sanpodo In Drosophila, asymmetric division occurs during proliferation of neural
precursors of the central and peripheral nervous system (PNS), where a
membrane-associated protein, Numb, is asymmetrically localized during cell
division and is segregated to one of the two daughter cells (the pIIb cell)
following mitosis. numb has been shown genetically to function as an antagonist
of Notch signaling, and also as a negative regulator of the
membrane localization of Sanpodo, a four-pass transmembrane protein required for
Notch signaling during asymmetric cell division in the central nervous system
(CNS). lethal giant
larvae (lgl) is required for Numb-mediated inhibition of Notch in the
adult PNS. Sanpodo is
expressed in asymmetrically dividing precursor cells of the PNS and Sanpodo
internalization in the pIIb cell is dependent cytoskeletally-associated Lgl. Lgl
specifically regulates internalization of Sanpodo, likely through endocytosis,
but is not required for the endocytosis Delta, which is a required step in the
Notch-mediated cell fate decision during asymmetric cell division. Conversely,
the E3 ubiquitin ligase Neuralized is required for both Delta endocytosis and
the internalization of Sanpodo. This study identifies a hitherto unreported role
for Lgl as a regulator of Sanpodo during asymmetric cell division in the adult
PNS (Roegiers, 2005).
This analysis of Sanpodo function in the adult PNS suggests that, as in the embryo,
Sanpodo is expressed only in asymmetrically dividing precursor cells and is required for
cell fates dependant on high levels of Notch signaling,
perhaps through the direct interaction between Sanpodo and the full length Notch
receptor. Sanpodo also interacts directly with Numb
in vivo, and in both the embryonic CNS and the adult
PNS, numb inhibits plasma membrane association of Sanpodo. Therefore, it
appears that Sanpodo plays a similar role in asymmetrically dividing precursor cells in
both the CNS and PNS in Drosophila (Roegiers, 2005).
Although there are many similarities between the mechanisms of asymmetric
cell divisions in embryonic neuroblasts and adult sensory organ precursor cells,
one difference involves the role of lgl. In neuroblasts, lgl is
required along with another cortical tumor suppressor, dlg, to target
Numb to a basal crescent during mitosis, whereas in pI cells, only dlg is required for Numb crescent
formation. While
lgl is dispensable for segregation of Numb to the pIIb cell following pI
cell mitosis, lgl is required for the inhibition of Notch signaling in
the pIIb cell. Based on the current study, it is proposed
that Lgl functions with Numb to remove Sanpodo from the membrane, leading to
down regulation of the Notch signaling pathway in the pIIb cell. Through what
mechanism might Lgl regulate Sanpodo localization? Studies in Drosophila, yeast,
and vertebrate cells have implicated Lgl as both a regulator of exocytosis,
through its interaction with t-SNARES, and as cytoskeletal effector.
In this study, no phenotypes suggesting gross defects in exocytosis were detected;
in fact, increased
accumulation of the membrane protein Sanpodo at the plasma membrane is seen in
lgl mutants. Accumulation of Sanpodo at the plasma membrane in lgl
mutants resembles the phenotype of three endocytic proteins Numb, alpha-Adaptin, and
Shibire, suggested that lgl may have a broader role in vesicle traffic.
Although a potential role for Lgl in endocytosis is observed, this role appears to
be specific to Sanpodo, since endocytosis of Delta occurs normally in lgl
mutants, suggesting that Lgl is not required for bulk endocytosis. Increasingly,
selective endocytosis is being implicated as an important regulator of signaling
pathways. Two recent studies demonstrate that Liquid facets,
an endocytic epsin participates
in the Neuralized-mediated Delta endocytosis, apparently by targeting
mono-ubiquitinated Delta to a specific, activating, endocytic compartment.
The Notch receptor is also subjected to an ubiquitin-mediated
endocytic step required for activation via the E3 ubiquitin ligase Deltex, which
targets Notch to the late endosome. However, the roles of
Liquid facets and Deltex have not been explored in asymmetrically dividing
neural precursors. One possible function for Lgl could be to direct Sanpodo
toward a specific endocytic compartment. Alternatively, Lgl may be involved
indirectly, by targeting molecules required for Sanpodo endocytosis to the
membrane region. This scenario would be more consistent with Lgl's role as an
exocytic regulator. An alternative hypothesis may be that Lgl regulates Sanpodo
localization through its interaction with the cytoskeleton. Lgl functions
as an inhibitor of non-muscle myosin II function in both Drosophila and yeast.
The data suggests that cytoskeletal association of Lgl is required for
regulating Sanpodo localization, because phosphorylation of Lgl by aPKC, which
causes an autoinhibitory conformational change in Lgl that disrupts the
association with the cytoskeleton, causes membrane
accumulation of Sanpodo. It remains to be determined if Sanpodo endocytosis
requires inhibition of myosin II activity (Roegiers, 2005).
Previously, Numb and Neuralized had been implicated in two complementary, and
possibly independent, mechanisms to determine cell fate in PNS precursor cells.
Numb functions to inhibit Notch autonomously by internalizing Sanpodo in the
pIIb cell: while Neuralized acts on Delta in the pIIb cell to induce Notch
signaling non-autonomously in the pIIa cell. Both neuralized-dependant uptake of
Delta and Sanpodo internalization
require dynamin function, suggesting that these steps rely on endocytosis.
Unexpectedly, it was found that loss of neuralized function affects both Delta
internalization and Sanpodo internalization.
