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First published online 2 October 2008
doi: 10.1242/dev.024349
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Institute of Neuroscience, Institute of Molecular Biology, Howard Hughes Medical Institute, University of Oregon, Eugene, OR 97403, USA.
* Author for correspondence (e-mail: cdoe{at}uoneuro.uoregon.edu)
Accepted 4 September 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Castor, Pdm (Nubbin), Cell fate, Lineage, Temporal identity, Timer, Hunchback, Kruppel
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Cepko, 1999;
Doe and Skeath, 1996;
Harris, 1997;
Livesey and Cepko, 2001;
Rapaport et al., 2001;
Reid et al., 1997;
Walsh and Reid, 1995). In the
mammalian cerebral cortex, individual neural stem cells generate progeny
capable of populating all laminar layers
(Reid et al., 1997;
Walsh and Reid, 1995).
Birth-dating studies have revealed that each layer is occupied by neurons of
similar birth-order, such that early-born neurons occupy the deepest layers
and late-born neurons occupy the most-superficial layers
(McConnell, 1995). It has been
shown in mouse that the transcription factor Foxg1 is required at a precise
moment during neural stem cell proliferation to repress the first-born cell
fates and allow progenitors to specify later-born cells in the lineage
(Hanashima et al., 2007;
Hanashima et al., 2004). These
findings highlight the importance of temporal queues during the development
and diversification of the nervous system.
The Drosophila embryonic CNS has emerged as a powerful model
system for dissecting the temporal control of neurogenesis
(Cleary and Doe, 2006;
Grosskortenhaus et al., 2005;
Grosskortenhaus et al., 2006;
Isshiki et al., 2001;
Kanai et al., 2005;
Maurange et al., 2008;
Novotny et al., 2002;
Pearson and Doe, 2004;
Pearson and Doe, 2003). Neural
stem cells in the embryonic nerve cord, called neuroblasts, delaminate from
the epithelium to the interior of the embryo, marking the start of neural
differentiation. Individual neuroblasts can be identified based on the time at
which they are formed, their position within each hemisegment (NB7-1, for
example, is positioned in the seventh row, first column, of the neuroblast
array), and their pattern of gene expression
(Broadus et al., 1995;
Doe, 1992). In addition, each
neuroblast generates a unique and invariant cell lineage
(Bossing et al., 1996;
Karcavich and Doe, 2004;
Lundell and Hirsh, 1998;
Pearson and Doe, 2003;
Schmid et al., 1999;
Schmidt et al., 1997)
resulting from a series of asymmetric cell divisions in which neuroblasts
`bud-off' ganglion mother cells (GMCs) that typically undergo an additional
division to produce two post-mitotic neurons. In this manner, neurons
resulting from first-born GMC division are pushed to deep positions in the CNS
and express molecular markers for early-born cells, whereas later-born neurons
occupy more-ventral positions and express late-born fate markers
(Isshiki et al., 2001). The
early- and late-born markers are excellent candidates for genes that specify
birth-ordered cell fate, also called temporal identity
(Grosskortenhaus et al., 2006;
Isshiki et al., 2001;
Novotny et al., 2002;
Pearson and Doe, 2003).
Two transcription factors, Hunchback (Hb) and Kruppel (Kr), are known to
have crucial roles in specifying temporal identity. Hb is an Ikaros-type
zinc-finger protein that is expressed in newly formed neuroblasts and in their
early-born GMCs and neuronal progeny; it is necessary and sufficient to
specify the first temporal identity in multiple neuroblast lineages
(Cleary and Doe, 2006;
Isshiki et al., 2001;
Kambadur et al., 1998;
Novotny et al., 2002;
Pearson and Doe, 2003). Note
that we define the `first' temporal identity as the neuronal fates specified
during the window of Hb expression. This can be just one GMC and its sibling
neurons as in the NB7-3 lineage, or two GMCs and their neuronal progeny as in
the NB7-1 lineage. In the latter case, high Hb levels specify the first GMC/U1
neuron fate, and low Hb levels specify the second GMC/U2 fate (reviewed by
Pearson and Doe, 2004). Kr is
a zinc-finger protein that is detected at low levels together with Hb, and at
high levels in neuroblasts and their progeny immediately following Hb
downregulation; it is necessary and sufficient to specify the second temporal
identity in both the NB7-1 and NB7-3 lineages
(Isshiki et al., 2001). We
define the `second' temporal identity to be that following the Hb-dependent
first temporal identity; this can be the second-born or the third-born GMC in
a lineage.
