The Interactive Fly

Genes involved in tissue and organ development

The Drosophila Brain

  • The Adult Brain - Index to brain structure and function
  • Genes expressed in Adult Brain Development and Function
  • The Larval Brain and Development of the Adult Brain
  • Odor detection and Processing - Odorant receptors and olfactory receptor neurons, and olfactory learning
  • The Visual System - Optic lobe and optic glomeruli
  • The Central Body Complex - Ellipsoid body, superior arch, fan shaped body and the protocerebral bridge
  • Mushroom Bodies - The site of olfactory and other learning
  • Neuroendocrine complex - Ring gland: prothoracic gland, corpus allatum, and corpus cardiac
  • Subesophageal Ganglion - Site of the taste system and feeding behavior
  • Behavioral Paradigms - Sexual Behavior, motor Behavior, photoperiod response and others

    Embryonic origin of the brain

    The brain consists of supraesophageal and subesophageal ganglia. The supraesophageal ganglia is made from three neuromeres, or neural segments. These neuromeres are formed during neurogenesis, coincident with the formation of the rest of the central nervous system (CNS). The blastoderm anlage of the brain forms part of three modified head segments (labrum, antennal segment, intercalary segment) and the acron. The acron and labrum contribute to the protocerebrum, the antennal segment gives rise to the deuterocerebrum and the intercalary segment to the tritocerebrum. Together, these three neuromeres form the brain hemisphere of the larva, which becomes the supraesophageal ganglion of the adult. orthodenticle directs the formation of the first neuromere, while development of the second and third neuromeres is directed by empty spiracles and buttonhead. These three head gap genes are expressed in partially overlapping domains of the head neuroectoderm where they are required to turn on and/or maintain the proneural gene lethal of scute. In loss of function mutations of each of the head gap genes, l'sc expression is reduced or absent in the domain of expression of the corresponding head gene. Failure of l'sc expression is followed by the absence of neuroblasts and, at later stages, defects in specific parts of the brain that are normally produced by these neuroblasts. Loss of tailless function leads to the absence of all protocerebral neuroblasts (Younossi-Hartenstein, 1997). The subesophageal ganglion develops from neuromeres of the three gnathal segments (mandible, maxilla and labium). Antennal and optic lobes are derived from the eye-antennal disc. Formation of these lobes awaits the development of the eye-antennal disc in the third instar larva.

    Gene expression in subdomains of the brain

    The neuroblasts that give rise to the brain (see Views of cephalic lobe neuroblasts) segregate from the procephalic neurectoderm and form three neuromeres: the protocerebrum (P), deuterocerebrum (D) and tritocerebrum (T). The first two neuromeres can each be further subdivided into three regions: the anterior, central and posterior protocerebral domains (Pa, Pc and Pp) and the anterior central and posterior deuterocerebral domains (Da, Dc and Dp). With respect to their position and the expression of the markers asense and seven-up, 23 small groups of neuroblasts consisting of from one to five neuroblasts per group have been identified. In Drosophila there are a total of 75-80 neuroblasts, 19 identified in Pa, 14 in Pc and 18 in Pp. There are 22 identified in the deuterocerebral domain and 6 in the tritocerebrum. The first seven groups of cells that segregate (Pc1 to 4, Dc1 to 3 and Dp1; collectively called SI/II) arise from the central domain of the protocerebral and deuterocerebral neurectoderm, respectively. Later groups form anterior and posterior to the earlier ones, leading to a centrifugal increase in the procephalic neuroblast population. SIII neuroblasts (Pa1 to 4, Pp1 and 2, and Dp2) arise during stage 10. SIV neuroblasts (Pa5 and 6, Pp3 and 4, Da1 and T1 and 2) arise during early stage 11, and SV neuroblasts (Pp5, Pdm) during stage 11 and early stage 12 (Younossi-Hartenstein, 1997).

    Early tailless expression (blastoderm stage) covers the anlage of the entire brain. Beginning approximately with the onset of gastrulation, an anterior-dorsal region with a high expression level (called HL domain) can be distinguished from a posterior-ventral domain expressing tll at a somewhat lower level. The HL domain coincides with part of the central and anterior protocerebral neurectoderm. The LL domain covers the remaining part of the protocerebral neuroectoderm. orthodenticle is expressed in a circumferential domain of the cellular blastoderm but during gastrulation becomes restricted to a domain that encompasses most of the protocerebral neurectoderm and an adjacent part of the deuterocerebral neurectoderm. All neurobasts segregating from this domain transiently express otd during stages 10 and 11; they clearly include groups Pc3 and 4, Pa3 and 6, Pp1 and 2, Pp5, Da, Dp2 and possibly Dc1, Dp1 and part of Dc2. buttonhead is initially expressed in a wide domain including the anlagen of the antennal, intercalary and mandibular segments, as well as the acron. With the beginning of gastrulation, expression disappears from most of the procephalon, except for small domains of the posterior part of the deuterocerebral and tritocerebral neurectoderm and a dorsoanterior patch that partially overlaps with the dorsoanterior protocerebrum. Both the late deutocerebral and tritocerebral btd domains contain few, if any neuroblasts. empty spiracles is in an asymmetric circumferential domain of the cellular blastoderm. During gastrulation, this pattern resolves into two stripes that occupy anterior portions of the deuterocerebral neuroectoderm and the mandibular metamere, respectively. In addition, a small circular domain corresponding to the tritocerebral neurectoderm appears ventral to the deuterocerebral stripe (Younossi-Hartenstein, 1997).

    Fasciclin II, an adhesion molecule expressed by neurons and glia defines coherent domains of the embryonic brain. One large, coherent domain encompasses part of the tritocerebrum and deuterocerebrum (D/T). Four others are defined (P1, P2, P3 and P4) in the protocerebrum. The formation of the neuropile of the brain is initiated from these centers. Axons extending ventrally from the D/T domain form the cervical connective and the subesophageal (tritocerebral) commissure. In the protocerebrum, ventrally directed axons from P4 and P3 (dorsal to P2), as well as dorsally directed axons from P1 (ventral to P2), converge onto cluster P2. The P2 cluster subsequently elongates medially and links up with its contralateral counterpart, thereby forming a track for the supraesophageal commissure (Younossi-Hartenstein, 1997).

    The expression of the proneural gene lethal of scute is required for the development of the majority of the procephalic neuroblasts. lethal of scute expression patterns correspond to many of the identifiable 23 groups of neuroblasts described above. l'sc expression in the procephalic neurectoderm is controlled in partially overlapping domains of the neurectoderm. Loss of function of a given head gap gene results in the absence of l'sc expression in its domain, followed by the absence of neuroblasts that would normally segregate from this domain (Younossi-Hartenstein, 1997).

