BIOLOGICAL OVERVIEW

Neural differentiation in Drosophila requires mechanisms to distinguish one cell from another. This is necessary because the nerves in the fly have very specific identities, and usually only a single cell, or just a very few per segment, are fated to assume a particular task and identity. Each unique cell fate is carried out by a specific combination of transcription factors. Pox neuro is one of a number of transcription factors that combine to determine cell fate in both the central and peripheral nervous system. Other genes determining cell fate in this specific fashion include even-skipped, fushi tarazu, Krüppel and runt.

Illustrative of Pox neuro's role in neuroblast differentiation is the analysis of poly-innervated external sense organs (p-es), known as the campaniform sensilla. These p-es bristles are innervated by several chemosensory neurons and one mechanosensory neuron. In contrast to p-es, the trichoid sensilla, another variety of external sense organs, are mechanosensory and monoinnervated.

Pox neuro is expressed in mother cells that give rise to p-es organs, and embryos deleted for Poxn have no p-es. Ubiquitous expression of Poxn leads to supernumary p-es and induces the transformation of trichoid sensilla to p-es.

In some of these mutants, new p-es induced by Poxn overexpression are found in positions where no trichoid sensilla are normally found. The new p-es are derived from transformed chordotonal neurons (internal stretch neurons). The induction of new p-es are accompanied by the induction of cut, a gene not normally expressed in chordotonal neurons. Both cut and Poxn are normally expressed in p-es neurons. Although cut is considered to be downstream of achaete-scute, it is apparent that cut is also responsive to Poxn. This illustrates the importance of cross-regulatory interactions in cell fate determination. The pathways are not linear (Vervoort, 1995).

The function of Poxn in the development of the larval peripheral nervous system (PNS) and in other developmental aspects has been analyzed using a loss-of-function mutant of Poxn. In addition to the transformation of p-es into m-es organs in the mutant embryo, the external structure of the trichome-like sensilla (hairs) misdifferentiates into that of the campaniform-like sensilla (papillae) in the second and third larval instars. Poxn is expressed in a cell associated with the external structure of the trichome-like sensilla in the first and second instar larvae. These results imply that Poxn is required in two distinct steps in the development of the larval PNS: (1) development of the larval p-es organs during embryogenesis and (2) re-formation of larval sensory hairs after each larval molt. In addition to its expression in the developing PNS, Poxn is also expressed in concentric domains of the leg and antennal imaginal discs of early third instar larvae, and in the region of the wing disc that will form the wing hinge. The loss of Poxn function results in defects of segmentation of the tarsus and antenna and in a distortion in the wing hinge. These results indicate that the Poxn gene plays crucial roles in the morphogenesis of the appendages, in addition to its role in the early specification of neuronal identity (Awasaki, 2001).

Various types of sensory organs are arranged in a highly reproducible pattern on the thoracic and abdominal segments of Drosophila embryo and larva. In the wild type, the thoracic (T) segments bear two p-es organs, a lateral kolbchen (lk) and a ventral kolbchen (vk). The abdominal (A) segments also bear two p-es organs, a hair (h3) and a papilla (p6). The external sensory structures were examined in the first instar larva of the Poxn^- null mutant, Poxn⁷⁰. In the T segments, the lk is missing and an extra papilla-like structure forms in a more ventral position, just dorsal to p4. The vk is also missing and an extra papilla forms more dorsally, adjacent to p3. In the A segments, the ventral p-es organ p6 is absent and a new hair forms near the position of h1, while the dorsal p-es organ h3 is substituted by a papilla-like structure at a position dorsal to h2. These results confirm the observations on homozygous Df(2R)WMG embryos, where the Poxn locus is deleted, and demonstrate that Poxn is involved in the development of the external structures of larval p-es organs (Awasaki, 2001).

In order to determine whether the transformation of p-es organs in Poxn mutants affects the number of neurons as well as the external morphology of the organs, the development of the embryonic PNS was examined with a neuron specific antibody, MAb22C10. Peripheral neurogenesis is essentially complete and the arrangement of the neurons is highly invariant in the mid-embryonic stage. In the meso- and meta-thoracic hemisegments (T2 and T3) of the wild-type embryos, the two p-es organs, vk and lk, are innervated by three neurons, les3 and v'es3. In the Poxn^- mutant, the les3 neurons are substituted by a single neuron with a solely extending dendrite. The v'es3 neurons are also missing and instead a single neuron and an accompanying md neuron are arranged just below the lateral cluster. In the first to seventh abdominal hemisegments (A1-A7) of wild-type embryos, two p-es organs, a hair (h3) and a papilla (p6), are each innervated by two neurons, des2 and v'es2, respectively. In the Poxn^- mutant, the des2 neurons are missing in the dorsal cluster. Instead, a single neuron forms dorsal to lch5. Likewise the v'es2 neurons are substituted by a single es neuron accompanied by a md neuron, at a position near that of the lesA and ldaA neurons. Thus, in the Poxn^- mutant, the neurons that normally innervate the p-es organs are absent, and single neurons are found associated to the transformed external structures (Awasaki, 2001).

It was also noted that the axons of the neurons associated to the transformed structures fasciculate with those of the nearest neurons, rather than retracing the pathway that p-es neurons would normally have followed (Awasaki, 2001).

The SMCs of the p-es organs arise in stage 11 and they and their progeny were monitored using an enhancer trap line A37 or anti-Cut antibody. The expression pattern of A37 or Cut in early steps of PNS development were compared between the wild-type and Poxn⁷⁰ /Poxn⁷⁰ embryos. There are no differences in the pattern between them, indicating that the early steps in PNS development are normal in the mutant (Awasaki, 2001).

This loss of function analysis supports the idea that Poxn plays a central role in the specification of the p-es organs. The inactivation of Poxn transforms the external structures of p-es organs into those of m-es organs. In the absence of Poxn the number of p-es neurons is correspondingly reduced from 2-3 to 1, consistent with the observation that the mutation of Poxn results in a transformation of poly-innervated gustatory bristles into mono-innervated tactile bristles in the adult. Thus, the gene Poxn is functionally required for the development of both external and internal structures of p-es organs in embryos as well as in adults (Awasaki, 2001).

The position of the transformed organs is not the same as that of the original ones, indicating that the identity of the sensory organs influences the positions where they will eventually differentiate. In wild-type embryos, the thoracic p-es neurons (les3 and v'es3) are present in the lateral and ventral clusters, respectively, while the abdominal p-es neurons (des2 and v'es2) belong to the dorsal and ventral clusters. In the mutants, all transformed es neurons are present in the lateral clusters (Awasaki, 2001).

The early steps in PNS development, such as the formation of the SMCs and their progeny, are normal in the mutant. Therefore, the misplacement of the transformed es organs occurs during later development, most probably as a result of the change in organ identity that abolishes the programmed migration. The transformed SMCs will therefore keep their lateral position, and the organs will correspondingly appear displaced relative to the expected position of the untransformed p-es organs. A similar situation has been documented in the case of the chordotonal (ch) organs. The lateral ch organs (lch5) in the abdominal segments form dorsolaterally and migrate to a lateral position during their development. The homologous thoracic organs, lch3, do not undergo this migration and remain dorsolateral. When the precursors of the lch5 are transformed into those of es organs by the ubiquitous expression of Cut, they keep their original position and develop in a dorsolateral position. Thus, neural specification genes such as cut and Poxn, in combination with segmental identity genes such as Ubx, are involved in the final pattern of the sensory organs: the pattern depends not only on the position where precursors form, but also on subsequent migration during morphogenesis (Awasaki, 2001).

The external structure of es organs were examined in the second and third instar larvae of the Poxn^- mutants to confirm the above results. Surprisingly, it was found that the external structures of es organs in the second and third instar mutant larvae are different from those in the first instar larvae. In addition to the transformation of the p-es into m-es organs, all hairs disappear and are replaced by papilla-like structures both in T and A segments. The positions of the organs, however, do not change. This indicates that Poxn is required for the formation of hair-like external structures after the first instar (Awasaki, 2001).

To confirm the involvement of Poxn in hair formation, its expression during larval life was examined. Anti-Poxn immunostaining shows that Poxn is expressed in a cell closely associated with each hair, presumably the hair-secreting trichogen cell, during the first and second instars. This supports the idea that Poxn plays a role in specifying the external structure of larval hairs after each molt (Awasaki, 2001).

At each molt, the entire cuticle of the insects, including many specialized cuticular structures such as external sensory organs, is shed and has to be rebuilt again. During the reconstruction process, the trichogen and tormogen cells of the mechanosensory hairs synthesize material for the formation of the new shaft and socket, respectively. In the absence of Poxn function all hair shafts disappear and are replaced by papilla-like structures in the second and third instar larvae. Poxn is expressed during the first and second instars in individual cells closely associated with each hair, possibly the trichogen cells. These observations indicate that Poxn is required for the reconstruction of the hair shaft after each molt, although it is unclear whether the papilla-like structure in the mutant results from a transformation from hair into papilla or from the loss of the hair structure itself. First instar mutant larvae do not show this defect in hair structure, indicating that the formation of hair shaft during embryogenesis does not need Poxn. Thus, the initial differentiation of hair shafts during embryogenesis and their reformation after each larval molt appears to depend on distinct mechanisms (Awasaki, 2001).

The transformation of the external structures of larval es organs has been reported in the analysis of the BarH1 and BarH2 genes. When both Bar genes are deleted, the papillae are transformed into hairs, while the ubiquitous expression of one of them suffices to produce the reciprocal transformation of hairs into papillae. These results have been documented on embryos, however, and it is not known whether the BarH genes are also involved in the reconstruction of the hair/papilla structure after molting. An examination of possible interactions between Poxn and BarH at various stages would be interesting (Awasaki, 2001).

In order to discover other functions of Poxn, the pattern of expression of Poxn was examined in imaginal discs. In the leg discs of mid-third instar larvae, Poxn is expressed in two concentric circles in a region corresponding to the prospective tarsus. This expression is transient and disappears in the late third instar (wandering) larvae. In late third instar larvae, Poxn expression is also observed in the developing adult chemosensory bristles in the leg discs (Awasaki, 2001).

In the eye-antennal discs, Poxn is expressed at the mid-third instar larval stage in the antennal but not in the eye region. The pattern of expression is very similar to the pattern in the leg discs: two concentric circles in the region of the prospective arista, which is homologous to the leg tarsus. This expression is also transient and disappears by the late third larval stage. In the wing discs of mid-third instar larvae, Poxn is expressed in four stripes corresponding to regions of the prospective wing hinge area, in a quadrant pattern. This expression is maintained at least until mid pupal stages. As the dorsal and ventral cells of the wing disc become faced during early pupation, the dorsal and ventral regions expressing Poxn become superimposed and the labeling appears localized to two regions of the putative wing hinge. In the anterior margin of the wing disc, Poxn is also expressed in two rows corresponding to the formation of the adult chemosensory bristles (Awasaki, 2001).

The pattern of expression of Poxn in leg discs suggests that Poxn might play a role in leg morphogenesis. The morphology of the mesothoracic leg of Poxn^- mutants and of wild-type flies was compared. The tarsus is normally composed of five segments, T1-T5 (proximal to distal), which are separated from each other by joints. In the mutants, the tarsus is reduced to three segments. The reduction in the number of tarsal segments results from a fusion rather than from a loss of the segments. The intermediate segment of the mutant tarsus includes the bristle patterns of the T2 and T3 segments of wild-type flies, which is marked by a gradual increase of bristle length from proximal to distal in each tarsal segment. Also, in many cases, an incomplete ball and socket is seen where the prospective joint should have formed. However, the intersegmental membrane is never seen on the ventral side. Thus, T2 and T3 segements would be replaced by a single T2/3 segment in the mutants (Awasaki, 2001).

A similar situation occurs in the distal segment of the mutant tarsi, where the pattern of bristles corresponds to the juxtaposition of the patterns in the normal T4 and T5 segments. It is concluded that T4 and T5 are fused into a single T4/5 segment. In this case, however, no incomplete joints are seen. Thus Poxn is required for formation of the joints between T2 and T3, and between T4 and T5 (Awasaki, 2001).

The length of the tarsal segments is reduced in the mutants. The T2/3 segment is reduced by 34% relative to the combined length of T2+T3, while for T4/5 the reduction is about 56% relative to that of T4+T5. A 20% reduction is also observed in the mutant T1 segment. The size or patterning around the circumference of the tarsus appears normal, based on cuticular and bristle pattern. Other leg segments (tibia, femur, trochanter and coxa) are unaffected in the mutants (Awasaki, 2001).