The best candidate for a multi-lineage third temporal identity factor is
Pdm [which refers to a pair of co-expressed, redundantly functioning
POU-domain proteins, Pdm1 (Nubbin) and Pdm2]. Pdm is expressed immediately
after Kr in many neuroblasts and is known to specify the third temporal
identity (U4 neuron) within the NB7-1 lineage
(Grosskortenhaus et al.,
2006). However, the analysis of just one neuroblast lineage does
not resolve whether Pdm has a specific function in specifying U4 motoneuron
identity, or a more general function as a multi-lineage third temporal
identity factor. This is a crucial distinction because many transcription
factors are likely to regulate the specification of different neuronal
subtypes without having any connection with temporal patterning. In fact, Pdm
is also required for specification of the first-born progeny in the NB4-2
lineage (Yang et al., 1993;
Yeo et al., 1995), raising
some doubt as to its role as a multi-lineage temporal identity gene.
The best candidate for a multi-lineage fourth temporal identity factor is
the zinc-finger protein Castor (Cas), which is detected in neuroblasts just as
Pdm levels fade away, and which together with Pdm specifies the fourth
temporal identity (U5 neuron) in the NB7-1 lineage
(Grosskortenhaus et al., 2006;
Isshiki et al., 2001). As with
Pdm, it is impossible to know whether Cas has a specific role in specifying U5
identity or a general role as a fourth temporal identity gene without
analyzing its function in additional neuroblast lineages. This has been
difficult because most neuroblast lineages have not been characterized past
the first or second cell division and few molecular markers are known for
late-born neurons. For example, NB7-3 generates neurons with
well-characterized molecular markers
(Isshiki et al., 2001;
Lundell et al., 1996;
Lundell and Hirsh, 1998;
Novotny et al., 2002), but it
divides only three times and never expresses Cas. By contrast, NB2-4 divides
many times and expresses Pdm and Cas
(Isshiki et al., 2001), but
there are no molecular markers available to identify late-born neurons in this
lineage. Thus, to test the role of Pdm and Cas as multi-lineage late temporal
identity factors, and to test any new candidate late-born temporal identity
factors, it is necessary to characterize a new neuroblast lineage for both
birth-order lineage data and neuronal molecular markers.
Here we trace the birth-order of the first four divisions in the NB3-1 lineage and develop molecular markers to distinguish early-born and late-born neuronal identity, allowing us to use this lineage to assay late temporal identity gene expression and function. We find that Hb and Kr specify early temporal identity in this lineage, extending their role as multi-lineage temporal identity factors to a different spatial domain of the CNS. Surprisingly, we find that Pdm is not required to specify the third temporal identity, but rather that Pdm is required to repress Kr and thus close the second temporal identity window. Similarly, we find that Cas is required to close the third temporal identity window in this lineage. We conclude that Hb and Kr are multi-lineage temporal identity factors, whereas Pdm and Cas are timing factors that close successive temporal identity windows in the NB3-1 lineage.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Isshiki et al.,
2001); Kr1, KrCD/CyO hb-lacZ to
remove Kr CNS expression (Isshiki et al.,
2001; Romani et al.,
1996); Df (2L)ED773, which removes both pdm1 and
pdm2 (Grosskortenhaus et al.,
2006), cas24/TM3 ftz-lacZ [formerly called
ming24 (Cui and Doe,
1992)], red e spdoZZ27/TM3 and
numb2 pr cn Bc/Cyo ftz-lacZ
(Skeath and Doe, 1998);
cas-lacZ (Cui and Doe,
1992); and svpe22/svpz4
(Miller et al., 2008;
Mlodzik et al., 1990). Unless
otherwise noted, for misexpression experiments we crossed insc-gal4
(1407-gal4, Bloomington Stock Center) on chromosome II to
UAS-hb on chromosomes II and III
(Wimmer et al., 2000),
UAS-Kr on chromosomes II and III
(Hoch and Jackle, 1998),
UAS-HA:pdm2 on chromosomes II and III
(Grosskortenhaus et al., 2006)
and UAS-cas on chromosomes II and III (W. Odenwald, NIH, Washington
DC) at 29°C. Recombinant clones were generated using flies with the
following genotype: y w hs-FLP/+; X-15-33/X-15-29
(courtesy of Allan C. Spradling, Carnegie Institute, Washington DC).