    Loss of tll function results in the absence of all protocerebral neuroblasts and loss of all four coherent domains of Fas II expression in the protocerebrum. Also missing is the optic lobe. orthodenticle functions in a domain that includes a large part of the protocerebrum and a smaller part of the adjacent deuterocerebrum. Loss of otd results in loss of P1, P2 and P4 coherent domains of Fas II expression. Also missing is a nerve that carries axons from the antennal organ. In buttonhead mutation the D/T cluster is missing; consequently a cervical connection is missing that normally sends nerves to the labral sensory organ, the hypopharyngeal sensory organ and the stomatogastric nervous system (Younossi-Hartenstein, 1997).

    Neuroblasts delaminate from the procephalic neurectoderm in a stereotyped spatiotemporal pattern that is tightly correlated with the expression of l'sc. The pattern of neuroblasts was reconstructed by using the marker asense; similar to its expression in the ventral neuroblasts, asense labels all brain neuroblasts. seven-up, expressed in specific subsets of neuroblasts making up approximately one-third of the total, is also used as a marker. For most, if not all, of these clusters the number of neuroblasts and the time of onset of svp expression are absolutely invariant. Neuroblast groups expressing svp are the following (number of cells in each group is given): Pa1 (1), Pa3 (2), Pa5 (2), Pc1 (1), Pc3 (4-6), Pp1 (1-2), Pp3 (3), Dc1 (1), Dc3 (5-6), Dp1 (2-3), and T1 (2) (Younossi-Hartenstein, 1996).

    E(spl)-C expression in the head starts during stage 8 in the Pc domain, followed slightly later by the Dc domain. E(spl)-C genes remain expressed at a high level in the Pc and Dc domains for several hours after neuroblasts have delaminated from these regions. During stage 10, the expression progresses and ultimately covers most of the procephalic ectoderm (Younossi-Hartenstein, 1996).

    The proneural genes achaete and scute and the segment polarity genes wingless and engrailed each have limited expression in only a few identifiable and stereotyped clusters of the head. For example, sc appears exclusively in a small part of the Pc domain, followed by transient expression in one to two Pc2 neuroblasts. wg is expressed in a total of three patches and engrailed is expressed in domains that are posterior and ventral to the adjacent wg domains. en is expressed in one patch in both the protocerebrum and the deuterocerebrum (Younossi-Hartenstein, 1996).

    Procephalic neuroblasts divide in a stem cell mode giving rise to progeny neuroblasts and ganglion mother cells. Procephalic ganglion mother cells and neurons are arranged in elongated vertical chains attached to the basal neuroblast surface. Each new ganglion mother cell pushes the previous one away from the neuroblast. In the ventral cord, the first three to four ganglion mother cells born by each neuroblast lie side by side, directly contacting the neuroblast. This difference results in a comparatively thick cortex of the brain hemispheres measuring in some places five to eight cell diameters in thickness. It is likely that the neuroblasts of the brain that are born during embryogenesis and proliferate during embryonic development are activated again during the larval period to produce adult-specific neurons (Younossi-Hartenstein, 1996).

    Specification and development of the pars intercerebralis and pars lateralis, neuroendocrine command centers in the Drosophila brain

    The central neuroendocrine system in the Drosophila brain includes two centers, the pars intercerebralis (PI) and pars lateralis (PL). The PI and PL contain neurosecretory cells (NSCs) which project their axons to the ring gland, a complex of peripheral endocrine glands flanking the aorta. This paper presents a developmental and genetic study of the PI and PL. The PI and PL are derived from adjacent neurectodermal placodes in the dorso-medial head. The placodes invaginate during late embryogenesis and become attached to the brain primordium. The PI placode and its derivatives express the homeobox gene Dchx1 and can be followed until the late pupal stage. NSCs labeled by the expression of Drosophila insulin-like peptide (Dilp), FMRF, and myomodulin form part of the Dchx1 expressing PI domain. NSCs of the PL can be followed throughout development by their expression of the adhesion molecule FasII. Decapentaplegic (Dpp), secreted along the dorsal midline of the early embryo, inhibits the formation of the PI and PL placodes; loss of the signal results in an unpaired, enlarged placodeal ectoderm. The other early activated signaling pathway, EGFR, is positively required for the maintenance of the PI placode. Of the dorso-medially expressed head gap genes, only tailless (tll) is required for the specification of the PI. Absence of the corpora cardiaca, the endocrine gland innervated by neurosecretory cells of the PI and PL, does not affect the formation of the PI/PL, indicating that inductive stimuli from their target tissue are not essential for early PI/PL development (de Velasco, 2007).

    The insect neuroendocrine system consists of several populations of neurosecretory cells (NSCs) with peripheral axons terminating in contact with specialized neurohemal glands where the neurohormones are released. The majority of NSCs are found in the dorso-medial protocerebrum, the so-called pars intercerebralis (PI) and pars lateralis (PL). These NSCs project their axons towards a set of small glands, the corpora cardiaca (CC), and corpora allata (CA). In Drosophila, the CC and CA, along with a third neuroendocrine gland, the prothoracic gland (PTG), are fused into a single complex, the ring gland, which surrounds the anterior tip of the aorta. The PI-PL/ring gland complex of insects has been repeatedly compared to the hypothalamus-pituitary axis in vertebrates, based on clear similarities between the two, anatomically and functionally (i.e., their shared role in energy metabolism, growth, water retention, and reproduction). Previous studies of the insect neuroendocrine system have focused on the neurosecretory cells of the PI and PL; however, not much is known about the different types of non-secretory PI neurons, and even less information exists about the development of this important part of the insect brain. This paper focused on the formation of the PI during Drosophila embryonic and larval development (de Velasco, 2007).

    The PI is histologically recognized as the unpaired antero-medial domain of the protocerebral cortex that is located anterior of the calyces of the mushroom bodies and dorsal of the central complex. Beside innervating the neuroendocrine glands and thereby acting as the uppermost center of endocrine release, the neurites of PI neurons are structurally and functionally integrated into the medial compartments of the protocerebrum. Some of the cells of the PI were shown to play a role in various behaviors, including locomotor activity and flight behavior. Moreover, an early role of certain PI cells as pioneer neurons during the formation of protocerebral axon tracts has also been reported (de Velasco, 2007).