The effect of the Poxn^- mutation on the morphology of the antenna was examined. In wild-type flies, the antenna comprises three segments plus the arista, which stands on a basal cylinder. The basal cylinder, which consists of two small segments, has been shown to be homologous to segments T2-T4 of the tarsus. In the mutant, the joint between the basal cylinder and arista does not form properly. The other structures of the antenna are not affected in the mutants. Thus, the loss of Poxn function causes homologous defects in the leg and antenna (Awasaki, 2001).

The wing structure at the hinge region in wild-type and mutant flies was compared. Two regions, which correspond to the regions of expression of Poxn in the prospective wing, are deformed in the mutants. Anteriorly, the proximal part of the radius is thick and shortened in the mutants. Posteriorly, the first vannal vein and the postcubitus are fused and the region anterior to the alula is reduced and misformed (Awasaki, 2001).

Leg formation occurs through concentric folding of leg discs and subsequent segmentation of epithelia of leg discs along the proximodistal axis to generate concentric domains. Leg segmentation requires repeated subdivision occurring in multiple phases. The decapentaplegic and wingless genes encoding secreted signaling molecules are expressed dorsally and ventrally, respectively, along the anteroposterior compartment boundary. They define the concentric expression of Distal-less and dachshund in the leg disc. Dll is expressed in the distal region of leg discs and is required for the development of all distal structure other than coxa. Several genes, such as aristaless, spineless, bric a brac and BarH1 and BarH2, act downstream of Dll to establish the tarsal segments. The evidence that Poxn is expressed in the leg in regions that correspond exactly to the tarsal region where the morphological defects were seen in the mutant indicates the involvement of Poxn in tarsal formation. Poxn expression is abolished in the Dll mutants, supporting the view that Poxn also acts downstream of Dll in tarsal formation (Awasaki, 2001 and references therein).

Similar losses of tarsal joints or segment borders are known in other mutants such as four-jointed and bab. The fj minus mutation causes a fusion of T3 to T2 in the tarsal segment, whereas one class of bric a brac minus mutations causes a complete fusion of T4 to T5 and a partial fusion of T4 to T3 and of T3 to T2. These phenotypic similarities present an interesting problem as to whether these genes act upstream or downstream of Poxn. The epistatic relationships among these genes is currently being examined (Awasaki, 2001).

The wing of Drosophila is composed of two regions, the proximal hinge and the distal wing blade. The proximodistal patterning of the wing differs from that of legs. Different spatial and temporal interactions between Notch, wg and vestigial specify proximal and distal pattern elements of the wing. After specification of a wing primordium, cells that are exposed to the activity of both wg and vg will become wing blade and those that are continuously under the influence of wg alone will develop as hinge. Another gene, nubbin, is also required for the proximodistal specification in the wing disc. The expression of nub is restricted to the wing pouch (the region of the wing disc that corresponds to the prospective wing blade) and mutations in the gene cause a severe reduction of the wing with transformation of distal elements into proximal ones. Poxn is required for the formation of the wing hinge region. Preliminary experiments have shown that the region of expression of Poxn is expanded in nub mutants, suggesting that the expression of the nub gene in the distal region is necessary to restrict the expression of Poxn to the proximal region (Awasaki, 2001).

The Pax gene family consists of tissue-specific transcriptional regulators that contain the DNA-binding 'paired' domain. Numerous Pax genes have been identified in various animals. They are classified into four groups each of which shares a specific motif and a highly conserved paired domain. The Poxn gene belongs to group II. In addition to Poxn, the other known member of Pax group II in Drosophila is shaven, which plays a role in the development of the PNS. Interestingly, shaven is also known to function in the differentiation of the shaft. The overall structure of shaven is closely related to that of the other group II genes, whereas Poxn is more distantly related to the other genes of this group. It is therefore tempting to speculate that the functions apparently shared between Poxn and shaven, i.e. their role in hair formation, may correspond to conserved ancestral functions, while the function uniquely associated to Poxn, namely the specification of p-es organs, may have been acquired more recently by this gene. In this context, it will be interesting to know how Poxn acquired its specific functions in the development of the wing hinge and in the segmentation of tarsus and antenna. The identification and analysis of genes related to Poxn and shaven in other insects may give answers to this question and shed some light on the relationship between gene variation and the evolution of development (Awasaki, 2001).

Pox neuro control of cell lineages that give rise to larval poly-innervated external sensory organs in Drosophila

The Pox neuro (Poxn) gene of Drosophila plays a crucial role in the development of poly-innervated external sensory (p-es) organs. However, how Poxn exerts this role has remained elusive. This paper analyzes the cell lineages of all larval p-es organs, namely of the kolbchen, papilla 6, and hair 3. Surprisingly, these lineages are distinct from any previously reported cell lineages of sensory organs. Unlike the well-established lineage of mono-innervated external sensory (m-es) organs and a previously proposed model of the p-es lineage, this study demonstrate that all wild-type p-es lineages exhibit the following features: the secondary precursor, pIIa, gives rise to all the three support cells - socket, shaft, and sheath, whereas the other secondary precursor, pIIb, is neuronal and gives rise to all neurons. It was further shown that in one of the p-es lineages, that of papilla 6, one cell undergoes apoptosis. By contrast in Poxn null mutants, all p-es lineages have a reduced number of cells and their pattern of cell divisions is changed to that of an m-es organ, with the exception of a lineage in a minority of mutant kolbchen that retains a second bipolar neuron. Indeed, the role of Poxn in p-es lineages is consistent with the specification of the developmental potential of secondary precursors and the regulation of cell division but not apoptosis (Jiang, 2014).

Lineage-specific determination of ring neuron circuitry in the central complex of Drosophila

The ellipsoid body (EB) of the Drosophila central complex mediates sensorimotor integration and action selection for adaptive behaviours. Insights into its physiological function are steadily accumulating, however the developmental origin and genetic specification have remained largely elusive. This study identified two stem cells in the embryonic neuroectoderm as precursor cells of neuronal progeny that establish EB circuits in the adult brain. Genetic tracing of embryonic neuroblasts ppd5 and mosaic analysis with a repressible cell marker identified lineage-related progeny as Pox neuro (Poxn)-expressing EB ring neurons, R1-R4. During embryonic brain development, engrailed function is required for the initial formation of Poxn-expressing ppd5-derived progeny. Postembryonic determination of R1-R4 identity depends on lineage-specific Poxn function that separates neuronal subtypes of ppd5-derived progeny into hemi-lineages with projections either terminating in the EB ring neuropil or the superior protocerebrum (SP). Poxn knockdown in ppd5-derived progeny results in identity transformation of engrailed-expressing hemi-lineages from SP to EB-specific circuits. In contrast, lineage-specific knockdown of engrailed leads to reduced numbers of Poxn-expressing ring neurons. These findings establish neuroblasts ppd5-derived ring neurons as lineage-related sister cells that require engrailed and Poxn function for the proper formation of EB circuitry in the adult central complex of Drosophila (Bridi, 2019).

The Drosophila central complex is a composite of midline neuropils that include the protocerebral bridge, the fan-shaped body, the ellipsoid body (EB), the noduli and the lateral accessory lobes. These neuropils are interconnected in a modular way whereby columnar projection neurons leading to and from the central complex connect all its components that are themselves intersected by tangential layers of neural processes, which together form functional modules, each representing a segment of sensory space. Functional studies have identified specific roles for the central complex in higher motor control, courtship and orientation behaviours, visual memory and place learning, as well as sleep, attention, arousal and decision-making (Bridi, 2019).

In contrast to expanding insights into the physiological role of the central complex in regulating behaviour, its developmental origin and genetic specification has largely remained elusive. Earlier work described a primordial central complex at late larval/early pupal stages, which becomes fully formed by 48 h after puparium formation. Genetic studies have identified several alleles of as-yet unidentified genes, as well as orthodenticle, Pax6/eyeless, Pox neuro (Poxn), tay-bridge, roundabout, Pdm3 and semaphorin as genes involved in normal formation of central complex sub-structures (Bridi, 2019).

This study investigate the origin and formation of EB ring neurons R1-R4 in the developing and adult brain of Drosophila. Bilateral symmetric neuroblasts ppd5 were identified in the embryonic procephalic neuroectoderm as founder cells of neuronal progeny that constitute R1-R4 subtypes of tangential ring neurons in the adult EB. Mutant analysis and targeted genetic manipulations reveal a lineage-specific requirement of engrailed (en) and Poxn activity that determines the number and identity of ppd5-derived progeny and their EB ring-specific connectivity pattern in the adult central complex of Drosophila (Bridi, 2019).

Previous studies suggested the Drosophila EB -- as part of the central complex -- develops from precursor cells that differentiate during larval development and during pupal stages generate the EB neuropil. Lineage analysis demonstrates that at least part of its origin can be traced back to the embryonic procephalic neuroectoderm. This study identified Engrailed-expressing neuroblasts ppd5 as embryonic stem cells that give rise to Poxn-expressing progeny, which ultimately differentiate into EB ring neurons. Genetic tracing with en-Gal4 identified R1-R4 ring neurons, suggesting that embryonic neuroblasts ppd5 are the major source of Poxn-expressing progeny leading to EB ring neurons detected in this study. Based on their position, morphology, gene expression patterns and axonal fasciculation, these findings suggest that ppd5-derived larval lineages correspond to previously described larval lineages variously called 'EB-A1/P1', 'DALv2/3', 'MC1' or 'DM'. It was previously demonstrated that these larval lineages express Poxn and give rise to gamma-amino butyric acid (GABA)-ergic ring neurons in the central complex of the adult brain. It therefore is proposed to (re-) name them according to their embryonic origin (Bridi, 2019).

Subclass-specific Gal4 lines together with Poxn expression identifies these lineage-related, ppd5-derived sister cells as R1-R4 ring neurons. Moreover, brain-specific Poxn-Gal4 mediated labelling identifies ring neurons and their axonal projections covering all layers of the EB neuropil, thus suggesting neuroblasts ppd5 give rise to the majority, if not all, of ring neuron subtypes. The ontogenetic relationship between Engrailed-expressing neuroblasts ppd5 and Poxn-expressing EB ring neurons is affirmed by the fact that en-Gal4 and Poxn-Gal4-targeted RNAi-mediated knockdown of Poxn causes similar EB neuropil-specific phenotypes. Together, these data establish that ppd5-derived progeny are clonal units contributing to the EB ring neuron circuitry in the central complex in Drosophila (Bridi, 2019).

How are these units specified? In both insects and mammals, the patterning and specification of neural lineages is regulated by genetic programs from neurogenesis to neuronal differentiation. This study in Drosophila shows that the development and specification of EB-specific circuit elements is likewise dependent on the lineage-specific activity of developmental regulatory genes. Early formation and maintenance of Poxn-expressing ppd5 lineages requires engrailed function as revealed with a deficiency removing both engrailed orthologues, en and invected. Previous studies showed that, engrailed/invected are required for the specification of neuroblast identity in the developing nervous system, suggesting that engrailed is also required for the specification of ppd5. A later, lineage-specific function of engrailed was found in the specification of ring neuron numbers, onsistent with its transient expression in Poxn+ lineages in the embryonic brain but not at later developmental stages nor in adult ring neurons. engrailed codes for a homeodomain transcription factor mediating the activation and suppression of target genes, regulatory interactions that are required for neural lineage formation and specification in the procephalic neuroectoderm. In contrast, no function for Poxn in embryonic brain development has been reported, suggesting that Poxn is only during later stages of development required for lineage and/or neuronal specification in the central brain (Bridi, 2019).

Indeed, experiments identify a postembryonic requirement of Poxn in the specification of ppd5-derived progeny. Previous studies showed that zygotic mutations of Poxn perturb EB neuropil formation, in that presumptive ring neurons are unable to project their axons across the midline and as a consequence, the EB ring neuropil is not formed. In the present study, en-Gal4-targeted knockdown of Poxn reveals Engrailed-expressing cells that project across the midline and form a ring-like neuropil instead of their normal ipsilateral projections to the SP. Significantly, no ppd5-derived GFP-labelled cells were observed that project ipsilaterally towards the SP, neurons that are normally detectable with en-Gal4 targeted GFP expression in the adult brain. Furthermore, en>Poxn-IR-targeted, EB neuron-like projections do not form a torroidal ring but are rather characterised by a ventral cleft. These en>Poxn-IR cells aberrantly retain Engrailed expression even though their axonal projection and connectivity pattern clearly identify them as ring neurons that are normally devoid of Engrailed but instead express Poxn. Together these data suggest that, based on their morphology, Engrailed expression, axogenesis and ring-specific projection patterns, en>GFP cells normally projecting to the SP have been transformed into EB ring neurons in en>mCD8::GFP,Dcr2,Poxn-IR flies (Bridi, 2019).