Failure to internalize Delta into the pIIb cell causes a cell fate
transformation of the pIIa cell into a pIIb cell in neuralized mutants,
and this transformation occurs despite the accumulation of Sanpodo at the membrane,
suggesting that accumulation of Sanpodo at the membrane is not sufficient to
induce Notch signaling in the pIIb cell in the absence of neuralized. It is
unclear whether membrane accumulation of Sanpodo in neuralized
mutants is due to
a direct interaction between Neuralized and Sanpodo, perhaps through
ubiquitination of Sanpodo, or through an indirect mechanism. Regardless, the
data show that regulation of Sanpodo membrane localization is not completely
independent of neuralized function. In summary, this study suggests that Sanpodo
is regulated by both neuralized and lgl, while Delta is regulated by
neuralized independently of lgl. In addition, this study shows that lgl
appears to contribute to the endocytosis of Sanpodo, which suggests a broader
role for lgl in vesicle trafficking, which may have important
implications for its role as a tumor suppressor. Could the
regulation of Notch signaling by Sanpodo, Lgl and Numb be conserved across
species? Sequence analysis did not reveal any homologues of Sanpodo beyond other
insect species. However, loss of function
studies of the mouse homologues of Drosophila numb and lgl in the
developing brain show strikingly similar phenotypes. Targeted numb/numblike
knockouts in dorsal forebrain and Lgl1
knockouts cause profound disorganization of the layered regions of the cortex
and striatum and formation of rosettelike accumulations of neurons. These
phenotypes may indicate that Numb and Lgl function together to regulate Notch
signaling in mouse neurogenesis as well as in Drosophila PNS development, but
a functional homologue sanpodo has yet to be identified in the mouse (Roegiers, 2005).
During asymmetric cell division in Drosophila sensory organ precursors (SOPs), the Numb protein segregates into one of the two daughter cells, in which it inhibits Notch signalling to specify pIIb cell fate. Numb acts in SOP cells by inducing the endocytosis of Sanpodo, a four-pass transmembrane protein that has been shown to regulate Notch signalling in the central nervous system. In sanpodo mutants, SOP cells divide symmetrically into two pIIb cells. Sanpodo is cortical in pIIa, but colocalizes with Notch and Delta in Rab5- and Rab7-positive endocytic vesicles in pIIb. Sanpodo endocytosis requires alpha-Adaptin, a Numb-binding partner involved in clathrin-mediated endocytosis. In numb or alpha-adaptin mutants, Sanpodo is not endocytosed. Surprisingly, this defect is observed already before and during mitosis, which suggests that Numb not only acts in pIIb, but also regulates endocytosis throughout the cell cycle. Numb binds to Sanpodo by means of its phosphotyrosine-binding domain, a region that is essential for Numb function. These results establish numb- and alpha-adaptin-dependent endocytosis of Sanpodo as the mechanism by which Notch is regulated during external sensory organ development (Hutterer, 2005; full text of article).
This analysis shows that Sanpodo regulates Notch signalling during Drosophila ES organ development. In the pIIa cell, Sanpodo is localized at the plasma membrane and is required for Notch activation. In the pIIb cell, Sanpodo is removed from the plasma membrane by Numb- and alpha-Adaptin-dependent endocytosis. This correlates with the inability of this daughter cell to activate Notch signalling, suggesting that it is the plasma-membrane-localized Sanpodo protein that activates the Notch receptor. Previous epistasis experiments have suggested that Sanpodo acts during the intramembranous (S3) cleavage of the Notch receptor. Assuming that this cleavage occurs at the plasma membrane, it is possible that Notch needs to bind to Sanpodo to become a substrate for the protease Presenilin, which carries out the S3 cleavage (Hutterer, 2005).
Although this model is attractive, it does not explain why Sanpodo colocalizes with Notch in endocytic vesicles and why these vesicles are found in both pIIa and pIIb cells. Furthermore, it was found that ectopic expression of Sanpodo during neurogenesis (where Numb is expressed but not asymmetric) causes a neurogenic phenotype. Thus, Sanpodo can both activate and inhibit Notch signalling depending on the absence or presence of Numb. These observations are more consistent with an alternative model in which Sanpodo regulates the endocytosis of Notch. It was recently shown that ubiquitination and subsequent endocytosis can downregulate Notch. Conversely, endocytosis can also positively influence Notch signalling and was shown to be required for Notch activation in vertebrates. It is speculated that Sanpodo might have a general role in Notch endocytosis. In the absence of Numb, endocytosis could be required for Notch signalling, whereas in its presence, the inhibitory endocytic pathway could prevail. Although this model is speculative, it would also explain why expression of Numb in tissues that do not express Sanpodo has little or no influence on Notch signalling (Hutterer, 2005).
To follow the expression and subcellular localization of Spdo, antibodies specific to two overlapping regions of the predicted cytoplasmic domain of Spdo were generated. Using either antibody, it was found that Spdo is expressed in all NBs, all GMCs, and transiently in most, if not all, neurons in the CNS. In the PNS, Spdo is expressed in all SOPs and their progeny. In the mesoderm, Spdo is expressed in heart and somatic muscle precursors that undergo spdo-dependent asymmetric divisions. Spdo is also expressed in the asymmetrically dividing cells of the posterior midgut. Thus, all embryonic cells known to undergo asymmetric divisions, even those thought to divide asymmetrically in a spdo-independent manner, appear to express Spdo. Consistent with Spdo playing a role to regulate asymmetric NB divisions, a weak, but consistent, duplication of GMC1-1a is observed in spdo mutant embryos (O'Connor-Giles, 2003).
Asymmetric division of sensory organ precursors (SOPs) in Drosophila
generates different cell types of the mature sensory organ. In a genetic screen
designed to identify novel players in this process, a mutation
was isolated in Drosophila sec15, which encodes a component of the exocyst,
an evolutionarily conserved complex implicated in intracellular vesicle transport.
sec15− sensory organs contain extra neurons at the
expense of support cells, a phenotype consistent with loss of Notch signaling. A
vesicular compartment containing Notch, Sanpodo, and endocytosed Delta
accumulates in basal areas of mutant SOPs. Based on the dynamic traffic of
Sec15, its colocalization with the recycling endosomal marker Rab11, and the
aberrant distribution of Rab11 in sec15 clones, it is proposed that a defect
in Delta recycling causes cell fate transformation in
sec15− sensory lineages. The data indicate that Sec15
mediates a specific vesicle trafficking event to ensure proper neuronal fate
specification in Drosophila (Jafar-Nejad, 2005).
