Cell intrinsic and cell extrinsic factors mediate asymmetric cell divisions during neurogenesis in the
Drosophila embryo. In one of the well-studied neuronal lineages in
the ventral nerve cord (the NB4-2->GMC-1->RP2/sib lineage), Notch (N) signaling interacts with asymmetrically localized Numb (Nb) to
specify sibling neuronal fates to daughter cells of GMC-1. The NB4-
2 is delaminated in the second wave of NB delamination during
mid stage 9 (~4.5 hours) of embryogenesis and is located in the 4th row along the
anteroposterior axis and 2nd column along the mediolateral
axis within a hemisegment. The NB4-2 generates its first GMC
(GMC-1, also known as GMC4-2a) ~1.5 hours after formation.
The GMC-1 divides ~1.5 hours later to generate two cells, the
RP2 and the sib. The RP2 cell eventually occupies its position
in the anterior commissure along with the other RP neurons
(RP1, RP2, RP3 and RP4) and projects its anteroipsilateral
axon to the intersegmental nerve bundle (ISN) and innervates
muscle #2 on the dorsal musculature. The sib cell migrates to
a position posterior and more dorsal to RP2. The DiI tracing of the NB4-2
lineage indicates that the sib has no axonal projection at mid
stage 17 of embryogenesis; thus, its
ultimate fate has not been determined. In this study, loss-of-function
mutations in N and nb, cell division mutants cyclinA (cycA), Regulator of cyclin A1
(rca1) and string/cdc25
phosphatase (stg/cdc25
phosphatase), and the microtubule destabilizing agent, nocodazole, were all used to investigate
asymmetric cell fate specifications by N and Nb in the context of cell cycle. Mutation in rca1 gene was initially identified as a dominant
suppressor of roughex (rux) eye phenotype. In rux, the cells enter
S-phase precociously due to ectopic activation of a CycA/Cdk
complex in early G1 (Dong, 1997). In embryos lacking the
rca1 activity, the cells appear to arrest in G2 of the cell cycle (at stages 15-
16) similar to cycA mutants (Wai, 1999 and references).
The loss of cycA, rca1 or stg leads to a block in the division of GMC-1, however, this GMC-1
exclusively adopts an RP2 identity. The requirement of cycA or rca1 for cell division in the
CNS is lineage specific.
Anti-Eve staining of cycA or rca1 mutant embryos indicates
that loss of these gene products does not affect all the Eve-positive
lineages in the ventral nerve cord. Eve is expressed in
other neuronal lineages such as the CQs, the Us and the ELs. The CQs are formed from NB7-1, an S1 neuroblast. The GMC for these
neurons are formed at the same time as the GMC for the
aCC/pCC neurons (generated from another S1 neuroblast,
NB1-1) and divide at the same time as GMC for the aCC/pCC
lineage. The NB7-1 in cycA or rca1 mutants
does not divide to generate an Eve-positive GMC for the CQs.
However, the effect on CQs is partially penetrant in both
the mutants. Thus, ~75% of the hemisegments had missing CQs in cycA mutants; in rca1
mutants, this figure is ~50%. The effect on the generation of U
neurons is as follows: in cycA mutants, the effect is fully
penetrant; whereas, in rca1 mutants, 65% of the hemisegments
were missing the Us. It must be pointed out that in those
hemisegments where these neurons (Us and CQs) are formed,
the number of these neurons is fewer than normal. Finally, the
effect of the loss of cycA or rca1 on another Eve-positive lineage,
the EL neurons, is minimal. The EL neurons are formed from
NB3-3, an S4
neuroblast (the formation of this neuroblast extends between
S3-S5). None of the hemisegments have
missing EL neurons, in either the cycA mutants or the rca1 mutants.
The above result indicates that the loss of rca1 or cycA does
not affect the division of all neuroblasts. One possibility for this result is that
the maternal deposition of these gene products is masking the
zygotic loss of these gene products in these lineages. However,
this seems unlikely since the GMCs for the
aCC/pCC or the RP2/sib lineages are generated earlier than the
GMCs for the EL neurons. Moreover, the maternal deposition
of CycA, for example, is completely exhausted before stage 7 and none of the
neuroblasts have delaminated from the neuroectoderm at this
stage of development. Thus, these results
indicate that the effect of loss of cycA or rca1 is lineage specific
and every neuronal lineage is not sensitive to the loss of these
cell division genes. It is most likely that some other cyclins
(i.e., Cyclin B) complement the loss of CycA in these lineages (Wai, 1999).
While the loss of N leads to the specification of RP2 fates to both
progeny of GMC-1 and loss of nb results in the specification of sib fates to these daughter cells, the GMC-1
in the double mutant between nb and cycA assumes a sib fate.
While the GMC-1 fails to divide to generate two
cells in these double mutants, the GMC-1 assumed a sib fate.
About ~35% of the hemisegments show this
phenotype. This penetrance of the phenotype is slightly higher
than the phenotype observed in nb single mutants alone. This
suggests that cycA mutation has an enhancing effect on the nb
phenotype. This would argue that normally a small amount of
the Nb protein segregates into a sib cell and that, in the absence
of cell division, all of Nb is accumulated in one cell, and therefore, is much
more effective in blocking the N signaling. Moreover, since the
nb phenotype is epistatic to the cell division mutant phenotype,
Nb must be acting downstream of these genes. This result is
consistent with the finding that Nb becomes localized during
metaphase and is not localized in stg mutants. Thus, in rca1 or cycA mutants, the absence of a
localized Nb prevents the N signaling from specifying sib fate
and, as a result, the GMC-1 assumes an RP2 fate.
These epistasis results indicate that both N
and nb function downstream of cell division genes and that progression through cell cycle is required for the
asymmetric localization of Nb. In the absence of entry into metaphase, the Nb protein prevents the N signaling
from specifying sib fate to the RP2/sib precursor. These results are also consistent with the finding that the
sib cell is specified as RP2 in N;nb double mutants. Finally, these results show that nocodazole-arrested
GMC-1 in wild-type embryos randomly assumes either an RP2 fate or a sib fate. This suggests that
microtubules are involved in mediating the antagonistic interaction between Nb and N during RP2 and sib
fate specification (Wai, 1999).