    The extremely limited information about the development of the PI stems mainly from studies on grasshopper and Drosophila. For grasshopper, the dorso-medial subpopulation of brain neuroblasts, measuring approximately 20 in number, was tentatively assigned to the formation of the PI, although detailed lineage studies have not yet been carried out. Three groups of cells that form part of the adult PI have been followed in more detail from embryonic stages onward. These include (part of) the NSCs, a group of unpaired cells derived from a cell called the 'dorsal midline progenitor', and a small set of early differentiating neurons expressing the antigen TERM-1 that act as pioneers of the brain commissure (de Velasco, 2007).

    Cells situated along the antero-dorso-medial edge of the population of brain neuroblasts can be assumed to produce neuronal offspring that gets incorporated into the PI, although specific lineages have not yet been followed. The neurectoderm of the so-called 'head midline', which gives rise to at least part of (if not the entire) PI was described as a specialized region with morphological and molecular similarities to the ventral (trunk) midline (mesectoderm). Both trunk and head midline domains contain neuronal progenitors that do not delaminate as individual cells like the neuroblasts of the lateral neurectoderm, but that invaginate as an elongated furrow (ventral midline) or as several separate placodes (head midline). Furthermore, both trunk and head midline require the activity of EGFR signaling for survival and fate specification. Another molecular characteristic shared by the midline of the trunk and head is the extended expression of the neurogenic genes of the E(spl) complex. These genes are activated in the neurectoderm (in general) by Notch signaling, and are responsible for inhibiting proneural gene expression, thereby mediating the lateral inhibition process that delimits the number of neuroblasts delaminating at any given position from the neurectoderm. At the stage when no more neuroblasts are born, Notch signaling and the expression of E(spl) transcripts cease. This happens around stage 11 in the lateral neurectoderm of the trunk and the head. However, in the midline domains, expression continues far beyond that stage, up until stage 14. This finding was interpreted to indicate a temporally extended neurogenic potential of the midline cells. In other words, neurectodermal cells along the head midline are still neurogenic at a stage when other ectodermal cells are already on their way to become epidermal cells (de Velasco, 2007).

    A comprehensive developmental study of the Drosophila pars intercerebralis and pars lateralis has been undertaken with the aim of understanding their embryonic origin from the dorsal head midline, developmental morphogenesis, and architecture. The PI, marked by its continued expression of the homeobox gene Dchx1, is derived from a placode located anteriorly in the neurectoderm of the dorso-medial head. Three additional placodes are situated posterior to the PI placode; one, marked by the expression of FasII, gives rise to a cluster of neurons that form the pars lateralis (PL), which represents the second domain within the brain that contains neurosecretory cells). The remaining two placodes express the homeobox gene drx) and become part of the protocerebum surrounding the PI and PL. This study further investigated the role in PI development of early acting signaling pathways (Dpp, EGFR) and transcription factors expressed in the dorso-medial head neurectoderm [tailless (tll), ventral nerve cord defective (vnd), single minded (sim); orthodenticle (otd)] and in the corpora cardiaca [sine oculis (so), glass (gl), forkhead (fkh)] in the PI placode. The findings provide a developmental genetic framework for the study of the Drosophila central neuroendocrine system (de Velasco, 2007).

    Pars intercerebralis: structure and development during the postembryonic phase: To analyze the PI in Drosophila larvae, pupae, and adult, brains were used in which GFP was driven by Chat-Gal4, which is expressed in most differentiating neurons, including their axons. This staining visualized most, if not all neuronal cell bodies and their proximal axons that comprise the PI. The PI of the adult and 3 day pupa appears as an unpaired cluster of cell bodies filling the cleft between the brain hemispheres. At most positions along the antero-posterior axis, it is well demarcated from the lateral cortex by a glial lamella. The following will distinguish the anterior PI (PIa), located around the medial lobes of the mushroom body, from the central PI (PIc; dorsal to the central complex) and posterior PI (in between central complex and protocerebral bridge). Most PIa neurons are small and weakly Chat-GFP-positive; in addition, one can distinguish a subset of large, strongly Chat-GFP-positive somata with axons that pass through the superior medial protocerebrum and form the NccI nerve that projects to the ring gland. Based on their trajectory, these large neurons comprise the neurosecretory cells of the PI. The central PI (PIc) is comprised of neuronal somata that form a rather homogenous population in regard to size and Chat-GFP expression levels. Axons of PIc neurons turn laterally and appear to branch in the posterior part of the superior-medial protocerebrum (called DP compartment in the larva). Neurons of the posterior PI (PIp) are small and densely packed; many PIp neurons form axons that fasciculate in a fiber tract projecting straight ventrally and then anteriorly into the fan-shaped body. One cannot define a clear boundary between the PIp and the laterally/posteriorly adjacent protocerebral cortex (de Velasco, 2007).

    It is clear from the above said that the PI is formed by neurons that contribute to many different brain circuits, and that much work is needed to elucidate the exact structure and function of these cells. Do all of these cells share a common origin? In other words, can one define one or more specific neural lineages whose cells exclusively contribute to the PI? And is there an ontogenetic relationship between the PI and the PL, the second cluster of neurosecretory cells that, without specific markers, cannot be recognized within the brain of the larva, pupa or adult? To approach these questions, the development of the PI was followed backward in time. In the early pupa and larva, all components of the PI defined in the previous section for the late pupa and adult can be recognized; however, they do not form an unpaired cluster in the brain midline, but are split into bilaterally symmetric clusters. Thus, as an anatomical entity, the pars intercerebralis does not exist in the larva, but evolves during the pupal period (de Velasco, 2007).

    In the one day-old pupa, cells with the characteristics of the anterior PI (strongly Chat-GFP-positive neurons located dorsal of the medial lobes) form axons that branch in the DA and CA compartments and continue peripherally as the NccI nerve (Ncc stands for nervus corporis cardiaci). In the central PI, large, strongly Chat-GFP-positive cells cover the developing ellipsoid body and fan-shaped body. The same picture presents itself in the wandering larva. Besides their large size and high expression level of Chat-Gal4, neurons of the PIa and PIc are distinguished from the lateral cortex by lacking any Neurotactin expression. Neurotactin expression defines a distinct, transient stage in neural differentiation. Thus, all primary neuroblasts and neurons of the late embryonic brain express neurotactin, to then lose it in the early larva. When neuroblasts become reactivated and produce secondary neurons, these also express Neurotactin until mid-pupal stages. The fact that the larval PIa/c has no Neurotactin-positive cells says that this domain consists only of primary neurons, or is characterized by a distinct mode of Neurotactin expression that sets it apart from other neurons. In either case, it serves as a suitable global marker of the larval PIa/c. Using the neuron-specific marker Elav supports the view that the PIa/c consists mainly of primary neurons which can be distinguished from undifferentiated secondary neurons by their large cell and nuclear size: anti-Elav reveals large neurons in the PIa/c which contrast sharply from the small secondary neurons of the laterally adjacent cortex (de Velasco, 2007).

