drifter
During imaginal development, vvl is required for cell proliferation and the differentiation of the wing veins (de Celis, 1995).
vvl is expressed in cells fated to be tracheal placodes and wing veins before they undergo differentiation. Its absence prevents tracheal elongation and vein differentiation. Tracheal tree formation occurs by cell migration and cell fusion from the tracheal placodes. Migration of cells from the tracheal placodes is dependent on the breathless (btl) gene, the Drosophila homolog of FGF receptor. The same tracheal phenotype occurs in vvl and btl mutants, suggesting vvl and btl may act through similar downstream effector genes (de Celis, 1995). But the two genes are expressed independently so they appear to function in parallel pathways.
ventral veins lacking is expressed in future wing veins in both dorsal and ventral surfaces before the surfaces make contact. Therefore, vvl would appear to be required in vein formation to define or implement a vein differentiation program (de Celis, 1995). Integrins and laminin A may be involved. The torpedo receptor-tyrosine-kinase/rolled pathway, is involved in this instance in maintaining vvl expression.
Axonal selection of synaptic partners is generally believed to determine wiring specificity in the nervous system. However, evidence has been found for specific dendritic targeting in the olfactory system of Drosophila: second order olfactory neurons (Projection Neurons) from the anterodorsal (adPN) and lateral (lPN) lineages send their dendrites to stereotypical, intercalating but non-overlapping glomeruli. POU domain transcription factors, Acj6 and Drifter, are expressed in adPNs and lPNs respectively, and are required for their dendritic targeting. Moreover, misexpression of Acj6 in lPNs, or Drifter in adPNs, results in dendritic targeting to glomeruli normally reserved for the other PN lineage. Thus, Acj6 and Drifter translate PN lineage information into distinct dendritic targeting specificity. Acj6 also controls stereotypical axon terminal arborization of PNs in a central target, suggesting that the connectivity of PN axons and dendrites in different brain centers is coordinately regulated (Komiyama, 2003).
Prior to this study of PNs, it was generally believed that synaptic connection specificity is conferred by selection of synaptic partners by presynaptic axons. Systematic lineage analysis strongly suggests that PN dendrites play an active role in establishing connection specificity. Specifically, a given PN's lineage and birth order predicts its glomerular target. However, the position of a given PN's target glomerulus is correlated neither with its neuroblast lineage nor with birth order. Thus, it is unclear how a PN's lineage contributes to its dendritic targeting specificity (Komiyama, 2003).
Molecular genetic evidence is provided that this active dendritic targeting is controlled by transcriptional programs within PNs. The data suggest that the observed dendritic targeting specificity is achieved in two steps: specification of a particular lineage and further intra-lineage specification. The POU domain transcription factors Acj6 and Dfr play critical roles in the first step (Komiyama, 2003).
Several lines of evidence support the idea that Acj6 and Drifter play analogous roles in translating lineage information into dendritic targeting specificity of adPNs and lPNs. (1) Acj6 and Dfr are mutually exclusively expressed in adPNs and lPNs; this lineage-specific expression could be used to regulate the distinct wiring specificity of these two PN lineages. (2) Loss-of-function phenotypes in neuroblast clones demonstrate that Acj6 and Dfr are required for proper dendritic targeting of at least a subset of PNs in their respective lineages. The neuroblast clone phenotypes likely underestimate the requirement of Acj6 or Dfr in PN dendritic targeting. Since each glomerulus is innervated by an average of 3 PNs, it might not be possible to detect inappropriate targeting if 1 or 2 PNs in the same class still innervate the glomerulus properly. This possibility is supported by the study of DL1 PNs. In neuroblast clone analysis, 11 out of 19 acj6-/- clones exhibited no detectable defects in DL1 glomerular innervation; in single-cell clone analysis with a higher resolution, each of the 11 clones showed significant phenotypes. Results from single-cell clone analysis of other PN classes support the generality of the DL1 phenotype -- failure to innervate one specific glomerulus (Komiyama, 2003).
(3) Misexpression of Acj6 in lPNs, or Dfr in adPNs, leads to dendritic targeting defects. In the case of Acj6 misexpression in lPNs, where the phenotypes are stronger (possibly due to a higher ratio of transgene to endogenous Acj6 expression than could be observed for Dfr transgene/endogenous Dfr), there are two qualitatively different mistargeting phenotypes. The first is non-specific accumulation of dendrites in the lateral part of the antennal lobe with associated glomerular organization defects. This phenotype is analogous to the non-specific accumulation of adPN dendrites in the dorsal part of the antennal lobe in acj6-/- adPN clones and may reflect a default response of dendrites deprived of targeting information. The second class of phenotypes is more revealing. In this case, lPN dendrites are mistargeted to well-defined dorsal landmark glomeruli distant from lPN cell bodies and areas of non-specific accumulation. Certain inappropriate glomeruli are specifically targeted, while their neighbors remain uninnervated; this observation argues against the alternative interpretation that misexpression simply causes non-specific dendritic spillover. The specificity of the mistargeting phenotypes caused by misexpression is further supported by the following two observations: (1) overexpression of Acj6 in adPNs, or Dfr in lPNs, never results in any phenotypes; and (2) specific mistargeting is not observed in loss-of-function mutants (Komiyama, 2003).
Taken together, these results strongly suggest that Acj6 and Dfr participate in instructing adPNs and lPNs to innervate a set of glomeruli appropriate to each lineage. At present, it remains probable that other transcription factors act in concert with Acj6 and Dfr to completely specify these lineage-dependent wiring programs. The existence of these other factors -- in addition to the likely underestimation of phenotypes in
neuroblast clone analysis, or perdurance in the case of Dfr -- may explain why both loss-of-function and gain-of-function experiments affect only specific subsets of glomeruli (Komiyama, 2003).
It is important to note that Acj6 and Dfr alone cannot specify a particular PN to target its dendrites to a particular glomerulus. All adPNs express Acj6, yet they project their dendrites to a series of different glomeruli according to their birth order. There must be timing factors, probably also transcription factors, which further distinguish PNs within the same lineage based on their birth order. An elegant mechanism to specify different progeny from a common neuroblast has recently been described in the Drosophila embryonic CNS, where neuroblasts exhibit asymmetric cell division patterns similar to those giving rise to PNs. In the embryonic CNS, the neuroblast changes its transcription factor profile as a function of time, thereby specifying the fate of neurons born at different stages. It is suspected that analogous timing factors might exist in PN lineages. These timing factors, in collaboration with lineage-specific factors, will ultimately specify the expression of a repertoire of cell surface molecules that allow PNs to target their dendrites precisely to specific glomeruli (Komiyama, 2003).
Could the same hypothetical timing factors be used in both lineages? This was tested by attempting to switch the DL1 class of adPN to its lPN equivalent by simultaneously removing Acj6 and misexpressing Dfr. If the only differences between the DL1 adPN and its lPN equivalent are the POU domain lineage factors, it might be expected that the DL1 PNs lacking Acj6 but expressing Dfr now would target to a novel glomerulus. These PNs indeed acquire novel features compared to simple loss of Acj6. They no longer even partially innervate DL1. In a subset of clones, their axons also acquired novel branching patterns and terminal fields. However, a clear switch is not observed based both on these dendritic or axonal phenotypes. This could be due to inappropriate level and/or timing of transgene expression; it could also be because: (1) the hypothetical timing factors are not exactly the same in adPNs and lPNs; (2) Acj6 and Dfr are not the only factors distinguishing these two lineages, or (3) cell-cell interaction among PNs from the same lineage may play a role in determining targeting specificity (Komiyama, 2003).
Acj6 is necessary not only for PN dendritic targeting, but also for establishing highly stereotyped PN axon branching patterns and terminal fields in a higher olfactory center. This is best exemplified by the analysis of DL1 single-cell clones. acj6-/- DL1 PNs are defective specifically in the dorsal branch without affecting general axon growth and guidance. This specific phenotype suggests that Acj6 plays a role in selecting synaptic connections with specific third order neurons. Axon terminal arborizations of other classes of PNs are also likely to be regulated by Acj6, as revealed by phenotypes from neuroblast clones containing ~13 classes of adPNs. As for Dfr, there is no evidence from loss-of-function studies that it plays a role in PN axon terminal arborization because there is no equivalent in the lateral lineage to the DL1 PN, which can be unambiguously identified independent of its dendritic innervation. However, the fact that simultaneous loss of Acj6 and gain of Dfr in DL1 clones result in qualitatively different axonal phenotypes compared with simple loss of Acj6 suggests that Dfr also plays a role in regulating axon terminal arborization in the lateral horn (Komiyama, 2003).
