2.2 is sufficient in the most ventral regions of the rostral
hindbrain, but that additional Shh-activated transcription factors are
required for the induction of 5-HT neurons in more lateral positions.
|
Further support for the involvement of Nkx6.1 in rostral 5-HT development is revealed by ectopic expression of Nkx2.2 caudal to r1. In contrast to in r1, in r2-7 misexpression of Nkx2.2 activates expression of Nkx6.1 in the ventricular zone (Fig. 6B, bottom; and data not shown). Concurrent with this ability, Gata factors and subsequently 5-HT neurons are induced in regions of high Nkx6.1 expression (Fig. 6A,B, bottom; and data not shown). Thus there is a strict dependence on the combined expression of Nkx2.2 and Nkx6.1 for the activation of Gata2 and specification of early born 5-HT neurons.
As a requirement for Nkx6.1 in 5-HT neuron development has not previously
been described, we examined whether Nkx6.1 is necessary for endogenous 5-HT
development within r1 of the hindbrain by misexpressing the class I HD protein
Dbx2. Dbx2 inhibits Nkx6.1 expression in the spinal cord
(Briscoe et al., 2000);
however, neither Dbx2 nor the closely related Dbx1 represses Nkx6.1 expression
in r1, and 5-HT neuron development is unaffected (data not shown).
Interestingly, the Nkx6.1 and Dbx2 expression domains are not adjacent in r1
of the hindbrain, and neither are the expression domains of Nkx2.2 and its
corresponding class I HD protein Pax6 (data not shown). This suggests that the
mutual repression between class I and II HD protein pairs that are adjacent in
the spinal cord (Briscoe et al.,
2000
; Muhr et al.,
2001
) may not act in r1 to establish progenitor domains, or is
lost at later stages of development.
Thus we used morpholino-modified antisense oligonucleotides to interfere
with Nkx6.1 expression (Kos et al.,
2001; Heasman,
2002
). Electroporation of fluorescently-labeled morpholinos
targeted against a slice junction in chick Nkx6.1 (6.1 MO; see Fig. S1 at
http://dev.biologists.org/supplemental/)
results in the loss of both Gata and Pet1 expression on the electroporated
side of the rostral hindbrain (Fig.
7A and data not shown). The disruption of downstream transcription
factors is specific as Nkx2.2 expression is maintained in cells that fail to
activate Gata (Fig. 7B and data
not shown). Subsequently, 5-HT neurons fail to develop in the presence of the
Nkx6.1 morpholino despite the continued expression of Nkx2.2
(Fig. 7C-E). An inverted
control morpholino has no affect on Gata expression or 5-HT development (data
not shown). Thus evidence from both gain- and loss-of-function studies support
the hypothesis that Nkx2.2 and high levels of Nkx6.1 make up the HD code for
the specification of 5-HT neurons within r1.
|
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Discussion |
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Gata2 is sufficient to induce 5-HT neurons only in r1
(Fig. 2), despite being induced
by Nkx2.2 in r2-7 preceding the induction of 5-HT neurons
(Fig. 6), and despite being
necessary for the proper specification of all 5-HT neurons in explants from
Gata2 null embryos. Unlike 5-HT neurons in r1, the development of
caudal populations follows that of visceral motoneurons (vMN) from common
progenitors (Pattyn et al.,
2003). Underlying the switch between early born vMN and later born
5-HT neurons is the downregulation of Phox2b expression, and Gata2, though not
sufficient, may be required for this process. Thus, a key factor for the
development of caudal 5-HT neurons that is regulated by Nkx2.2 is suggested
but not identified by this study. Interestingly, in the spinal cord, Gata2 has
been implicated in the development of Chx10-positive V2 interneurons, which
are reduced in number in Gata2 null mice
(Zhou et al., 2000
). However,
in r1 of the hindbrain, Gata2 is not sufficient to specify V2 interneurons,
but rather transforms them to a 5-HT cell fate.