    As expected from its adult and late pupal morphology, the posterior PI (PIp) is difficult to define with any confidence in the larval brain. Neurons in the posterior-medial cortex, around the emerging commissure of the lateral horn which served as a topological landmark for the PIp at later stages, have axons directed anteriorly towards the central complex primordium. These neurons, which should include the cells comprising the later PI, belong to several lineages of secondary neurons of the DPM group. Without more specific markers, it is not possible to ascertain exactly which DPM lineages produce the posterior PI, and whether these lineages contribute only to the PI, or to laterally adjacent parts of the brain as well (de Velasco, 2007).

    Definition of the PIa/c as a expression domain of the homeobox gene Dchx: The vertebrate gene Chx10 (also called Vsx) appears in the anlage of the forebrain during neurulation and is later expressed and required for neurons of the retina. The Drosophila genome contains two closely associated Chx10 homologs, Dchx1 and Dchx2. Their expression profile shows similarities to the expression of their vertebrate counterparts. Dchx1 appears in the early anlage of the visual system (the optic lobe placode/optic anlage of the late embryo and larva), as well as in differentiating neurons of the optic lobe. In addition, Dchx1 is expressed in a dorso-medial domain of the brain that overlaps with the PIa/c, and at least in part, the PIc as defined above. Thus, large Dchx-positive primary neurons are located anterior and dorsal of the medial lobe of the mushroom body and are flanked by the secondary lineages of the DAM and DPMl groups, respectively. Based on these criteria, the Dchx-positive neurons are recognized as the larval PIa/c as described above. Dchx-positive primary neurons of the PIa/c can be followed backward in time through early larval stages. Posterior to the group of large PIa/c neurons, a cluster of small Dchx-positive cells are found that overlap with the group of DPMm lineages. It is speculated that these neurons represent the primordium of the posterior PI. As expected, the Dchx-positive secondary neurons of the PIp are born in the late larva, when DPM lineages (along with most other lineages of the central brain) proliferate. In early larval and embryonic brains, only few scattered Dchx-positive cells appear in the postero-medial brain (de Velasco, 2007).

    The expression of GFP driven by a Dchx promoter construct visualizes the projection pattern of the Dchx-positive neurons populating the PI. Most, if not all of these cells conform to a relatively simple commissural pattern, whereby axons cross in the anterior part of the supraesophageal commissure. Axonal and dendritic branches form a dense plexus that fills out the DA and CA compartments. A subset of PI neurons, notably those that co-express Drosophila insulin-like peptide (Dilp) form axons that leave the dorso-medial brain and project to the ring gland, as well as the subesophageal ganglion/tritocerebrum (de Velasco, 2007).

    Neurosecretory cells targeting the ring gland lie within the PIa/c: The larval PI includes most, if not all, of the neurosecretory cells that project towards the corpora cardiaca/corpora allata through the NccI. Larval brains were labeled with antisera against Dilp, FMRFamide, and myomodulin. In all of these experiments, peptidergic neurons with axons to the ring gland were located in the Dchx-positive/neurotactin-negative PIa/c domain. Peptidergic axons branch in the CA/DA compartments, cross the midline, and then extend posterior, passing underneath the supraesophageal commissure. This nerve connection constitutes the NccI nerve. The axonal marker FasII reveals the NccI has already developed in the late embryo. A second connection between the dorso-lateral protocerebrum and the ring gland, called NccII, also expresses FasII from embryonic stages onward. The FasII-positive neurons that give rise to the NccII are not part of the PI, but instead turn out to represent the Pars lateralis (PL), which occupies a position in the dorsal brain cortex, anteriorly adjacent to the calyx of the mushroom body. The NccII root emitted by the PL forms a conspicuous tract of the larval brain that passes the calyx and peduncle medially before reaching the medial edge of the protocerebrum where it joins the NccI on its way to the ring gland (de Velasco, 2007).

    The PI and PL are derived from a series of neuroepithelial placodes in the embryonic head: The Dchx-expressing cells of the PIa/c can be followed into the early embryonic period when they form a narrow cluster of approximately 40-50 cells in the antero-medial procephalon, right behind the furrow that separates the procephalon from the clypeolabrum. Two similar-sized domains are labeled by FasII and the Drosophila Rx homolog, Drx. Together, these three markers define linearly arranged, non-overlapping domains along the dorsal midline of the procephalon. The Dchx domain will be called 'embryonic PIa/c'. During later embryogenesis, a global morphogenetic movement shifts all of these domains posteriorly; at the same time, the FasII and Drx-positive domains also move laterally. Dchx remains strongly expressed in a cohesive cluster that develops into the larval PIa/c. It is currently unclear whether the secondary neuroblast(s) that later give rise to the neurons of the PIp are derivatives of the PIa/c, or whether they are recruited from outside this domain. In late embryos, faintly Dchx-positive neurons and neuroblasts can be recognized outside the PIa/c (not shown) (de Velasco, 2007).

    Cells of the FasII-positive domain give rise to the NccII and therefore constitute the embryonic primordium of the PL. In the late embryo, shortly before the somata of these neurons lose FasII expression, one can follow a FasII-positive axon tract from the FasII cluster towards the primordium of the ring gland. This tract represents the embryonic NccII, given that it exhibits the same topographical characteristics as the NccII of the larva (point of origin in postero-lateral protocerebrum; passing the mushroom body towards medially; joining nascent NccI towards ring gland) (de Velasco, 2007).

    The PI, PL and Drx domains visible in the mid-stage embryo (stages 11-13) form neuroepithelial placodes that split by invagination from the procephalic ectoderm. This mode of neurogenesis differs from the mechanism of delamination that produces all other brain neuroblasts within the surrounding neurectoderm. The use of anti-Crumbs (anti-Crb) as a marker for apical membrane domains of epithelial cells reveals a stereotypic pattern of small invaginations in the dorso-medial procephalon of stage 12/13 embryos. The anterior invagination ('1') is formed/surrounded by Dchx-positive cells and corresponds to the PIa/c; the second, intermediate invagination ('2') lies in the FasII-positive PL, and two posterior invaginations, one medially ('3'), one further laterally ('4'), flank the Drx domain. During stage 13/14, the invaginations pinch off the surface ectoderm and form small vesicles in between the outer epithelium (the nascent head epidermis) and the brain surface (de Velasco, 2007).