In a genetic screen designed to identify novel players in Drosophila sensory
organ development, a mutation in sec15 was isolated that caused a pIIa to pIIb transformation phenotype. Sec15 is a component of a multiprotein complex called the exocyst or Sec6/8 complex. Mutations in exocyst components were originally isolated in a yeast screen for secretion-defective mutants. Subsequent analysis of the exocyst complex in yeast and mammalian cell culture systems has indicated that it functions in intracellular vesicle transport. In yeast, the
exocyst mediates the post-Golgi to membrane targeting of exocytic cargo via an interaction with the Rab GTPase Sec4p. In Madin-Darby canine kidney (MDCK) epithelial cells, the exocyst localizes to areas of cell-cell contact and is involved in basolateral delivery of vesicles. However, none of the studies on the exocyst components have implicated these proteins in cell fate determination.
The data suggest that Sec15 mediates highly specific intracellular trafficking
events that promote N signaling and thereby ensure proper cell fate
specification in Drosophila mechanosensory organs (Jafar-Nejad, 2005).
The various cell types that form an adult sensory organ in Drosophila
are generated via asymmetric divisions of a pI and its progeny. Differential
activation of the N signaling pathway between the two daughter cells of each
division ensures that each sensory organ acquires the proper complement of cell
types necessary to function. Sec15, a component of the
evolutionarily conserved exocyst complex as reported here, is required for proper cell fate
specification of the pI progeny. Studies on sec15 mutations in the
eye did not reveal any fate change in the photoreceptors. Loss-of-function
mutations in three other exocyst components have been reported previously:
sec5 and sec6 in flies and sec8 in mice. sec8
mutant mice die at day E7.5, before the development of specific neuronal
populations can be studied. Also, sec5 and sec6
mutations are cell lethal in the Drosophila eye. Therefore, this
report is the first to identify a role for an exocyst component
in cell fate determination. At this point, it cannot be predicted if sec5 and
sec6 also play a role in neuronal cell fate specification. However, given
the data obtained from studies of the fly eye, the hypothesis is favored that
components of the exocyst may form more than a single functional unit and/or
have subunit-specific roles (Jafar-Nejad, 2005).
Live imaging of dividing pI cells indicates that Sec15 is
associated with a vesicular compartment that traffics between apical and
subapical areas. In sec15− SOPs, an expanded
compartment is observed that contains Spdo, N, and Dl.
Unlike wt pI and pIIb cells, in which
Spdo/N/Dl+ vesicles tend to reside at or above the level of septate
junctions, in mutant SOPs these puncta accumulate at the basal side of the cell.
Together, these observations suggest that Sec15 is involved in vesicle
trafficking to the apical parts of the cell. The defect in the apical
trafficking of proteins does not seem to be a general one, since localization of
E-Cad and Arm at the adherens junction is not disrupted in mutant tissue.
Therefore, the data link a specific vesicle trafficking event to a developmental
decision made by sensory precursor cells (Jafar-Nejad, 2005).
Genetic experiments and
immunohistochemical stainings strongly suggest that Sec15 and Spdo function in
the same pathway in sensory cell fate determination process.
It has been proposed, based on studies performed on
the asymmetric divisions of Drosophila embryonic neuroblasts, that Spdo
promotes N signaling at the membrane of the signal-receiving cell. In contrast,
Numb and α-Adaptin in the signal-sending cell might promote endocytosis of
Spdo and its removal from the membrane, thereby preventing the reception of
signal by this cell. The subcellular distribution of Spdo in pIIa and pIIb
cells is similar to its localization in embryonic neuroblast progeny, suggesting
that this model might also apply to adult bristle formation. Notably, however,
Spdo is observed at or close to the membrane of both pI progeny in sec15
clones. Therefore, while the
proposed role for Spdo in promoting N signaling at the membrane of the
signal-receiving cell cannot be ruled out, the data suggest a role for Spdo in Dl recycling in the
signal-sending cell. It should be noted, though, that these two models are not
mutually exclusive. Presence of a significantly higher number of vesicles
containing both Dl and N in pIIb compared to the pIIa in wt sensory precursors
has been implicated in the ability of the pIIb cell to send the Dl signal.
Colocalization of Spdo with Dl in a significant fraction of these vesicles
suggests that a defect in Spdo/Dl trafficking in pIIb contributes to the
sec15 loss-of-function phenotype (Jafar-Nejad, 2005).
Presence of endocytosed Dl in
vesicles that accumulate in sec15 clones implicates these vesicles in the
endocytic traffic of Dl. This notion is further supported by the observation
that in both wt and sec15− SOPs, the Spdo/Dl/N puncta
show a significant colocalization with the endosomal markers Rab5 and HRS. It
has recently been proposed that in order to signal, Dl needs to traffic through
a specific endocytic compartment, which will lead to recycling of the protein. A defect in Dl recycling is further suggested by the aberrant accumulation of the recycling
endosomal marker Rab11 in sec15 clones. The Rab11+ endosomal
compartment is thought to be a central trafficking intermediate in both exocytic
and endocytic pathways and is shown to control the traffic of cargo from the
perinuclear recycling endosomal compartment to the membrane. Interestingly, it has been shown that Sec15
meets the criteria of being an effector for Rab11 in mammalian cell lines: Sec15
physically binds Rab11 in a GTP-dependent manner; Sec15 colocalizes with Rab11
in the perinuclear region of the cells; Sec15 labels structures containing an
endocytosed protein in immuno-EM experiments. Similarly, Drosophila Sec15
and Rab11 interact physically and show a high level of colocalization in SOPs.
Altogether, these data are compatible with a model in
which Sec15 regulates the traffic of a subset of endocytosed Dl to the membrane
of the pIIb cell via a Rab11+ recycling endosomal compartment. Sec15 traffics symmetrically in pIIa and pIIb.
Therefore, it is proposed that an intrinsic difference between the endocytic traffic
of Dl in pIIa and pIIb allows the pIIb cell to employ the Sec15-Rab11 machinery
differentially from the pIIa cell and thereby assume the role of signal-sending
cell. The most likely mechanisms for the proposed intrinsic difference are
unequal segregation of Neur into the pIIb, which promotes Dl endocytosis in this
cell, and asymmetric distribution of the Rab11+ recycling
endosomes in the pIIb versus pIIa, which is thought to specifically mediate Dl
recycling in the pIIb (Jafar-Nejad, 2005).