The resulting additional ring neurons in en>mCD8::GFP,Dcr2,Poxn-IR flies are accompanied with a ventrally open EB ring neuropil. A comparable phenotype is seen in brains of Poxn(757)>Poxn-IR flies which are characterised by an increased number of Poxn(757)-Gal4-targeted ring neurons, suggesting that increasing numbers of EB ring neurons lead to an arch-like neuropil reminiscent of the arch-like EB seen in the majority of arthropods. In support of this notion, previous work has demonstrated that in vivo amplification of ppd5-derived progenitor cells can lead to fully differentiated supernumerary GABAergic ring neurons that form functional connections often characterised by a ventrally open EB ring neuropil. Together, these data identify differential roles of Poxn activity during neuroblast lineage formation, in that Poxn is required for cell identity determination of ppd5-derived progeny, as well as for the specification of cell numbers and terminal neuronal projections of EB ring neurons (Bridi, 2019).

These Poxn functions in ppd5-derived brain lineages are reminiscent of Poxn activity in the peripheral nervous system (PNS) which mediates the specification of sensory organ precursor (SOP) cell lineages giving rise to external sense organs, the tactile and gustatory bristles, respectively. In these SOP lineages, differential Poxn activity determines progeny fate between chemosensory (gustatory) or mechanosensory (tactile) neuronal identities. Furthermore, SOP lineage-specific Poxn function specifies the number of these neurons and their connectivity pattern. The apparent functional commonalities between Poxn-mediated specification of ppd5 neuroblast-derived lineages in the brain and SOP lineages in the PNS, suggest that evolutionarily-conserved mechanisms underlie the development and specification of clonal units as cellular substrates for neural circuit and sensory organ formation (Bridi, 2019).

The cytoarchitecture of both the insect and mammalian brain are characterised by neural lineages generated during development by repeated asymmetric divisions of neural stem and progenitor cells. These ontogenetic clones are thought to constitute building blocks of the insect and mammalian brain. In support of this notion, lineage-related progeny constitutes sets of circuit elements of the mushroom bodies and antennal lobes in Drosophila. Clonal relationship also characterises the lineage-dependent circuit assembly in the mammalian brain, where stem cell-like radial glia give rise to clonally-related neurons that synapse onto each other, as has been shown for cortical columns and GABAergic interneurons in the neocortex and for striatal compartments of the basal ganglia. The current study in Drosophila shows that a pair of bilateral symmetric, engrailed-expressing embryonic stem cells, neuroblasts ppd5, give rise to R1-R4 subtypes of tangential ring neurons that contribute to the layered EB neuropil. Thus, ppd5 neuroblast lineages constitute complete sets of circuit elements intrinsic to the adult central complex in Drosophila (Bridi, 2019).

It has been suggested that clonal expansion of neural lineages contributed to the evolution of complex brains and behaviours. Key to this hypothetical scenario are ancestral circuit elements in the form of genetically encoded stem cell-derived clonal units, like the ones described in the current study. In such a scenario, lineage-related ancestral circuit elements might have been multiplied and co-opted or diversified during the course of evolution. Multiplication and co-option have been suggested for the evolution of the multiple-loop architecture of the basal ganglia that allows processing of cognitive, emotional and motor information. In line with this hypothesis, quantitative control of the transcription factor Prospero is sufficient to cause clonal expansion of ring-neuron circuitry in Drosophila (Shaw, 2018), which has been implicated in cognitive and motor information processing and resembles extensive correspondences to vertebrate basal ganglia, ranging from comparable developmental genetics to behavioural manifestations and disease-related dysfunctions (Bridi, 2019).

In contrast to multiplication and co-option, the diversification of stem cell lineages can equally contribute to neural circuit evolution. The current results identify differential and tightly regulated spatio-temporal functions of engrailed and Poxn that lead to the differentiation of ppd5 progeny into hemi-lineage specific identities in the adult brain. Loss of engrailed affects the formation of precursors cells, whereas its lineage-specific knockdown affects the number of Poxn expressing ring neurons. Correspondingly, en-Gal4-driven lineage-specific knockdown of Poxn results in an identity transformation of Engrailed-expressing neurons in the adult brain in that they no longer project to the SP, but instead reveal an EB ring-neuron identity. These data indicate a binary switch of hemi-lineage identities as the result of a feed-forward mechanism between engrailed and Poxn. engrailed may activate transcription (directly or indirectly) of Poxn, which in turn represses engrailed to permit differentiation of R1-R4 neurons, thereby regulating the specification of neuronal identities in ppd5 hemi-lineages. This hypothesis is consistent with lineage tracing and MARCM experiments, as well as the transient expression of engrailed in embryonic ppd5 lineages but not in adult EB ring neurons. However, further studies are required to elucidate the nature and extend of these putative regulatory interactions between Engrailed and Poxn (Bridi, 2019).

In summary, these findings establish a causal relationship between a pair of bilateral symmetric embryonic stem cells, neuroblasts ppd5 and the lineage-related assembly of their EB ring neuron progeny as structural units of the central complex in Drosophila. Based on these observations it is proposed that amplification and diversification of ontogenetic clones together with the repurposed use or exaptation of resulting circuitries, is a likely mechanism for the evolution of complex brains and behaviours (Bridi, 2019).

Genomic length - 8 kb

Bases in 5' UTR - 747+

Exons - four

Bases in 3' UTR - 338

PROTEIN STRUCTURE

Amino Acids - 425

Structural Domains

Poxn and Pox-m share presence of paired domains with paired but unlike paired they have no homeodomains (Bopp, 1989).

Evolutionary Homologies

Poxn and POX-M, paired box regions, are homologous to the paired box regions of paired and of the two gooseberry genes. Unlike the gooseberry genes and the engrailed-invected pair, Poxn and pox-m are not linked and therefore have differing developmental roles. pox-m is involved in mesodermal differentiation.

The many paired-box containing genes in mice fall into six classes. The Drosophila Poxn by itself falls into class V, while POX-M falls into class I with human Pax-1 and HuP48. Drosophila Paired, Gooseberry-proximal and Gooseberry-distal fall into class II (Walther, 1991).

Pax proteins play a diverse role in early animal development and contain the characteristic paired domain, consisting of two conserved helix-turn-helix motifs. In many Pax proteins the paired domain is fused to a second DNA binding domain of the paired-like homeobox family. By amino acid sequence alignments, secondary structure prediction, 3D-structure comparison, and phylogenetic reconstruction, the relationship between Pax proteins and members of the Tc1 family of transposases, which possibly share a common ancestor with Pax proteins, has been examined. It is suggested that the DNA binding domain of an ancestral transposase (proto-Pax transposase) was fused to a homeodomain shortly after the emergence of metazoans about one billion years ago. Using the transposase sequences as an outgroup the early evolution of the Pax proteins was examined. This novel evolutionary scenario features a single homeobox capturing event and an early duplication of Pax genes before the divergence of porifera, indicating a more diverse role of Pax proteins in primitive animals than previously expected (Breitling, 2000).

An attemp has been made to reconstruct the phylogeny and to reliably root the phylogenetic tree of Pax proteins. Since homeodomains, which have been compared for that purpose, are only present in some of the Pax proteins and are conspicuously absent in the PaxA/neuro and Pax1-9 group, the analysis was restricted to the paired box itself. This was facilitated by the introduction of a novel outgroup. Comparison of the X-ray structures of the paired box of Drosophila Paired (1PDN) and human Pax6 (6PAX) within the database of 3D-structures has revealed that the N-terminal subdomain (PAI domain) is closely related to the DNA binding domain of Tc3 transposase of Caenorhabditis elegans (1TC3). A general similarity between transposase DNA binding domains and the paired domain has been reported and their structural relationship has been observed during the analysis of the transposase structure. Initial Blast searches identified a group of transposases from C. elegans whose DNA binding domain seems to be more closely related to the paired box than to most other transposases. The DNA binding domain of these C. elegans transposases (proteins K03H6.3, W04G5.1, F26H9.3, F49C5.8, and C27H2.1; accession numbers T33011, T26169, T21438, T22423, and T19530) shows highly significant similarity only to Bmmar1, a transposase from Bombyx mori [accession number AAB47739, E-score (E)=2e-27 compared to K03H6.3], and to many Pax proteins (e.g. Hydra magnapapillata Pax2/5/8, E=9e-05; Phallusia mammilata Pax6 E=3e-04; or Paracentrotus lividus Pax1/9 E=6e-04). The DNA binding domains of other transposases yield E-scores worse than 1e-03 (e.g. Anopheles albimanus transposase AAB02109, E=9e-03). It is supposed that the transposases of C. elegans and B. mori might represent molecular fossils (proto-Pax) from the time before a homeobox capturing event took place, during which the catalytic domain of the transposase was lost and the DNA-binding domain was fused to a homeobox yielding the first PAX protein. If this is indeed the case, the proto-Pax transposases should also contain the C-terminal subdomain (RED domain) of the paired box. This subdomain is less conserved among Pax proteins than the PAI domain and does not show significant homology in sequence alignments between transposases and Pax proteins. A secondary structure analysis of the proto-Pax transposases was performed using a consensus method (Jpred2), which predicted that they indeed contain two helix-turn-helix motifs, homologous to both the PAI and the RED domain of Pax proteins (Breitling, 2000).

The observation that the DNA binding domain of transposases is in fact closely related to the paired box indicates that it should be possible to use them as an outgroup in the phylogenetic analysis of Pax proteins to determine the most likely evolutionary sequence. The transposase sequence (C. elegans K03H6.3, E = 2e-27) with the highest Blast score was compared to Pax proteins to generate a multiple sequence alignment of Pax-like transposases using the JPred2 server. The JPred2 algorithm was also used to generate a multiple sequence alignment for Pax proteins. Both alignments were combined and realigned by using ClustalW. The resulting data set contains transposases of the Tc1 and mariner families, as well as a wide range of Pax proteins from all known subgroups. The complete alignment was then used for phylogenetic analysis (Breitling, 2000).

Neighbor-joining and parsimony analysis reliably subdivides the Pax proteins into five large groups, which correspond to the classical subfamilies Pax1-9/Pax meso, PaxD/3-7/Gooseberry/Paired, PaxB/2-5-8/Sparkling, Pax4-6/Eyeless and PaxA/Pax neuro. The internal topology of the subfamilies agrees fairly well with the accepted evolutionary relationship of the organisms. One exception is the Pax4-6/Eyeless subfamily which is extremely conserved, so that an unambiguous determination of the internal branching order was not possible. The position of Drosophila Eyegone is also unreliable, because this protein contains only a partial paired domain. In both trees PaxC is significantly associated with the PaxA/Pax neuro subfamily, although PaxC carries a homeobox, and PaxA/Pax neuro proteins do not. Neighbor-joining and parsimony tree reconstruction place the Pax family within the Tc1 family of transposases, while it was not possible to identify a single closest relative of the paired box. The supposed proto-Pax transposases from C. elegans and B. mori, as identified by Blast searches, are not reliably placed as a sister-group of the Pax proteins. This might be due to the general difficulty of reconstructing well-resolved phylogenetic trees of the transposase family (Breitling, 2000).

This focus on the paired box as a descendant of a Tc1- like transposase DNA binding domain allowed for a reevaluation of the early evolution of the paired domain. These results show that the evolutionary scenario proposed by Galliot and Miller (2000) is unlikely to correctly represent the evolution of Pax proteins. This hypothesis was based mainly on the assumption that PaxA, which consists only of a paired box, resembles the probable ancestor of Pax proteins. Contrary to that idea, the scenario developed here is based on the assumption that the paired box is originally derived from a transposase and indicates that PaxA is probably derived by a secondary loss of the homeobox of a PaxC-like protein. These observations also make unlikely the hypothesis that there was more than one homeodomain capturing event. Furthermore, they suggest that the first duplication of Pax proteins occurred before the divergence of the porifera. This consequently implies that sponges, which lack nerve cells and most of the organs patterned by Pax genes in higher animals, already contained (at least) two Pax genes. The function of these early Pax proteins remains a mystery (Breitling, 2000).