    Two additional markers, a reporter construct of the E(spl)m5 gene and the neuronal differentiation marker Elav, were used to further characterize the placodeally derived PI/PL and Drx domain. The 'dorsal head midline', a domain that, along with the midline of the trunk, is set apart from the lateral neurectoderm by the prolonged expression of the Notch target E(spl), as well as the expression of/dependence on EGFR signaling. Triggered by Notch activation, E(spl) is expressed in the neurectoderm as long as this layer is 'active', i.e., produces neuroblasts. The phenomenon of prolonged E(spl) expression was interpreted as an indication for an extended period of neurogenic potential (de Velasco, 2007).

    The previously defined dorsal head midline coincides with the PIa/c and PL, as evident from the double labeling experiments using the E(spl)-m5 reporter, anti-FasII and anti-Crb. E(spl)m5 is expressed in these domains throughout late embryogenesis into the early larval period. Furthermore, double-labeling with anti-Elav demonstrates that neuronal differentiation begins relatively late in these domains. Throughout stages 12, 13 and much of 14, the E(spl)m5-positive domains remain Elav-negative; Elav signal comes up faintly during stage 15, and only in the stage 16 embryo do most neurons of these domains express Elav (de Velasco, 2007).

    The placodeally derived, Crb-positive vesicles remain visible at the brain surface until late embryogenesis (stage 16). Subsequently, the cells lose Crb expression; it is assumedd that the intermediate and posterior vesicles ('2-4') convert into neurons that become incorporated in the brain, although this needs to be definitively shown. The anterior pair of vesicles ('1') appears to become incorporated into the corpora allata, the dorsal most part of the ring gland. The early development of this endocrine gland which secretes juvenile hormone during larval stages has not been clearly documented for Drosophila. In other insects, ectodermal placodes that invaginate from the ectoderm of gnathal segments were observed to give rise to both prothoracic glands (source of ecdysone) and corpora allata. The present data, which need to be substantiated by additional markers, indicate that at least part of the CA is derived from the PIa/c. Thus, in the late embryo, the anterior vesicles, still expressing Crb and E(spl)m5, approach each other and eventually fuse in the midline, forming a cluster of cells that moves posteriorly, behind the level of the brain commissure, and becomes incorporated into the dorsal part of the ring gland (de Velasco, 2007).

    Genetic specification of the PI: The dorsal procephalic ectoderm that gives rise to the PI is patterned by several signaling pathways, in particular the Dpp and DER pathway. Furthermore, the head gap genes tailless (tll) and orthodenticle (otd) are expressed in the dorsal procephalon. The Tll expression domain includes the PI/PL placodes; these placodes, on the other hand, are not part of the Otd domain. The midline determinant Single minded (Sim) is expressed faintly from stage 13 onward in a subset of cells in the PI primordium. Ventral nerve cord defective (Vnd) is expressed in a longitudinal medial domain of the ventral neurectoderm flanking the mesectoderm. The paramedian stripe of Vnd expression continues in the head, but ends laterally/ventrally of the PI/PL (de Velasco, 2007).

    Loss of Dpp results in the absence of the dorso-medial head epidermis that in wild-type separates the bilateral PI anlagen from each other. The PI anlagen are enlarged and fused in the dorsal midline. In DER mutants, Dchx expression is virtually absent, supporting the previously reported finding that the dorso-medial procephalic ectoderm ('dorsal head midline') requires DER signaling. Dchx is also eliminated in tll mutants, but is present, if possibly reduced, in embryos mutant for otd and other head gap genes (de Velasco, 2007).

    Aside from these genes, which are expressed in the PI anlage, the possibility was tested of inductive interactions between the PI and neighboring tissues, in particular the foregut and stomatogastric nervous system (which transiently contacts the dorso-medial procephalic ectoderm) and the corpora cardiaca, which receive axons from the PI derived neurons. To remove the foregut, a null mutation was used in the fkh gene; the stomatogastric nervous system was eliminated by a mutation in so, and the corpora cardiaca by a mutation in gl. The results indicate that none of these genetic manipulations grossly affects the formation of the PI. The gl null mutation survives until the late larval period, which gave an the opportunity to analyze the structure and innervation of the ring gland that lacks the corpora cardiaca. Both Dilp and FasII-positive axons reach the ring gland and send axons towards the corpora allata, the dorsal part of the ring gland, which is unaffected in gl mutants. The only abnormality in ring gland innervation was the defasciculation and aberrant projection of FasII and FMRFamide-positive axons. In particular, FasII-positive axons frequently followed Dilp fibers onto the dorsal vessel, a behavior not observed in wild-type. The number of FMRFamide-positive axons reaching the corpora allata was reduced (de Velasco, 2007).

    Definition of the pars intercerebralis and pars lateralis in Drosophila: According to the classical anatomical definition that applies to adult insect brains, one can recognize the PI as an unpaired cluster of neuronal cell bodies located along the dorsal midline of the brain. In its anterior and intermediate part (PIa/c), the pars intercerebralis is clearly set apart from the adjacent lateral cortex by a glial lamella; at posterior levels, the boundary between the PI and neighboring cortex domains is fluid. The PI is comprised of several hundreds of neuronal cell bodies that include as a relatively small minority large NSCs with axonal projections to the ring gland. The PI as an unpaired midline structure appears first during the late pupal phase. Before that stage, cells of the PI form bilateral clusters in the dorso-medial cortex of both brain hemispheres. Two criteria allowed recognition the PI at these stages and follow it backward into the embryonic period. One was the expression of the homeobox gene Dchx1, the other the idiosyncratic proliferatory properties of the PI primordium. Dchx1 is expressed in bilateral placodes in the antero-medial neurectoderm of the early embryonic head. These placodes form the early primordium of the PI. In the late embryo, they move interiorly and become part of the dorso-medial brain cortex. During later larval stages, the PI primordium, aside from the continued expression of Dchx1, sets itself apart from the lateral cortex by the absence of stem cell-like neuroblasts producing secondary lineages. Instead, the PI primordium appears to grow, at a rather slow rate, by symmetric cell division. During metamorphosis, the cortex and neuropile of the brain hemispheres fuse, giving rise to the unpaired median PI and central complex (de Velasco, 2007).

    As often in development, it is difficult to state with any certainty whether the boundaries of the PI as defined in the adult coincide precisely with those visible earlier. In other words, it cannot be presently stated with any certainty that the sharp PI boundary defined, in the adult and late pupal brain, by glial septa coincides with the Dchx-positive cluster of the embryo and larva. It can be said, however, that many, if not all, of neurosecretory cells of the classically defined PI fall within the Dchx-expressing cell cluster. This was demonstrated in this study for Dilp, FMRFamide, and myomodulin; it can be said confidently that the NSCs of the PI expressing other peptides will also be included within the Dchx-positive domain (de Velasco, 2007).