These data suggest that at least some of the Spdo/N/Dl-containing vesicles that accumulate in the basal areas of sec15− SOPs are of a mixed exo-endocytic nature. This is not unprecedented, since traffic from the TGN to an endosomal compartment has been documented. Accordingly, it has been proposed that some exocytic cargo might pass through the recycling endosome on its way from the TGN to the plasma membrane. Recently, it has been shown that upon exit from the Golgi apparatus, newly synthesized E-Cad fuses with a Rab11+ recycling endosomal compartment before it reaches the plasma membrane. It is interesting to note that members of the exocyst complex have been shown to localize to both the TGN and recycling endosomes in polarizing epithelial cells. Although the
recycling endosome has been proposed as an intermediate to transfer the exocytic
cargo to the plasma membrane, it is possible that passing through these vesicles
somehow enhances the signaling ability of internalized Dl. In other words,
presence of Spdo might be part of the specific environment that Dl needs
to traffic through. Although Dl endocytosis and recycling are also implicated in
N signaling during lateral inhibition, no lateral inhibition defects are observed in sec15 clones. It is proposed that the link to Spdo results in the
specificity of the sec15 phenotype to the asymmetric divisions, since loss
of spdo similarly does not affect lateral inhibition (Jafar-Nejad, 2005).
In summary, the data indicate that one component of the highly conserved exocyst complex affects the asymmetric division of the sensory precursors in the Drosophila PNS through specific vesicle trafficking events. Components of the exocyst complex are conserved from yeast to human, and several reports have shown parallels between the contribution of asymmetric divisions to Drosophila and vertebrate neurogenesis. Therefore, it is conceivable that Sec15, and perhaps other members of the exocyst complex, are involved in neural cell fate determination in other species (Jafar-Nejad, 2005).
The function of the gene sanpodo (spdo) is illustrative of a fundamental process in developmental biology: the specification of cell fate through asymmetric cell division. Before looking at spdo in some detail, a brief outline of cell fate specification in the peripheral nervous system (PNS): the event is mediated in part through asymmetric cell divisions, a process in which one cell divides to give rise to two daughter cells with distinctly different fates. In the external sensory (es) organ lineage, the primary sensory organ precursor (SOPI) divides asymmetrically to produce two daughter cells, SOPIIa and SOPIIb. These two divide, producing (respectively) a hair and socket cell, and a neuron and glial cell. Many genes that play a role in specifying the fate of PNS cells and neurons have been found to be located in the Notch signaling pathway: Notch, Delta, Suppressor of Hairless, Enhancer of split, Mastermind and Kuzbanian. Loss of Delta, Notch, Supressor of Hairless or Kuzbanian leads to an increase in the number of neurons, the neurogenic phenotype. Another component, Numb, has been integrated into the Notch signaling pathway. Numb is a cytoplasmic protein that has been shown to bind to the intracellular domain of
Notch and to repress Notch function. Interestingly, in the SOPI of es organs the Numb protein is localized to a crescent at the cell surface. When SOPI divides, the Numb protein segregates into the SOPIIb daughter cell. The resulting daughter-cell-specific repression of Notch signaling is necessary for the SOPIIa and b cells to adopt different fates (Dye, 1998 and references).
Where does sanpodo fit in a discussion of cell fate specification? The gene was identified by Salzberg (1994), as well as in subsequent screens (Salzberg, 1997) for mutations altering the developmental pattern of the peripheral nervous system. All mutants are embryonic lethal. spdo embryos
display an approximate doubling of the number of neurons in the PNS when compared to wild-type embryos. This phenotype is the basis for the name Sanpodo, the Korean word for mountain grapes. In spite of the increased number of neurons, PNS neurons remain distributed among the four typical clusters within each segment, but, unlike other neurogenic mutants (e.g. Notch, Delta), the overall morphology of spdo embryos is not affected. In addition, no defects in axon pathfinding were observed in the PNS (Dye, 1998).
An increased number of multiple dendritic (md) or external sensory (es) neurons, as observed in spdo mutants, can be obtained by any of several different mechanisms: an increased recruitment of sensory organ precursors (SOPs), extra divisions of the progeny of SOPs (SOPI and
SOPII) or extra divisions of the neuron, or transformation of the es hair, socket or glial cells into neurons. It has been shown that in spdo mutants there are no extra SOPs recruited, and that the total number of cells within the dorsal and lateral clusters of the PNS is not increased
significantly. It is therefore concluded that the supernumerary
neurons must result from a fate change of cells in the same lineage and it is proposed that the two sibling cells of the SOPIIb (neuron and glia) take on the same fate, i.e. neurons (Salzberg, 1994). Since spdo mutant embryos lack virtually all Prospero and Bar-positive glial support cells in the PNS, it is concluded that the two sibling cells derived from the es SOPIIb take the same neuronal fate in spdo mutants (Dye, 1998).
The dorsal bipolar dendritic (dbd) lineage is the most simple and well-characterized asymmetric cell division in the PNS: the precursor cell divides asymmetrically to give rise to an md
neuron and a glial cell. The dbd neuron is often duplicated in spdo embryos. Glial markers show no glia associated with the duplicated dbd neurons. Hence the sibling cell is transformed into an md neuron. This transformation is the opposite of the numb phenotype, which
consists of two glial cells instead of two dbd neurons. This suggests that in spdo
mutants, for those md neurons that have a lineage-related sibling, the non-neuronal
cells as well as the neuronal cell adopt the neural fate. Staining with an md-specific marker reveals
another feature of spdo mutants. Es neurons are programmed to become md neurons in spdo mutants. This observation is particularly interesting because the same phenotype occurs in Notch mutants, suggesting that Notch signaling is impaired in spdo mutants. These observations suggest that spdo is a neurogenic gene and when mutated results in too many neurons, the hallmark of the neurogenic phenotype (Dye, 1998).