Preceding the induction of ectopic 5-HT neurons, Gata2 activates Lmx1b and
Pet1 (Fig. 3), consistent with
the timing of their developmental expression
(Fig. 1). Both Lmx1b and Pet1
are required for the specification of 5-HT neurons
(Hendricks et al., 2003;
Ding et al., 2003
;
Cheng et al., 2003
), yet we
find that they not sufficient to activate one another or to generate 5-HT
neurons. Thus Gata2 may activate additional transcription factors and/or
cooperate directly with Lmx1b and/or Pet1 to specify these neurons
(Fig. 8).
Upstream of Gata2, the HD proteins Nkx2.2 and Nkx6.1 act together to
mediate the Shh signal for the induction of 5-HT neurons in r1 (Figs
6,
7). In the ventral spinal cord,
Nkx6.1 is uniformly expressed and is not required for the development of V3
interneurons (Briscoe et al.,
1999; Briscoe et al.,
2000
; Sander et al.,
2000
). Yet in r1 of the hindbrain, expression of Nkx6.1 is more
complex, and we detect a region of higher expression near the ventral midline
that overlaps with the Nkx2.2-positive p3 domain, and from which
Gata2-positive 5-HT progenitors arise (Fig.
6). Furthermore, high levels of Nkx6.1 expression are required to
induce 5-HT neurons, and Nkx2.2 only generates ectopic 5-HT neurons in
conjunction with high expression levels
(Fig. 6). Conversely, a
reduction in functional Nkx6.1 disrupts 5-HT development
(Fig. 7). Thus, we identify
Nkx6.1 as an essential component in the p3 domain of r1, and identify graded
expression along the dorsoventral axis as a novel mechanism of HD code
diversity.
In contrast to the importance of Nkx6.1 in rostral 5-HT neuron development
in the chick, work by others suggests that Nkx6.1 is not essential for their
development in the mouse (Pattyn et al.,
2003). In Nkx6 null embryos, in which both Nkx6.1 and the
closely related Nkx6.2 are disrupted, Pet1 expression is maintained throughout
the hindbrain, suggestive of normal 5-HT development
(Pattyn et al., 2003
).
Rostrocaudal patterning of the hindbrain diverges between species in many
respects, including Hox gene expression, neuronal migration, axonal
projections, and the rhombomeric development of neuronal populations including
5-HT neurons (Glover, 2001
).
For example, 5-HT neurons do not develop in r4 in the mouse, where their
development appears to be inhibited by Nkx6 via Hox1b
(Pattyn et al., 2003
), but
5-HT neurons do develop in chick r4 where both Nkx6 and Hox1b are expressed
(Fig. 3) (S.E.C., unpublished)
(Glover, 2001
). Furthermore,
the Nkx6.2 expression domain differs between chick and mouse
(Vallstedt et al., 2001
), and
no Nkx6.1 protein gradient is detected in the mouse hindbrain (S.E.C.,
unpublished). Together, these differences suggest the possibility that an
altered HD code with respect to Nkx6 and 5-HT development occurs across
species.
To conclude, we have identified a Shh-activated transcriptional cascade
that controls the specification of a specific subset of 5-HT neurons: early
born 5-HT neurons in r1 that send ascending projections into the brain. This
cascade is only sufficient in r1, but because these neurons also depend on
Fgf8 secreted from the MHB (Ye et al.,
1998), this cascade may be spatially limited by the requirement
for this Fgf signal. Although components of this cascade, including Gata2, are
required for the specification of most or all 5-HT neurons, there appears to
be differences in their specific roles in distinct 5-HT populations
(Briscoe et al., 1999
;
Hendricks et al., 2003
;
Ding et al., 2003
), and
additional factors that contribute to the development of caudal populations
and affect the switch between Phox2b-positive vMN and 5-HT development remain
to be identified. Though it is clear that Gata3 has a specific role in caudal
5-HT neuron development (van Doorninck et
al., 1999
), it alone does not explain the difference in
specification requirements. Thus, overlapping but distinct signaling cascades
are implicated in the development of rostral versus caudal 5-HT neurons and
may underlie the individual functions of these related neuronal
populations.
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ACKNOWLEDGMENTS |
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Footnotes |
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