    The pars lateralis (PL) has been defined as a cluster of NSCs that lie outside the PI, and whose peripherally projecting axons form the NccII. The NccII can be recognized from early embryonic stages onward by its expression of the adhesion molecule FasII. The FasII-positive cells that give rise to the NccII, and that therefore should be considered as the primordium of the PL, are derived from a neurectodermal placode located posteriorly adjacent to the placode that forms the PI. It may be significant that both PI and PL, the centers including neurosecretory cells, are derived from placodeal neurectoderm (de Velasco, 2007).

    A third set of placodes appear in the dorso-medial protocerebral neurectoderm, posterior to the FasII-positive PL placode. The posterior placodes, at least partially, overlap with the main expression domain of the homeobox gene Drx. Drx-positive cells forming within this region can be followed into the larval stage. They spread out over a relatively large area of the dorso-posterior cortex. The expression of Drx may be significant given the fact that the vertebrate homolog of this gene is expressed and required in the primordium of the hypothalamus. However, in Drosophila, the relationship of the posterior placodes and their Drx-positive derivatives to the PI/PL is not clear. It is possible that non-ring gland-associated NSCs are derived from it; in addition, the drx-positive neurons may be functionally and anatomically closely connected to the PI/PL (de Velasco, 2007).

    Origin of PI/PL from neurectodermal placodes: This paper has shown that the PI/PL originate as placodes from the dorso-medial neurectoderm of the head, in a way that is similar to the formation of the optic lobe. In all of these cases, small domains of the neurectoderm are seen which, during stages 10 or early 11 of development, adopt the shape of placodes, with cells elongating in the apico-basal axis and expressing a higher level of apical markers such as Crb at their apical surface. Eventually, all of these placodes invaginate and sever their connection to the ectoderm several hours after their initial appearance (stages 12-13). Subsequently, cells of the placodes lose their epithelial phenotype and directly turn into neural cells (as in the case of the PI placode, or the SNS placodes), or give rise to neuroblasts (as in the case of the optic lobe). In addition, during the interval between their first appearance and invagination, the placodes give rise to 'early neural progenitors' which delaminate from the surface and move inside. For example, the optic lobe placode gives rise to at least four neuroblasts that delaminate during stage 11 and then proliferate, like all other neuroblasts, in a stem cell-like manner. Similarly, individual neurons delaminate from the SNS placodes before these structures invaginate. It is considered likely that the dorso-medial placodes described in this study also give rise to several neuroblasts (de Velasco, 2007).

    Most neurons of the insect brain are formed as part of fixed lineages, each lineage being produced by a stem cell-like neuroblast. Neuroblasts delaminate as individual cells, or small clusters of cells, from the neurectoderm, leaving behind other cells that then become specified and differentiate as epidermal cells. This peculiar mode of neural cell birth and proliferation is a derived feature found in insects and many crustaceans; it is not present in taxa considered basal in the arthropods, and taxa outside the arthropods. Early neurogenesis in chelicerates, myriapods and chilopods have recently been analyzed, and led to the interesting discovery that in these animals, the neurectoderm produces a large array of small placodes which subsequently invaginate and (after some additional rounds of mitosis) turn into the neurons and glial cells of the ventral nerve cord and brain. In terms of number and pattern, the placodes are comparable to the array of neuroblasts in insects, leading to the speculation that one might be able to define homologies between individual placodes and neuroblasts. To go a step further, one could speculate that at the root of arthropods, the neurectoderm was subdivided into a mosaic of small domains, each of which invaginated as a placode to then give rise to a specific part of the CNS. In time, this mode of neurogenesis was supplanted by the 'invention' of stem cell-like neuroblasts: instead of the entire placode invaginating, a single (or a few) cell(s) was selected from the placode at an early stage which then delaminated and continued to proliferate in an asymmetrical, stem cell-like manner. If this interpretation of neuroblasts vs. placodes among arthropods is correct, one would have to conclude that the occurrence of placodes along the ventral midline and head midline (as well the stomatogastric nervous system and optic lobe) of insects represents the phylogenetically older mode of neurogenesis. Likewise (and this notion is of course even more speculative), one could argue that molecular mechanisms at work in these placodes or the function of brain parts derived from them is phylogenetically more ancient compared to structures developing from neuroblasts. The same rationale has traditionally been put forward to argue that the 'fringe domains' of the cerebral cortex, including the archicortex (hippocampus) and paleocortex (entorhinal cortex) constitute the phylogenetically older regions of the mammalian brain (de Velasco, 2007).

    Pars intercerebralis and hypothalamus: Similarities between the neuroendocrine system of vertebrates and arthropods on the structural, functional and developmental level have been emphasized in many previous studies. In both vertebrates and arthropods, the highest command center of the neuroendocrine system is comprised of groups of NSCs located in the brain; these cells, besides innervating brain centers and thereby influencing neural circuits as 'neuromodulators', send their axons to peripheral neurohemal glands in which the hormones produced by the NSCs are stored and released. In vertebrates, neurosecretory cells are located in the hypothalamus. The endocrine gland they act upon is the pituitary. The corresponding structures in arthropods would be the PI/PL and their peripheral targets, the CC/CA, respectively. The main hormone produced by the CC is adipokinetic hormone (AKH) that mobilizes lipids and carbohydrates from the fat body. AKH shares common functions with the vertebrate hormone glucagon that is produced in endocrine cells of the pancreas, as well as peptidergic neurons in the brain. AKH also shows some sequence similarity with the N-terminus of glucagons. Similar and possibly homologous to the relationship between Drosophila insulin-like peptides and AKH, the function of glucagon in vertebrates is antagonized by insulin. Insulin itself is expressed like glucagon in the endocrine pancreas, but a whole family of insulin-like growth factors is found in hypothalamic NSCs. Other neuropeptides found in NSCs of hypothalamus and PI alike are FMRFamides and tachykinins. Also, the sequence similarity between vertebrate CRF and insect CRF-like diuretic hormone deserves attention in this context. Here a scenario might be considered in which ancestrally a peptide directly exerted a diuretic effect, a condition maintained in the arthropod line of evolution; in the line of evolution leading up to chordates, other hormones (ACTH, aldosterone) were 'interpolated' between the original peptide (CRF) and the action on excretory cells (de Velasco, 2007).