Promoter

The entire cis-regulatory region of the Drosophila Pox neuro gene has been dissected with regard to its enhancers, and their functions have been analyzed by the selective addition to Pox neuro null mutant flies of one or several functions, each regulated by a complete or partial enhancer. At least 15 enhancers have been identified with an astounding complexity in arrangement and substructure; they regulate Pox neuro functions required for the development of the peripheral and central nervous system and of most appendages. Many of these functions are essential for normal male courtship behavior and fertility. Two enhancers regulate the development of the penis, claspers and posterior lobes of male genitalia. Three enhancers, two of which overlap, control the development of chemosensory bristles in the labellum, legs and wings, some or all of which are required for the transmission of gustatory signals elicited by female pheromones. An additional enhancer regulates in the developing brain the connectivity of two specific neuronal clusters entrusted with processing olfactory pheromone signals from the antennal nerve. Finally, functions crucial for the ability of the male to copulate depend on an enhancer that activates Pox neuro expression in the embryonic ventral cord. In addition to these male courtship and fertility functions of Pox neuro, there have been identified enhancers that regulate proper segmentation of tarsal segments in the leg disc and in homologous segments of the antennal disc and proper development of the wing hinge, and hence the ability of the fly to fly (Boll, 2002).

Previous studies have shown that Poxn plays an essential early role in the specification of the larval poly-innervated external sensory organs and their adult homologs, the chemosensory taste bristles. In an exhaustive search for all enhancers, a plethora of additional Poxn functions have been found to be under the control of these enhancers. Intriguingly, many of these newly discovered functions, such as those required for the specification of taste bristles, are directly or indirectly linked to male courting behavior and fertility. Therefore, Poxn might be considered a male 'courtship gene' (Boll, 2002).

These male courtship and fertility functions of Poxn include functions required for the development of (1) taste bristles on tarsal segments and tibia, anterior wing margin and labellum, whose chemosensory neurons in part respond to female pheromone signals; (2) a ventral and dorsolateral cluster of neurons in the brain, entrusted with targeting the antennal lobe, ellipsoid body, lateral triangle and at least three additional centers in the brain and processing signals, some of which presumably originate from stimulatory olfactory signals propagated by the antennal nerve; (3) specific neurons in the larval ventral nerve cord during embryogenesis, on which the copulation behavior of the male depends; and (4) male genitalia, including penis, posterior lobes and claspers. The multitude of these courtship functions emphasizes the redundancy in the exchange of sensory information between males and females during courtship, an essential feature common to all communication systems, and obviously crucial for the survival of the species and its evolutionary success (Boll, 2002).

In addition, enhancers have been identified for previously reported Poxn functions affecting the segmentation of legs and antennae and the structure of the wing hinge. This analysis correlates the activity of enhancers not only with the expression patterns that they control, but also with the partial and complete rescue of mutant phenotypes of structural and behavioral nature. This approach thus reveals not merely the size, but also the complex arrangement and substructures of enhancers. The amazing complexity of the organization and substructures of the enhancers reflects their evolutionary history and thus may provide insights into the origin of their present functions (Boll, 2002).

The overall arrangement of the enhancers reveals an astounding density and complexity. Eleven Poxn enhancers have been delimited in this study and it is estimated that there is a total of at least 15, if enhancers active mainly in the embryo are included. Thus, at least nine enhancers are located in the upstream region, five in the introns and one in the downstream region of Poxn. Not all enhancers are separable from each other, but some overlap or interdigitate. For example, the enhancers for taste bristle development on legs and wings overlap to a large extent, but are not identical. Moreover, the region over which they extend includes completely the enhancers for (1) embryonic ventral cord expression, (2) penis development and (3) larval p-es organ development. Two extreme models for the arrangement of enhancers are conceivable. They truly overlap by sharing all or in part some of the same transcription factor binding sites, or they interdigitate without sharing any binding sites. The two models are, of course, not mutually exclusive, and in the case of Poxn enhancers a mixed model may be required. Thus, the leg and wing taste bristle enhancers, located in an XbaI-HindIII fragment, might share binding sites at both ends, while the central region might include part of an interdigitating wing bristle enhancer and might not be required for the development of leg taste bristles. Interestingly, this region also includes the enhancers for penis development, that thus might be interdigitating or overlapping with this part of the wing taste bristle enhancer (Boll, 2002).

The leg and wing taste bristle enhancers exhibit a complex substructure. They both depend at their proximal end on binding sites in the XbaI-PstI fragment, which cannot activate Poxn transcription sufficiently to support taste bristle development. Only the addition of the adjacent upstream region supports taste bristle development in distal parts of both legs and wings, while further addition of a large central region affects taste bristle development only in the wing. However, it is not known if this central region is required, together with the most distal region of the XbaI-PstI fragment, to support the development of taste bristles in the tibia and in the first and third tarsal segments. Both proximal and distal parts of the leg or wing taste bristle enhancer are active only in cis, but not in trans, with each other or with the central region: this implies that the leg and the wing bristle enhancer, included in the 5.2 kb XbaI-HindIII fragment, are both single enhancers rather than each being composed of several independent enhancers (Boll, 2002).

An additional complication of the leg taste bristle enhancer is the fact that, if intron and downstream control regions are absent, it produces in the tibia a large excess of ectopic taste bristles at the expense of mechanosensory bristles, an effect that is more pronounced in the male than in the female. Thus, the balance between taste and mechanosensory bristles in the tibia, yet not in other leg segments, clearly depends on the presence of additional intron and downstream elements. Interestingly, one to three ectopic taste bristles, located in the proximal region of the wing, are similarly suppressed by the additional presence of introns. This situation is further complicated by preliminary results with null allele Poxn^DeltaM22-B5 flies rescued by a Poxn transgene that completely lacks the upstream enhancers for wing and leg taste bristles, but includes all downstream and intron enhancers. As expected, all labellar taste bristles of these flies are rescued. Surprisingly, however, some of the leg taste bristles are rescued only in male forelegs, but none in female legs, while all ventral and about a third of the dorsal wing taste bristles are rescued in both males and females. It appears, therefore, that the downstream labellar taste bristle enhancer shows considerable redundancy with the upstream wing and leg taste bristle enhancers, yet not vice versa. Future detailed analysis of which binding sites are part of these enhancers is expected to shed light on their intricate structure and function relationships and to reveal insights into their evolutionary origin (Boll, 2002).

In addition to the enhancers that control male courtship functions, two enhancers were identified whose function is required in the male genital disc for the development of the penis, claspers and posterior lobe. Moreover, in leg and antennal discs, Poxn is expressed in, and required for the development of, two segment primordia that give rise to homologous segments. Their homology is reflected at the molecular level by the fact that their expression in leg and antennal discs is regulated by the same enhancer. It has been proposed that the genital disc is a ventral disc that behaves in a manner similar to the leg and antennal discs. However, identification of two enhancers required for the development of penis, claspers and posterior lobes that are different from the leg/antennal enhancer argues that these structures are not homologous to the leg/antennal segments and that the genital and leg/antennal discs may exhibit only a distant evolutionary relationship. A similar argument can be made for the Poxn enhancer, the function of which is required in the wing disc for proper development of the wing hinge. It therefore appears that the wing is not homologous to the leg or antenna, but only distantly related to it, a notion in agreement with the current model. Nevertheless, it is intriguing that Poxn has acquired during evolution enhancers that regulate functions in leg/antennal, wing and genital discs. Rather than homology of the structures derived from the different parts of the discs expressing Poxn, Poxn activity may reflect the close relationship of the gene networks in which Poxn participates, in agreement with the gene network hypothesis (Boll, 2002).

This analysis of enhancer functions was not limited to their more direct effects such as, for example, the regulation of Poxn expression in developing taste bristles and specification of their chemosensory fate. The main interest in this study was rather to assess and measure the indirect effects of separate Poxn enhancers and functions on male fertility and courting behavior. The first step in this communication system was the focus of study: female signals arouse the interest of males, which then react by extending and vibrating a wing and thus initiate courting of the female. The male receives through its sensory organs, and reacts to, many types of signals, which are additive and, at least under laboratory conditions, redundant -- i.e., as long as the combined input signal exceeds a threshold, the male begins courtship. This approach to analyze the various Poxn functions by a dissection of its enhancers allows a determination of which Poxn functions are involved in the reception and processing of these signals. These partially characterized courtship functions of Poxn can be divided into primary functions of the peripheral nervous system, which receives and propagates the different signals, and secondary functions of the CNS and brain, on which processing and integration of the signals depends (Boll, 2002).

The following sensory modalities play a role in courtship: (1) visual input received by the photoreceptors of the eye; (2) pheromone signals received by gustatory input from receptors in the neurons of the taste bristles on legs, wing and labellum; (3) pheromone signals received by olfactory input from receptors in the neurons of the olfactory sensilla in the third antennal segment and maxillary palp; (4) auditory input from signals innervating neurons of the chordotonal organs of Johnston's organ in the second antennal segment, and (5) mechanosensory input innervating neurons of mechanosensory bristles. Courting tests with Poxn mutant males some of whose Poxn functions have been rescued showed that these are important only for the reception of signals by neurons of taste bristles. Since Poxn is never expressed in developing and adult olfactory or chordotonal organs and mechanosensory bristles, it might have functions in the reception of only the first two types of signals. However, an essential function of Poxn in the reception of light input is ruled out by two observations: Poxn^DeltaM22-B5 males are able to initiate courtship at daylight, but not under red light, and w; Poxn^DeltaM22-B5 double mutants also fail to initiate courtship at daylight. This result further supports the notion that visual and chemosensory taste and olfactory inputs play the major role in the initiation of male courtship behavior, while mechanosensory and auditory inputs play a subordinate role. Moreover, it demonstrates that the visual input alone is sufficient to trigger male courtship, though at a much reduced efficiency when compared with the use of all sensory modalities affecting courtship initiation. Finally, it follows that Poxn plays an important role in the reception of pheromones by gustatory receptors as evident from the observation that the latency times of courtship initiation at daylight and under red light are considerably prolonged by the selective removal of taste bristle functions. The fact that the selective removal of all taste bristles does not eliminate courtship in the dark strongly suggests that Poxn has functions crucial for the processing of signals elicited by female pheromones in the olfactory receptors. It further follows that not only visual, but also olfactory input alone is sufficient to trigger male courtship, though also at reduced efficiency, illustrating the redundancy of the system (Boll, 2002).

At present, the contributions of leg, wing and labellar taste bristles in the reception of the female pheromone signals are not known. It is not known, for example, if wing taste bristles recognize pheromones or are redundant for this function because no significant change in courtship behavior is noticed between males rescued by two specific fragments despite a considerable difference in the number of wing, but not leg or labellar, taste bristles. These and related questions are now amenable to an experimental approach if future analysis of the taste bristle enhancers permits a selective removal of the different taste bristle functions (Boll, 2002).

It may be important that Poxn is expressed in chemosensory neurons of prothoracic legs that connect contralaterally in the male, but not in the female. This and the additional sexual dimorphism that males have about 50% more chemosensory bristles on their forelegs than females suggest that pheromone receptors on male leg taste bristles are restricted to the forelegs (Boll, 2002).

Two Poxn enhancers have been identified that regulate secondary courtship functions of Poxn, one active in the developing brain (brain enhancer), the other in the embryonic ventral CNS (ventral cord enhancer). The results suggest that Poxn expression under control of the brain enhancer in the developing and adult brain is crucial for the proper processing of courtship signals elicited by female pheromones in the olfactory receptors. This conclusion is further supported by comparing Poxn males without taste bristle and ventral cord functions in the presence (Poxn^DeltaM22-B5; XK) and absence of the brain function (Poxn^DeltaM22-B5; E77). In the absence of the brain function, these males do not initiate courtship under red light, while no difference in courting between the two types of males is apparent in daylight. Similarly, if Poxn males without taste bristle functions, but with the ventral cord function, are compared in the presence (Poxn^DeltaM22-B5; XK) and absence of the brain function (Poxn^DeltaM22-B5; C1), it is found that the brain function is crucial for courting under red light, but not in daylight. Since these males have no taste bristle input, they are able to court only in the absence of proper visual input if the olfactory input is processed by the brain function of Poxn. These results, therefore, demonstrate that the Poxn brain function is necessary for the processing of olfactory input, to which the ventral cord function does not contribute. This conclusion is also consistent with the observation that the antennal lobe, which receives the olfactory signal through the antennal nerve, is targeted by the Poxn-expressing ventral and ventrolateral neuronal clusters in the brain. In the Poxn mutant, however, the Poxn-expressing neurons in the brain fail to make their proper connections (Boll, 2002).

In summary, Poxn includes two courtship functions involved in the reception and processing of sensory input: (1) the reception and propagation of female pheromone signals through taste bristles, and (2) the processing in the brain of olfactory signals elicited by female pheromones. A third function of Poxn during copulation is also considered. Because the brain enhancer has not been removed completely in any of the Poxn rescue constructs that include taste bristle enhancers, it is possible that the brain enhancer is also required for the processing of signals received from taste bristles. Similarly, a role of the brain enhancer in the processing of mechanosensory and auditory input cannot be excluded (Boll, 2002).