Spdo functions downstream of Numb, a protein known to be involved in the Notch pathway. Within the es lineage, numb function is required in both the SOPIIb and its daughter cells to correctly specify cell fate. spdo function is also required within the es lineage, but in a manner just the
opposite of that in numb. To place spdo in the Numb/Notch
pathway, an analysis of numb;spdo double mutants was undertaken. spdo function is likely
downstream of numb, because numb;spdo embryos have many
more neurons than those that only lack numb. Alternatively, Spdo functions as an antagonist of Numb. Based on the epistatic interaction studies, it is proposed that the transformation of SOPIIb into SOPIIa brought about by loss of numb is often blocked in the absence of spdo. Because loss of Numb in the SOPIIb leads to increased Notch signaling, Notch activation and/or signaling may require Spdo function. A role for Spdo in Notch signaling is supported by the finding that reducing Notch protein function at the time of SOPIIa division decreases the number of es glial cells and increases the number of es neurons. The es neurons express md-specific markers in spdo mutants, as is observed in Notch mutants. It is therefore unlikely that Spdo functions in an independent pathway, although at the present time this possibility can not be formally excluded (Dye, 1998).
sanpodo mutation reveals an additonal role for Sanpodo in external sensory organ hair differentiation. Examination of cuticle preparations of pharate first instar larvae reveal
that the three hairs of Keilin organs, a cluster of external
sensory organs in the thoracic segments, are almost always affected in spdo mutants. When compared to wild type, the hairs are most often reduced in size or absent. The morphology of the socket cells is often abnormal: in approximately 10% of the mutant embryos there appear to be extra hair cells and a
corresponding lack of socket cells, suggesting that the socket
cells are transformed into hair cells. The typical structures corresponding to the ventral Kolbchen do not appear to be present; the Kolbchen are refractive club-like sensory structures found in the thoracic
segments. Hairs in the abdominal
segments are often present, albeit reduced in size. It has been concluded that the differentiation of the
external structures of many PNS organs are affected in spdo
mutants and that these defects resemble those associated with mutations in genes encoding actin binding proteins. Staining with phalloidin reveals a severe decrease in F-actin staining in Keilin organs and Kolbchen organs in spdo mutants when compared to wild type. This decrease in F-actin staining is observed for the external structures of the entire PNS in mutants (Dye, 1998).
Although most glial CNS cell lineages are not characterized, it can be demonstrated that CNS glia are affected by mutations in sanpodo. Staining with anti-RK2/REPO, a glial marker, reveals a severe loss of glial cells in the CNS. Analysis of the expression of another glial marker, glial cells missing (gcm) , shows that lack of spdo causes a severe decrease in the number of Gcm-positive cells in stage 15 embryos. Because gcm loss-of-function mutations transform glial cells into neurons and overexpression of gcm transforms neurons into glia, gcm is considered to be a glial identity gene. Thus, the absence of gcm expression in spdo mutants suggests that a subset of CNS glia have not adopted a glial fate. The lineages of most CNS glia are not well defined and it is not known if these cells have siblings. As a result, it could not be determine if the absence of gcm expression in spdo mutants results from a cell fate switch, as is occurring in the PNS, or from a failure to maintain gcm expression (Dye, 1998).
Staining of axons in the CNS with monoclonal antibodies mAb BP102 and mAb 1D4 shows that mutations in spdo cause very noticeable disruptions of the longitudinal tracts. The anterior and posterior commissures form and separate properly, but they appear thicker and less condensed than normal. The phenotype in the ventral nerve cord in spdo mutants is strikingly similar to the phenotype reported for gcm loss-of-function mutations. These observations suggest that loss of spdo alters cellular identity in the CNS as well (Dye, 1998).
sanpodo mutation has been shown to disrupt external sensory organ hair differentiation. The hairs of external sensory organs have been shown to be reduced in size and altered in morphology in singed and chickadee mutants. These genes encode, respectively, an actin-bundling protein (Fascin), and G-actin binding protein (Profilin). A determination was made whether lack of spdo caused any defects in the morphology of the extra sensory (es) organ hairs of first instar larvae. Examination of cuticle preparations of pharate first instar larvae reveals that the three hairs of Keilin organs, a cluster of external sensory organs in the thoracic segments, are almost always affected in spdo mutants. When compared to wild type, the hairs are most often reduced in size or absent. In addition, the morphology of the socket cells is often abnormal. In approximately 10% of the mutant embryos extra hair cells are observed and a corresponding lack of socket cells, suggesting that the socket cells are transformed into hair cells. Absent as well from the thoracic segments are the typical structures corresponding to the ventral Kolbchen, a refractive club-like sensory structure that appear as round structures in the tops of wild-type embryos. Hairs in the abdominal segments are often present, albeit reduced in size. It is therefore concluded that the differentiation of the external structures of many PNS organs are affected in spdo mutants and that these defects resemble those associated with mutations in genes encoding actin binding proteins. Staining with phalloidin in spdo mutants reveals a severe decrease in F-actin staining in Keilin organs and Kolbchen organs, when compared to wild type. This decrease in F-actin staining is observed for the external structures of the entire PNS in such mutants (Dye, 1998).
It is thought that in numb mutant embryos there are increased levels of Notch signaling, resulting in a transformation of SOPIIb cells into SOPIIa cells. Consequently, loss of numb reduces the number of PNS neurons. Because this phenotype is the opposite of that in spdo mutants, the epistatic relationship between numb and spdo was determined. numb;spdo double homozygous embryos were analyzed with both neuronal and glial markers to determine if the phenotype caused by loss of numb requires the presence of Spdo or vice versa. When compared to wild-type, spdo mutants exhibit roughly twice the normal amount of neurons; embryos that lack only numb function have 3-8 neurons per hemisegment (compared to approximately 35 in wild-type). Double mutants never exhibit the numb phenotype; rather, the number of neurons is more similar to that seen in spdo embryos, showing that loss of spdo partially suppresses the numb phenotype. Analysis of the epistatic interactions between numb and spdo within a single PNS lineage also demonstrates that spdo is epistatic to numb. The dbd lineage of the PNS consists of one neuron and one glial cell derived from a single precursor cell. In the absence of numb, two dbd glial cells are seen; in spdo mutants, the dbd glial cell is absent. In double mutants, the dbd glial cell is missing, as is seen in spdo embryos. These observations suggest that spdo functions downstream of, or antagonistically to, numbto specify cell fate in this cell lineage (Dye, 1998).