Molecular markers and immunostaining
Antibody staining was performed according to standard methods. Primary
antibodies, dilutions and sources were: rabbit HB9 (Exex - FlyBase) 1:1000
(Odden et al., 2002); guinea
pig HB9 1:500, and rat Islet (Isl; Tailup - FlyBase) 1:500
(Broihier and Skeath, 2002);
mouse Islet 1:200, mouse Fasciclin 2 (FasII) 1:100, mouse FasIII 1:5
(Developmental Studies Hybridoma Bank, University of Iowa); rabbit Hb 1:200
(this work); guinea pig Kr 1:500 [East Asian Distribution Center for
Segmentation Antibodies (EADC), Mishima, Japan]; rat Pdm2 1:10
(Grosskortenhaus et al.,
2006); rabbit Cas 1:1000
(Kambadur et al., 1998); rat
Zfh2 1:400 (M. Lundell, University of Texas at San Antonio); mouse En 4D9 1:5
(Patel et al., 1989); mouse
Late Bloomer 1:4 (C. Goodman, University of California, Berkeley); and mouse
β-galactosidase 1:500 (Promega, Madison, WI). Secondary antibodies were
purchased conjugated to Alexa 488, Rhodamine RedX or Cy5 (Jackson, West Grove,
PA), biotin (Vector, Burlingame, CA) or alkaline phosphatase (Southern
Biotechnology, Birmingham, AL) and used at 1:400. Confocal image stacks were
collected using a Leica SP2 confocal microscope, processed using ImageJ (NIH)
and displayed as two-dimensional projections. Histochemical preparations were
acquired using a Zeiss Axioplan microscope.
Identification of NB3-1 and the RP motoneurons
We staged embryos using standard methods
(Campos-Ortega and Hartenstein,
1985), and identified NB3-1 by spatial and morphological features
along with the expression of Engrailed (which marks neuroblasts in rows six
and seven). RP1, RP4, RP3 and RP5 were identified as Isl+
HB9+ neurons in the dorsal-medial region of each hemisegment. The
only other nearby Isl+ HB9+ neurons are the
more-posterior/lateral EW neurons from the NB7-3 lineage, which can be
distinguished from the RP neurons by their expression of Engrailed
(Isshiki et al., 2001;
Lundell et al., 1996). RP1 and
RP4 are often shown as insets as they appear directly ventral to RP3 and hence
are often obstructed from view.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Landgraf et al., 1997;
Schmid et al., 1999). We first
characterized the expression of the known and candidate temporal identity
genes Hb, Kr, Pdm and Cas in NB3-1 as it begins its cell lineage. We detected
Hb and Kr expression in the newly formed NB3-1 at stage 10, Kr alone during
early stage 11, Pdm alone at mid stage 11, and Cas expression from late stage
11 into stage 12 (Fig. 1A,B).
We conclude that NB3-1 sequentially expresses Hb/Kr 
Kr Pdm
Cas during the initial phase of its cell lineage, and that this lineage
is appropriate for investigating the role of all four genes in specifying
temporal identity.
Next, we characterized the birth-order of the RP neurons and determined
which of the known or candidate temporal identity genes was expressed at the
time of their birth. We used both molecular markers and cell body position to
identify and distinguish the RP neurons
(Fig. 2A,B; see Materials and
methods for details). Motoneuron backfills have shown that RP1/4 are the most
dorsal, RP3 is intermediate, and RP5 is most ventral in position within the
CNS (Landgraf et al., 1997;
Schmid et al., 1999).