The function of the ventral cord enhancer also appears to be required for the processing of sensory signals that affect male courting behavior. However, in contrast to the brain enhancer, the ventral cord enhancer controls a Poxn function that does not influence the initiation of courtship, but somehow affects the success of copulation. In the absence of the ventral cord function, males attempt to copulate, but are unable to attach themselves to the female genitalia or soon fall off after copulation and remain on their back shivering for several minutes before they recover. By contrast, the ventral cord function is dispensable for the processing of input received through taste bristles. This is evident from the courtship behavior of males that lack the ventral cord but not the brain function: Poxn^DeltaM22-B5; SaK males, many of whose leg and wing taste bristles are rescued, initiate courtship at a considerably enhanced frequency both at daylight and under red light when compared with Poxn^DeltaM22-B5; XK males, which have no taste bristles. It follows that the ventral cord function is not primarily required for the reception of signal(s) received from sensory organs, but rather for signal processing and input into the efferent nervous system, such as for the activation of certain motorneurons required to initiate and maintain copulation (Boll, 2002).

To evaluate how the ventral cord function influences copulation, Poxn^DeltaM22-B5; DeltaSH males, whose genitalia are rescued and thus are able to copulate, were compared in the presence and absence of the ventral cord function carried by C1, which cannot contribute to taste bristle development in trans. The ventral cord function dramatically enhances successful copulation and hence male fertility. Thus, the ventral cord function of Poxn is not responsible for the processing of courtship signals, except during its last phase to initiate and maintain copulation (Boll, 2002).

What could the ventral cord function of Poxn be? Most probably it is required to orchestrate the complex movements of copulation. Thus, the male initiates copulation by bending the abdomen forward, attaching its end to the female genitalia through its claspers, mounting the female and inserting the penis into the vagina. It maintains this position by anchoring the penis within the vagina. Apparently, this complex coordinated movement of male abdomen and genitalia, which is regulated by efferents of motorneurons, is disturbed by the absence of the ventral cord function of Poxn. Specifically, mechanosensory signals in the male genital region may not be properly processed and thus impair the coordinated movement regulated by motorneurons (Boll, 2002).

An intriguing feature of the ventral cord function of Poxn is its early expression during embryogenesis, whereas its mutant phenotype becomes apparent only in the adult. A probable explanation is that many of the larval ventral cord neurons specified during embryogenesis persist to the adult stage. Although they constitute only a small fraction of about 5%-10% of the adult CNS, they contribute disproportionately to certain neuronal classes and thus may provide clues that are important for the proper organization of the adult CNS. Future studies investigating the function of Poxn in these neurons might not only shed light on their role in adult courtship behavior, but also on the complex developmental changes in the neurons of the larval CNS that are conserved during metamorphosis (Boll, 2002).

Transcriptional Regulation

Both Poxn and pox-m are regulated by paired (Bopp, 1989).

target of Poxn is a potential target of Paired box neuro. Poxn is expressed in two clusters of cells in each segment, one dorsal and one ventral. The dorsal-most cluster is displaced laterally in the second and third thoracic segments, a pattern typical of the chemosensory organs. The expression of tap follows the same pattern, with two important differences. (1) While Poxn is expressed in the sensory mother cell (SMC) and throughout the lineage until shortly before the progeny undergo differentiation, tap is expressed only at or near the onset of differentiation. Thus tap expression is very transient, lasting probably for less than an hour. (2) While Poxn is expressed in most or all of the progeny of the SMC, tap is expressed in only one cell of each organ (Gautier, 1997).

It has been confirmed that tap depends (directly or indirectly) on Poxn by inducing the ectopic expression of Poxn early during embryogenesis. the overexpression of Poxn has been shown to result in the development of ectopic chemosensory organs, both in the larva and in the adult. Additional cells expressing tap were observed embryos were the ectopic expression of Poxn was induced at 4-6 h after egg laying. Conversely, in embryos homozygous for a deficiency removing Poxn, the expression of tap is completely abolished and largely but not completely so in the CNS. Poxn binds to polytene chromosomes at 74B, the same location that codes for tap (Gautier, 1997).

Targets of Activity

The gene cut is expressed in the external sense organs. This expression differentiates external sense organs from chordotonal neurons. Among the external sense organs, Poxn is expressed in only poly-innervated organs where it induces cut and differentiates these from the mono-innervated organs. Poxn expression does not depend on cut, while pox-m can induce cut in poly-innervated external sex organs. The identity of the cut regulation region controlled by Poxn has been established (Verwoort, 1995).

abd-A regulates the segmental identity of neural elements in the peripheral nervous system. Anti-Poxn stains cells in the PNS that give rise to poly-innervated sensory organs. Some of these stain-accepting cells produce structures that are homologous to one another yet still different from one another, depending on their location (thorax or abdomen). A dorsal row of Poxn-positive cells become kölbchen in the thorax (dorsal pits), but become small sensory hairs in the abdomen. These sense organs differ in both their position and in their differentiation. In thoracic segments T2 and T3, the dorsal Poxn-positive cells migrate to a more ventral position than do those in the abdomen. Both differential migration and the terminal differentiation of these precursors are determined by abdominal-A (Castelli-Gair, 1994).

DEVELOPMENTAL BIOLOGY

Embryonic

Poxn expression is restricted to two neuronal stem cells (neuroblasts) of the CNS, and two sensory mother cells in the peripheral nervous system (Dambly-Chaudière, 1992). The pattern of pox- neuro expression becomes more complicated as more neurons are generated. It appears however that cells expressing Poxn are clonally related (Bopp, 1989).

The expression in the PNS is confined to precursors of poly-innervated external sensory organs, the chemically sensitive campaniform sensilla. There is additional expression in gnathal segments (Dambly-Chaudière, 1992).

Larval

Expression of Poxn in the wing disc is restricted to the sensory mother cells of the poly-innervated sense organs, suggesting that Poxn also determines the lineage of poly-innervated adult sense organs (Dambly-Chaudière, 1992).

The gene Poxn codes for a transcriptional regulator that specifies poly-innervated (chemosensory), as opposed to mono-innervated (mechanosensory), organs in Drosophila. The ectopic expression of Poxn during metamorphosis results in a transformation of the morphology and central projection of adult mechanosensory organs toward those of chemosensory organs. Poxn also controls the number of neurons. To determine whether Poxn can transform not only the sense organ precursor cells but also their daughter cells, the effects of the ectopic expression of Poxn were examined at different stages of the lineage. Poxn can act at a late stage to affect the fate of the undifferentiated neuron (Nottebohm, 1994).

Adult

Overexpression of Poxn induces the morphological transformation of bristles on the adult leg from mechanosensory to chemosensory. The neurons innervating the transformed bristles follow pathways and establish connections appropriate for chemosensory bristles (Nottebohm, 1992). Behavioural tests show that these neurons establish connections appropriate to taste-mediating bristles (Nottebohm, 1994).

Effects of mutation or deletion

Deletion of Poxn results in morphological transformation of chemosensory into mechanosensory organs (Nottebohm, 1992).

A large family of putative odorant-binding protein (OBP) genes has been identified in the genome of Drosophila. Some of these genes are present in large clusters in the genome. Most members are expressed in various taste organs, including gustatory sensilla in the labellum, the pharyngeal labral sense organ, dorsal and ventral cibarial organs, as well as taste bristles located on the wings and tarsi. Some of the gustatory OBPs are expressed exclusively in taste organs, but most are expressed in both olfactory and gustatory sensilla. Multiple binding proteins can be coexpressed in the same gustatory sensillum. Cells in the tarsi that express OBPs are required for normal chemosensation mediated through the leg, since ablation of these cells dramatically reduces the sensitivity of the proboscis extension reflex to sucrose. OBP genes expressed in the pharyngeal taste sensilla are still expressed in the poxneuro genetic background, while OBPs expressed in the labellum are not. These findings support a broad role for members of the OBP family in gustation and olfaction and suggest that poxneuro is required for cell fate determination of labellar but not pharyngeal taste organs (Galindo, 2001).

poxneuro is a paired domain transcriptional regulator. Mutants defective for poxneuro have an abnormal number of leg segments and conversion of labellar gustatory sensilla to mechanosensory bristle phenotype. RT-PCR experiments reveal that poxneuro mutants fail to express most, but not all, putative gustatory receptors. To determine if poxneuro transforms the support cells that make OBPs, it was determined whether reporter genes regulated by the promoter for OBP56g are still expressed in the poxneuro mutant background (Galindo, 2001).

LacZ expression was examined in labellar sensilla in wild-type flies expressing LacZ under control of the OBP56g promoter. When the P element carrying this construct is crossed into the poxneuro genetic background, LacZ expression is completely absent in the labellum. These results indicate that the cells in labellar sensilla do not express OBP56g in the poxneuro genetic background. This supports the notion that poxneuro acts early in the development of chemosensory sensilla to delegate chemosensory identity on all cells in the sensillum, including the cells that synthesize and secrete OBP56g (Galindo, 2001).

To assess the role of poxneuro in the differentiation of the pharngeal chemosensory organs, flies carrying the OBP56b promoter driving LacZ expression in specific pharyngeal organs were crossed into the poxneuro genetic background. poxneuro does not disrupt expression of LacZ regulated by the pharyngeal OBP promoter OBP56b. Together, these data indicate that poxneuro is required for expression of OBP56g in the labellar gustatory sensilla, but not for OBP56b expressed in pharyngeal gustatory organs. This suggests that different developmental mechanisms are required for the proper specification of pharyngeal and labellar gustatory sensilla (Galindo, 2001).

The specification of bract cells in Drosophila legs has been analyzed. Mechanosensory bristles induce bract fate in neighboring epidermal cells, and the RAS/MAPK pathway mediates this induction. Spitz and EGF receptor have been identified as the ligand and the receptor of this signaling; by ubiquitous expression of constitutively activated forms of components of the pathway it has been found that the acquisition of bract fate is temporally and spatially restricted. The role of the poxn gene in the inhibition of bract induction in chemosensory bristles has also been studied (del Álamo, 2002).

Drosophila legs are covered by a constant and leg-specific pattern of different types of external sensory organs, mainly mechanosensory (MB) or chemosensory (ChB) bristles. Bristles on the legs can be classified by the presence of bracts. Bracts are small epidermal structures that appear associated to MB in specific places on adult femur, tibia and the tarsal segments of all legs. Bracts appear on the proximal side of the bristles, and share the same polarity. Bracts are also present in the proximal costa of the wing, showing the same morphology as in the leg (del Álamo, 2002).

Are bristle and bract related by lineage? Sensory organ precursors (SOPs) undergo a specific pattern of cell divisions that give rise to four cells: two epidermal cells, the shaft and socket, and two neural cells, a neuron and a sheath cell. Previous clonal analyses of leg disc have suggested a lack of lineage relationship between bristles and bracts. These results were confirmed by labelling clones of cells induced in early third instar larvae; the bract cell does not belong to the SOP lineage (del Álamo, 2002).

How is bract fate specified? The results indicate that the acquisition of bract fate is controlled at three levels. One level of control takes place in the receptor cell, where the competence to acquire bract fate is spatially and temporally controlled. Ubiquitous expression of activated Raf provided in short pulses of time indicated that the competence to acquire bract fate is spatially restricted to specific regions of imaginal discs. There is also a temporal restriction to early pupal development, with peak competence between 8-12 hours APF. These results are consistent with there being a temporally and spatially restricted expression of a tissue-specific regulator that gives the receptor cell the competence to activate bract fate (del Álamo, 2002).

The expression of the poxn gene is both necessary and sufficient for the specification of bract-less ChBs. Since ChBs are specified before epidermal cells are competent for bract induction this provides an explanation for the bract-less phenotype of ChBs. Nevertheless, Poxn overexpression suppresses bracts, and the result of the combined overexpression of Poxn and activated Raf or sSpi indicates that Poxn acts in the SOP cells to repress Spi signaling. Poxn and Rho1 are co-expressed in the SOP. Therefore, these results do not allow the molecular mechanisms by which poxn expression represses Spi signaling to be deduced. Nevertheless, as the result of Star and Rho overexpression indicates that they are not sufficient to induce bracts, and that at least one other component present in the SOP cell is required, it can be tentatively suggested that poxn may act upstream of this other gene (del Álamo, 2002).