In the central nervous system (CNS) of Drosophila embryos lacking either cyclin A or regulator of cyclin A (rca1) several
ganglion mother cells (GMCs) fail to divide. Rca1 is novel 412 amino acid protein required for both mitotic and meiotic cell cycle progression, Whereas GMCs normally produce two sibling neurons that acquire different fates ('A/B'), non-dividing GMCs differentiate exclusively in the manner of one of their progeny ('B'). The rca1 mutation was initially identified and characterized
from a screen for aberrant expression patterns of Even-
skipped (Eve) protein in the embryonic CNS (I. Orlov, R.
Saint, N. Patel, unpublished results cited by Lear, 1999). Eve is normally
expressed in the nuclei of several cells in the CNS; these
include GMC 1-1a and its progeny, aCC and pCC; GMC 4-2a and one of its progeny, RP2, as well as the EL, U, and CQ neurons. In cycA and rca1 mutants, Eve is
expressed in fewer cells per hemisegment than wild-type. In
the position where the siblings aCC and pCC normally sit,
a single Eve-positive nucleus that is larger than
the wild-type aCC or pCC is observed. In the
position of RP2, there is still one Eve-positive nucleus,
but again it often appears larger than normal. A loss of Eve expression is also observed where
the U and CQ neurons normally sit and a decrease in the
number of Eve-positive EL neurons (Lear, 1999).
The GMC 4-2a and
GMC 1-1a lineages recieved the closest focus because of their well-characterized
development and because various molecular markers exist
that label these GMCs and their progeny. In wild-type embryos, GMC 4-2a divides early in stage 11, and two
Eve-expressing nuclei are initially observed upon this division. Eve expression is quickly shut off in
the smaller RP2 sibling nucleus but remains on in RP2. In
cycA or rca1 mutants, Eve expression turns on normally in
GMC 4-2a; however, two nuclei are rarely observed during
stage 12, and the single Eve-expressing nucleus remains large. Likewise, GMC 1-1a
normally divides during stage 10 in wild-type embryos to
generate the Eve-positive neurons aCC and pCC. In cycA or rca1 mutants, GMC 1-1a expresses
Eve as in wild-type but rarely divides. Instead, this GMC comes to reside in the same
dorsal plane and posterior position where aCC and pCC sit
in wild-type embryos. Other Eve-expressing lineages, including the U/CQ neurons and the
EL neurons, appear to be affected as well in
cycA and rca1 mutants. Notably, even the most severe
alleles of cycA and rca1 examined do not show complete
expressivity of CNS phenotypes in all lineages (Lear, 1999).
Having observed that GMCs acquire the fate of the 'B'
sibling neuron in cycA or rca1 mutants, it was next determined whether GMCs could acquire the 'A' fate through
activation of the Notch pathway. If Delta signal must be
provided from a sibling neuron, then GMCs, which lack a
true 'sibling', may not have the potential to acquire the 'A'
fate through extracellular signaling. The rca1
mutation was combined with either a zygotic numb mutation or an activated form of Notch in order to
examine this question. In zygotic numb mutants, sibling neuron fate alterations
('A/B' to 'A/A') occur infrequently or do not occur in some sibling pairs; depletion of both maternal and zygotic numb causes sibling neurons to acquire equalized fates ('A/A') with near-complete expressivity. In rca1;numb double mutant embryos,
binary cell fate undergoes a change ('B' to 'A') in several GMCs as well.
GMC 4-2a frequently adopts the 'A' fate of RP2 sibling in
rca1;numb or hs-N
intra;rca1 embryos. In contrast, GMC 4-2a always acquires the 'B' fate of RP2 in rca1 mutants alone. Notably, it was observed that the 'B' to 'A' fate
change (RP2 to RP2 sibling) occurs with greater frequency
in rca1;numb double mutants than the RP2/sib ('A/B') to
sib/sib ('A/A') fate change that occurs in numb mutants alone (Lear, 1999).
Thus GMCs in cycA and rca1
mutants differentiate as neurons: they assume the 'B' fate
normally taken by one of their sibling progeny. These
GMC fate decisions correspond to Notch pathway mutants
('B/B'), and they oppose the fate changes observed in
embryos lacking numb ('A/A').
The loss of zygotic numb or constitutive activation of Notch in a rca1 background allows for a
binary fate switch in GMCs: GMCs often differentiate as the
'A' sibling in the context of these mutations. These results
indicate that activation of the Notch pathway causes
GMCs to adopt the 'A' neuronal fate. Thus, fate choice in
non-dividing GMCs appears to occur in much the same way
that binary fate decisions occur in sibling neurons.
In some
models of asymmetric division, a specific factor required
to attain one of the sibling fates is produced only upon
progression of the cell cycle. The observation that GMCs
can attain the fate of either sibling neuron indicates that
gene products dependent upon GMC division are not
required in this fate decision (Lear, 1999).
Finally, expression of Delta in the mesoderm is
sufficient to attain both sibling fates. In Dl
mutant embryos, misspecification of neuroectodermal cells results in an excess of neuroblasts and their resulting neuronal progeny. Additionally, binary cell fate alterations are
observed at the sibling neuron level. In wild-type embryos, Vnd protein is expressed in
pCC but not aCC; in Dl mutants, the numerous GMC 1-1a progeny all lack Vnd
expression, indicating that all
of these neurons acquire the aCC fate. The
twist-GAL4 line was used to drive Dl expression in the
embryonic mesoderm. In a wild-type background, expression of ectopic Dl using the twist-
GAL4 line appears to have little effect on the embryo;
significantly, no effect in CNS cell fate specification is observed. When Dl is expressed in the embryonic
mesoderm of a Dl
homozygous mutant using twist-GAL4,
many sibling neuron pairs attain differential fates ('A/B'). Specifically, at least one
aCC and one Vnd-expressing
pCC is observed in each thoracic/abdominal
hemisegment of these embryos. Thus, cell fate specification
of the aCC/pCC sibling pair is rescued by expression of Dl
in the mesoderm. Thus, these results indicate that the intrinsic determinant Numb is absolutely required to attain differential
sibling neuron fates. While the extrinsic factors Notch and Delta are also required to attain both fates, these results indicate that Delta signal can
be received from outside the sibling pair (Lear, 1999).