Early-born neurons occupy deeper layers
(Isshiki et al., 2001),
consistent with a birth-order of RP1/RP4 RP3 RP5. Here, we used
molecular markers to identify the NB3-1-derived RP neurons, and assayed their
Hb, Kr, Pdm and Cas expression profile. We found that RP1/4 are Hb+
Kr+, RP3 is Hb- Kr+, and RP5 is
Hb- Kr- (Fig.
2A). This precisely matches the sequence of gene expression within
NB3-1 as it progresses through the early portion of its lineage
(Fig. 1). We conclude that
RP1/4 are born during the early Hb+ Kr+ neuroblast
expression window, RP3 is born during the Hb- Kr+
neuroblast expression window, and RP5 is generated after Hb and Kr expression
is lost from the neuroblast.
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), raising the possibility that they are sibling neurons. To
determine whether RP1 and RP4 are sibling neurons, we used sanpodo
and numb mutants to equalize sibling cell fate
(Skeath and Doe, 1998). We
found that sanpodo mutants typically generate a pair of RP1 neurons
and a pair of RP4 neurons, whereas numb mutants show the opposite
phenotype (Fig. 2B,
Table 1). These results show
that the RP1 and RP4 neurons are not siblings, but rather that they each have
a non-RP sibling that assumes the RP fate in sanpodo mutants.
Furthermore, we generated clones by mitotic recombination that label the
entire NB3-1 lineage except RP1 (n=4;
Fig. 2C). As all four neurons
are definitively produced by NB3-1 based on DiI labeling
(Bossing et al., 1996;
Schmid et al., 1999), this
proves that RP1 is derived from the first-born GMC in the lineage. We conclude
that NB3-1 sequentially generates the RP1 RP4 RP3 RP5
neurons (and their non-RP siblings), followed by a pool of local interneurons
(Fig. 2D).
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). We tested whether Hb has a similar function in the
NB3-1 lineage, which is located in the anterior region of the neuromere. We
used hb mutants that were rescued for Hb segmentation expression
(Isshiki et al., 2001), and
found a loss of the early-born RP1 and RP4 neurons, whereas the later-born RP3
and RP5 neurons were unaffected (Fig.
3A, Table 1). Thus,
Hb is required for the specification and/or survival of the early-born RP1 and
RP4 neurons. To determine whether Hb is sufficient to induce the first-born
RP1/RP4 temporal identity, we used insc-gal4 UAS-hb to prolong
expression of Hb in NB3-1 beyond its normal expression window. We observed as
many as nine RP neurons per lineage, and all appeared to take the early-born
RP1/RP4 identity based on molecular markers
(Fig. 3B,C,
Table 1). Consistent with the
increase in RP1/RP4 motoneurons, we observed a thickening of the
FasII+ and FasIII+ motor axon fascicles exiting the CNS
and entering the ventral longitudinal muscle fields (see Fig. S1 in the
supplementary material). Late-born RP3 and RP5 neurons expressing Zfh2 or Cut
were never detected. We conclude that Hb is necessary and sufficient to
specify early-born RP1/RP4 temporal identity within the NB3-1 lineage,
paralleling its role in specifying the first temporal identity in the NB7-1
and NB7-3 lineages (Isshiki et al.,
2001; Novotny et al.,
2002).
Kruppel specifies the second temporal identity in the NB3-1 lineage
Kr is known to specify the second temporal identity in the NB7-1 and NB7-3
lineages (i.e. the fate of the GMC born immediately after Hb downregulation)
(Cleary and Doe, 2006;
Isshiki et al., 2001). We
tested whether Kr has a similar function in the NB3-1 lineage. We used
Kr mutants that were rescued for early segmentation expression
(Isshiki et al., 2001), and
found a loss of the RP3 neuron, whereas the early-born RP1/RP4 neurons and the
late-born RP5 neuron were mostly unaffected
(Fig. 4A,
Table 1). Thus, Kr is required
for the specification and/or survival of the RP3 neuron, i.e. of the RP neuron
born during the Kr neuroblast expression window. To determine whether Kr is
sufficient to induce the RP3 identity, we used insc-gal4 UAS-Kr to
prolong expression of Kr in NB3-1 for the entire length of its cell lineage.