These observations bring back the question of why PNs are prespecified to project their dendrites to specific glomeruli and thereby receive specific olfactory input, and to have axons exhibiting specific branching patterns and terminal fields, presumably allowing stereotyped connections with third order neurons. By making PNs genetically distinct at the outset, it is possible to coordinate the dendritic choices of different glomeruli and the specific connections made by axons in higher centers. This coordination may contribute to innate behavioral responses to odorant stimuli by allowing a highly stereotyped relaying of olfactory information from the periphery to higher olfactory centers. Mechanistically, it is possible that PNs use similar cell surface molecules, whose expression depends on specific transcription factors such as Acj6 and Dfr, to guide both dendrites and axons to appropriate targets. The dual Acj6 phenotypes (both axonal and dendritic) provide support for this hypothesis. In ongoing forward genetic screens and candidate tests to identify genes necessary for PN dendritic and axonal connectivity, additional mutants have been found with simultaneous defects in dendritic targeting and axonal arborization (Komiyama, 2003).
In theory, the dual phenotypes in dendrites and axons could be caused by primary defects in dendritic targeting, with axon arborization defects as a secondary consequence, or vice versa. However, two lines of evidence argue against such possibilities: (1) developmental studies indicate that there is not a sequential development of dendritic and axonal arborization; (2) different mutants exhibit different ranges and specificity in their axonal and dendritic phenotypes -- even for individual PNs with the same mutant genotype, there was no clear correlation between the severity of dendritic and axonal phenotypes. The possibility is thus favored that the correct targeting of PN axons and dendrites are both directly regulated events rather than a sequential process in which, for example, the correct targeting of dendrites then instructs the corresponding axonal arborization (Komiyama, 2003).
POU domain transcription factors are used widely in C. elegans, Drosophila, and mammalian development. In particular, classes III and IV POU domain proteins play a variety of important roles in neural development. C. elegans UNC-86, the founding member of the POU IV class, is expressed shortly after asymmetric division in one of the two daughter cells. In unc-86 mutants, the daughter neuroblast that usually expresses UNC-86 now acquires the fate of its parental neuroblast, resulting in reiterations of cell lineage. UNC-86 also regulates differentiation of a number of neuronal classes such as touch sensory neurons or HSN motor neurons. In mammals, 3 class IV and 4 class III POU domain proteins are widely expressed in the nervous system during development. Knockout experiments demonstrate their important functions in different developmental processes. Because there is genetic redundancy between members of the same class, however, phenotypes resulting from single gene knockouts tend to reflect defects in cells that uniquely express that particular POU domain protein (Komiyama, 2003).
Acj6 and Dfr are respectively the single existing members of the class IV and class III POU domain proteins in Drosophila. Both genes have been shown to play a variety of roles in development. In particular, photoreceptor axon targeting is disrupted in acj6 mutants, however this phenotype is not cell autonomous (Acj6 is not expressed in photoreceptors) and is probably due to a requirement for Acj6 in the target lamina neurons. By restricting genetic manipulations to a small subset of neurons with well-defined connection specificity, the requirement of Acj6 and Dfr in other developmental events is bypassed and focus was placed on their function in olfactory projection neurons. This study assigns a new function for POU domain proteins: regulating lineage-dependent wiring specificity down to specific synapse formation. Interestingly, PNs from two lineages utilize two POU domain proteins of different classes for analogous functions. It remains to be seen whether the large number of mammalian POU domain proteins could be used in this way to regulate the specificity of numerous connections necessary to assemble the mammalian nervous system (Komiyama, 2003).
Lastly, Acj6 functions in a subset of ORs to regulate the expression of olfactory receptors; it is possible that it also regulates other molecules including putative ORN axon targeting molecules (which are likely to be distinct from the ORs themselves). The demonstration that Acj6 is necessary for dendritic targeting specificity of a subset of PNs raises an intriguing possibility that Acj6 may regulate matching ORNs and PNs destined to form synaptic connections. In fact, Acj6 is also expressed in a subset of neurons whose cell bodies are located near the lateral horn, one of the two central targets of PN axons. Thus, it is even feasible that Acj6 also regulates matching of synaptic partners in the next olfactory center. Molecular markers and other genetic tools are currently being developed to test these intriguing possibilities (Komiyama, 2003).
Adaptation to diverse habitats has prompted the development of distinct
organs in different animals to better exploit their living conditions. This is
the case for the respiratory organs of arthropods, ranging from tracheae in
terrestrial insects to gills in aquatic crustaceans. Although
Drosophila tracheal development has been studied extensively, the
origin of the tracheal system has been a long-standing mystery. Tracheal placodes and leg primordia arise from a common pool of cells in
Drosophila, with differences in their fate controlled by the
activation state of the wingless signalling pathway. Early events that trigger leg specification have been elucidated and it is shown
that cryptic appendage primordia are associated with the tracheal placodes
even in abdominal segments. The association between tracheal and appendage
primordia in Drosophila is reminiscent of the association between
gills and appendages in crustaceans. This similarity is strengthened by the
finding that homologues of tracheal inducer genes are specifically expressed
in the gills of crustaceans. It is concluded that crustacean gills and insect
tracheae share a number of features that raise the possibility of an
evolutionary relationship between these structures. An evolutionary
scenario is proposed that accommodates the available data (Franch-Marro, 2006).
The Drosophila tracheal system has a clearly metameric origin,
arising from clusters of cells, on either side of each thoracic and abdominal
segment, that express the tracheal inducer genes trachealess
(trh) and ventral veinless (vvl). Conversely, the leg
precursors can be recognized as clusters of cells that express the
Distal-less (Dll) gene, on either side of each thoracic
segment; these will give rise both to the Keilin's Organs (KOs, the
rudimentary legs of the larvae) and to the three pairs of imaginal discs that
will give rise to the legs of the adult fly (Franch-Marro, 2006).
To investigate whether there is a direct physical association between the
leg and tracheal primordia, Drosophila embryos co-stained
for the expression of trh and early markers of leg primordia were examined.
Although Dll is one of the most commonly used markers for the leg
primordia, it is not the earliest gene required for their specification.
Instead, a couple of related and apparently redundant genes,
buttonhead (btd) and Sp1, act upstream of
Dll in the specification of these primordia (Estella, 2003). Examining the specification of tracheal cells with respect to btd expression, tracheal cells were observed to appear in close apposition to btd-expressing cells, from the earliest stages of their appearance (by stage 9/early stage 10). Interestingly, unlike Dll, btd is initially expressed both in the thoracic and abdominal segments, and its expression is restricted to the thoracic segments later, under the influence of the BX-C. Thus, the cells of the respiratory system in Drosophila always arise in close proximity to the cells that are fated to give rise to the legs (Franch-Marro, 2006).
To fully endorse this conclusion it is necessary to show that the
btd-expressing cells in the abdomen correspond to cryptic leg
primordia. This may be a key point because, although many of the genes
required for leg development are already known, it has not yet been possible
to induce leg development in abdominal segments (except by transforming these
segments into thoracic ones). In particular, although the Dll
promoter contains BX-C binding sites that repress its expression in the
abdominal segments, no ectopic appendage has been reported by misexpressing
Dll in the abdomen. These observations have lead to some doubts as to
whether a leg developmental program is at all compatible with abdominal
segmental identity (Franch-Marro, 2006).