Pax2 and Poxn define the presumptive deutocerebral-tritocerebral boundary in Drosophila

Studies on expression and function of key developmental control genes suggest that the embryonic vertebrate brain has a tripartite ground plan that consists of a forebrain/midbrain, a hindbrain and an intervening midbrain/hindbrain boundary region, each of which are characterized by the specific expression of the Otx, Hox and Pax2/5/8 genes, respectively. The embryonic brain of Drosophila expresses all three sets of homologous genes in a similar tripartite pattern. Thus, a Pax2/5/8 expression domain is located at the interface of brain-specific otd/Otx2 and unpg/Gbx2 expression domains anterior to Hox expression regions. This territory is identified as the deutocerebral/tritocerebral boundary region in the embryonic Drosophila brain. Mutational inactivation of otd/Otx2 and unpg/Gbx2 result in the loss or misplacement of the brain-specific expression domains of Pax2/5/8 and Hox genes. In addition, otd/Otx2 and unpg/Gbx2 appear to negatively regulate each other at the interface of their brain-specific expression domains. These studies demonstrate that the deutocerebral/tritocerebral boundary region in the embryonic Drosophila brain displays developmental genetic features similar to those observed for the midbrain/hindbrain boundary region in vertebrate brain development. This suggests that a tripartite organization of the embryonic brain was already established in the last common urbilaterian ancestor of protostomes and deuterostomes (Hirth, 2003).

In the embryonic CNS of vertebrates, the Pax2, Pax5 and Pax8 genes are expressed in specific domains that overlap in the presumptive MHB region. Drosophila has two Pax2/5/8 orthologs, Pox neuro (Poxn) and Pax2 (Hirth, 2003).

The embryonic brain of Drosophila can be subdivided into the protocerebrum (PC or b1), deutocerebrum (DC or b2) and tritocerebrum (TC or b3) of the supra-esophageal ganglion and the mandibular (S1), maxillary (S2) and labial (S3) neuromeres of the sub-oesophageal ganglion. Expression of engrailed (en) delimits these subdivisions by marking their most posterior neurons. Because of morphogenetic processes, such as the beginning of head involution, the neuraxis of the embryonic brain curves dorsoposteriorly within the embryo. Accordingly, anteroposterior coordinates will here henceforth refer to the neuraxis rather than the embryonic body axis (Hirth, 2003).

It was first determined whether Pax2 is expressed in specific domains of the Drosophila brain, by analyzing its expression pattern using in situ hybridization, immunolabelling and lacZ reporter gene expression. Pax2 transcripts initially appear during gastrulation and at stage 9/10 are observed in a segmentally reiterated pattern of the developing procephalic and ventral neuroectoderm, with its anteriormost expression domain located at the future deutocerebral-tritocerebral boundary. Expression of Pax2 transcripts in the developing brain begins at stage 10/11 and is most prominent in a longitudinal stripe at the medial part of the protocerebrum and in a transversal stripe at the posterior border of the deutocerebrum. Immunolabelling with a Pax2-specific polyclonal antibody reveals that Pax2 protein distribution resembles that of Pax2 transcripts, as does a Pax2-lacZ reporter gene expressing ß-galactosidase. In addition to its expression in the developing anterior brain, Pax2 expression is also seen in six to eight cells located at the lateral margin of each hemisegment throughout the more posterior CNS regions of the sub-oesophageal ganglion and ventral nerve cord (Hirth, 2003).

To determine the expression of the second Drosophila Pax2/5/8 ortholog, Poxn expression was characterized using immunolabelling and lacZ reporter genes. Poxn protein is first detected in the developing brain at the end of germband extension (stage 10/11) in two stripes of the procephalic neuroectoderm, that subsequently become restricted to the posterior protocerebrum and the posterior deutocerebrum. Poxn expression in more posterior regions of the CNS also occurs in segmentally reiterated patterns. A comparison between Pax2 and Poxn expression domains reveals, that Pax2 and Poxn are never co-expressed in the same cells of the CNS. Moreover, and with one exception, expression of Pax2 and Poxn does not occur at a comparable anteroposterior position along the neuraxis. The exception is in the posterior deutocerebrum where adjacent Pax2 and Poxn expression domains define a transversal domain immediately anterior to the tritocerebral brain neuromere. This transversal domain of adjacent Pax2 and Poxn expression is distinguishable from segmentally reiterated expression in more posterior regions by the fact that it is the only position along the neuraxis where expression of both genes coincides with a neuromere boundary. This transversal domain of adjacent Pax2/5/8 ortholog expression is referred to as the deutocerebral-tritocerebral boundary (DTB) region (Hirth, 2003).

It is important to note that the DTB is located anterior to the expression domain of the Drosophila Hox1 ortholog labial (lab), which is expressed in the posterior tritocerebrum. Moreover, the DTB is located posterior to the expression domain of the Drosophila Otx orthologue otd in the protocerebrum and anterior deutocerebrum. Thus, in Drosophila as in vertebrates, a Pax2/Poxn (Pax2/5/8) expression domain is located between the anterior otd/Otx2 and the posterior Hox-expressing regions. This raises the question of whether the DTB in the embryonic Drosophila brain might have developmental genetic features similar to those observed for the MHB in vertebrate brain development (Hirth, 2003).

In the embryonic vertebrate brain, Otx2 is expressed anterior to and abutting Gbx2. The future MHB as well as the overlapping domains of Pax2, Pax5 and Pax8 expression are positioned at this Otx2-Gbx2 interface. To investigate if comparable expression patterns are found in the embryonic fly brain, the brain-specific expression of the Drosophila Gbx2 ortholog unplugged (unpg) was determined in relation to that of otd, using immunolabelling and an unpg-lacZ reporter gene that expresses ß-galactosidase like endogenous unpg. The otd gene is expressed in the protocerebrum and anterior deutocerebrum of the embryonic brain, as well as in midline cells in more posterior regions of the CNS. Expression of unpg-lacZ in the embryonic CNS is first detected at stage 8 in neuroectodermal and mesectodermal cells at the ventral midline, with an anterior limit of expression at the cephalic furrow. Subsequently, the unpg expression domains in the CNS widen and have their most anterior border in the posterior deutocerebrum. Double immunolabelling of Otd and ß-galactosidase reveal that the posterior border of the brain-specific otd expression domain coincides with the anteriormost border of the unpg expression domains along the anteroposterior neuraxis. There is no overlap of otd and unpg expression in the brain or in more posterior regions of the CNS (Hirth, 2003).

These findings indicate that the otd-unpg interface is positioned at the anterior border of the DTB. This was confirmed by additional immunolabelling studies examining unpg-lacZ, otd, Poxn and en expression in the protocerebral/deutocerebral region of the embryonic brain. Thus, double immunolabelling of Otd and En confirms that the posterior border of otd expression extends beyond the protocerebral en-b1 stripe into the anterior deutocerebral domain. Labelling Otd and Poxn confirms that the Poxn expression domain of the DTB is posterior to this deutocerebral otd expression boundary. Labelling En and ß-galactosidase (indicative of unpg expression), confirms that the anteriormost unpg expression domain overlaps with the en-b2 stripe. Finally, labelling ß-galactosidase and Poxn confirms that this anteriormost unpg expression domain overlaps with the Poxn expression domain of the DTB. Therefore, in terms of overall gene expression patterns, it is found that a transversal domain of adjacent Pax2/Poxn expression defines the DTB region of the embryonic Drosophila brain. Furthermore, this region is located between an anterior otd expression domain and a posterior Hox expression domain. Moreover, it is located abutting and posterior to the interface of otd and unpg expression along the anteroposterior neuraxis (Hirth, 2003).

In mammalian brain development, homozygous Otx2-null mutant embryos lack the rostral brain, including the MHB-specific Pax2/5/8 expression domain, whereas Gbx2 null mutants misexpress Otx2 and Hoxb1 in the brain. Moreover, Otx2 and Gbx2 negatively regulate each other at the interface of their expression domains. To test if similar regulatory interactions occur in the embryonic brain of Drosophila, the expression of the corresponding orthologs was analyzed in otd and unpg mutant embryos. In otd-null mutant embryos, the protocerebrum is absent because protocerebral neuroblasts are not specified. Analysis of unpg, en and Poxn expression in otd-null mutant embryos reveals that the anteriormost border of unpg expression shifts anteriorly into the anterior deutocerebrum, while Poxn fails to be expressed in the deutocerebrum. In contrast to inactivation of otd, inactivation of unpg does not result in a loss of cells in the mutant domain of the embryonic brain, as is evident from the expression of an unpg-lacZ reporter construct in unpg-null mutant embryos. Analysis of otd expression in unpg-null mutants shows that the posterior limit of brain-specific otd expression shifts posteriorly into the posterior deutocerebrum, thus extending into the DTB. This was confirmed by additional immunolabelling studies examining otd, Poxn and en expression in the protocerebral/deutocerebral region of the embryonic brain in unpg-null mutants. Double immunolabelling of Otd and En in unpg-null mutants confirms that the posterior border of brain-specific otd expression extends posteriorly to the deutocerebral en-b2 stripe into the posterior deutocerebrum. In addition, double immunolabelling of Otd and Poxn in unpg-null mutants confirms that the posterior border of brain-specific otd expression extends posteriorly into the Poxn expression domain of the DTB. Moreover, analysis of lab expression in unpg-null mutants shows that brain-specific lab expression shifts anteriorly into the anterior tritocerebrum. Thus, in both Drosophila and mammals, mutational inactivation of otd/Otx2 and unpg/Gbx2 results in the loss or misplacement of the brain-specific expression domains of orthologous Pax and Hox genes. Moreover, otd and unpg appear to negatively regulate each other at the interface of their expression domains (Hirth, 2003).

In vertebrate brain development, the Pax2 gene, and subsequently the Pax5 and Pax8 genes, are among the first genes expressed at the Otx2/Gbx2 interface, followed by the overlapping expression of En1 and Fgf8 genes. Inactivation of Pax2, Pax5, En1 or Fgf8 results in the loss of the midbrain and cerebellum because of a failure to maintain development of this brain region. In Drosophila, no obvious brain phenotypes were seen after mutational inactivation of Pax2, Poxn, en/inv or the Drosophila Fgf homolog branchless (bnl). The absence of brain phenotypes in these mutants contrasts with those observed in the vertebrate brain following mutational inactivation of the orthologous Pax2, Pax5, En1 and Fgf8 genes (Hirth, 2003).

Comparative developmental studies in urochordates and vertebrates have led to the notion that the basic ground plan for the chordate brain consists of a forebrain/midbrain region characterized by Otx gene expression, a hindbrain region characterized by Hox gene expression, and an intervening boundary region characterized by expression of Pax2/5/8 genes. This suggests that a corresponding, evolutionarily conserved, tripartite organization also characterized the brain of the last common ancestor of insects and chordates. A comparison of the brain-specific topology of gene expression patterns that define this tripartite organization in Drosophila and in mouse suggests that the vertebrate midbrain/hindbrain boundary (MHB) region corresponds to the insect deutocerebral-tritocerebral boundary (DTB) region. If this is the case, one might expect that other patterning genes that characterize the MHB region are also expressed at the insect DTB. Although this expectation is fulfilled for the segment-polarity genes en and wingless (wg) in Drosophila, these two genes are expressed at the borders of all CNS neuromeres, as well as at parasegmental boundaries in the epidermis; hence, their expression may not be indicative of brain-specific requirements (Hirth, 2003).

In addition to remarkable similarities in orthologous gene expression between insects and chordates, this study also shows that several functional interactions among key developmental control genes involved in establishing the Pax2/5/8-expressing MHB region of the vertebrate brain are also conserved in insects. Thus, in the embryonic brains of both fly and mouse, the intermediate boundary regions, DTB and MHB, are positioned at the interface of otd/Otx2 and unpg/Gbx2 expression domains. These boundary regions are deleted in otd/Otx2-null mutants and mispositioned in unpg/Gbx2-null mutants. Moreover, otd/Otx2 and unpg/Gbx2 genes engage in crossregulatory interactions, and appear to act as mutual repressors at the interface of their brain-specific expression domains. However, not all of the functional interactions among genes involved in MHB formation in the mouse appear to be conserved at the Drosophila DTB. Thus, in the embryonic Drosophila brain, no patterning defects are observed in null mutants of Pax2, Poxn, en or bnl. It remains to be seen if these genes play a role in the postembryonic development of the Drosophila brain (Hirth, 2003).