In the Drosophila CNS glial cells
are known to be generated from glioblasts, which produce
exclusively glia or neuroglioblasts that bifurcate to produce
both neuronal and glial sublineages. The
genesis of a subset of glial cells, the subperineurial glia
(SPGs), involves a new mechanism and requires Notch. SPGs share direct sibling
relationships with neurons and are the products of
asymmetric divisions. This mechanism of specifying glial
cell fates within the CNS is novel and provides further
insight into regulatory interactions leading to glial cell
fate determination. Furthermore, Notch
signaling positively regulates glial cells missing
expression in the context of SPG development (Udolph, 2001).
In order to better understand how a complete lineage of a
specific NGB with all its progeny, including its glial cells,
might be created, NB1-1 was chosen for a detailed analysis. NB1-
1 has been extensively used for cell fate specification studies
and a sound basis of information about this NB lineage is
available. NB1-1 is a NB that develops differential lineages
in the thoracic versus the abdominal segments. Focus was placed on the abdominal NB1-1A because only
these abdominal NB1-1 lineages contain glia. In addition to the
aCC/pCC sibling neurons, which are the progeny of the first
GMC produced from this lineage, NB1-1A generates 2 to 3
glial cells and 4 to 5 clustered interneurons (cN), yielding a
total of 9 to 10 cells. The three glial cells belong to the group
of subperineurial glia (SPG) that lie at the periphery of the
nerve cord and enwrap the entire ventral nervous system. Two of the glia, the A- and B-SPGs, can be found in dorsal positions, with a third
glia, the LV-SPG, located at ventral positions of the nerve cord.
All SPGs, including the A- and B-SPG and LV-SPG of NB1-1A, are specifically labelled by two enhancer trap lines, M84
and P101 (Udolph, 2001 and references therein).
As a first step toward elucidating the origin of the glial cells
of the NB1-1A lineage, the effects of loss of
function mutants in several genes, Notch, mastermind (mam)
and numb, which are known to affect the resolution of distinct
sibling cell fates, were tested for their effect on the development of A-, B- and
LV-SPGs. Embryos hemizygous/homozygous for a conditional Notch allele, Nts1, and also carrying one copy each of M84 and P101 (Nts1/M84/P101) were subjected to the non-permissive temperature of 29°>C after 6 hours of development. This regime allows Notch to function during the singling out of NBs and removes Notch during the crucial period when it is required for sibling cell fate resolution. Double staining with
anti-Eve and anti-ß-gal was performed. As expected,
in most hemisegments, Nts1/M84/P101 embryos duplicate the RP2 neuron at the expense of its sibling cell. Moreover, in
96% of the hemisegments, M84/P101+ cells could not
be found in typical dorsal or ventral positions. It is concluded that Notch function is required for the specification of the M84/P101 positive A-, B- and LV-SPGs. In wild-type embryos, M84/P101 is expressed in about eight SPGs per hemisegment, including the A- and B-SPGs and the LV-SPG (Udolph, 2001).
mastermind, which has been linked to the Notch signaling pathway by its
genetic interactions with Notch and its strikingly similar
phenotype in early and late neurogenesis, was tested. mam acts downstream of Notch during sibling cell fate
specification in the embryonic nervous system. The hypomorphic mam345 allele used in this
study shows only a mild hypertrophy of the nervous system but
clearly has an effect on sibling cell fate specification. A severe reduction (94%) of P101+ cells was observed in mam345;P101 embryos
similar to that seen with Nts1/M84/P101 embryos.
These data suggest that both genes are strictly required for the
specification of SPGs, most likely in a linear pathway.
However, it is unclear how Notch acts in the specification of
the SPGs. The possibility is considered that SPG glial cells could
arise from a series of asymmetric cell divisions, with Notch
being required to specify the glial daughters of these divisions (Udolph, 2001).
Based on its function as a negative regulator of Notch
signaling, the expected numb phenotype is opposite that of Notch
in terms of sibling cell fate transformation. The P101
expression pattern was tested in the background of a strong numb
mutation. In contrast to Notch and mam, additional
P101+ cells were found in the vicinity of the aCC/pCC position. In most
of the examined hemi-neuromeres, up to four ß-gal-positive cells were detected in dorsal positions close to aCC/pCC. This is indicative of a duplication of the A- and B-SPGs. Additional
P101+ cells with glial morphology were found in lateral and ventral
positions of the nerve cord, presumably duplications of other
SPGs. These findings are consistent with an
asymmetric cell division model for the genesis of the SPGs (Udolph, 2001).
The bipotential ganglion mother cells, or GMCs, in the
Drosophila CNS asymmetrically divide to generate two
distinct post-mitotic neurons. The midline repellent Slit (Sli), via its receptor Roundabout (Robo), promotes the terminal asymmetric division of
GMCs. In GMC-1 of the RP2/sib lineage, Slit promotes
asymmetric division by down regulating two POU proteins,
Nubbin and Mitimere. The down regulation of these
proteins allows the asymmetric localization of Inscuteable,
leading to the asymmetric division of GMC-1. Consistent
with this, over-expression of these POU genes in a late
GMC-1 causes mis-localization of Insc and symmetric
division of GMC-1 to generate two RP2s. Similarly,
increasing the dosage of the two POU genes in sli mutant
background enhances the penetrance of the RP2 lineage
defects whereas reducing the dosage of the two genes
reduces the penetrance of the phenotype. These results tie
a cell-non-autonomous signaling pathway to the
asymmetric division of precursor cells during neurogenesis (Mehta, 2001).