    Developmental similarities between vertebrate hypothalamus and arthropod PI are also strong. The anlagen of the pituitary and hypothalamus are neighboring structures within the anterior neural plate. Cells that will give rise to the anterior lobe of the pituitary (adenohypophysis) are anteriorly adjacent to the cells which will become the hypothalamus and the posterior pituitary. Numerous signals were found to be involved in delimiting the anlage of the neuroendocrine system within the anterior neural plate; they include Sonic hedgehog (Shh), members of the bone morphogenetic protein family (BMP7), and fibroblast growth factor family (FGF4/8). Among the molecular determinants that are switched on by these signaling pathways are the homeobox genes six3/6 and Rx, the paired-box genes pax6 and Nkx2.1/2, the PAS-bHLH gene sim1, and the orphan nuclear receptor Tlx. Genes acting further downstream in determining specific hypothalamic cell fates are POU III-related homeobox genes Brn-1, 2, and 4. It has been shown that loss-of-function mutant mice lacking Brn-2 do not develop part of the hypothalamus; Sim1 knock-out mutations in mice cause a similar phenotype to that one for Brn-2 (de Velasco, 2007 and references therein).

    This study has shown that homologs of three of the transcription factors expressed in the anlage of the vertebrate hypothalamus also appear in or adjacent to the anlage of the Drosophila PI/PL: the Nkx2.1/2 homolog vnd, the Sim1 homolog sim, and the Rx homolog Drx. In addition, the Six3/6 homolog optix also appears to overlap with the PI/PL of the st.11 embryo. The role of these Drosophila genes in PI development awaits further study. A previous paper (DeVelasco, 2004; see Embryonic development of the corpus cardiacum, a component of the ring gland) has shown that the Six1 homolog sine oculis (so) is expressed and required for the formation of the CC and the ontogenetically closely related stomatogastric nervous system. In contrast, counterpart(s) of Dchx1, the gene that is expressed strongly and continuously throughout Drosophila PI development, apparently do not play a role in neuroendocrine development in vertebrates. Detailed expression studies of Chx10/Vsx1 documented that this gene is expressed in numerous tissues outside of the retina, notably the ventral hindbrain, the diencephalic-mesencephalic boundary, and the epithalamus. However, neither expression nor function of Chx10/Vsx1 in the ventral diencephalon has been reported (de Velasco, 2007).

    In conclusion, this study presents evidence for a number of conserved properties in the way the progenitors of the central neuroendocrine system in vertebrate and Drosophila embryos are spatially laid out, and employ cassettes of signaling pathways and fate determinants. One may speculate that there existed in the common bilaterian ancestor a simple anterior brain with which sensory afferents and groups of neurosecretory cells were associated. These cells might have played pivotal roles in feeding behavior (olfactory/gustatory perception of food sources; feed back information from the intestinal tract and body cavity regarding the degree of urgency of feeding) and reproductive behavior, and could have evolved into the much more complex neuroendocrine systems that is found in today's highly derived bilateria, such as insects and vertebrates (de Velasco, 2007).

    Origin of the mushroom body

    The mushroom body (MB) has its origin as a quadruple structure of clonal units each of which contains a virtually identical set of neurons and glial cells. The MB is a paired neuropile structure. Each neuropile consists of about 2500 Kenyon cells (Kenyon cells were first described by F. C. Kenyon in 1896). The cell bodies lie in the dorsoposterior cortex of the protocerebrum, well removed from the anterior protocerebrum. They send fibers inwards to form the calyx neuropil (also distant from the anterior protocerebrum), where the fibers receive signals from the antennal lobe interneurons and other interneurons. From the calyx, the fibres extend anteriorly via the pedunculus to reach the anterior protocerebrum, where they form the alpha, alpha', beta and gamma lobes. It is in the protocerebral lobes that olfactory learning takes place. The genes dunce, rutabaga and DCO are expressed preferentially in the MB. The MB is also involved in courtship behavior.

    How are the rudimentary mushroom bodies generated? Although the embryonic Kenyon cells (KCs) are thought to be generated from only four neuroblast precursors per hemisphere, little is known about how the characteristic structure of the MBs arises, mainly because morphogenesis occurs within the highly differentiated and complex neuropil of the embryonic protocerebrum. Advantage is taken of the spacial specificity of selected P[GAL4] lines to investigate the embryonic patterning of the MBs. In these lines, a gene coding for an enzyme marker is inserted randomly into the Drosophila genome. Insertion of such a marker, whose expression is regulated by a neural promoter, in proximity to a transcriptional enhancer, causes the reporter to be expressed in a pattern that reflects the enhancer's regulatory properties. Reporter gene expression in a particular P[GAL4] line (called 238Y) first becomes visible at embryonic stage 13 and is restricted to just one of the four mushroom body neuroblasts (MBNbs). Shortly thereafter, a second MBNb becomes visible, though with reduced intensity. Soon thereafter, reporter gene expression in line 238Y also becomes visible within the KC progeny of the two labelled MBNb. Initially, these progeny remain in the vicinity of their MBNb of origin, thus giving rise to two clusters of KCs per hemisphere. Axonal growth of KCs is first observed at stage 14. From the outset, growth is directed along the neural axis, toward the main neuropil of the protocerebrum. By stage 15, a small bundle of fibers is observed growing from each KC cluster into the protocerebrum. As outgrowth proceeds, further labelled fibers from the KC cluster enlarge the initial bundle by fasciculating with it. By late stage 17, a prominent KC fiber tract is observed entering the main protocerebral neuropil. Although reporter gene expression in line 238Y is restricted to just two MBNBs and their progeny, it is likely that each of the two unlabelled MBNbs also generate a KC cluster and a fiber tract (Tettamanti, 1997).

    Further analysis of reporter gene expression in line 238Y shows how the major intrinsic axonal elements of the MB are pioneered. KC axons belonging to the adult MBs project rostrally through the protocerebum as a massive fiber tract known as the pedunculus. At the frontal margin of the brain, this gives rise to four lobular projections. The alpha and delta lobes project dorsally, while the beta and gamma lobes project medially. The adult MB is also notable for an extensive region of dendritic arborization known as the calyx. By embryonic stage 15, fascicles from each labelled cluster of KCs extend beyond the cortical layer of cell bodies into the protocerebral neuropil, where they begin to fasciculate with each other. By stage 16, this compound fascicle has formed a pedunculus-like structure, extending through the entire embryonic protocerebrum and reaching to the posterior end (according to neuraxis), where it turns medially. By stage 17, two closely apposed sets of axonal projections have extended further medially, from the tip of the pedunculus along the posterior margin. These medially projecting elements appear topologically equivalent to the adult beta/gamma projection. By late state 17, a further out-projection, extends anteriorly, orthagonal to the beta/gamma-like structure. Throughout this process, axons from each of the two labelled KC clusters appear to contribute to each of the new projections. At no stage during embryogenesis of line 238Y is evidence of a structure resembling the adult calix seen (Tettamanti, 1997).