We observed a maximum of three RP3 neurons per lineage
(Fig. 4B,
Table 1). We saw no deleterious
effect on the specification of RP1 and RP4, but the Cut+ RP5 neuron
was typically missing (Fig.
4B). We conclude that Kr is necessary, within a competence window,
to specify the second temporal identity in the NB3-1 lineage (RP3), similar to
its role in specifying the second temporal identity in the NB7-1 and NB7-3
lineages (Isshiki et al.,
2001).
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). To determine whether Pdm is a multi-lineage
temporal identity gene, we assayed its loss-of-function and misexpression
phenotype in the NB3-1 lineage. We assayed embryos homozygous for the
deficiency Df(2L)ED773, which eliminates both pdm1 and
pdm2 (henceforth referred to as pdm mutant embryos). In
pdm mutant embryos, we observed normal timing of Hb expression in
NB3-1 and other neuroblasts (data not shown), a modest extension of Kr
expression, and a similar delay in Cas expression
(Fig. 5A). Consistent with this
change in neuroblast gene expression, pdm mutant embryos showed
normal specification of the early-born Hb+ RP1 and RP4 neurons, but
possessed extra Kr+ RP3 neurons, followed by an apparently normal
Cut+ late-born RP5 (Fig.
5C, Table 1). We
conclude that Pdm is not required to specify the third temporal identity (the
Cut+ RP5 neuron), but is required to limit Kr expression in the
neuroblast and thus close the second temporal identity window after the birth
of just one Kr+ RP3 neuron. We next determined whether the continuous expression of Pdm in NB3-1 was sufficient to induce ectopic RP5 neurons (i.e. extend the third temporal identity window). We used insc-gal4 UAS-pdm2 to generate continuous Pdm expression in neuroblasts, and observed normal timing of Hb expression in NB3-1 and other neuroblasts (data not shown), but premature loss of Kr expression and precocious Cas expression (Fig. 5B). Consistent with this change in neuroblast gene expression, we observed normal specification of the early-born Hb+ RP1 and RP4 neurons, but a lack of Kr+ RP3 neurons; there was also a loss of the Cut+ late-born RP5 neuron (Fig. 5D, Table 1). We conclude that Pdm is not sufficient to specify the third temporal identity (RP5), but rather it acts as a timer element to define the window of Kr expression and thus the length of the second temporal identity window. The precocious expression of Cas in these Pdm misexpression embryos may result in the precocious formation of Cas+ interneurons at the expense of the RP5 neuron (see below).
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) and found
no residual β-galactosidase expression in any NB3-1-derived RP neurons
(data not shown). This suggests that Cas expression is initiated after NB3-1
has made its fourth GMC, at the time when it shifts to producing local
interneurons (Fig. 2D). Thus,
although we can test whether Cas is important for closing the third (RP5)
temporal identity window, owing to the lack of interneuronal markers we are
unable to assay for a Cas function in specifying the fourth (interneuron)
temporal identity.
To test whether Cas is required to close the third temporal identity
window, we assayed cas-null mutant embryos
(Cui and Doe, 1992). We found
that cas mutants have normal Hb and Kr expression in neuroblasts
(data not shown), but prolonged Pdm expression
(Fig. 6A), consistent with
previous work showing that Cas is required to repress pdm
(Grosskortenhaus et al., 2006;
Kambadur et al., 1998). At the
neuronal level, we found that cas mutants have normal early-born RP1,
RP4 and RP3 neurons but possess ectopic RP5 neurons
(Fig. 6B,
Table 1), consistent with a
prolonged third temporal identity window. The ectopic RP5 neurons are not
specified by the persistent Pdm protein because pdm mutants still
formed apparently normal RP5 neurons (Fig.
