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First published online 6 August 2008
doi: 10.1242/dev.024802
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Research Report |
National Institutes of Natural Sciences, Okazaki Institute for Integrative Bioscience, National Institute for Physiological Sciences, Higashiyama 5-1, Myodaiji, Okazaki, Aichi 444-8787, Japan.
* Author for correspondence (e-mail: shigashi{at}nips.ac.jp)
Accepted 21 July 2008
SUMMARY
The p2 progenitor domain in the ventral spinal cord gives rise to two interneuron subtypes: V2a and V2b. Delta-Notch-mediated cell-cell interactions between postmitotic immature neurons have been implicated in the segregation of neuron subtypes. However, lineage relationships between V2a and V2b neurons have not been reported. We address this issue using Tg[vsx1:GFP] zebrafish, a model system in which high GFP expression is initiated near the final stage of p2 progenitors. Cell fates were followed in progeny using time-lapse microscopy. Results indicate that the vast majority, if not all, of GFP-labeled p2 progenitors divide once to produce V2a/V2b neuron pairs, indicating that V2a and V2b neurons are generated by the asymmetric division of pair-producing progenitor cells. Together with evidence that Notch signaling is involved in the cell fate specification process, our results strongly suggest that Delta-Notch interactions between sister cells play a crucial role in the final outcome of these asymmetric divisions. This mechanism for determining cell fate is similar to asymmetric divisions that occur during Drosophila neurogenesis, where ganglion mother cells divide once to produce distinct neurons. However, unlike in Drosophila, the divisional axes of p2 progenitors in zebrafish were not fixed. We report that the terminal division of pair-producing progenitor cells in vertebrate neurogenesis can reproducibly produce two distinct neurons through a mechanism that may not depend on the orientation of the division axis.
Key words: Neural development, Asymmetric division, Spinal cord, Zebrafish, V2 neuron
INTRODUCTION
Neuronal diversity, one of the fundamental features of the nervous system,
occurs as a result of several developmental mechanisms. In the developing
vertebrate spinal cord, neural progenitor cells initially group together into
distinct domains along dorsoventral axis in response to signals emanating from
local organizing centers. In the ventral spinal cord, five distinct domains,
p3, pMN, p2, p1 and p0, are known to be formed
(Goulding and Lamar, 2000
;
Jessell, 2000). The next level
of complexity arises within each specific domain. One mechanism that underlies
this second level of neuronal diversity involves specific cell-cell
interactions via Delta-Notch signaling. For example, the p2 progenitor domain
generates two distinct neuronal subtypes, V2a and V2b
(Karunaratne et al., 2002), and
recent studies have shown that Delta-Notch signaling plays a crucial role in
this segregation process (Del Barrio et
al., 2007; Peng et al.,
2007). Analyses of gene expression patterns, together with BrdU
incorporation studies, strongly suggest that this Delta-Notch signaling occurs
in postmitotic young neurons.
The role of Delta-Notch signaling in postmitotic young neurons has been
extensively investigated in Drosophila. In this organism, neuronal
precursor cells divide asymmetrically to produce proliferative precursor cells
and ganglion mother cells (GMC). GMCs then undergo a terminal asymmetric
division to produce two distinct neurons
(Roegiers and Jan, 2004).
Delta-Notch-mediated cell-cell interactions between these two sibling neurons
have been shown to be crucial for proper asymmetric division
(Buescher et al., 1998). A
similar mechanism may facilitate the generation of V2a and V2b neurons, with
these two distinct types of neurons specified via Delta-Notch signaling
between two sibling neurons arising from a single precursor. However, it is
also possible that Delta-Notch signaling occurs between populations of young
neurons that come from different precursors.
Distinguishing between these two potential mechanisms requires lineage analysis from the p2 progenitor stage through to final neuronal differentiation, which is difficult to accomplish in amniotes. Indeed, it is not known whether V2 neurons in amniotes are generated from the division of pair-producing progenitor cells or from cell divisions that produce progenitors and neurons. The paucity of available neuronal lineage information is a current limitation in the study of CNS development in amniotes. In our present study, we have addressed this issue using zebrafish as a model system.