Since the initial expression of btd in the abdominal segments is
downregulated by the BX-C genes, it was reasoned that sustained expression of
btd might overcome the repressive effect of the BX-C genes and force
the induction of leg structures in the abdomen. To test this, a
btd-GAL4 driver was used to drive btd expression, expecting that the
perdurance of the GAL4/UAS system would ensure a more persistent expression of
btd in its endogenous expression domain. No sign
was ever obtained of ectopic Dll expression or KOs in the abdominal segments, but the increased expression of btd had an effect on the
KOs of the thoracic segments, which had more sensory hairs than the three
normally found in wild-type KOs. Thus, on its own, btd seems unable to overcome BX-C repression of leg development (Franch-Marro, 2006).
One possibility would be that the BX-C genes could suppress appendage
development in the abdomen by independently repressing both btd and
Dll in this region. To assess this possibility, the same
btd-GAL4 driver was used to simultaneously induce the expression of both
btd and Dll. Under these circumstances, it was observed that KOs
develop in otherwise normal abdominal segments; as in the
previous experiment, the newly formed KOs have more than three sensory hairs.
These results suggest that expression of btd and Dll in the
btd-expressing abdominal primordia is sufficient to induce the
development of leg structures in the abdomen, overcoming the repressive effect
of the BX-C genes. Furthermore, these results demonstrate that these clusters
of btd-expressing cells in the abdomen are indeed cryptic leg primordia. These results clearly show that tracheal cells are specified in close proximity to the leg primordia, in both thoracic and abdominal segments (Franch-Marro, 2006).
Previous results have shown that the leg primordia are specified straddling
the segmental stripes of wingless (wg) expression in the
early embryonic ectoderm, whereas tracheal cells are specified in between these
stripes. To investigate whether wg might play a role in
determining the fate of these primordia, what happens when the
normal pattern of wg expression is disrupted was studied. In
wg mutant embryos, trh and vvl from the earliest
stages of their expression are no longer restricted to separate clusters of
cells; instead larger patches of expression add up to a continuous band of
cells running along the anteroposterior axis of the embryo, while
btd expression is suppressed in this part of the embryonic ectoderm.
Conversely, ubiquitous expression of wg suppresses trh expression, while causing an expansion of btd expression along the embryo. Restricted
activation or inactivation of the wg pathway by the expression of a
constitutive form of armadillo or a dominant-negative form of
dTCF, respectively, are also able to specifically induce or repress
trh and btd expression. trh/vvl and btd seem to respond independently to wg signalling and there is no sign of cross-regulation among them, since btd expression is normal in trh vvl double mutants, and trh and vvl expression is normal in mutants for a deficiency uncovering btd and Sp1 (Franch-Marro, 2006).
The role of wg as a repressor of the tracheal fate is further
illustrated by looking at the behaviour of transformed cells: the clusters of
cells that have lost btd expression and gained trh and
vvl expression in wg mutant embryos begin a process of
invagination that is characteristic of tracheal cells. Furthermore, these
cells also express the dof (stumps) gene, a
target gene of both trh and vvl in the tracheal cells. Although further development of these cells is hard to ascertain
because of gross abnormalities in wg- embryos, these
results indicate that they have been specified as tracheal cells. Thus,
wg appears to act as a genetic switch that decides between two
mutually exclusive fates in this part of the embryonic ectoderm: the tracheal
fate, which is followed in the absence of wg signalling; and the leg
fate, which is followed upon activation of the wg pathway. Given that there are no cell lineage restrictions setting apart the cells of the tracheal and leg
primordia, these two cell populations could be considered as a single
equivalence group, with the differences in their fate controlled by the
activation state of the wg signalling pathway (Franch-Marro, 2006).
A link between respiratory organs and appendages is also found in many
primitively aquatic arthropods, like crustaceans, where gills typically
develop as distinct dorsal branches (or lobes) of appendages called epipods.
Following the current observations, which suggest a link between respiratory organs
and appendages in Drosophila, whether further
similarities could be found between insect tracheal cells and crustacean
gills was examined. Specifically, whether homologues of the tracheal inducing
genes might have a role in the development of appendage-associated gills in
crustaceans was considered (Franch-Marro, 2006).
RT-PCR was used to clone fragments of the vvl and trh
homologues from Artemia franciscana and from Parhyale
hawaiensis, representing two major divergent groups of crustaceans
(members of the branchiopod and malacostracan crustaceans, respectively). In
the case of Artemia vvl, a fragment was cloned that corresponds to the
APH-1 gene and an antibody was generated for immunochemical staining in developing Artemia larvae. It was observed that Artemia Vvl is initially absent from early limb buds; it becomes weakly and uniformly expressed while the limb is developing its characteristic branching morphology, and becomes
strongly upregulated in one of the epipods as its cells begin to differentiate. Uniform weak expression persists in mature limbs, but expression levels in the epipod are always significantly higher. Expression of the trh homologue from Artemia appears to be restricted to the same epipod as Vvl.
Similarly, homologues of vvl and trh were cloned from Parhyale hawaiensis and their expression was studied by in situ hybridization. Both genes are specifically expressed in the epipods of developing thoracic appendages. Besides epipods, the Artemia trh and vvl homologues are also expressed in the larval salt gland, an organ with osmoregulatory functions during early larval stages of Artemia development (Franch-Marro, 2006).
What is the significance of the two Drosophila tracheal inducer
genes being specifically expressed in crustacean epipods/gills? One
possibility is that the expression of these two genes was acquired independently in insect tracheae and in crustacean gills. Alternatively, tracheal systems and gills may have inherited these expression patterns from a common evolutionary precursor, perhaps a respiratory/osmoregulatory structure that was already present in the common ancestors of crustaceans and insects (Franch-Marro, 2006).
The latter possibility is considered unlikely by conventional views,
because of the structural differences between gills and tracheae (external
versus internal organs, discrete segmental organs versus fused network of
tubes), and the difficulty to conceive a smooth transition between these
structures. Yet, analogous transformations have occurred during arthropod
evolution: tracheae can be organized as large interconnected networks or as
isolated entities in each segment (as in some apterygote insects),
invagination of external respiratory structures is well documented among
groups that have made the transition from aquatic to terrestrial environments
(terrestrial crustaceans, spiders and scorpions), and conversely evagination
of respiratory surfaces is common in animals that have returned to an aquatic
environment (tracheal gills or blood gills in aquatic insect larvae). A
very similar (but independent) evolutionary transition is, in fact, thought to
have occurred in arachnids, where gills have been internalised to give rise to
book lungs, and these in turn have been modified to give rise to tracheae in
some groups of spiders. Thus, a relationship between insect tracheae and crustacean
gills is plausible (Franch-Marro, 2006).
A particular type of epipod/gill has also been proposed as the origin of
insect wings, a hypothesis that has received support from the specific
expression in a crustacean epipod of the pdm/nubbin (nub) and apterous
(ap) genes - that have wing-specific functions in Drosophila. In
fact, the Artemia nub and ap homologues are expressed in the
same epipod as trh and vvl, raising questions as to the
specific relationship of this epipod with either tracheae or wings. A
resolution to this conundrum becomes apparent when one considers the different
types of epipods/gills found in aquatic arthropods, and their relative
positions with respect to other parts of the appendage (Franch-Marro, 2006).
The primary branches of arthropod appendages, the endopod/leg and exopod,
develop straddling the anteroposterior (AP) compartment boundary, which
corresponds to a widely conserved patterning landmark in all arthropods. Different types of epipods/gills, however, differ in their
position with respect to this boundary. For example, in the thoracic
appendages of the crayfish, some epipods develop spanning the AP boundary
[visualized by engrailed (en) expression running across the
epipod], whereas others develop exclusively from anterior cells (with no
en expression). Given that wing primordia comprise cells from both the
anterior and posterior compartments, wings probably derived from structures
that were straddling the AP boundary. Conversely, given that tracheal
primordia arise exclusively from cells of the anterior compartment (anterior
to en and even wg-expressing cells), it seems probable that tracheal cells evolved from a population of cells that was located in the anterior compartment. In this respect, it is interesting to note that the former type of epipods express nub, whereas the latter do not (Franch-Marro, 2006).