Eleven alleles of spdo were isolated based on dramatic alterations in even-skipped expression in the CNS. Embryos homozygous for the null spdo ZZ27 allele, subsequently called 'spdo embryos,' show normal Eve + GMCs but an equalization of sibling neuron identity as detected by Eve and other markers. The RP2 motoneuron (derived from GMC 402a) is duplicated at the expense of the RP2sib, as shown by staining for Eve, Zfh-1 and 22C10. The aCC motoneuron (derived from GMC1-1a) is duplicated at the expense of the pCC interneuron, as shown by staining for Zfh-1, 22C10 and by following axonal projections. The Usib (derived from GMC7-1a) fates are duplicated at the expense of the U neurons, as shown by Eve staining. dMP2 is duplicated at the expense of vMP2, as shown by Odd-skipped and 22C10 staining. Although the spdo sibling neuron phenotype is identical to the Notch sibling neuron phenotype, none of the 11 spdo alleles show the excess neuroblast formation characteristic of Notch mutations. spdo germline clones yield an Eve CNS phenotype identical to embryos that lack only zygotic spdo function. Thus, spdo does not appear to function during Notch-mediated lateral inhibition in the neuroectoderm (Skeath, 1998).
Numb is known to bind to the intracellular domain of Notch and antagonize Notch signaling but, with the exception of the dMP2/vMP2 neurons, it has not been reported to play a role in sibling neuron development in the CNS. However, due to the widespread role of Notch in specifying asymmetric sibling neuron identity, the CNS function of numb was re-investigated. Sibling neuron development was assayed using four different numb alleles and a deficiency that uncovered the numb locus. In embryos homozygous for the strongest numb allele (nb 2 ), an equalization of sibling neuron phenotype is observed for all siblings tested, with the exception of aCC/pCC. RP2 is transformed into RP2sib approximately 50% of the time; three Usibs are transformed into three U neurons; and dMP2 is transformed into vMP2. The numb phenotypes for RP2, Usib and dMP2 neurons are reciprocal to those observed in spdo, Delta, Notch or mam embryos. This is consistent with studies showing that Numb antagonizes Notch function and extends this interaction to a diverse array of CNS sibling neurons. In addition, a strong decrease in the number of Eve + EL neurons is observed in numb mutant embryos. There is clear evidence of maternal numb function during CNS development, which may account for the lack of a fully penetrant numb sibling neuron phenotype. Changing the dose of maternal numb product directly affects CNS development and suggests that numb may have earlier CNS functions in addition to sibling neuron specification (Skeath, 1998).
spdo and numb have opposite sibling neuron phenotypes and so the epistatic relationship between the two genes was determined by examining the phenotype of a numb;spdo double mutant. The numb;spdo double mutant phenotype is identical to embryos lacking spdo alone. Thus, spdo is genetically downstream of numb, just as has been observed for Notch pathway mutations in other lineages. sanpodo and numb exhibit dosage-sensitive interactions, as gene products that act in the same biochemical pathway often do. The sibling neuron phenotype in numb embryos is sensitive to the level of spdo. For example, homozygous nb 2 embryos show a loss of EL and RP2 neurons, but reducing the dosage of spdo by one-half in nb 2 embryos leads to a recovery of Eve + EL and RP2 neurons. Thus, halving the dosage of spdo strongly suppresses the numb CNS phenotype. These results show that the numb phenotype is extremely sensitive to the dosage of spdo, consistent with the two proteins acting in the same biochemical pathway (Skeath, 1998).
In numb embryos, there is a striking decrease in the number of Eve + EL neurons. Notch, Delta, mam and spdo single mutants do not alter the number of Eve + EL neurons. Importantly, numb;spdo double mutant embryos show a complete rescue of Eve + EL neurons , suggesting that Numb acts to prevent Spdo-mediated downregulation of eve expression (i.e. in the absence of Spdo, the loss of Numb is irrelevant). These data are consistent with a model in which Numb blocks Notch/Spdo-mediated downregulation of eve in the neurons of the EL lineage (Skeath, 1998).
Spdo regulates Notch-mediated sibling cell fate decisions but
is not involved in Notch-mediated lateral inhibition. Notch functions in the neurogenic ectoderm to limit the number of cells adopting a neural fate. spdo does not alter the number of neuroblasts that delaminate from the ectoderm, but instead is involved only in regulating sibling cell fate in the progeny of neuroblasts. Although the spdo sibling neuron phenotype is identical to the Notch sibling neuron phenotype, none of the 11 spdo alleles show the excess neuroblast formation
characteristic of Notch mutations. Mutations in two other genes, Delta (10 alleles)
and mastermind (1 allele) have been identified that yield similar equalization of sibling neuron fates. Because both Delta and mastermind are in the well-characterized Notch signaling pathway, null and hypomorphic alleles of several 'Notch pathway'
genes have been tested: Delta, Notch, mam, neuralized and E(spl). Mutations in all these genes result in an excess of neuroblasts due to failure of lateral inhibition within the neuroectoderm. However, mutations in neuralized and E(spl) have no effect on the identity of the sibling neurons that were assayed, despite strong
defects in the earlier process of neuroblast formation. In contrast, Delta, Notch and mam mutations all yield similar sibling neuron phenotypes, in addition to excessive neuroblast formation. These results can be illustrated using embryos homozygous for a hypomorphic mam allele in which neuroblast formation is essentially normal but sibling neuron fates are equalized. Loss of mam does not affect eve expression in GMCs, but leads to the duplication of RP2, Usib, aCC and dMP2 fates at the expense of the RP2sib, U, pCC and vMP2 fates, respectively. Thus, mutations in three genes (Delta, Notch and mam) have precisely the same sibling neuron phenotype as spdo mutations, suggesting that spdo, Delta, Notch and mam act together to specify asymmetric sibling neuron fate (Skeath, 1998).
Asymmetric cell division is a widespread mechanism in developing tissues that leads to the generation of cell diversity. For the most part the basis of asymmetric cell division has been analyzed in neuroblasts in the process by which neuroblast division yields another neuroblast and a secondary precursor cell: the ganglion mother cell (GMC). In the embryonic central nervous system of Drosophila melanogaster, GMCs divide and produce postmitotic neurons that take on different cell fates. The current study analyses the process of binary fate decision of two pairs of sibling neurons that occurs during cell division in GMCs. This process is accomplished through the intrinsic fate determinant, Numb. GMCs have apical-basal polarity; Numb localization and the orientation of division are coordinated to segregate Numb to only one sibling cell. The correct positioning of Numb and the proper orientation of division require Inscuteable (Insc). Loss of insc results in the generation of equivalent sibling cells. These results provide evidence that sibling neuron fate decision is nonstochastic and normally depends on the presence of Numb in one of the two siblings. Moreover, the data suggest that the fate of some sibling neurons may be regulated by signals that do not require lateral interaction between the sibling cells (Buescher, 1998).