The symmetric division of GMCs in sli mutants is similar to
that observed in insc, Notch or rapsynoid (raps; also known as pins) mutants and opposite that of nb.
Previous results show that the cytoplasmic adaptor protein Insc
is required for the asymmetric division of GMC-1 into RP2 and
sib. During GMC-1 division, Insc protein localizes to the apical side and Nb to the basal side. The Nb-negative daughter cell becomes specified as
sib by Notch signaling whereas the cell that inherits Nb
becomes an RP2 owing to the blocking of Notch signaling by
Nb. Thus, in insc mutants, both cells inherit Nb and are
specified as RP2 while in nb mutants both progeny becomes
sib. Given the similarity of sli, sim and insc mutant phenotypes, the relationship between Sli and Insc was examined. First, in sli mutants the localization of Insc in GMC-1, when examined,
is not asymmetric. About 7% of the hemisegments show this phenotype. A similar non-localization of Insc was also observed in GMC1-1a of the aCC/pCC lineage. In raps mutant embryos, Insc is also not localized
and as in sli the GMC-1 divides symmetrically to generate two
RP2s. Thus, failure to localize Insc in these GMCs in sli mutants is responsible for their symmetric mitosis. In insc;nb double mutants both the daughters of GMC-1 are specified as sib by Notch signaling. In sli;nb (or sim;nb) double mutant embryos also, both the progeny of GMC-1 adopt a sib fate. Thus, Sli is required upstream of Nb
during the asymmetric division of GMC-1. Since the GMC-1
symmetrically divides to yield two RP2s in Notch;nb double
mutants, and two sibs in sli;nb double
mutants, Sli is also upstream of Notch signaling during the
asymmetric division of GMC-1. These results also indicate that when the GMC-1 in sli mutants symmetrically divides, both daughters inherit Nb (Mehta, 2001).
In Drosophila, neuroblasts undergo typical asymmetric divisions to produce another
neuroblast and a ganglion mother cell. At mitosis, neural fate determinants, including
Prospero and Numb, localize to the basal cortex from which the ganglion mother cell buds
off; Inscuteable and Bazooka, which regulate spindle orientation, localize apically. Lethal (2) giant larvae (Lgl) is essential for asymmetric
cortical localization of all basal determinants in mitotic neuroblasts, and is therefore
indispensable for neural fate decisions. Lgl, which itself is uniformly cortical, interacts with
several types of Myosin to localize the determinants. Another tumor-suppressor protein,
Lethal discs large (Dlg), participates in this process by regulating the localization of Lgl. The
localization of the apical components is unaffected in lgl or dlg mutants. Thus, Lgl and Dlg
act in a common process that differentially mediates cortical protein targeting in mitotic
neuroblasts, and that creates intrinsic differences between daughter cells (Ohshiro, 2000).
In mitotic neuroblasts, the Prospero transcription factor and Numb, an antagonist of Notch
signaling, associate with their respective adapter proteins, Miranda and Partner of numb
(Pon), and thereby localize to the basal cortex. In contrast, Inscuteable (Insc), Bazooka (Baz)
and Partner of Inscuteable (Pins) form a ternary complex at the apical cortex independently of
the basal determinants. However, the mechanisms that underlie the asymmetric protein
sorting in neuroblasts are not known. To address this issue, chromosomal
deficiencies have been sought that affect the subcellular distribution of Miranda. Such screening identified the
lgl tumor-suppressor gene that encodes a protein containing WD40 repeats. In
wild-type neuroblasts, Miranda, which localizes apically during interphase, accumulates at the
basal cortex upon mitosis after a transient spread into the cytoplasm. In germline
clone embryos lacking both maternal and zygotic lgl activity (lglGLC embryos), Miranda does
not localize asymmetrically in mitotic neuroblasts, but rather is distributed uniformly
throughout the cortex as well as in the cytoplasm, where it is concentrated along microtubule
structures. Consequently, Miranda segregates into both the daughter neuroblast
and the ganglion mother cell (GMC). Numb and Pon are also
distributed uniformly at the cortex and in the cytoplasm (Ohshiro, 2000).
It would be expected that the abnormal distribution of Numb and Miranda in lgl mutant neuroblasts
results in incorrect determination of neural cell fate. Given the difficulty of monitoring neural cell
fate in severely distorted lglGLC embryos, this prediction was tested by analyzing the lineage of
the external sensory organ in the notum, in which all cell divisions are asymmetric and sibling
cells adopt distinct fates as a result of the asymmetric inheritance of Numb.
Sensory organ precursor cells in this lineage segregate Numb into a daughter cell pIIb, which
subsequently generates three inner cells (a glial cell, a neuron and a sheath cell). The sibling
pIIa cell divides into two outer cells constituting the external sensory structure, a hair and a
socket. Exposure of lglts3 mutant larvae to 29°C during external sensory organ development
mislocalizes Numb in mitotic precursor cells, as observed in neuroblasts, and
often transforms inner cells into outer cells resulting in duplicated external sensory structures,
a phenotype expected from loss of numb function. Indeed, this notum phenotype
is enhanced by reducing the numb gene dosage by half. Equal partition of Numb between sibling cells would result in numb gain
of function phenotypes because the half dose of numb is enough for correct cell-fate
decisions. The observed numb loss-of-function phenotype therefore suggests that a reduction
in lgl activity does not only equalize Numb distribution between sibling cells but also
attenuates numb function, consistent with the observation of cytoplasmic Numb in the lgl
mutants. Conversely, the presence of an extra numb gene induces opposite phenotypes under
the lgl mutant condition. The outer cells are frequently transformed into the
inner cells, resulting in the loss of the external sensory structure. This appearance of
the numb gain of function phenotypes is simply explained by the fact that the partition of
additional Numb from the transgene into both sibling cells raises numb activities over the
threshold necessary to suppress Notch function in both cells. These data thus indicate that Lgl is
essential in neural fate decisions through cortical targeting of cell-fate determinants (Ohshiro, 2000).