In summary, it is suggested that the ancestors of arthropods had
specific areas on the surface of their body that were specialized for
osmoregulation and gas exchange. Homologues of trh and vvl
were probably expressed in all of these cells and played a role in their
specification, differentiation or function. Some of these structures were
probably associated with appendages, in the form of epipods/gills or other
types of respiratory surfaces. A particular type of gill, straddling the AP
compartment boundary, is likely to have given rise to wings,
whereas respiratory surfaces arising from anterior cells only may have given
rise to the tracheal system of insects. Confirmation of this hypothetical
scenario may ultimately come from the discovery of new fossils, capturing
intermediate states in the transition of insects from an aquatic to a
terrestrial lifestyle (Franch-Marro, 2006).
Proper information processing in neural circuits requires establishment of specific connections between pre- and postsynaptic neurons. Targeting specificity of neurons is instructed by cell-surface receptors on the growth cones of axons and dendrites, which confer responses to external guidance cues. Expression of cell-surface receptors is in turn regulated by neuron-intrinsic transcriptional programs. In the Drosophila olfactory system, each projection neuron (PN) achieves precise dendritic targeting to one of 50 glomeruli in the antennal lobe. PN dendritic targeting is specified by lineage and birth order, and their initial targeting occurs prior to contact with axons of their presynaptic partners, olfactory receptor neurons. A search was performed for transcription factors (TFs) that control PN-intrinsic mechanisms of dendritic targeting. Two POU-domain TFs, acj6 and drifter have been identified as essential players. After testing 13 additional candidates, four TFs were identified, (LIM-homeodomain TFs islet and lim1, the homeodomain TF cut, and the zinc-finger TF squeeze) and the LIM cofactor Chip, that are required for PN dendritic targeting. These results begin to provide insights into the global strategy of how an ensemble of TFs regulates wiring specificity of a large number of neurons constituting a neural circuit (Komiyama, 2007).
For technical simplicity, larval born GH146-Gal4-positive PNs, originating from three neuroblast lineages, anterodorsal (adPNs), lateral (lPNs), and ventral (vPNs), were studied. Out of ~25 classes defined by their glomerular targets, focus was placed on 17 classes whose target glomeruli are reliably recognized across different animals. The MARCM technique allows visualization and genetic manipulation of PNs in neuroblast and single-cell clones in otherwise heterozygous animals, so PN-intrinsic programs can be studied for dendritic targeting. GH146 is expressed only in postmitotic PNs (Komiyama, 2007).
acj6 and drifter have been identified as lineage-specific regulators of PN dendritic targeting. To identify additional transcription factors (TFs) that regulate dendritic targeting of different PN classes, candidates were tested that have been shown to regulate neuronal subtype specification and targeting specificity and have available loss-of-function mutants. The following was tested; (1) the expression of candidate genes in PNs at 18 hr after puparium formation (APF) when PN dendrites are in the process of completing their initial targeting, and/or (2) their requirement in PNs by examining dendritic targeting in homozygous mutant MARCM clones (Komiyama, 2007).
DL1 adPN expresses Acj6, an adPN lineage factor, but not Drifter or Cut. acj6−/− DL1 PNs typically have diffuse dendrites that always innervate, but are not limited to, DL1. drifter misexpression alone did not affect their dendritic targeting. However, when loss of acj6 and gain of drifter were combined, the dendrites completely missed DL1 and targeted anterior glomeruli (Komiyama, 2007).
Misexpression of Cut alone caused DL1 PNs to target part of DL1 and the vicinity, similar to acj6−/−. Notably, this diffuse phenotype was directional, because most mistargeted dendrites targeted medially to DL1 (Komiyama, 2007).
cut misexpression combined with loss of acj6 caused severe mistargeting of DL1 adPNs. The dendrites completely missed DL1 and occupied the medial to dorsomedial AL, typically VM2, DM6, and DC1. Interestingly, these glomeruli are all adPN targets near DM1 and DM2, the two glomeruli that most frequently fail to be innervated by cut−/− lPNs. One interpretation is that loss of acj6 made the DL1 adPN more sensitive to the instructive information of cut to target the medial AL, but the remaining lineage information kept the dendrites within the adPN glomeruli in the area. If this were true, adding a lPN lineage factor drifter may bring the dendrites to DM1 or DM2, since this might recreate, based on partial knowledge of the TF code, a code for targeting these glomeruli. Loss of acj6 and misexpression of cut and drifter were combined simultaneously in DL1 adPNs. Under this condition, the dendrites again mostly targeted the medial to dorsomedial AL. However, glomerular preferences were strikingly different: they frequently innervated 1, DM2, and DA2. Notably, DA2 and DM2 are lPN targets (Komiyama, 2007).
These results suggest that cut and drifter have qualitatively different instructive information, with cut controlling global targeting and drifter controlling local glomerular choice according to their lineage (Komiyama, 2007).
ventral veins lacking mutants show a disruption of the developing tracheal tree as well as commissural defects in the developing CNS. These defects appear to be caused by a failure in proper migration of tracheal cells and midline glia (Anderson, 1995).
Since SOX2 can rescue Dichaete mutant phenotypes and is known to interact
with the POU-domain transcription factor OCT-3, the possibility of a genetic interaction between
Dichaete and the Drosophila POU-domain gene ventral
veinless was examined. vvl is expressed in the midline and is required for
correct MGL development. In embryos homozygous for the vvl ZM allele, which
has reduced but detectable levels of Vvl, anterior and posterior
commissures fail to separate correctly, the longitudinals are
thinner and there are regions where they collapse towards the
midline. As with Dichaete hypomorph, only a small number
of neuromeres are affected. However, in Dichaete;vvl double mutants all phenotypes are far more pronounced and occur in
almost every hemisegment demonstrating a strong synergistic effect on the development of the nerve cord. Staining with anti-Fasciclin II shows that most of the longitudinal axons
cross the midline many times with a roundabout-like phenotype. The midline of the
double mutant embryos was tested with anti-Slit and very few cells were found
at stage 16. The few remaining cells stain very weakly and are
found ventral to the commissures. Similar results are
obtained with Argos mRNA. In single Dichaete and vvl
mutants, Slit expression is only weakly affected. However, glial
cells with reduced expression and aberrant morphology have been
found. To support the contention that Dichaete and vvl interact, the consequences of ectopic expression of Dichaete
and Vvl were examined. Ectopic expression of Vvl in segregating neuroblasts alone does
not disrupt the neuropile. Expression of
Dichaete alone causes weak defects in the commissures
mainly thinning of posterior commissures and thickening of the
anterior commissure. When Dichaete and Vvl are
expressed together in neuroblasts, however, the neuropile phenotypes are far
more severe. The longitudinals collapse toward the midline
throughout the neuropile and, in some segments, commissures
appear fused. In this case, unlike the Dichaete;vvl double
mutant, anti-Fasciclin II staining shows collapse of the
longitudinals toward the midline but they do not cross the
midline. Taken together, these data suggest that, as in the
mouse, SOX and POU domain transcription factors interact to
regulate the expression of target genes (Soriano, 1998).
The Drosophila tracheal system arises from clusters of ectodermal cells that invaginate and migrate to originate a network of epithelial
tubes. Genetic analyses have identified several genes that are specifically expressed in the tracheal cells and are required for tracheal
development. Among them, trachealess (trh) is able to induce ectopic tracheal pits and therefore it has been suggested that it would act
as an inducer of tracheal cell fates; however, this capacity appears to be spatially restricted. The expression of the tracheal
specific genes in the early steps of tracheal development and their crossinteractions have been examined. There is a set of primary genes including trh
and ventral veinless (vvl) whose expression does not depend on any other tracheal gene and a set of downstream genes whose expression
requires different combinations of the primary genes. The combined expression of primary genes is sufficient to induce some
downstream genes but not others. These results indicate that there is not a single master gene responsible for the appropriate expression of the
tracheal genes and support a model where tracheal cell fates are induced by the cooperation of several factors rather than by the activity of a
single tracheal inducer (Boube, 2000).
trh and vvl appear to initiate or to act very early in the genetic hierarchy specifying tracheal development. vvl expression in the tracheal cells is independent of trh function.