The focus for the analysis of the roles of insc, numb, and components of the N-signaling pathway in fate specification, was on the only two pairs of GMC-derived neurons for which sibling relationships have been established: the RP2/RP2sib and the aCC/pCC neurons. These neurons are derived from two GMCs that can be identified unambigously by their specific expression of the nuclear protein Even-skipped (Eve). GMC1-1a divides into the aCC/pCC neurons that have approximately equal size and continue to express Eve. However, at later stages of development, aCC is distinguished from pCC by the expression of Zfh-1 and 22C10 (a membrane associated antigen). aCC is a motoneuron and forms an ipsilateral projection that pioneers the intersegmental nerve. GMC4-2a divides to form the sibling neurons RP2/RP2sib that are morphologically distinguishable. In 88% of the hemisegments, the newborn siblings show a significant difference in the size of their nuclei and cell bodies. This asymmetry appears to be initiated during cell division. In GA1019 mutant embryos, in which GMC4-2a fails to complete cytokinesis, cells are formed that contain one large and one small nucleus. This strongly suggests that the difference in size is generated early, prior to the completion of cytokinesis. The larger cell always adopts the RP2 fate, which is characterized by the expression of Eve, Zfh-1, and 22C10. RP2 forms an antero-ipsilateral projection. The smaller sibling always adopts the RP2sib fate, which is characterized by a further decrease in cell and nuclear size and the loss of Eve immunreactivity. Zfh-1 and 22C10 expression have not been shown in RP2sib. These observations suggest that the cell and nuclear size difference may serve as an early physical marker that will allow one to differentiate between the two progeny of GMC4-2a, irrespective of the molecular markers they express later (Buescher, 1998).
Mutations in mastermind (mam),sanpodo, and Notch equalize aspects of sibling cell fate but retain the difference in cell and nuclear size of sibling neurons. In mam mutant embryos, both progeny of GMC4-2a can adopt the RP2 fate with respect to Eve, Zfh-1, and 22C10 expression. However, despite this apparent change from the RP2sib to the RP2 cell fate, the unequal sizes of the GMC4-2a daughter cells remain; that is, their sizes are unaffected. mam is required for the correct fate specification of RP2sib and pCC but not for that of RP2 and aCC. The requirement for mam suggests that N signaling may be involved in the resolution of distinct sibling neuron cell fate. Mutations in mam and N result in similar defects and support the notion that N signaling is required for the resolution of sibling neuron fate. In inscuteable mutant embryos, GMC1-1a and GMC4-2a are correctly formed and express normal levels of Eve (and in the case of GMC4-2a, also Pdm-1). However, GMC1-1a divides to form two sibling neurons that both adopt the aCC fate (94%) with respect to marker gene expression. Similarly, GMC4-2a division results in two sibling cells, both of which adopt the RP2 fate (96%) with respect to expression of Eve, Zfh-1, and 22C10, as well as axon morphology. This strongly suggests that in wild-type embryos, the divisions of GMC1-1a and GMC4-2a are asymmetric in an insc-dependent manner and produce sibling cells that are intrinsically different; loss of insc function leads to the generation of sibling neurons with equivalent cellular identities. Moreover, in contrast to mam, sanpodo, and Notch mutant embryos, the duplicated RP2s seen in insc mutants are equal with respect to their cell and nuclear size. These observations are consistent with the idea that the size difference seen in wild-type embryos is generated by an insc-dependent process during the GMC cell division and occurs prior to the events mediated by mam, spdo, and N that presumably act at the level of the postmitotic sibling cells. No size asymmetry between the sibling neurons should be generated in an insc background regardless of whether the other functions (e.g., spdo) are present or not (Buescher, 1998).
In Drosophila, much has been learned about the specification of neuronal cell fates but little is known about the lineage of mesodermal cells with different developmental fates. During development, individual mesodermal precursor cells are initially singled out to become the founder cells for specific muscles. The selection of muscle founder cells is thought to employ a Notch-mediated process of lateral inhibition, similar to what is observed for the specification of neural precursors. These muscle founder cells then seem to fuse with the surrounding, uncommitted myocytes, inducing the formation of muscle fiber syncytia. In contrast, the differentiated progeny of neural precursor cells are usually the result of a fixed pattern of asymmetric cell divisions that are directed, in part, by interactions among Numb (a localized intracellular-receptor protein); Sanpodo, and Notch (a transmembrane receptor protein). The roles of these neural lineage genes have been examined in the cell fate specification of muscle and heart precursors. numb and spdo mutations have opposite effects in the specification of muscle founder cells. In all numb mutant embryos examined, the number of Kruppel and S59/NK1 expressing muscle cells is dramatically reduced or absent in stage 12/13 embryos. In spdo mutant embryos the number of S59 and Kr expressing muscle founders is increased (Park, 1998).