MATERIALS AND METHODS
Transgenic zebrafish
Tg[vsx1:GFP] was constructed from zC67N1 BAC
(Kimura et al., 2006;
Lee et al., 2001).
Tg[huC:Gal4-VP16] was constructed using the huC promoter
(Higashijima et al., 2003) and
Gal4-VP16 (Koster and Fraser,
2001). Tg[UAS:myc-notch intra]
(Scheer et al., 2001) was
obtained from the Zebrafish International Resource Center.
Antibodies
Primary antibodies used were guinea pig anti-Chx10, rat anti-Vsx1 (these
were generated in our laboratory using bacterially-expressed proteins), rabbit
anti-Scl (Qian et al., 2007),
rabbit anti-GFP (Invitrogen) and mouse anti-PH3 (Upstate).
Time-lapse observations
Tg[vsx1:GFP] embryos at 16 hpf (hours post fertilization) were
mounted in 2% low-melting-point agarose. Spinal segments 2-10 were observed
every 10-30 minutes under a confocal microscope (Zeiss LSM510) at room
temperature. A solution of 0.003% 3-amino benzoic acid ethyl ester was
periodically added to the specimens to stop spontaneous movement. After 4 to 6
hours of observation, embryos were fixed and processed for
immunohistochemistry. For figure preparation, several optical sections were
stacked from which montages were generated.
RESULTS AND DISCUSSION
Tg[vsx1:GFP] zebrafish
During the course of our studies on V2 neuron development, we observed that
a homeobox gene, vsx1 (also known as chx10.1), was expressed
in the V2 region of the spinal cord
(D'Autilia et al., 2006). As
shown in Fig. 1A,B (22 hpf),
vsx1 mRNA was detected in a small number of cells located near the
lateral region of the spinal cord, just dorsal to the motoneuron domain (V2
region). To visualize vsx1-expressing cells in live animals, we
generated Tg[vsx1:GFP] zebrafish. As shown in
Fig. 1C (2.5 dpf), GFP was
expressed in the spinal cord, as well as in the brain and eye, consistent with
a previous report showing vsx1 expression in these regions
(Passini et al., 1998).
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As GFP is stable protein, we anticipated that vsx1-expressing cells and their progeny would be labeled in Tg[vsx1:GFP]. Vsx1-GFP cells frequently presented as side-by-side pairs (arrow in Fig. 1D). As development proceeded, more vsx1-GFP cells were present (Fig. 1E; 19 hpf), and the number of labeled, paired cells increased (arrows in Fig. 1E). Vsx1 expression was transient, so some vsx1-GFP cells had lost Vsx1 expression at this stage (see Fig. S1 in the supplementary material). GFP expression was also observed in single cells (arrowheads in Fig. 1E). The fluorescence intensity of single vsx1-GFP cells tended to be weaker than that of paired cells, suggesting that these cells had recently initiated GFP expression.
Occasionally, single spherical vsx1-GFP cells showed weak Vsx1 immunoreactivity (arrowhead in Fig. 1D), suggesting that Vsx1 proteins in these cells had spread within the soma due to the collapse of the nuclear membrane during mitosis. Consistent with this view, phospho-histone H3 (PH3) immunoreactivity (a marker of mitotic cells) was occasionally detected in these spherical cells (see Fig. S2 in the supplementary material). These observations suggested that GFP expression was initiated in single p2 progenitor cells and that the paired cells were their progeny.
Cell fate of vsx1-GFP cells
To verify this possibility, and to further investigate the ultimate fate of
the paired vsx1-GFP cells, we performed time-lapse imaging followed by
immunohistochemistry for markers of differentiated V2 neurons. The markers
used were Chx10/Alx/Vsx2 (V2a marker) and Scl (V2b marker)
(Smith et al., 2002). Cells
were analyzed in the time-lapse experiments that met the following criteria:
(1) initial GFP expression occurred in singly isolated cells (earlier forming
pairs were excluded); (2) daughter cells expressed at least one of the V2
markers (later forming pairs without marker expression were excluded); and (3)
intermingling did not occur between the two different lineages.