5) and pdm cas double mutants still formed
Cut+ RP5 neurons (data not shown). Interestingly, cas
mutants had a few RP-like (Islet+ HB9+) neurons that
lacked expression of the motoneuron marker Late Bloomer and thus might have a
mixed interneuron/RP motoneuron identity (see Fig. S2 in the supplementary
material). We next examined insc-gal4 UAS-cas embryos, which have
continuous expression of Cas in NB3-1. We found that RP5 was often missing,
but the early-born RP1, RP4 and RP3 were normal
(Fig. 6C,
Table 1). We conclude that the
precocious expression of Cas is sufficient to close the third temporal
identity window, in which RP5 is specified. Taken together, our results
suggest that Cas is necessary and sufficient to close the third temporal
identity window in the NB3-1 lineage.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Hunchback and Kruppel are multi-lineage temporal identity factors
We have shown that Hb and Kr are necessary and sufficient to specify the
first and second temporal identities, respectively, in the NB3-1 lineage. We
can now conclude that Hb and Kr function as temporal identity factors in many
spatial domains of the CNS [anterior-medial (NB3-1), posterior-medial (NB7-1)
and posterior-lateral regions (NB7-3)], showing that temporal identity and
spatial identity are independent with regards to Hb and Kr. Furthermore, Hb
and Kr maintain similar functions in neuroblasts that form at distinct times
during embryogenesis [early (NB7-1), middle (NB3-1) and late (NB7-3)], thus
confirming that temporal identity is a lineage-autonomous event that is not
coordinated by embryo-wide timing events
(Brody and Odenwald, 2000;
Grosskortenhaus et al., 2005).
Overall, our data strongly support the conclusion that Hb and Kr are
multi-lineage temporal identity genes.
Our data also provide insight into neuroblast competence. When we
misexpressed Hb in the NB3-1 lineage, we were able to generate up to nine RP
motoneurons; if each has a non-RP sibling, it would be close to the expected
number of cells for the entire lineage
(Schmid et al., 1999). Thus,
Hb seems capable of maintaining at least three very different neuroblast
lineages (NB3-1, NB7-1 and NB7-3) in a `young' state for their entire lineage.
By contrast, misexpression of Kr produces only a few RP3 motoneurons before
NB3-1 proceeds to make the later-born neurons. The inability of Kr to maintain
a second temporal identity state might be due to the initiation of progressive
restriction in neuroblast competence in NB3-1, as occurs in NB7-1
(Cleary and Doe, 2006;
Pearson and Doe, 2003).
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;
Mettler et al., 2006) and in
the NB3-1 lineage (data not shown). It should be noted that Svp represses Hb
expression in all neuroblasts tested to date, whereas Pdm represses Kr
expression in some but not all neuroblasts.
Pdm does not act as a timer element in all neuroblast lineages. For
example, pdm mutants do not show extended Kr expression in the NB7-1
or NB7-3 lineages, as judged from the lack of ectopic Kr+ neurons
in these lineages (Grosskortenhaus et al.,
2006) (see Fig. S3 in the supplementary material). These results
suggest that the spatial identity of a neuroblast can alter its response to
timing factors such as Pdm. Although this is counter to the simple model that
spatial and temporal factors are independent and act combinatorially to
specify birth-order identity within each lineage
(Pearson and Doe, 2004), it is
consistent with the finding that spatial identity occurs at the time of
neuroblast formation (Chu-LaGraff and Doe,
1993; Prokop and Technau,
1994; Skeath et al.,
1995), prior to the expression of temporal factors. Taken
together, these data suggest that spatial cues allow individual neuroblasts to
respond differently to a temporal identity factor expressed at a similar time
in all lineages.
The prior expression of early temporal identity factors is also likely to
alter the response of a neuroblast to later temporal identity factors.
Previous work has shown that misexpression of later temporal factors such as
Kr, Pdm or Cas, has no detectable effect on the fate of first-born
Hb+ neurons in the NB7-1 lineage
(Cleary and Doe, 2006;
Grosskortenhaus et al., 2006;
Isshiki et al., 2001;
Pearson and Doe, 2003).
Consistent with these results, we find that in the NB3-1 lineage, Pdm
misexpression cannot repress Kr or activate Cas during the early
Hb+ expression window (Fig.