A progenitor cell that generates both a pericardial heart cell and a somatic muscle founder cell was the focus of investigation. The two sibling cells studied were a single dorsal muscle, DA1, and a non-muscle pericardial cell (termed EPC), which is associated with the heart. Both cell types express Eve, however, they can be distinguished from one another morphologically. The precursor for both the EPCs and the putative founders of DA1 muscle emerges from a small cluster of mesodermal Eve-expressing cells in each hemisegment at mid-stage 11. At first, these mesodermal Eve cells are indistinguishable from one another, and they co-express Mef2, which is expressed in the entire early mesoderm and later in all (contractile) muscle types. Subsequently Mef2 expression ceases in the future EPCs as they begin to differentiate as non-muscle, pericardial cells. The putative DA1 founder seems to maintain Mef2 expression and begins fusing with surrounding myocytes. In Mef2 mutants, no fusion occurs but the putative muscle founders maintain expression of their muscle precursor markers, such as Eve. The asymmetric segregation of Numb into one of these
daughter cells antagonizes the function of Notch and Spdo by preventing the presumptive muscle
founder from assuming the same fate as its cardiac sibling. In numb mutants, most DA1 muscles are physically absent and the remaining ones lack Eve (and Kr) expression; in addition, the number of EPCs is doubled. These data suggest that in numb mutants, the putative DA1 founders are transformed into EPCs, because the Eve progenitor cells that normally give rise to DA1 founders and EPCs now only produce EPCs. Overexpression of numb or loss-of-spdo-function result in a failure to generate EPCs but allow for the formation of DA1 muscles. Similarly, expression of constitutively active Notch leads to a failure of DA1 muscle formation and an increase in the number of EPCs. Studies of double mutants indicate that spdo is epistatic to numb (in numb;spdo double mutant embryos, the spdo phenotype is apparent), suggesting that it acts downstream of numb. These results suggest that asymmetric cell
divisions, in addition to the previously-documented inductive mechanisms, play a major role in cardiac
and somatic muscle patterning. In addition, the cytoskeleton may have a role in the
asymmetrical localization of cell fate determinants (Park, 1998).
The Drosophila heart is a simple organ composed of two
major cell types: cardioblasts, which form the simple
contractile tube of the heart, and pericardial cells, which
flank the cardioblasts. A complete understanding of
Drosophila heart development requires the identification of
all cell types that comprise the heart and the elucidation
of the cellular and genetic mechanisms that regulate
the development of these cells. A new population of heart cells is reported here: the Odd
skipped-positive pericardial cells (Odd-pericardial cells).
Descriptive, lineage tracing and genetic
assays were used to clarify the cellular and genetic mechanisms that
control the development of Odd-pericardial cells. Odd
skipped marks a population of four pericardial cells per
hemisegment that are distinct from previously identified
heart cells. Within a hemisegment,
Odd-pericardial cells develop from three heart progenitors
and these heart progenitors arise in multiple
anteroposterior locations within the dorsal mesoderm. Two
of these progenitors divide asymmetrically such that each
produces a two-cell mixed-lineage clone of one Odd-pericardial
cell and one cardioblast. The third progenitor
divides symmetrically to produce two Odd-pericardial
cells. All remaining cardioblasts in a hemisegment arise
from two cardioblast progenitors, each of which produces
two cardioblasts. Furthermore, numb
and sanpodo mediate the asymmetric divisions of the two
mixed-lineage heart progenitors noted above (Ward, 2000).
Having established a wild-type profile of Odd-pericardial cell
development it was of interest to identify the genetic regulatory
mechanisms that govern Odd-pericardial cell development.
Genes that control
asymmetric divisions regulate Eve-pericardial cell
development. Thus, whether loss of
sanpodo or numb function affect Odd-pericardial cell and
cardioblast development was examined. Normally 4.2 Odd-pericardial cells and 6.0 cardioblasts develop within each
abdominal hemisegment of late-stage embryos. In
numb mutant embryos, 6.0 Odd-pericardial
cells and 4.2 cardioblasts were detected per
hemisegment. Conversely, 7.6 cardioblasts
and 2.7 Odd-pericardial cells per hemisegment were detected in
sanpodo mutant embryos. Thus, in numb mutant embryos roughly two extra Odd-pericardial cells and two fewer
cardioblasts were detected per hemisegment. Conversely, in sanpodo mutant
embryos roughly two fewer Odd-pericardial cells and two
additional cardioblasts form per hemisegment (Ward, 2000).
These results demonstrate that sanpodo promotes Odd-pericardial
cell development and opposes cardioblast
development. Conversely, numb opposes Odd-pericardial cell
development and promotes cardioblast development. In
addition, they suggest that two cardioblasts and two Odd-pericardial
cells arise via the asymmetric divisions of
numb/sanpodo dependent heart progenitors. These results are
consistent with the known requirement for Notch in pericardial cell development. Loss of numb function disrupts the precise alignment of
cardioblasts leading to 'broken rows' of cardioblasts in numb
mutant embryos (Ward, 2000).
Multiple models can explain the reciprocal effects of sanpodo
and numb on cardioblast and Odd-pericardial cell
development. For example, one model predicts that two mixed-lineage
heart progenitors each divide to yield one cardioblast
and one Odd-pericardial cell. A second model predicts the
existence of four progenitors: two would divide with each
producing one Odd-pericardial cell and one cell of unknown
fate; the other two progenitors would divide each producing
one cardioblast and one cell of unknown fate. In these and other
models, loss of numb or sanpodo function would equalize all
asymmetric divisions and could result in the observed Odd-pericardial
cell and cardioblast phenotypes (Ward, 2000).
An enhancer trap in the seven-up gene identifies the
two mixed-lineage heart progenitors.
Towards the end of the lineage analyses it was discovered
fortuitously that an enhancer trap in the gene seven-up labels
four heart cells in each abdominal hemisegment. This enhancer trap is referred to as svp-lacZ). Two of these cells reside
at the dorsal midline and are cardioblasts since they express Mef2. The other two cells reside just lateral and slightly
ventral and anterior to the svp-lacZ cardioblasts. These two
cells are Odd-pericardial cells because they express Odd. The relative
positioning of the svp-lacZ cardioblasts and Odd-pericardial
cells closely resembles that of the sibling cardioblasts and Odd-pericardial
cells marked by the mixed lineage heart clones. This suggests that the svp-lacZ heart cells may
identify the four progeny of the two mixed lineage heart
progenitors that arise in each hemisegment. If the four svp-lacZ
heart cells are the progeny of these two progenitors, then loss
of sanpodo function should convert all svp-lacZ heart cells to
cardioblasts and loss of numb function should convert all svp-lacZ
heart cells to Odd-pericardial cells. In sanpodo mutant
embryos, all four svp-lacZ cells acquire the cardioblast fate and in numb mutant embryos all four svp-lacZ cells
acquire the Odd-pericardial cell fate. The results
from these experiments demonstrate that svp-lacZ identifies the
progeny of the two mixed lineage heart progenitors and that
numb and sanpodo mediate the asymmetric divisions of these
mixed-lineage heart progenitors (Ward, 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).
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).
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).
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).
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