Representative examples from these analyses, which involved of a total of 120 cell lineages, are shown in Fig. 2A (lateral view; see Movie 1 in the supplementary material) and Fig. 2B (dorsal view; see Movie 2 in the supplementary material). The results are summarized in Fig. 2C. Two trends became evident. First, the vast majority (118 out of 120) of individual cell lineages resulted in paired cells. Second, the vast majority of these pairs (112 out of 118) consisted of V2a/V2b pairs. In six cases, one of the cells in the pair did not have detectable levels of one of the V2 neuronal markers (two V2a/ND and four V2b/ND). The absence of V2 marker expression in one of the pairs might be attributable to the difference in the timing of protein expression. In two cases, the vsx1-GFP cells directly differentiated into Chx10-positive cells without any sign of division. Thus, there were a few examples of V2a neurons that were not produced by asymmetric division, but the observation frequency of these neurons was extremely low (two out of 120). Thus, the vast majority, if not all, of the V2 neurons were generated by pair-generating progenitors that undergo a single division to produce V2a/V2b pairs.
Although vsx1-GFP expression was initiated in the p2 progenitors, it is
important to note that intensely labeled cells were located near the lateral
surface of the spinal cord, where the final divisions of these cells occurred
(Fig. 2B). These cells thus
appeared to be qualitatively distinct from general proliferative precursor
cells, which have an elongated shape in the medial-lateral dimension and
maintain contact with luminal side of the spinal cord
(Kimmel et al., 1995). This
suggests that laterally located p2 progenitors with a high level of
vsx1 expression represent an intermediate state, such that they are
committed to become pair-producing progenitors.
These putative intermediate progenitors, referred to as p2 intermediate
progenitors hereafter, might be analogous to the basal progenitors in
mammalian cortex development, as both cell types are located in a relatively
basal (outer) region of the CNS and divide once to produce neuron pairs
(Haubensak et al., 2004;
Noctor et al., 2004). However,
this is not fully certain as basal progenitors in the cortex are derived from
divisions of apical-surface-contacting neuroepithelial cells, whereas p2
intermediate progenitors may be directly derived from neural plate cells
without divisions, given that they arise as early as 16 hpf (around the stage
at which the neural rod is just formed). The elucidation of how p2
intermediate progenitors arise is an important issue for future studies.
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; Peng et
al., 2007). These findings, along with the lineage information
described above, suggest that Delta-Notch-mediated cell-cell interactions
might occur between the two p2 sibling neurons and facilitate the adoption of
distinct cell fates. Indeed, upon forced activation of Notch signaling, both
paired vsx1-GFP cells adopted the V2b fate (see Fig. S3 in the supplementary
material), consistent with the following developmental scheme: in p2 sibling
neurons, one cell has relatively high-level of Notch signaling and adopts the
V2b fate, whereas the other cell, containing lower activity, adopts the V2a
fate.
There appears to be species-specific involvement of delta genes in V2
neuron differentiation. In amniotes, Delta-like4 (Dll4) has
been shown to be crucial (Del Barrio et
al., 2007; Peng et al.,
2007). In zebrafish, dll4, the ortholog of amniote
Dll4, appeared not to be expressed in the V2 lineage (data not
shown), whereas deltaC was expressed in p2 intermediate progenitors
(see Fig. S4 in the supplementary material). Following cell division,
deltaC was expressed at higher levels in the cell from each pair that
would ultimately adopt the V2a fate (see Fig. S4 in the supplementary
material). This suggests that deltaC is involved in cell fate
specification in the zebrafish V2 lineage.