It is also found that trh expression in the tracheal cells is
independent of vvl function indicating that the
two genes act in parallel in the control of tracheal cell development. btl, a gene encoding an FGF receptor homolog required for tracheal migration, is a target of trh: btl requires vvl for
the maintenance of its transcription. Transduction of
the FGF signalling also requires the Downstream of FGF (Dof) protein, which is
specifically expressed in the tracheal cells. However, dof is
not a target gene activated as a result of FGF signaling as its
expression is not affected in btl mutant embryos. Conversely, the results show that the specific expression of dof in the tracheal cells is dependent on trh and vvl activity. Thus, trh and vvl enable the
tracheal cells to be competent to FGF signaling by regulating
the expression of at least two elements (btl and dof) acting at
different steps in the Btl pathway (Boube, 2000).
In contrast to the general requirement of the Btl pathway,
the Dpp and EGF pathways are required for migration of
certain branches of the tracheal system; competence of the
tracheal cells to those signals depends on the specific
tracheal expression in the tracheal cells of tkv and rho,
respectively. Similarly to the btl pathway, rho expression in the tracheal cells depends both on trh
and vvl function.
However, while tracheal expression of tkv also depends on
vvl, it appears to be independent of trh. The opposite
appears to be the case for two other tracheal genes, tracheal defective (tdf) and pebbled (peb) [also known as hindsight
(hnt)], which code for two putative transcription factors. Both genes
appear to be targets of trh but they are present in the tracheal cells of vvl mutant embryos. Thus, some tracheal genes
seem to be common targets of vvl and trh but others seem
to depend only on one of them (Boube, 2000).
Whether knirps (kni) and knirps
related (knrl) would fit in this genetic hierarchy was also analyzed. Both genes code for putative transcription factors that are expressed in
overlapping patterns and share redundant functions during
tracheal development; both genes have an early expression in
the tracheal placodes and a later expression in a particular
subset of tracheal branches. Quite surprisingly, it was found that the early tracheal expression of kni is not abolished in either trh or vvl mutant embryos and also that all the tracheal genes mentioned above are
expressed in the tracheal cells of embryos that are mutant for
a deficiency that uncovers both kni and knrl.
In summary, there are some primary tracheal genes whose expression appears not to be regulated by any other tracheal gene. Subsequently, one or more of
those primary genes are necessary for the appropriate expression of other downstream genes in the tracheal cells (Boube, 2000).
The complexity of regulatory interactions described
above indicates that more than one gene can act as an inducer of the expression of downstream tracheal genes. This seems to contrast with earlier results suggesting that the trh gene acts as the master gene for tracheal fate. Evidence for
this comes from experiments of ubiquitous expression of trh,
which generates new tracheal pits at the correct position in
anterior and posterior segments that normally do not form
pits. The same result is observed when an
UAS-trh construct is specifically expressed in the embryonic terminal regions by means of a sal-Gal4 line. In both
cases, induction of extra tracheal pits can be visualized very
early in development by the appearance of additional clusters of cells that express btl in more anterior and posterior
segments. However, the capacity of trh to induce
btl expression appears restricted to specific positions in the
embryo. The restricted activation of btl by trh is not due to low levels of trh since the use of the Gal4/UAS system induces high
levels of trh transcripts. Conversely, the
alternative conclusion is favored: the activity of trh alone is not
sufficient to induce btl expression, probably because other
factors are also required in combination with trh.
vvl is a good candidate for such a factor. vvl is required to
induce some of the tracheal genes. In addition, vvl is
expressed independent of trh in the tracheal placodes
and in the analogous location within the segments that do
not form tracheal pits. These are precisely the positions where trh can induce additional tracheal pits. Thus, it was asked whether both trh and vvl are
required to instruct those cells to adopt a tracheal fate. On inducing an UAS-trh construct in the terminal regions of vvl
mutant embryos (with the same sal-Gal4 line as above),
ectopic induction of btl in new patches of cells is suppressed. This result indicates that it is the localized expression of vvl that accounts for the restricted induction of btl in
a particular set of cells upon general expression of trh. The situation is different in the normal tracheal placodes where vvl is dispensable for induction of btl expression and is only required for its maintenance (Boube, 2000).
The above experiments indicate that vvl is required for the
induction of extra tracheal pits in additional segments.
However, general expression of vvl with an UAS-vvl
construct does not induce additional tracheal pits or ectopic
expression of btl. It was asked whether the co-expression of vvl and trh would be sufficient to induce tracheal fates, as monitored by induction of btl expression. Indeed, simultaneous expression of an
UAS-vvl and an UAS-trh construct in the embryonic terminal regions under the common control of the sal-Gal4 line
induces btl expression throughout both regions.
Also, co-expression of vvl and trh in unrelated regions such
as the distal leg primordia (directed by a Dll-Gal4 line) is
sufficient to induce btl expression. Thus, vvl and
trh are both required and their co-expression is sufficient to
ectopically induce btl expression (Boube, 2000).
On the contrary, vvl and trh appear not to be sufficient for
the expression of the remaining tracheal genes. While
tracheal expression of dof, tdf, peb, tkv and rho require either vvl or trh, or both, no induction of
any of these genes is observed upon ectopic expression of vvl and/or trh. Therefore, these results raise the possibility that full induction of tracheal fates requires one or more
additional factors. In this regard it is worth noting that the
tracheal branches generated from ectopic trh in vvl expressing cells are abnormal and do not fuse with the normal tracheal tree (Boube, 2000).
Expression of trh is repressed by sal in the terminal
regions leading to the suggestion that this is the
mechanism that accounts for the confinement of tracheal
placodes to the central segments of the embryo. In contrast, vvl is expressed at the correct positions in segments that normally do not form
tracheal placodes, although its expression in those sites is
much weaker. Whether sal could also
regulate vvl expression was investigated. Indeed, vvl expression
is strongly increased in those sites in sal mutant embryos suggesting that vvl is downregulated by sal in the
segments that do not form tracheal pits. Similarly, kni expression is also upregulated in the same sites in sal mutant embryos. Repression of vvl and kni by sal could in principle be attributed to the downregulation of trh by sal. However, this seems not to be the case
because expression of vvl and early expression of kni in the
tracheal placodes does not depend on trh. Therefore, sal seems to independently downregulate trh, vvl and
kni in the most anterior and posterior embryonic regions (Boube, 2000).
Because trh and vvl are sufficient to activate btl, additional patches of btl expression were found in sal mutant
embryos. dof and rho are expressed in additional patches of cells in sal mutant embryos. Repression of dof and rho by sal could also be attributed to the downregulation of trh and vvl by
sal. However, this seems not to be the case because co-expression of trh and vvl in the sal domain is not sufficient to
induce either dof or rho expression. Instead, sal
could directly repress dof and rho or, alternatively, it could
repress an additional factor necessary for their induction.
In summary, many tracheal genes appear to be independently downregulated by sal in the terminal regions. Besides, the lack of sal expression does
not have the same effect on the tracheal genes. In particular,
some of the additional patches of trh expression are much
weaker than the normal ones. This difference is not so
pronounced in the case of vvl expression in sal mutant
embryos. Also, one additional anterior pair of cell clusters for rho and dof expression is observed. Therefore, not all the tracheal placodes are equivalent in sal mutant embryos (Boube, 2000).
In the embryonic epidermis, dacapo expression starts during G2 of the final division cycle and is required for the arrest of cell cycle progression in G1 after the final mitosis. The onset of dacapo transcription is the earliest event known to be required for the
epidermal cell proliferation arrest. To advance an understanding of the regulatory mechanisms that terminate cell proliferation at the appropriate stage, the control of dacapo transcription has been analyzed.
dacapo transcription is not coupled to cell cycle progression. It is not affected in mutants where proliferation is
arrested either too early or too late. Moreover, upregulation of dacapo expression is not an obligatory event of the cell cycle
exit process. During early development of the central nervous system, Dacapo cannot be detected during the final division
cycle of ganglion mother cells, while it is expressed at later stages. The control of dacapo expression therefore varies in
different stages and tissues. The dacapo regulatory region includes many independent cis-regulatory elements. The elements
that control epidermal expression integrate developmental cues that time the arrest of cell proliferation (Meyer, 2002).