It is conceivable that the similarities of orthologous gene expression patterns and functional interactions in brain development evolved independently in insects and vertebrates. However, a more reasonable explanation is that an evolutionary conserved genetic program underlies brain development in all bilaterians. This would imply that the generation of structural diversity in the embryonic brain is based on positional information that has been invented only once during evolution and is provided by genes such as otd/Otx2, unpg/Gbx2, Pax2/5/8 and Hox, conferring on all bilaterians a common basic principle of brain development. If this is the case, comparable orthologous gene expression and function should also characterize embryonic brain development in other invertebrate lineages such as the lophotrochozoans. This prediction can now be tested in lophotrochozoan model systems such as Platynereis or Dugesia (Hirth, 2003).

Taken together, these results indicate that the tripartite ground plan that characterizes the developing chordate brain is also present in the developing insect brain. This implies that a corresponding tripartite organization already existed in the brain of the last common urbilaterian ancestor of insects and chordates. Therefore, an urbilaterian origin of the tripartite brain is proposed (Hirth, 2003).

Reproduction in higher animals requires the efficient and accurate display of innate mating behaviors. In Drosophila, male courtship consists of a stereotypic sequence of behaviors involving multiple sensory modalities, such as vision, audition, and chemosensation. For example, taste bristles located in the male forelegs and the labial palps are thought to recognize nonvolatile pheromones secreted by the female. A putative pheromone receptor, GR68a, is expressed in chemosensory neurons of about 20 male-specific gustatory bristles in the forelegs. Gr68a expression is dependent on the sex determination gene doublesex, which controls many aspects of sexual differentiation and is necessary for normal courtship behavior. Tetanus toxin-mediated inactivation of Gr68a-expressing neurons or transgene-mediated RNA interference of Gr68a RNA leads to a significant reduction in male courtship performance, suggesting that GR68a protein is an essential component of pheromone-driven courtship behavior in Drosophila (Bray, 2003).

Upon analysis of about a quarter of the 70 Gr genes, a Gr gene, Gr68a, was identified exhibiting the hallmarks of a putative pheromone receptor. In adults, Gr68a is exclusively expressed in neurons of about ten male-specific taste bristles in the forelegs. No expression is observed in females or any other organ or structure of males. Identical β-gal or GFP expression patterns were observed with four independent transgenic p[Gr68a]-Gal4 driver lines, indicating that male-specific expression reflects an intrinsic property of the Gr68a promoter. To verify that the β-gal-positive cells are indeed neurons and not support cells associated with taste bristles, antibody staining was performed; β-gal immunoreactive cells were found to have the typical structure of sensory neurons and express ELAV protein, a pan-neuronal marker not expressed in other cell types. To verify that the Gr68a gene is expressed in one of the chemosensory neurons and not in the single mechanosensory neuron present in taste bristles, its expression was analyzed in a pox-neuro (poxn) mutant background. Poxn is necessary for specification of chemosensory neurons, and poxn mutant flies show a complete transformation of all chemosensory neurons into mechanosensory neurons. Indeed, the p[Gr68a]-Gal4 driver is not expressed in these flies: this confirms that Gr68a is expressed in chemosensory neurons in the male foreleg (Bray, 2003).

Control of bract formation in Drosophila: poxn, kek1, and the EGF-R pathway

In Drosophila, the sensory organs are formed by cells that derive from a precursor cell through a fixed lineage. One exception to this rule is the bract cell that accompanies some of the adult bristles. The bract cell is derived from the surrounding epidermis and is induced by the bristle cells. On the adult tibia, bracts are associated with all mechanosensory bristles, but not with chemosensory bristles. The differences between chemosensory and mechanosensory lineages are controlled by the selector gene pox-neuro (poxn). This study shows that poxn is also involved in suppressing bract formation near the chemosensory bristles. The gene kek1, described as an inhibitor of the EGF-R signaling pathway, has been identified in a screen for poxn downstream genes. kek1 can suppress bract formation and can interfere with other steps of sensory development, including SMC determination and shaft differentiation (Layalle, 2004).

Misexpression of poxn at a late stage of mechanosensory bristle development has no effect on the morphology of the organ, but results in a suppression of bract formation. Misexpression of poxn and alteration of the EGF-R pathway affect bract formation during the same time window. It is concluded that poxn is responsible for the absence of a bract near the organs where it is expressed (Layalle, 2004).

kek1, a gene defined as an inhibitor of the EGF-R signaling pathway, is represented in a subtractive library enriched in genes that are specifically expressed in the chemosensory lineage. kek1 is not expressed in cells of the mechanosensory lineage at the time when bract induction takes place, and is expressed at a high level by the outer cells of the chemosensory organs. Its presence in the subtractive library, and differential pattern of expression between mechanosensory and chemosensory lineages, make kek1 a putative target of poxn (Layalle, 2004).

This point was confirmed by demonstrating that the expression of kek1 is modified following ectopic expression of poxn. Specifically, the ubiquitous expression of poxn results in the activation of kek1 expression in the outer cells of the mechanosensory lineage, where kek1 is normally silent. The activation of kek1 in mechanosensory cells is not complete in the experimental conditions that were used. It should be noted, however, that the repression of bract formation is also partial, suggesting that the overexpression of poxn is not complete. Altogether, this dataset reveals that kek1 is a target of poxn (although not necessarily a direct one) (Layalle, 2004).

The difference of expression of kek1 in the chemosensory and mechanosensory lineages, and the role of kek1 in modulating the EGF-R pathway, suggest a role for this gene in the control of bract formation. kek1 mutants do not show any abnormality in bract formation or in sensory organ development, however. More generally, the complete viability and wildtype phenotype of flies deleted for kek1 is a surprise, given the importance of the EGF-R pathway in many aspects of development (Layalle, 2004).

One obvious explanation for this absence of phenotype in kek1 mutant flies would be the existence of a functional redundancy between kek1 and another inhibitor of the EGF-R pathway. This possibility is supported by the identification of five kekkon-like genes in the Drosophila genome. Therefore this study relied on a gain-of-function analysis to decide whether kek1 might play a role in the control of bract induction (Layalle, 2004).

The EGF-R pathway has been implicated in the formation of the precursor cells for at least some of the macrochaetae on the notum. Since kek1 acts as an inhibitor of the EGF-R signaling pathway in ovary development, it might also inhibit this pathway in the notum and thereby interfere with the determination of macrochaetae. The overexpression of kek1 eliminates those macrochaetae that are most dependent on EGF-R signaling (Layalle, 2004).

Macrochaetae suppression was observed when the expression of kek1 was forced in the proneural cluster (using the sca-Gal4 driver), but not when its expression was forced after the SMC had been determined (using the neu-Gal4 driver). This shows that kek1 interferes with the formation of the precursor cells but not with subsequent steps of the lineage. It is concluded that, with respect to SMC determination, kek1 acts as an inhibitor of the EGF-R pathway, much as it does in the ovary. kek1 is expressed in the notum region of third instar wing discs. It may be, therefore, that kek1 plays a role in defining the position where SMCs are formed, or in defining the time window during which they are determined. The expression of kek1 in wing discs is very dynamic, however, and it has not been possible to determine whether this expression overlaps that of the proneural genes during normal development (Layalle, 2004).

The overexpression of kek1 induces a loss of mechanosensory shafts in the legs. At the latest step of the lineage the socket cell was found to express kek1 at a high level, whereas the shaft cell does not. The loss of shafts could therefore be due to a transdetermination of shaft towards socket fate. No socket duplication was observed however, and anti-Cut labeling demonstrated the absence of one of the two support cells at a frequency similar to that of shaft disappearance. It is concluded that the shaft cell has been lost rather than transformed (Layalle, 2004).

The ectopic expression of kek1 can prevent bract induction near mechanosensory bristles. This observation is entirely consistent with the idea that the control of kek1 expression contributes to the control of bract formation. In the sca-Gal4 line, bracts may be absent even when a shaft is formed, suggesting a direct effect of kek1 on bract formation. Since in this line kek1 expression is driven not only in the mechanosensory lineage but also in epidermal cells, it may be that this epidermal expression contributes to bract suppression. Whatever the case, the effect demonstrates that kek1 is capable of interfering with bract formation (Layalle, 2004).

The effect of Kek1 on EGF-R signaling has been shown to involve a direct interaction between the extracellular domains of the two proteins. At least part of the effect may be mediated by heterodimerization, implying that the two genes are expressed in the same cell. The observation that kek1 is expressed in the chemosensory support cells and affects bract formation by ectodermal cells suggests that the Kek1 protein may also interfere with the functioning of EGF-R proteins carried by an adjacent cell (Layalle, 2004).

Bract induction involves the activation of the EGF-R pathway in an epidermal cell, presumably through the expression of the EGF-R ligand, Spitz, by the outer cells of the sensory lineage. In the case of chemosensory lineages, or after ectopic expression of poxn at a late stage of the mechanosensory lineage, the presence of Poxn protein activates the expression of kek1 (and presumably of other members of the kek family). The Kek1 protein binds to the EGF-R and prevents the formation of a bract. The expression of a dominant-negative form of the receptor mimics this effect. When the dominant-negative is expressed both in bristle cells and in epidermal cells the inhibition of bract formation could be due to the inactivation of the EFG-R in the cells that receive the Spitz signal, i.e., in the epidermal cells. An absence of bracts is also observed when the dominant-negative form of the EGF-R is overexpressed only in bristles cells. In this case, it is proposed that the supernumerary receptors sequester the ligand and thereby prevent bract induction. Ligand sequestration would also account for the absence of bract cells when the normal EGF-R is overexpressed in the bristle outer cells (Layalle, 2004).

Taste-independent detection of the caloric content of sugar in Drosophila

Feeding behavior is influenced primarily by two factors: nutritional needs and food palatability. However, the role of food deprivation and metabolic needs in the selection of appropriate food is poorly understood. This study shows that the fruit fly selects calorie-rich foods following prolonged food deprivation in the absence of taste-receptor signaling. Flies mutant for the sugar receptors Gr5a and Gr64a cannot detect the taste of sugar, but still consumed sugar over plain agar after 15 h of starvation. Similarly, pox-neuro mutants that are insensitive to the taste of sugar preferentially consumed sugar over plain agar upon starvation. Moreover, when given a choice between metabolizable sugar (sucrose or D-glucose) and nonmetabolizable (zero-calorie) sugar (sucralose or L-glucose), starved Gr5a; Gr64a double mutants preferred metabolizable sugars. These findings suggest the existence of a taste-independent metabolic sensor that functions in food selection. The preference for calorie-rich food correlates with a decrease in the two main hemolymph sugars, trehalose and glucose, and in glycogen stores, indicating that this sensor is triggered when the internal energy sources are depleted. Thus, the need to replenish depleted energy stores during periods of starvation may be met through the activity of a taste-independent metabolic sensing pathway (Dus, 2011).

This taste-independent sugar-sensing pathway has several distinctive characteristics. First, this pathway is specifically associated with a starved state; taste-blind flies execute food-choice behavior after prolonged food deprivation of between 10 and 15 h of starvation. This time frame coincides with the onset of starvation-induced sleep suppression, indicating that these two behaviors might share a common metabolic trigger. Second, the taste-independent pathway operates on a different timescale from the gustatory pathway. Whereas WT flies made a food choice almost instantly, taste-blind flies chose sugars only after the ingestion of food. Third, this pathway responds to the nutritional content of sugars, but not to their orosensory value. Taste-blind flies chose metabolizable sugars over nonmetabolizable sugars and never consumed nonmetabolizable sugars. Furthermore, the fact that WT flies failed to distinguish a metabolizable sugar from a nonmetabolizable sugar, but shifted their preference to the metabolizable sugar after starvation, indicates that the taste-independent pathway is not an artifact associated with taste-blind flies, but functions in WT flies. Finally, the ability to detect the caloric content of sugars correlated under multiple experimental conditions with drops in hemolymph glycemia (Dus, 2011).

These results demonstrate that starvation directs the selection of nutrient-rich foods in the fly in the absence of the gustatory cues. Thus, as previously suggested in mice, postingestive cues can drive feeding behavior independently of gustatory information. The physiological factors that triggered the taste-independent food choices in mice are, however, unknown. In Drosophila, the internal energy state and carbohydrate metabolism play crucial roles in the metabolic sensing of food according to the results. A possible evolutionary purpose of taste-independent metabolic sensing is to ensure that animals select calorie-rich foods to quickly replenish energy, especially in times of food shortage (Dus, 2011).