Mechanism of differential Notch signaling in V2 neurons
The apparent mode of V2 neuron differentiation in zebrafish described thus
far is strikingly similar to Drosophila neurogenesis, in which GMCs
divide once to produce two distinct neurons. Delta-Notch signaling between
postmitotic sibling pairs plays a crucial role in both systems. In
Drosophila, the division axes of GMCs are fixed, and this polarized
division is presumed to be important for proper uneven distribution of Numb
during GMC divisions (Buescher et al.,
1998). Notch signaling is inhibited in the progeny inheriting Numb
(Roegiers and Jan, 2004),
thereby biasing Notch signaling levels between the two sibling cells. To
determine whether a similar mechanism governs the division of p2 intermediate
progenitors, divisional orientation was examined by capturing images
immediately after division. The results showed that division axes were not
fixed (Fig. 3A1-A4, lateral
views; Fig. 3B1-B4, dorsal
views; embryos at around 16-17 hpf). Quantitative analyses are shown in
Fig. 3D,E. Although the
orientation of the mitotic axis was not completely random, the divisions
occurred with various orientations. We conclude from this that the regulation
of division in p2 intermediate progenitors differs from Drosophila
GMC divisions in that the division axes are not fixed.
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Apart from possible involvement of asymmetry, the presence of
deltaC in p2 intermediate progenitors suggests that genetic programs
for future Delta-Notch signaling in p2 progeny have been, to some extent,
predetermined in p2 intermediate progenitors. In mammals, foxn4 has
been shown to be important for the initiation of Delta-Notch signaling via the
activation of Dll4 expression, directly or indirectly
(Del Barrio et al., 2007).
foxn4, which is also expressed in p2 intermediate progenitors (see
Fig. S5A in the supplementary material), may be involved in activation of
deltaC expression in zebrafish.
In addition to the cell-cell interaction between sibling pairs, externally derived signals, such as those emanating from the floorplate, could influence cell fates. If so, there might be some correlation between cell fate and relative position of the cell in a pair. However, V2a/V2b pairs show evidence of various spatial configurations (see Fig. S6 in the supplementary material), suggesting that cell fates are not strongly influenced by extrinsic factors.
A model of V2 neuron differentiation is represented in
Fig. 4 (the expression patterns
of vsx1, gata2 and foxn4, presented in Figs S1 and S5 in the
supplementary material, have been incorporated). Although similar to previous
models (Del Barrio et al.,
2007; Peng et al.,
2007), our current model has the crucial distinguishing feature
that it represents one unit of lineage. An important issue that arises from
this is whether the same lineage relationship is present in mammalian V2
neurons. Based upon gene expression pattern similarities, it is possible that
mammalian V2 neurons might also originate from the division of pair-producing
progenitors. However, in subsequent Delta-Notch interactions, differences
could arise owing to the higher cell density in the mammalian spinal cord,
including possible Delta-Notch interaction between cells which are not lineage
related. Nonetheless, because sibling pairs have immediate exposure to each
other following division, they are perfectly positioned to initiate
Delta-Notch signaling. Hence, it is likely that binary fate determination
occurs between sibling cells. Clarification of this possibility will require
lineage analysis of mammalian V2 neurons.
The results of our current study are in agreement with previous reports
suggesting that Delta-Notch-mediated binary fate specification between sibling
postmitotic neurons could occur in other regions of the vertebrate spinal cord
(Mizuguchi et al., 2006;
Shin et al., 2007;
Wildner et al., 2006). Other
reports suggest that neuronal progenitor cells derived from the mammalian
cortex can divide asymmetrically to produce two uniquely differentiating
postmitotic neurons in a low-cell-density culture system
(Kawaguchi et al., 2004;
Shen et al., 2002). As
vsx1-expressing p2 progenitors are, to some extent, analogous to
basal progenitors in mammalian cortex, Delta-Notch-mediated binary fate
specification between the sibling cells of a single basal progenitor cell may
also occur there.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/18/3001/DC1
ACKNOWLEDGMENTS
We thank Z. Wen, A. Reugels, R. Tsien, S. Fraser, J. Lewis, M. Kobayashi, T. Boehm and M. Bortolussi for providing DNA/antibodies. We are also grateful to M. Ito, H. Iwasaki and Y. Okamura for helpful discussions. This work was supported in part by grants from MEXT Japan.
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