The pattern of stg expression anticipates and determines the embryonic cell division pattern. stg expression in the embryonic epidermis before mitosis 16, therefore, is highly similar to the pattern of dap expression, which also precedes this terminal mitosis 16. To analyze whether stg and dap expression before mitosis 16 are mechanistically coupled, the distribution of stg transcripts in ventral veins lacking (vvl) and trachealess (trh) embryos was examined. vvl and trh are expressed within the prospective tracheal pit regions and are known to co-operate for the
specification of tracheal cell fate. The characteristic early dap expression in tracheal pits is not detected in vvl
embryos and it is severely decreased in trh embryos. Interestingly, while the characteristic early expression of stg is not observed in trh embryos, it is normal in vvl mutants. As expected, progression through mitosis 16 also occurred in the normal pattern in these vvl mutants. The absence of the characteristic early dap expression in tracheal pits of vvl embryos, therefore, is not preceded by a change in the proliferation program. These findings indicate that the control of stg and dap expression is mechanistically distinct. Moreover, they suggest that regulators of developmental fates control dap expression directly and not indirectly by controlling the cell proliferation program via other cell cycle regulators (Meyer, 2002).
Most of the cells in the embryonic peripheral nervous system (PNS) of
Drosophila are born in their final location. One known exception is
the group of lateral chordotonal organs (lch5) whose precursors form in a
dorsal position, yet the mature organs are located in the lateral PNS cluster.
Mutations in the u-turn (ut) locus perturb the localization
of lch5 neurons and result in a 'dorsal chordotonals' phenotype.
ut is shown to be allelic to ventral veinless (vvl), also known
as drifter. Vvl, a POU-domain transcription factor, has been shown to
participate in the development of tracheae and CNS in the embryo, and in wing
development in the adult; however, its role in PNS development has not been
described. Characterization of the 'dorsal chordotonals' phenotype of
vvl mutant embryos has revealed that in the absence of Vvl, cell fates
within the lch5 lineage are determined properly and the entire organ is
misplaced. Based on the positions of lch5 cells relative to each other in
mutant embryos, and in normal embryos at different developmental stages, a two-step model is proposed for lch5 localization. lch5 organs must first rotate
to assume a correct polarity and are then stretched ventrally to their final position. In this process, Vvl function is required in the ectoderm and possibly in the lch5 organs as well (Inball, 2003).
Vvl is also expressed in developing external sensory organs in the embryo
and in the adult. In the embryo, loss of Vvl function results in increased
apoptosis in specific es organs. Analysis of vvl mutant clones in adults reveals a requirement for Vvl in the control of cell number within the bristle lineage (Inball, 2003).
The ut gene was identified in a chemical mutagenesis screen for mutations affecting the development of the embryonic PNS. It was mapped by
meiotic recombination to genetic position 3[26] on the left arm of the third
chromosome. The approximate cytological location was determined to be
65A2;65E1. Three embryonic lethal alleles of ut were
generated in that screen: utH599,
utH76 and utM638. One of the candidate genes in these genomic regions is
ventral veinless (vvl), which maps to 65C5. Two
observations strongly suggest that ut and vvl could be
allelic: (1) a cross between ut alleles and the
In(3LR)282 chromosome, which has a breakpoint in the 65C-D region,
yielded adult progeny that lacked the L4 wing veins (vvl
is known to be required for wing vein formation) and (2) vvl mutant embryos exhibit a collapse of the anterior
segments of the ventral nerve cord (VNC), a phenotype observed also in
ut mutant embryos. Complementation tests between the
embryonic lethal allele vvlGA3 and ut alleles
demonstrate that ut and vvl are indeed allelic (Inball, 2003).
The question of how lch5 organs reach from their dorsal place of origin to
their final lateral position has not been answered yet. All existing data
regarding the so called 'lch5 migration' come from descriptions of abnormally
positioned (and abnormally oriented) lch5 neurons in different mutant
backgrounds. Based on these data, it has been suggested that concomitant with moving ventrally, the lch5 neurons turn approximately 145° counter clockwise (Inball, 2003).
By visualizing all the cells of lch5 organs located in a variety of
positions between the dorsal and lateral clusters, it was possible to determine that the polarity of the neurons reflects the polarity of the whole organ. The mature ch organ is subepidermal and contains a neuron ensheathed by a scolopale cell, a ligament cell and a cap cell. The cap cells are connected to the ectoderm by attachment cells, which derive from the ch lineage. When the neurons point ventrally, the ligament cells are the most dorsal cells
of the organ and the cap cells are the most ventral, whereas for the dendrites
to point dorsally, the ligament cells must be ventral and the cap cells
dorsal. In normal stage 12 embryos, the ligament cells can be detected at the dorsal part of the organ, whereas in older embryos they migrate ventrally to become the ventralmost cells of the organ. Based on these observations, a two-step model is proposed for the lateral localization of lch5 organs. In the first step,
rotation of the organ takes place. The organ rotates around the attachment cells, which anchor it to the ectoderm and thus function as a pivot. The
rotation results in both bringing the organ to its correct orientation and placing it in a more ventral position, closer to the lateral cluster. This step occurs during stage 12 and perhaps early stage 13. Once in their correct orientation the lch5 organs go through the second step, which involves ventral
stretching into their final shape and position, as seen in stage 15 or older embryos. This model is further supported by the fact that when the thoracic dch3 are forced to descend to the lateral cluster by overexpressing abd-A, their orientation is reversed and the ligament cells are found at their ventral edge (Inball, 2003).
What makes the lch5 organs go through this process? It is possible that the
ligament cells respond to a signal and migrate ventrally, thereby pulling with
them all other cells of the organ. Interestingly, a similar change from a
dorsal to ventral position has recently been shown for the PG3 cell, which
like the ligament cells expresses the glial marker REPO. This may suggest that
a common mechanism governs the change in position of both types of
REPO-expressing cells. Another possibility is that the rotation step depends
on the neurons and that the ligament cells are required only for stretching.
Since the rotation of lch5 is completed by early stage 13, when axonal outgrowth
begins, it is possible that the growing axons serve as a guide for the
ligament cells. However, in vvl mutant embryos, the lch5 organs often fail to stretch even when their axonal outgrowth seems largely normal. This suggests that although the axons may play a part in guiding the ligament
cells, other factor/s are required as well. The morphogenetic movements
occurring during dorsal closure in stage 14 are likely to affect the
dorsoventral position of the lch5 ectodermal attachment cell. However, dorsal
closure alone cannot account for the stretching of lch5, which is not
completed before late stage 15, well after dorsal closure is completed (Inball, 2003).
Mutations in three loci, abd-A, hth and sal, have been
shown to perturb the lateral localization of lch5 organs.
Mutations in these three loci result in both abnormal localization and
abnormal number of lch5 organs. However, decisions of organ number and organ
localization are not always coupled. For example, mutations in the EGFR
pathway gene rhomboid and the EGFR pathway antagonist argos,
affect the number of lch5 organs but only rarely affect their position. vvl is the first gene that affects the
localization of the lch5 organs without affecting their number. The abnormal
localization of lch5 organs in vvl mutant embryos is similar to the
abnormal localization of these organs in hth and abd-A
mutant embryos, suggesting these genes are required in the same developmental
pathway. However, epistasis experiments did not provide evidence for direct genetic interactions between vvl and hth or vvl and abd-A (Inball, 2003).
Vvl, a class III POU-domain transcription factor, and its mammalian
homologs, have been shown to be required for cell migration. Brn1 and Brn2, the mouse homologs of Vvl, have a crucial role in the migration of cortical neurons. In the CNS of Drosophila embryos, Vvl is required for the migration of midline glial cells. In the
embryo, Vvl is also required for tracheal cell migration and in its absence the tracheal tree fails to form (Inball, 2003).