How do starved sugar-blind flies preferentially ingest metabolizable sugar over nonmetabolizable sugar? It is plausible that sugar-blind flies are equally attracted to and feed on both sugars, but those on nonmetabolizable sugar resume foraging because of the lack of nutritional value in this sugar. These foraging flies are again equally attracted to both sugars, but those on nonmetabolizable sugar continue to forage until they find the correct food substrate. Food choice in this model is mediated by random selection and 'trapping' of the flies on the metabolizable sugar. Alternatively, sugar-blind flies might readily detect the metabolizable sugar without ingesting a large amount of food because nutrient information is rapidly conveyed to the brain within minutes of ingesting food. In this model, the flies select for metabolizable sugar over nonmetabolizable sugar by a metabolic sensor that operates on a fast timescale to mediate discrimination between the two sugar substrates. Tracking and monitoring the locomotor activity and feeding behavior that generates a preference for metabolizable sugar will address this question (Dus, 2011).

It is intriguing to speculate on the molecular nature of the metabolic sensor. This sensor could be expressed in a subset of neural, digestive, or other tissues. Among the organs and cells that have been proposed for their involvement in feeding regulation in the fly are the fat body, the insulin-producing cells (IPC), and the corpora cardiaca/allata complex. These cells may respond to the metabolic value of sugars in circulation, as seen with the glucose-excited and glucose-inhibited neuropeptide neurons in the arcuate nucleus of the mammalian hypothalamus. A model that explains how changes in circulating glucose levels alter the electrical and secretory properties of the hypothalamic glucose-responsive neurons could also describe how metabolizable sugars trigger the metabolic sensor. In mammals, glucose-sensitive cells detect glucose availability by responding to metabolites of glycolytic enzymes such as hexokinase or the energy-sensing AMP-activated protein kinase (Dus, 2011).

Almost all crucial metabolic functions in mammals are also conserved in Drosophila. During the past decade, researchers using the fruit fly as a model system for studying feeding behaviors and feeding-related disorders, including obesity, have shed much light on the molecular mechanisms of metabolism. By revealing the possibility of a metabolic sensing pathway in Drosophila, this study has introduced the possibility of understanding the molecular mechanism underlying this pathway. Identification of the cellular and genetic nature of this sensor might reveal the identity of the master switch that regulates many hunger-driven behaviors (Dus, 2011).

The molecular basis for water taste in Drosophila

The detection of water and the regulation of water intake are essential for animals to maintain proper osmotic homeostasis. Drosophila and other insects have gustatory sensory neurons that mediate the recognition of external water sources, but little is known about the underlying molecular mechanism for water taste detection. This study identified a member of the Degenerin/Epithelial Sodium Channel family, Pickpocket 28 (Ppk28), as an osmosensitive ion channel that mediates the cellular and behavioral response to water. This study used molecular, cellular, calcium imaging and electrophysiological approaches to show that ppk28 is expressed in water-sensing neurons and loss of ppk28 abolishes water sensitivity. Moreover, ectopic expression of ppk28 confers water sensitivity to bitter-sensing gustatory neurons in the fly and sensitivity to hypo-osmotic solutions when expressed in heterologous cells. These studies link an osmosensitive ion channel to water taste detection and drinking behavior, providing the framework for examining the molecular basis for water detection in other animals (Cameron, 2010).

To uncover novel molecules involved in taste detection, a microarray-based screen was perfected for genes expressed in taste neurons. Proboscis RNA from flies homozygous for a recessive poxn null mutation was compared to RNA from heterozygous controls. poxn mutants have a transformation of labellar gustatory chemosensory bristles into mechanosensory bristles, and therefore lack all taste neurons. Whole genome microarray comparisons revealed that 256 of ~18,500 transcripts were significantly decreased in poxn mutants (>2 fold enrichment in control relative to poxn). These included 18 gustatory receptors (representing a 21-fold enrichment in the gene set) and 8 odorant binding proteins (13-fold enrichment) (Cameron, 2010).

In the mammalian gustatory system, ion channels mediate the detection of sour and salt tastes, suggesting that ion channel genes may also participate in Drosophila taste detection. Therefore the expression pattern of candidate taste-enriched ion channels was examined. The putative promoter of one gene, pickpocket 28 (ppk28), directed robust reporter expression in taste neurons on the proboscis. ppk28 belongs to the Degenerin/Epithelial sodium channel family (Deg/ENaC) and these channels are involved in the detection of diverse stimuli, including mechanosensory stimuli, acids and sodium ions. In the brain, ppk28-Gal4 drives expression of GFP in gustatory sensory axons that project to the primary taste region, the subesophageal ganglion. In situ hybridization experiments confirmed that transgenic expression recapitulates that of the endogenous gene, as 48/52 of ppk28-Gal4 neurons expressed endogenous ppk28 (Cameron, 2010).

Previous studies have identified different taste cell populations in the proboscis, including cells labeled by the gustatory receptor Gr5a that respond to sugars and cells marked by Gr66a that respond to bitter compounds. To determine whether these taste neurons express ppk28-Gal4, co-labeling experiments were performed with reporters for Gr5a and Gr66a. These experiments revealed that ppk28 did not co-label Gr5a cells or Gr66a cells, and is thus unlikely to participate in sweet or bitter taste detection. An enhancer-trap Gal4 line, NP1017-Gal4, labels water-sensing neurons in taste bristles on the proboscis and carbonation-sensing neurons in taste pegs. ppk28 is expressed in taste bristles but not in taste pegs. Interestingly, ppk28 showed partial co-expression with NP1017-Gal4, with the majority of ppk28-positive cells containing NP1017-Gal4 (22/30). This correlation suggested the intriguing possibility that ppk28 participates in water taste detection (Cameron, 2010).

To directly investigate the response specificity of ppk28-expressing neurons, the genetically encoded calcium sensor G-CaMP was expressed in ppk28-Gal4 cells, the proboscis was stimulated with taste substances, and activation of ppk28-Gal4 projections were monitored in the living fly by confocal microscopy. ppk28-Gal4 neurons were tested with a panel of taste solutions, including sugars, bitter compounds, salts, acids and water. ppk28-Gal4 neurons showed robust activity upon water stimulation. In addition, ppk28-positive cells responded to other aqueous solutions even in the presence of a wide range of chemically distinct compounds. This response diminished as a function of concentration. Taste compounds such as NaCl, sucrose and citric acid significantly decreased the response. In addition, compounds unlikely to elicit taste cell activity such as ribose, a sugar that does not activate Gr5a cells, N-methyl-D-glucamine (NMDG), an impermeant organic cation and the non-ionic high molecular weight polymer polyethylene glycol (PEG, 3350 average molecular weight), all blunted the response in a concentration-dependent manner. These data demonstrate that ppk28-expressing neurons respond to hypo-osmotic solutions. This response profile is consistent with previous electrophysiological studies that identified a class of labellar taste neurons activated by water and inhibited by salts, sugars and amino acids (Cameron, 2010).

To determine the function of ppk28 in the water response, a ppk28 null mutant was generated by piggybac transposon mediated gene deletion, removing 1.769kb surrounding the ppk28 gene. The water responses of ppk28 control, mutant and rescue flies were examined by extracellular bristle recordings of l-type labellar taste sensilla. These recordings monitor the responses of the four gustatory neurons in a bristle, including water cells and sugar cells. Control flies showed 12.0±0.9 spikes/sec when stimulated with water. Remarkably, ppk28 mutant cells had a complete loss of the response to water (spikes/sec=0.8±0.1). This response was partially rescued by reintroduction of ppk28 into the mutant background (spikes/sec=6.4±1.0), demonstrating that defects were due to loss of ppk28. Responses to sucrose were not significantly different among the three genotypes, arguing that the loss of ppk28 specifically eliminates the water response. These results were confirmed by G-CaMP imaging experiments that monitor the response of the entire ppk28 population. As expected, ppk28-Gal4 neurons in the mutant did not show fluorescent increases to water and transgenic re-introduction of ppk28 rescued the water response. Taken together, the electrophysiological and imaging data demonstrate that ppk28 is required for the cellular response to water (Cameron, 2010).

The detection of water in the environment and the internal state of the animal may both contribute to drive water consumption. To evaluate the degree to which water taste detection contributes to consumption, the behavioral responses were examined of ppk28 control, mutant and rescue flies to water. Drinking time rather than drinking volume was used to monitor consumption due to difficulty in reliably detecting small volume changes. When presented with a water stimulus, control flies drank on average 10.3±1.1 seconds, mutants drank 3.0±0.3 seconds and rescue flies drank 11.5±1.5 seconds. Additionally, control, mutant and rescue flies ingested sucrose equally, showing that ppk28 mutants do not have general drinking defects. Similar defects in water detection were seen when control, mutant and rescue flies were tested on the proboscis extension reflex to water or when genetically ablating ppk28-Gal4 neurons. Although ppk28 mutants lack water taste cell responses and drink less, they still do consume water, arguing that additional mechanisms must exist to ensure water uptake. These experiments reveal that water taste neurons are necessary for normal water consumption. Moreover, they establish a link between water taste detection in the periphery and the drive to drink water (Cameron, 2010).

Whether ppk28 is directly involved in water detection was examined. If ppk28 is the water sensor, then its expression in non-water sensing cells should bestow responsiveness to water. To test this, the Gal4/UAS system was used to ectopically express ppk28 in Gr66a-expressing, bitter-sensing neurons, and taste-induced responses were monitored by extracellular bristle recordings and G-CaMP imaging experiments. For extracellular bristle recordings, responses were recorded from i-type sensilla which contain bitter-sensing, Gr66a-positive neurons but lack water cells. Expression of ppk28 in Gr66a-Gal4 neurons did not significantly affect the response to denatonium (G-CaMP imaging) or caffeine, endogenous ligands for Gr66a-Gal4 neurons. In response to water, Gr66a-Gal4 neurons showed no significant activity consistent with previous studies. Notably, misexpression of ppk28 in Gr66a-Gal4 neurons conferred sensitivity to water, as seen by extracellular bristle recordings and G-CaMP imaging. Moreover, the response was blunted as solute concentration was increased. Both NMDG and sucrose (substances that do not activate Gr66a-Gal4 neurons) produced dose-sensitive response decreases. The finding that both activation by water and inhibition by other compounds are conferred by ppk28 strongly suggests that ppk28 senses low osmolarity (Cameron, 2010).

To determine if ppk28 requires a taste cell environment to function or confers responsiveness to other cell-types, ppk28 was expressed in HEK293 heterologous cells. A FLAG-tagged ppk28 (inserted after amino acid 222 in the extracellular domain) was expressed in HEK293 cells, confirming that the protein was made and trafficked to the cell surface. For calcium imaging experiments, an untagged version of ppk28 was cotransfected with dsRed. Cells expressing the mammalian trpv4 osmo-sensitive ion channel were used as a positive control and cells transfected with the vector alone as a negative control. Cells were grown in a modified Ringers solution at 303 mmol/kg, loaded with Fluo-4 to visualize calcium changes and challenged with Ringers solution of different osmolalities. Cells transfected with vector alone showed a modest increase at 60% osmotic strength, whereas cells transfected with mammalian trpv4 showed fluorescence increases to all hypo-osmotic solutions, as expected. Importantly, cells transfected with ppk28 significantly responded to decreased osmolality, with dose-sensitive responses elicited by osmolalities of 216 and 174 mmol/kg. These experiments reveal that ppk28 bestows sensitivity to hypo-osmotic solutions in a variety of non-native environments and argue that the channel itself senses low osmolarity. This work provides a foundation for future studies of the biophysical properties of channel activation. Moreover, the ability to express ppk28 in heterologous cells and study its function creates the opportunity to compare its mechanism of gating with other Deg/ENaC family members involved in mechanosensation or sodium sensing (Cameron, 2010).

Overall, these studies examined the molecular basis for water taste detection in Drosophila and identified an ion channel belonging to the Deg/ENaC family, pickpocket 28 (ppk28), as the water gustatory sensor. This work demonstrates that an ion channel responding to low osmolarity mediates cellular and behavioral responses to water. Although the taste of water has received relatively little attention as a classic taste modality, water-responsive taste neurons have been described in many other insects, such as the blowfly and mosquitoes, as well as in mammals, such as cats and rats. The identification of ppk28 as a water taste receptor provides a framework for examining water taste detection in other animals, including humans (Cameron, 2010).

Osmosensation is important not only for the detection of external water sources by peripheral neurons but also for monitoring the plasma osmolality by central neurons. Several studies have identified members of the transient receptor potential family as candidate peripheral and central osmosensors, but the role of members of the Deg/ENaC family in osmosensation has received little attention. The finding that ppk28 is an osmosensitive ion channel raises the possibility that Deg/ENaC ion channels may participate broadly in peripheral and central osmosensation (Cameron, 2010).

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date revised:   15 March 2015 

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