Apoptosis is prevalent during development of the central nervous system, yet very little is known about the signals that specify an apoptotic cell fate. The role of Numb/Notch signaling in the development of the serotonin lineage of Drosophila has been studied; it is necessary for regulating apoptosis. When Numb inhibits Notch signaling, cells undergo neuronal differentiation, whereas cells that maintain Notch signaling initiate apoptosis. The apoptosis inhibitor p35 can counteract Notch-mediated apoptosis and rescue cells within the serotonin lineage that normally undergo apoptosis. Furthermore, tumor-like overproliferation of cells is observed in the CNS when Notch signaling is reduced. These data suggest that the distribution of Numb during terminal mitotic divisions of the CNS can distinguish between a neuronal cell fate and programmed cell death (Lundell, 2003).
The segmented Drosophila nerve cord develops from stereotyped
division of 30 neuroblasts (NB) in each hemisegment. A pair
of serotonergic neurons in each hemisegment arises from NB7-3. The divisions of the NB7-3 lineage have recently been
determined using a combination of molecular markers and clonal analysis. NB7-3
produces three GMCs. GMC-1 produces two neurons: GW, a motoneuron, and EW1,
the more medial serotonergic neuron. GMC-2 produces EW2, the more lateral
serotonergic neuron. GMC-3 produces EW3, a neuron that synthesizes the
neuropeptide corazonin. The GW neuron projects an axon ipsilateral and
posteriorly, and the three EW interneurons all project axons anterior to the
posterior commissure (Lundell, 2003 and references therein).
Several genes have been shown to be essential in the differentiation of the
NB7-3 lineage. Sequential expression of the segmentation transcription
factors, Hunchback->Krüppel->Pdm1, within neuroblasts has been
shown to be important in the development of several lineages, including NB7-3. The subsequent GMCs and neuronal progeny maintain the expression of the transcription factors that are present in the NB at their birth. In the case
of NB7-3, Hunchback (Hb) is expressed only in GMC-1 and its progeny, and is
both necessary and sufficient to define the fates of these cells.
Krüppel (Kr) is expressed in both GMC-1 and GMC-2 and is necessary and
sufficient to establish the fate of the EW2 serotonergic neuron. Pdm1
is expressed primarily in EW2. Differentiation of the NB7-3 lineage is also affected by
mutations in wingless (wg) and other members of the Wingless
signaling pathway such as, engrailed (en), hedgehog
(hh) and patched (ptc). In
addition, mutations in the transcription factors eagle (eg) and
huckebein (hkb) also disrupt NB7-3 differentiation. eg has
been shown to suppress a rough eye phenotype caused by the overexpression of
Ras1, suggesting that eg may be involved in Ras signaling.
hkb is regulated by both en and hh in the NB7-3
lineage. The exact relationship between these genes and signal transduction pathways within the NB7-3 lineage remains to be determined (Lundell, 2003 and references therein).
The results of this study demonstrate that the intercellular Notch
signaling pathway can be modulated during terminal divisions of the CNS to
direct a choice between neuronal development and programmed cell death.
The division of GMC-1 produces two distinct neuronal cell fates: the EW1
interneuron and the GW motoneuron. In this division, genetic alteration in the
expression of Notch leads to switching between these two cell fates.
A loss of Notch activity in spdo mutants leads to two
Ddc/Hb-expressing EW1 cells and the overexpression of Notch leads to two Zfh-1
expressing GW cells. Therefore, Notch signaling must be inactivated during
development of the EW1 neuron. Numb appears to have a minor role in this
inactivation. In a numb1 mutant, 7% of the hemisegments do
not develop an EW1 neuron, and a similar number of numb1
hemisegments show two Zfh-1-expressing GW cells. This transformation from an
EW1 cell fate to a GW cell fate is what one would expect if Numb were
inhibiting Notch. However, most EW1 neurons develop normally in a
numb1 mutant and do not convert to the GW cell fate.
Therefore, inactivation of Notch signaling in EW1 is mostly independent of
Numb function. One possible explanation is that EW1 has a factor that is
redundant for Numb function, which can inhibit Notch signaling and is capable
of masking the effect of a numb1 mutation in most
hemisegments. The unique expression of Hb in GMC-1 progeny could be
responsible for establishing this redundancy. However, if a redundant
Numb-like factor does exist, it is insufficient to protect EW1 during
expression of the UAS-NotchACT transgene (Lundell, 2003).
The A8 segment is unique in that it has only a single serotonergic neuron
instead of the pair of serotonergic neurons found in the more anterior
segments. In a wild-type fly, this cell appears to be a derivative of GMC-2,
because it expresses Zfh-2, but in a numb1 mutant, this
cell appears to be a derivative of GMC-1, because it expresses Hb. A single
Hb/Ddc-expressing cell in A8 is identical to the phenotype in the more
anterior segments of a numb1 mutant. This suggests that in
a numb1 mutant an EW1 cell is the default developmental
pathway for this lineage. One possibility is that the redundant
Notch-inactivating mechanism proposed for EW1 is induced only in the
presence of a numb mutation. This would explain why the A8 EW1 cell
is seen only in the numb mutant and not in a wild-type animal. If
this were true, then the preservation of EW1 cells in a numb mutant
would be due to the mutation itself. Until a putative redundant factor is
identified it is impossible to determine whether it is expressed normally in
wild-type animals or is expressed only in numb mutant animals (Lundell, 2003).
Like GMC-1, most GMCs divide, producing two progeny cells. However, GMC-2
and GMC-3 of the NB7-3 lineage produce only one neuron. It has been
suggested that the mitotic sisters of EW2 and EW3 may undergo apoptosis. This
idea is supported by the detection of apoptotic cells with TUNEL in the
wild-type NB7-3 lineage and experiments with the apoptosis inhibitor p35,
which can produce ectopic Ddc and corazonin-containing cells. The origin of
the ectopic cells within NB 7-3 has not been formally determined by lineage
tracing; however, the hypothesis that they are mitotic sisters of EW2 and EW3
is supported by the observations that GMC-2 and GMC-3 progeny often appear as
mitotic pairs and that ectopic NB7-3 cells are immunoreactive for Zfh-2 (Lundell, 2003).