5B). Just as prior spatial patterning cues may alter the response
to a later temporal identity factor, so too may prior temporal identity factor
expression alter the response of a neuroblast to later temporal identity
factors. The mechanism by which spatial and temporal factors confer heritable
changes to neuroblasts remains a mystery. An entrypoint into this mechanism
could be the investigation of how Hb blocks Pdm from repressing Kr gene
expression.
If Pdm does not specify temporal identity in NB3-1, what is the third
temporal identity factor in this lineage? It has recently been reported that
the SoxB family member Dichaete is expressed immediately prior to Cas in many
embryonic neuroblast lineages (Maurange et
al., 2008). However, Dichaete is only transiently expressed in
medial column neuroblasts, such as NB3-1, at their time of formation
(Zhao and Skeath, 2002) and
thus does not have the proper timing for a third temporal identity factor in
this lineage. Alternatively, absence of Hb, Kr and Cas might specify the third
temporal identity, with Pdm acting solely as a timing factor to establish a
gap between Kr and Cas expression. Another possibility is that an as yet
unknown factor specifies the third temporal identity in the NB3-1 lineage.
Finally, Pdm might specify aspects of RP5 identity that we are not able to
detect with our limited number of markers; unfortunately, owing to severe
morphological defects in late-stage pdm mutant embryos, we have been
unable to assay the RP5 axon projection to its target muscle, which would
provide a sensitive read-out of its neuronal identity.
Castor closes the third temporal identity window
Cas is expressed right after Pdm in most neuroblasts, and at the time NB3-1
is generating its fourth temporal identity (interneurons). We find that
cas mutants have an extended window of Pdm neuroblast expression and
exhibit production of ectopic RP5 neurons. Thus, Cas is required to close the
third (RP5) temporal identity window. In addition, we find that precocious
expression of Cas can prematurely close the third temporal identity window and
repress the specification of RP5. We observed comparable phenotypes in the
NB7-1 lineage, in which loss of Cas leads to ectopic U4 formation and gain of
Cas results in the repression of the U4 identity
(Grosskortenhaus et al.,
2006). Based on these observations, we predict that Cas functions
in multiple neuroblast lineages to close the third temporal identity window.
Does Cas specify the fourth temporal identity? We cannot answer this question
in the NB3-1 lineage owing to a lack of interneuron markers, but Cas does
specify the fourth temporal identity (together with Pdm) in the NB7-1 lineage
(Grosskortenhaus et al.,
2006). In the future, the role of Cas in the NB3-1 lineage could
be examined by making CD8::GFP-marked cas mutant clones and assaying
neuronal identity by axon projections, or by developing molecular markers for
interneurons within the lineage.
Temporal identity genes, timing factors and neuronal cell-type specification
We propose that there are two classes of genes that regulate neuroblast
temporal identity (Fig. 7). One
class, of which Hb and Kr are good examples, encodes temporal identity factors
that are necessary and sufficient to directly specify a particular temporal
identity in multiple neuroblast lineages
(Isshiki et al., 2001). A
second class encodes timing factors that establish the timing of temporal
identity gene expression, but do not directly specify temporal identity.
Timing factors, however, may indirectly influence the specification of
temporal identities as seen in NB3-1, in which pdm is required to
restrict the specification of RP3 and properly advance the neuroblast to the
Cas-positive state (Fig. 5).
Seven up, the one timing factor identified previously, downregulates Hb
protein levels and, along with cytokinesis, closes the first temporal identity
window to facilitate the Hb Kr transition
(Grosskortenhaus et al., 2005;
Kanai et al., 2005). The Kr
Pdm Cas transitions are independent of cell-cycle progression
(Grosskortenhaus et al.,
2005). Here, we have shown that Pdm closes the second temporal
identity window by repressing Kr expression and activating Cas in NB3-1. Taken
together, our observations suggest that Kr and Pdm are involved in a
negative-feedback loop in which Kr activates Pdm, which in turns represses Kr
and activates Cas to advance neuroblast timing independent of cell-cycle
progression. Through its role as a regulator of Kr and Cas timing, Pdm can
restrict the production of neuronal cell types and advance the NB3-1
lineage.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/21/3491/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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