The mechanism by which Vvl affects the lateral localization of lch5 organs is not clear. Cell migration requires the existence of signals from the environment and the ability of the migrating cell to receive and respond to these signals. In tracheal development,
Vvl functions autonomously and it was suggested to regulate the expression of
cell surface molecules necessary for the migration of tracheal cells. In
lch5 organs, Vvl expression is detected in the neurons. However, expressing
Vvl under elav-Gal4 regulation in vvl mutant background
could not rescue the mutant phenotype. This result suggests that the neuronal expression of Vvl is either not required or not sufficient for lch5 lateral
localization. Driving Vvl expression with ato-Gal4 rescues the
mutant phenotype; however, this occurs with a much lower efficiency than when Vvl is
expressed under arm-Gal4 regulation. The major differences between these two drivers are that while ato-Gal4 drives strong expression in the lch5 lineage and in a small group of ectodermal cells, arm-Gal4 induces strong expression throughout the ectoderm and only weak expression in
lch5 organs. Thus, the results of these experiments cannot determine
unambiguously where Vvl is required during lch5 lateral localization, and suggest it could function in both lch5 organs and the surrounding ectoderm, or in the ectoderm alone. The more efficient rescue generated by arm-Gal4 may indicate that the ectodermal expression of Vvl is the main factor with regard to lch5 positioning. In the ectoderm, Vvl could be involved in the generation of a positional cue. Although the rescue of lch5 localization is achieved by ubiquitous expression of Vvl in the ectoderm, it should be noted that the normal ectodermal expression of Vvl during critical stages of lch5 positioning (stages 12 and early 13) is not uniform. Vvl is more strongly expressed in a dorsal domain of the embryo, from the position of the lateral cluster dorsally. Later during stage 13, Vvl expression becomes uniform throughout the ectoderm. It is not clear yet whether this differential expression is significant in the context of lch5 positioning. Vvl has been shown to interact with other transcription factors in the CNS and trachea. Thus, another possibility is that an unidentified partner of Vvl confers a spatial specificity to its activity (Inball, 2003).
Two additional cell types in the vicinity of the developing lch5 organs
express high levels of Vvl: tracheal cells and oenocytes. The trachea is
probably not involved in the process of lch5 localization, since
trachealess mutants do not exhibit a 'dorsal ch' phenotype. The possible role of oenocytes in lch5 migration is intriguing.
Impaired lch5 localization is many times accompanied by partial or complete
loss of oenocytes, as seen in embryos mutant for abd-A, hth and vvl.
However, rhomboid mutants lack oenocytes, yet
their lateral ch organs (which consist of three, instead of five, scolopidia) are almost always positioned properly. Thus, it seems more probable that lch5 organs and oenocytes are independently affected by the same mutations (Inball, 2003).
In Drosophila, loss of es organ cells has been attributed to one of two reasons. Either the organ completely fails to form because of interference with the function of the proneural genes, or cell fate
transformations occur between the cells comprising these organs. However, in vvl mutant embryos the decreased number of these cells is a result of increased apoptosis. Any of the cells of the organ could be affected, and the remaining cells express typical markers, suggesting that initial decisions of cell fates are not impaired. It is therefore possible that Vvl is required
for cell survival in the developing es lineages. Another possibility is that Vvl is required for the differentiation of these organs, and that in its absence some of the cells fail to differentiate properly and go through apoptosis (Inball, 2003).
In mammals, POU-domain transcription factors play significant
roles in survival of cells in the nervous system. Members of the class IV POU-factors are known to be essential for differentiation and survival of PNS cells. The most
interesting of those in the context of Drosophila es organ
development is Brn3c, which is required for maturation and survival of the inner ear hair cells. The vertebrate inner ear hair cells are mechanosensory organs, considered homologous to Drosophila bristles in many aspects.
The parallelism between the two types of organs has been shown at the levels of function, structure and the molecular mechanisms responsible for their development. Mice deficient for Brn3c fail to develop inner ear hair cells and are completely deaf. A mutation in the human homolog of this gene has been shown to
cause progressive hearing loss. The defects seen in the development of the hair cells
in Brn3c-null mice are limited to maturation and survival of these organs (Inball, 2003 and references therein).
Although there is not sufficient evidence to consider a functional homology between Vvl, a class III POU-factor, and the mammalian class IV POU-factors, it will be interesting to determine whether the similarity of their loss-of-function phenotypes extends further at the molecular level (Inball, 2003).
vvl mutant clones in adult head tissue caused defects in bristle development that typically result in supernumerary cells. One possible explanation for an increase in the number of bristle cells is that too many precursors are formed as a result of inefficient lateral inhibition. In such a case, the appearance of complete ectopic organs would be expected. However, the
supernumerary cells do not constitute separate organs, rather they increase the number of cells within a single es organ. This observation suggests that one or two extra cell divisions take place, resulting in the production of extra cells within the lineage. Thus, it is possible that Vvl is required in
these cells for exit from the cell cycle (Inball, 2003).
Many abnormalities are also observed in the structure of the external support cells of the mutant bristles, especially in the shaft. Whether the structural defects are secondary to the abnormal pattern of cell division, or
they represent another independent role for Vvl in the differentiation of these structures remains to be determined (Inball, 2003).
Acampora, D., et al. (1999). Progressive impairment of developing neuroendocrine cell lineages in the
hypothalamus of mice lacking the Orthopedia
gene. Genes Dev. 13: 2787-2800.
Agarwal, V. R. and Sato, S M. (1991). XLPOU 1 and XLPOU 2, two novel POU domain genes expressed
in the dorsoanterior region of Xenopus embryos.
Dev. Biol. 147: 363-73
Andersen, B., et al. (1997). Functions of the POU domain genes Skn-1a/i and
Tst-1/Oct-6/SCIP in epidermal differentiation. Genes Dev. 11(14): 1873-1884
Anderson, M.G., Perkins, G.L., Chittick, P., Shrigley, R.J. and Johnson, W.A. (1995). drifter, a Drosophila Pou domain transcription factor, is required for correct differentiation and migration of tracheal cells and midline glia. Genes Dev. 9(1): 123-37
Anderson, M.G., et al. (1996). Function of the Drosophila POU domain
transcription factor Drifter as an upstream
regulator of Breathless receptor tyrosine kinase
expression in developing trachea. Development 122: 4169-4178
Arbouzova, N. I., Bach, E. A. and Zeidler, M. P. (2006). Ken & barbie selectively regulates the expression of a subset of Jak/STAT pathway target genes.
Curr. Biol. 16(1): 80-8. 16401426
Bermingham, J. R., et al. (2002). Identification of genes that are downregulated in the absence of the POU domain transcription factor pou3f1 (Oct-6, Tst-1, SCIP) in sciatic nerve. J. Neurosci. 22(23): 10217-10231. 12451123
Billin, A., Cockerill, K., and Poole, S. (1991). Isolation of a family of Drosophila POU domain genes expressed in early development. Mech. Dev. 34: 75-84. Boube, M., Llimargas, M. and Casanova, J. (2000). Cross-regulatory interactions among tracheal genes support a co-operative
model for the induction of tracheal fates in the Drosophila embryo. Mech. Dev. 271-278.
Burglin, T. R. and Ruvkun, G. (2001). Regulation of ectodermal and excretory function by the C. elegans POU homeobox gene ceh-6. Development 128: 779-790. 11171402
Castro, D. S., et al. (2006). Proneural bHLH and Brn proteins coregulate a neurogenic program through cooperative binding to a conserved DNA motif. Dev. Cell 11(6): 831-44. Medline abstract: 17141158
Certel, K., et al. (1996). Distinct variant DNA-binding sites determine cell-specific autoregulated expression of the Drosophila POU domain transcription factor drifter in midline glia or trachea. Mol. Cell. Biol 16: 1813-23
Certel, K., et al. (2000). Restricted patterning of vestigial expression in Drosophila wing imaginal
discs requires synergistic activation by both Mad and the Drifter POU domain
transcription factor. Development 127: 3173-3183.
Certel, S. J. and Thor, S. (2004). Specification of Drosophila motoneuron identity by the combinatorial action of POU and LIM-HD factors. Development 131: 5429-5439. 15469973
de Celis, J.F., Llimargas, M. and Casanova, J. (1995). ventral veinless, the gene encoding the Cf1a transcription factor, links positional information and cell differentiation during embryonic and imaginal development in Drosophila melanogaster. Development 121: 3405-3416
Eisen, T., et al. (1995). The POU domain transcription factor Brn-2: elevated expression in
malignant melanoma and regulation of melanocyte-specific gene
expression. Oncogene 11: 2157-2164
Estella, C., Rieckhof, G., Calleja, M. and Morata, G. (2003). The role of buttonhead and Sp1 in the development of the ventral imaginal discs of Drosophila. Development 130: 5929-5941. 14561634
Estes, P., Fulkerson, E. and Zhang, Y. (2008). Identification of motifs that are conserved in 12 Drosophila species and regulate midline glia vs. neuron expression.