During the divisions of GMC-2 and GMC-3, genetic alterations in the
expression of Notch lead to a switching between a neuronal cell fate and
apoptosis. A reduction of Notch signaling with either
spdoG104 or UAS-Numb embryos produces ectopic
NB7-3 cells that express Zfh-2. Conversely, the overexpression of Notch in
either UAS-NotchACT or numb1 embryos
led to an increase in TUNEL labeling of GMC-2 and GMC-3 progeny. Additionally,
inhibiting apoptosis with UAS-p35 or reducing Notch activity with
spdoG104 can rescue the numb1
phenotype. It is hypothesized that during the divisions of GMC-2 and GMC-3, Numb
partitions asymmetrically into EW2 and EW3 where it inactivates Notch
signaling and leads to neuronal development. The mitotic sisters of EW2 and
EW3 do not receive Numb, maintain Notch signaling and undergo apoptosis. The
difficulty in detecting wild-type hemisegments that have more than four
immunoreactive Eg cells, suggests that any other cells produced during
divisions of the NB7-3 lineage quickly undergo apoptosis (Lundell, 2003).
Ectopic Eg cells in the NB7-3 lineage can be induced at stage 15 by
H99, UAS-Numb, spdoG104 and UAS-p35. However, the ability of these alleles to produce ectopic Ddc and corazonin-containing
neurons at later stages is variable. No significant
ectopic Ddc or corazonin-containing cells were detected in either H99 or
UAS-Numb CNS. For UAS-Numb it was shown that the ectopic Eg
cells detected at stage 15 can undergo apoptosis. spdoG104
mutants produce only ectopic Ddc cells, but the reduction in the number of
corazonin-containing cells in general suggests that either GMC-3 does not
consistently form in these mutants or that GMC-3 progeny may convert from a
corazonin-containing cell fate to a serotonergic cell fate. UAS-p35
mutants produce both ectopic Ddc and corazonin-containing cells at low
frequency, but the allele is much more efficient at rescuing the EW neurons in
numb1 and UAS-Notch mutants. Therefore, apoptosis
is harder to reverse in cells that normally undergo apoptosis, than in the
cells genetically induced to undergo apoptosis. The ability of these various
alleles to produce ectopic Ddc- and corazonin-containing cells could be
influenced by mutant effects they cause outside the NB7-3 lineage or may
reflect different roles they have in the apoptotic pathway. The mechanism by
which Notch induces apoptosis in the NB7-3 lineage remains to be determined,
but the apoptotic genes reaper, grim and hid may be involved
because all three of these genes are deleted in the H99 allele (Lundell, 2003).
Notch-induced apoptosis in the NB7-3 lineage will probably be regulated by
other factors in addition to Numb. The Ras signaling pathway has been shown to
inhibit Notch-induced apoptosis in the Drosophila pupal retina.
Wingless has been shown to mediate Notch signaling and
mutations in the Wingless pathway can lead to ectopic serotonergic cells. It will be a challenge to determine how these different signaling pathways interact to specify apoptosis within the NB7-3 lineage (Lundell, 2003).
The tumor-like expansion of Ddc-expressing cells observed in heterozygous
spdoG104 larvae suggests that Notch-induced apoptosis may
be essential for regulating cell proliferation. This spdo phenotype
is reminiscent of three tumor-suppressor genes; discs large
(dlg), lethal giant larvae (lgl) and
scribble (scrib), which produce tumors in the CNS and
imaginal disks. Interestingly, these three genes work in a common pathway that regulates cell polarity, and lgl and dlg have been shown to be essential for the distribution of Numb and other asymmetric determinants. Further
investigation will be necessary to determine if spdo is part of this
same mechanism and exactly how spdo mutants inhibit Notch signaling.
Spdo expression is ubiquitous throughout embryogenesis and persists through
the larval stages and into adults. If a spdo mutation can alter the response of
the Notch receptor to environmental cues that induce apoptosis, one would
expect to see overproliferation in additional tissues (Lundell, 2003).
Numb/Notch signaling is also known to affect development of the midline dopaminergic
cells. The expression of Ddc is essential to the biosynthesis of both
serotonin and dopamine. Anti-Ddc antibody detects not
only the serotonergic neurons, but also midline dopamine neurons. As a
consequence of using Ddc as a marker for the serotonin lineage, a number of
observations were made about the development of midline dopamine cells. In a numb1 mutant very few midline dopamine
cells are detectable with Ddc. spdoG104 mutants produce ectopic dopamine cells and can rescue dopamine cells in the numb1 mutant phenotype. Thus,
Numb/Notch signaling also has a role in the development of midline dopamine
cells, but further investigation into the significance and whether apoptosis
is involved in this lineage will require lineage analysis to determine the
origin of the midline dopamine cells (Lundell, 2003).
Loss of cell polarity and cancer are tightly correlated, but proof for a causative relationship has remained elusive. In stem cells, loss of polarity and impairment of asymmetric cell division could alter cell fates and thereby render daughter cells unable to respond to the mechanisms that control proliferation. To test this hypothesis, Drosophila larval neuroblasts were generated containing mutations in various genes that control asymmetric cell division and then their proliferative potential was assayed after transplantation into adult hosts. It was found that larval brain tissue carrying neuroblasts with mutations in raps (also called pins), mira, numb or pros grew to more than 100 times their initial size, invading other tissues and killing the hosts in 2 weeks. These tumors became immortal and can be retransplanted into new hosts for years. Six weeks after the first implantation, genome instability and centrosome alterations, two traits of malignant carcinomas, appeared in these tumors. Increasing evidence suggests that some tumors may be of stem cell origin. These results show that loss of function of any of several genes that control the fate of a stem cell's daughters may result in hyperproliferation, triggering a chain of events that subverts cell homeostasis in a general sense and leads to cancer (Caussinus, 2005).