Genetics 178(2): 787-99. PubMed Citation: 18245363
Franch-Marro, X., Martin, N., Averof, M. and Casanova, J. (2006). Association of tracheal placodes with leg primordia in Drosophila and implications for the origin of insect tracheal systems. Development 133(5): 785-90. 16469971
Hauptmann, G., and Gerster, T., et al. (1996). Complex expression of the zp-50 pou gene in the embryonic
zebrafish brain is altered by overexpression of sonic hedgehog. Development 122: 1769-1780
Herr, W. and Cleary, M. A. (1995). The POU domain: versatility in transcriptional regulation by a flexible two-in-one DNA-binding domain. Genes Dev. 9: 1679-93
Huang, S. and Sato, S. (1998). Progenitor cells in the adult zebrafish nervous system express a
Brn-1-related POU gene, tai-ji. Mech. Dev. 71(1-2): 23-35
Hussain, M. A., et al. (1997). POU domain transcription factor brain 4 confers pancreatic
alpha-cell-specific expression of the proglucagon gene through
interaction with a novel proximal promoter G1 element. Mol. Cell. Biol. 17(12): 7186-7194
Inball, A., Levanon, D. and Salzberg, A. (2003). Multiple roles for u-turn/ventral veinless in the development of Drosophila PNS. Development 130: 2467-2478. 12702660
Jaegle, M., et al. (2003). The POU proteins Brn-2 and Oct-6 share important functions in Schwann cell development. Genes Dev. 17: 1380-1391. 12782656
Johnson, W. and Hirsh, J. (1990). Binding of a Drosophila POU domain protein to a sequence element regulating gene expression in specific dopaminergic neurons. Nature 342: 467-470. Josephson, R., et al. (1998). POU transcription factors control expression of CNS stem
cell-specific genes. Development 125(16): 3087-3100
Kambadur, R., Koizumi, K., Stivers, C., Nagle, J., Poole, S. J. and Odenwald, W. F. (1998). Regulation of POU genes by castor and hunchback
establishes layered compartments in the Drosophila CNS. Genes Dev. 12(2): 246-60
. 9436984
Klemm, J., Rould, M., Aurora, R., Herr, W. and Pabo, C. (1994). Crystal structure of the Oct-1 POU domain bound to an octamer site: DNA recognition with tethered DNA-binding modules. Cell 77: 21-32
Komiyama, T., Johnson, W. A., Luo, L. and Jefferis, G. S. X. E. (2003). From lineage to wiring specificity: POU Domain transcription factors control precise connections of Drosophila olfactory projection neurons. Cell 112: 157-167. 12553905
Komiyama, T. and Luo, L. (2007). Intrinsic control of precise dendritic targeting by an ensemble of transcription factors. Curr. Biol. 17(3): 278-85. Medline abstract: 17276922
Llimargas, M. and Casanova, J. (1997). ventral veinless, a POU domain transcription factor, regulates different transduction
pathways required for tracheal branching in Drosophila. Development 124(17): 3273-3281
Llimargas, M. and Casanova, J. (1999).
EGF signalling regulates cell invagination as well as cell migration
during formation of tracheal system in Drosophila. Dev. Genes Evol. 209: 174-179
Ma, Y., et al. (2000). Functional interactions between Drosophila bHLH/PAS, Sox, and POU
transcription factors regulate CNS midline expression of the slit gene. J. Neurosci. 20(12): 4596-4605. 10844029
Mathis, J. M., et al. (1992). Brain 4: a novel mammalian POU domain transcription factor
exhibiting restricted brain-specific expression.
EMBO J. 11: 2551-61
Matsuzaki, T., Amanuma, H., and Takeda, H. (1992). A POU-domain gene of zebrafish, ZFPOU1, specifically expressed
in the developing neural tissues. Biochem. Biophys. Res. Commun. 187: 1446-53
Matsuo-Takasaki, M., Lim, J. H. and Sato, S. M. (1999). The POU domain gene, XlPOU 2 is an essential downstream determinant of neural induction. Mech. Dev. 89: 75-85.
Meier, S., Sprecher, S. G., Reichert, H. and Hirth, F. (2006). ventral veins lacking is required for specification of the tritocerebrum in embryonic brain development of Drosophila. Mech. Dev. 123(1): 76-83. 16326080
Meyer, C. A., et al. (2002). Drosophila p27Dacapo expression during embryogenesis is controlled by a complex regulatory region independent of cell cycle progression. Development 129: 319-328. 11807025
Michaud, J. L., et al. (1998). Development of neuroendocrine lineages requires the bHLH-PAS transcription factor SIM1. Genes Dev. 12(20): 3264-75
Murphy, A. M., et al. (1995).
The breathless FGF receptor homolog, a downstream
target of Drosophila C/EBP in the developmental control of
cell migration. Development 121: 2255-2263
Nakai, S., et al. (2003). Crucial roles of Brn1 in distal tubule formation and function in mouse kidney. Development 130: 4751-4759. 12925600
Okazawa, H., et al. (1996). Regulation of striatal D1A dopamine receptor gene transcription by Brn-4. Proc. Natl. Acad. Sci. 93: 11933-38
Reményi, A., et al. (2003). Crystal structure of a POU/HMG/DNA ternary complex suggests differential assembly of Oct4 and Sox2 on two enhancers. Genes Dev. 17: 2048-2059. 12923055
Roch, F., Jiménez, G. and Casanova, J. (2002). EGFR signalling inhibits Capicua-dependent repression during specification of Drosophila wing veins. Development 129: 993-1002. 11861482
Rorth, P., and Montell, D. J. (1992). Drosophila C/EBP: a tissue-specific DNA-binding protein
required for embryonic development. Genes Dev 6: 2299-311
Schonemann, M. D., et al. (1995). Development and survival of the endocrine hypothalamus and posterior
pituitary gland requires the neuronal POU domain factor Brn-2.
Genes Dev 9: 3122-3135
Soriano, N. S. and Russell, S. (1998). The Drosophila SOX-domain protein Dichaete is required for
the development of the central nervous system midline. Development 125(20): 3989-3996
Sotillos, S. and de Celis, J. F. (2006). Regulation of decapentaplegic expression during Drosophila wing veins pupal development. Mech. Dev. 123(3): 241-51. PubMed Citation: 16423512
Sotillos, S., Díaz-Meco, M. T., Moscat, J. and Castelli-Gair Hombría, J. (2008). Polarized subcellular localization of Jak/STAT components is required for efficient signaling. Curr. Biol. 18(8): 624-9. PubMed Citation: 18424141
Stuart, G. W., et al. (1995). POU-domain sequences from the flatworm Dugesia tigrina. Gene 161: 299-300
Sugitani, Y., et al. (2002). Brn-1 and Brn-2 share crucial roles in the production and positioning of mouse neocortical neurons. Genes Dev. 16: 1760-1765. 12130536
Treacy, M. N., et al. (1992). Twin of I-POU: A two amino acid difference in the I-POU homeodomain distinguishes an activator from an inhibitor of transcription. Cell 68: 491-505
Turner, E. E. (1996). Similar DNA recognition properties of alternatively spliced Drosophila POU factors. Proc. Natl. Acad. Sci. 93: 15097-15101
Verrijzer, C. P. and Van der Vliet, P. C. (1993). POU domain transcription factors. Biochim. Biophys. Acta 1173: 1-21
Witta, S. E., Agarwal, V. R. and S. M. (1995).
XIPOU 2, a noggin-inducible gene, has direct neuralizing activity.
Development 121: 721-730
Zelzer, E. and Shilo, B.-Z. (2000). Interaction between the bHLH-PAS protein Trachealess and the POU-domain protein Drifter, specifies tracheal cell fates. Mech. Dev. 91: 163-173.
drifter:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| Effects of Mutation
date revised: 10 June 2009
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