Malignant transformation and loss of cell polarity are tightly correlated in human carcinomas. Likewise, Drosophila larval tissues with mutations in dlg1, l(2)gl or scrib have impaired apicobasal polarity and neoplastic growth in the imaginal epithelia and nervous system. There are several hypotheses to explain how loss of polarity contributes to neoplastic transformation. Most of them involve models in which changes in cellular architecture impinge directly on the cell cycle either by inhibiting signals that restrain cell proliferation or by enhancing mitogenic pathways. An alternative hypothesis is that loss of polarity in stem cells that divide asymmetrically impairs the mechanisms that specify the fate of the resulting daughter cells. If these daughter cells are unable to follow their normal developmental program, they may not respond to the mechanisms that control proliferation in the wild-type lineage (Caussinus, 2005).
Drosophila neuroblasts are stem cells whose asymmetric cell-division machinery is fairly well characterized and thus provide a good model to test this hypothesis. In the embryo, Insc integrates into the apical cortex of two neuroblast protein complexes, Baz-DmPar6-aPKC and Gialpha-Raps, by associating with Baz and Raps. These two complexes mediate the basal localization of Mira and Pon and their interacting proteins, Pros and Numb, whose segregation into the ganglion mother cell (GMC) is required for the unequal fate of the two neuroblast daughter cells. The basal localization of Mira and Pros, as well as the spindle orientation and asymmetry of daughter-cell sizes, require the functions provided by dlg1, l(2)gl and scrib. Larval neuroblasts originate from quiescent embryonic neuroblasts, and their asymmetric division seems to be controlled by the same molecular complexes, although minor differences have been reported (Caussinus, 2005).
To assess the effect of disrupted stem-cell asymmetric division on cell proliferation, larval neuroblasts were generated with mutations in aPKC, raps, mira, pros or numb and their proliferation potential was assayed after transplantation into adult hosts. No substantial growth of 101 pieces of wild-type larval brains were observed 2 weeks after transplantation. Similar results were observed for 109 implants that carried homozygous aPKCk06403 clones, none of which grew to any noticeable extent. In contrast, pieces of brains from rapsP89/raps
P62 larvae or from larvae carrying homozygous numb03235, miraZZ176 or pros
17 clones grew to more than 100 times their original size, severely damaging and displacing the host's organs in the abdomen. Of the 103 flies studied in detail, 92% had one or more small tumor colonies derived from the implanted tissue but located at a long distance from the point of injection. The efficiency of tumor development ranged from 8% for numb03235 clones to 20% for rapsP89/rapsP62 tissue (Caussinus, 2005).
To assess further the growth potential of these tumors, they were cut into pieces and reimplanted into new hosts. More than 90% of these flies developed a tumor, even when they were implanted with numb 03235 tissue that had initially developed tumors in only 8% of implanted adults. This result suggests that the growing tumor mass adapts itself very rapidly to its new environment. Pieces of brain lobes from 9- to 12-d-old homozygous brat
k06028 and l(3)mbt
ts1 larvae, in which overgrowth was already apparent, developed tumors in 91% and 58%, respectively, of the implanted hosts (Caussinus, 2005).
All the tumors described here have been maintained in the laboratory, some for more than 2 years. This shows that the transformed cells became immortal and can proliferate without end, in contrast to cells of wild-type imaginal discs implanted into adult hosts, which remain alive after years of culture but very rarely proliferate. Among the established cell lines, substantial differences were observed in speed of growth, host lifespan or frequency or average number of additional tumor colonies, that could be attributed to the mutant background from which the tumors originated. Using the same criteria, these tumors were indistinguishable from dlg1, l(2)gl and scrib neuroblastomas (Caussinus, 2005).
Attempts were made to determine the kinds of cells that could be found in these tumors. Using green fluorescent protein as a clonal marker, it was observed that in tumors derived from tissue carrying numb
03235, miraZZ176 or pros17 clones induced by mitotic recombination, neither the wild-type twin nor the heterozygous background cells were able to proliferate upon implantation and were lost within 2 weeks. These cells accounted for most of the implanted mass, and so their inability to hyperproliferate provided a valuable internal control to substantiate the conclusion that tumor growth in this assay required the loss of the genes under study and was not just the result of dissection and transplantation into adult hosts. It also showed that the tumor growth induced by the loss of function of these genes was cell-autonomous (Caussinus, 2005).
Immunofluorescence staining for cell-specific markers identified the neuroblasts as relatively large cells, 8-12 microm in diameter, that expressed Mira. In miraZZ176 tumors, neuroblasts were identified by the expression of Wor. Ganglion cells were identified as small cells, 4-6 microm in diameter, that did not express Mira but did express Pros or, in pros
17-derived tumors, Numb. The intermediately sized cells that did not express Pros, some of which showed weak Mira staining, might be GMCs. Neuroblasts accounted for most of the mitotic activity observed in these tumors (86%). Daughter-cell size and Mira segregation during mitosis were symmetric in neuroblasts derived from rapsP89/rapsP62 tumors but asymmetric in those derived from numb03235 and pros
17 tumors. Daughter-cell size was also asymmetric in neuroblasts from miraZZ176 tumors (Caussinus, 2005).
Neither neuroblasts nor ganglion cells were markedly diluted or over-represented as the tumors aged from host to host. Therefore, like l(2)gl and dlg1 tumors, the tumors derived from numb03235, miraZZ176, pros17 and raps
P89/rapsP62 were neuroblastomas that resulted from the uncontrolled division of neuroblast stem cells and were largely composed of the undifferentiated cell types that belong to this lineage. The mechanism by which these tumors grew is not understood, but it must account for the observed continuous expansion of both the neuroblast and the ganglion cell populations. One plausible mechanism could be a low frequency of neuroblast divisions that generate two neuroblast daughters. Real-time analysis of cell proliferation in these tumors may provide an answer to this issue (Caussinus, 2005).
In most solid human tumors, malignancy is very often correlated with genome instability, which is thought to contribute to multistage carcinogenesis. As in most animal cells, the frequency of natural cases of genome instability in wild-type Drosophila neuroblasts and GMCs is low (less than 10-3). This is also the case in numb03235, miraZZ176, pros
17 and rapsP89/raps
P62 tumors shortly after transplantation. In 40-d-old tumors, however, 10%-15% of the cells presented different kinds of karyotype defects. Of the 340 karyotypes obtained from numb, mira, pros and raps tumors, 62% included segmental aneuploid; 9% were monosomic, trisomic or tetrasomic with respect to one or more chromosomes; 6% were triploid or tetraploid; and the remaining 23% included cells that could not be karyotyped owing to very high levels of ploidy, chromosome fragmentation or chromosome condensation (Caussinus, 2005).
The karyotypes obtained from cells in a single tumor were as different from one another as they were from the karyotypes of cells from other tumors, and none of the tumor lines that were established presented a distinct set of chromosome aberrations. Therefore, no substantial differences were observed attributable to the mutant condition that originated the tumor. In most tumor lines, the frequency of cells that contained abnormal karyotypes did not change noticeably over time, with one exception: 3 months after the first implantation, genome instability affected more than 95% of the cells in mirTF, a tumor line derived from miraZZ176. The absence or very low incidence of genome instability during the first round of implantation suggests that genome instability did not cause tumor formation in these tumor lines. But the onset of genome instability correlates well with a marked increase in the frequency of hosts that developed a tumor in later transplantations. Therefore, the possible contribution of genome instability to the evolution of these tumors remains to be assessed. Genome instability has also been reported in l(2)gl neuroblastomas (Caussinus, 2005).
In mammalian carcinomas, genome instability is tightly correlated with severe alterations of the centrosome cycle that affect the number of centrosomes per cell as well as centrosome size and shape. Supernumerary centrosomes can result in multipolar spindles and contribute to the generation of aneuploidy. Like the DNA cycle, the centrosome cycle is tightly controlled in wild-type neuroblasts, so that cells that have an abnormal number of centrosomes are exceptionally rare in wild-type tissue. This was not the case in numb03235, mira
ZZ176, pros17 or raps
P89/rapsP62 tumors: forty days after the first implantation, 15%-20% of those cells had more than two centrosomes. Some of these centrosomes were irregularly shaped, and their size range was much wider than that of control cells. A fraction of these could be centriole-less aggregates of pericentriolar material. The cells that had supernumerary centrosomes seemed to be hyperploid (Caussinus, 2005).
None of the mutant conditions from which these tumors originated has been reported to affect chromosome segregation or the centrosome cycle, which were both unaffected in early tumors. In addition, the cells of wild-type imaginal discs that have been kept for years in adult hosts maintain a stable genome and can differentiate into adult structures. Therefore, genome instability and impaired centrosome cycles in numb
03235, miraZZ176, pros17 and rapsP89/rapsP62 tumors cannot be considered a consequence of the mutant background or long-term exposure to the adult abdomen environment. Rather, the onset of genome instability and centrosome alterations suggests that once the mechanisms that control cell proliferation have been over-ridden, hyperproliferation triggers a chain of events that subverts cell homeostasis in a very general sense, including the DNA and centrosome cycles (Caussinus, 2005).
In summary, neoplastic transformation of Drosophila larval neuroblasts can be triggered by perturbing several of the functions that mediate asymmetric stem-cell division. In terms of growth rate, cell types, metastatic activity and extent of genome and centrosome instability, the resulting tumors are essentially indistinguishable from one another, regardless of the mutant from which they derive. The main conclusion that can be drawn from these data is that these tumors might have a common etiology: perturbation of neuroblast polarity and the resulting impairment of cell-fate determination. This argument is strengthened by the case of the homeobox-containing transcription factor Pros, which lies downstream of the other genes required for neuroblast asymmetric division (Caussinus, 2005).
The tumors in this study are practically indistinguishable from the neuroblastomas that arise in adults implanted with pieces of dlg1, l(2)gl or scrib mutant larval brains. Because these three neoplastic tumor suppressors are required for multiple aspects of neuroblast asymmetric cell division, including the basal localization of Mira, Numb and Pros, mislocalization of these proteins might explain, at least partially, the uncontrolled cell proliferation produced by loss of dlg1, l(2)gl or scrib function in larval neuroblasts (Caussinus, 2005).
The unequal segregation of cell-fate determinants resulting from asymmetric cell division is a fundamental mechanism for generating cellular diversity during development, organ homeostasis and repair. If impaired segregation of cell-fate determinants can cause the hyperproliferation of larval neuroblasts of Drosophila, it may similarly affect tissue stem cells in other species. At the moment, however, any parallel to stem-cell models of human cancer remains purely speculative. Consistent with this hypothesis, the inactivation of both Numb and Numb-like in the mouse dorsal forebrain leads to impaired neuronal differentiation, hyperproliferation of neural progenitors and delayed cell-cycle exit. In addition, loss of Lgl1 (also called Mlgl or Hugl), one of the two L(2)gl homologs in the mouse, results in a failure to asymmetrically localize Numb and leads to severe brain dysplasia (Caussinus, 2005).
In most human tumors, the identity of the first carcinogenic cell remains elusive. Indirect but growing evidence suggests that in some cases, the founders may be stem cells. Stem cells are self-renewing, have limitless replicative potential and produce differentiating cells, three features found in many cancers. Carcinomas occur in tissues that are maintained by a continuous supply of differentiating daughter cells originating from stem-cell division. Moreover, some of the signaling pathways that control stem-cell self-renewal, like the Notch, Wnt-ß-catenin and Hedgehog pathways, are known to have a role in carcinogenesis in these tissues. The results show that inactivation of any of several molecular mechanisms that control the asymmetry of the segregation of cell-fate determinants during stem-cell division may result in hyperproliferation of the stem-cell compartment and could contribute to cancer (Caussinus, 2005).
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