    Among the 85 neuroblasts in the larval central brain, four specific neuroblasts give birth to the neurons in the MB cortex above the calyx. These neuroblasts, called mushroom body neuroblasts (MBNbs), are four of the five neuroblasts that are mitotically active at the time of larval hatching. The four MBNbs give rise not only to the Kenyon cells but also to glial cells. Clonal analysis of the adult MB was performed using a genetic technique that marks individual clones. Clonal analysis reveals the following three features of MB organization: (1) the projection of the MBNb progeny is confined to the MB neuropile; (2) specific MB substructures are generated from individual MBNbs in a birth date-specific manner, and (3) the postembryonic progeny of any of the four MBNbs contributes to all the known substructures of the MB, rather than each MBNb being specialised to produce certain MB substructures (Ito, 1997).

    Clones induced just after larval hatching reveal that the progeny of a single MBNb project their axons to the entire MB neuropile. Clones of single MBNbs are arrayed in physically separated clonal units. Since there are four MBNbs, the adult Kenyon cells become arrayed in a four fold structure. Each clonal unit contains glia and all known types of Kenyon cells. Fibers running from the Kenyon cells arborize in the calyx; from here, the fibers continue on and eventually constitute the pedunculus. Fibers then defasciculate into the lateral and medial bundles. Fibers in the medial bundle form the alpha and beta lobes, while those in the lateral bundle contribute to the spur and alpha' and gamma lobes. Any one of the MBNbs can autonomously generate a clonal unit even when all other MBNs are absent (Ito, 1997).

    What are the consequences of the four fold mushroom body structure? Constructing such complex circuits as a quadriple structure of clonal units, each of which contains a complete set of MB cell types, might have been an efficient and economical means during evolution to improve the performance of information processing without fundamentally altering the process of MB development. The four-fold structuring might be integral to processing and resolution of input signals that convey various olfactory and other sensory information. However, this does not necessarily mean that the four clonal units are also identical in their function. For example, it is possible that the incoming neurons, such as the antennal lobe interneurons via the inner antenno-cerebral tract (also called antenno-glomerular tract), innervate the cells of the clones differently. Computer simulation suggests that even if there is no initial difference in input signals and neuronal connectivity, structurally identical neurons in a network array can develop different functions after 'training'. This sort of plasticity may well play an important role in the MB, since the volume of the neuropile, and thus the degree of complexity in the synaptic network will vary greatly, depending upon the post-eclosion experiences of each individual (Ito, 1997 and references).

    Generation of different types of mushroom body neurons

    The adult MBs appear as paired neuropils in the Drosophila brain. The gross morphology can be divided into discrete anatomical domains. The cell bodies are clustered at the dorsal posterior surface of the central brain and their dendrites form the calyx structure right below the cell body region. The axons form the peduncle, which extends ventrally toward the anterior surface of the brain, where it segregates into five terminal lobes (Crittenden, 1998). The alpha and alpha' lobes project toward the dorsal surface, while the beta, beta' and gamma lobes project toward the midline of the brain. As revealed by Golgi staining, MB neurons are unipolar; their dendrites branch into the calyx right below the cell body, and individual axons project through the peduncle and extend into the lobes. In the housefly, two types of axonal projections can be distinguished based on their branching patterns. Some axons bifurcate into dorsal and medial branches, while other axons only extend toward the midline without bifurcation. MB neurons with these stereotyped projection patterns have also been described in Drosophila. Interestingly, these five lobes can be grouped into three sets based on the expression levels of various MB-enriched antigens. For any given antigen, alpha and beta lobes always have comparable expression levels; alpha' and beta' lobes always share strong similarities, and the gamma lobe is distinct from the others. These findings have led to the proposal that there are three major projection configurations of MB axons. One type of MB neuron projects its axons only into the gamma lobe; the second type projects its axon branches into both alpha' and beta' lobes, while the third type projects its axon branches into both alpha and beta lobes (Crittenden, 1998; Lee, 1999).

    In each brain hemisphere, the MB is derived from four neuroblasts (Nbs). Labeling DNA replication by BrdU incorporation reveals continuous proliferation of MB Nbs from the embryonic stage to the late pupal stage. Interestingly, each MB Nb generates a similar set of neurons and glial cells, as evidenced by the 4-fold organization of marked neurons in various MB enhancer trap lines and the same gross morphology of marked neurons derived from random MB Nbs (Lee, 1999).

    Therefore, how MB neurons with different axonal projections are derived from the same Nb remains to be elucidated. Another interesting aspect regarding development of the MB is reorganization of larval MB neurons during metamorphosis. In the CNS, there are three known fates for most larval neurons during metamorphosis. Larva-specific neurons are removed by programmed cell death after puparium formation. Adult-specific neurons continue their morphogenesis through the pupal stage. In addition, some larval neurons that persist into adulthood withdraw their larval processes and extend new adult-specific processes during metamorphosis. Most MB neurons persist through metamorphosis and thus it is important to determine how MB neurons born at different stages reorganize their processes during metamorphosis (Lee, 1999).

    Using mosaic analysis with a repressible cell marker, Lee (1999) positively marked the axons and dendrites of multicellular and single-cell mushroom body clones at specific developmental stages. Systematic clonal analysis demonstrates that a single mushroom body neuroblast sequentially generates at least three types of morphologically distinct neurons. Neurons projecting into the gamma lobe of the adult MB are born first, prior to the mid-3rd instar larval stage. Neurons projecting into the alpha' and beta' lobes are born between the mid-3rd instar larval stage and puparium formation. Finally, neurons projecting into the alpha and beta lobes are born after puparium formation. Visualization of individual MB neurons has also revealed how different neurons acquire their characteristic axon projections. During the larval stage, axons of all MB neurons bifurcate into both the dorsal and medial lobes. Shortly after puparium formation, larval MB neurons are selectively pruned according to birthdays. Degeneration of axon branches makes early-born (gamma) neurons retain only their main processes in the peduncle, which then project into the adult gamma lobe without bifurcation. In contrast, the basic axon projections of the later-born (alpha'/beta') larval neurons are preserved during metamorphosis (Lee, 1999).

    Taken together with previous studies, neurogenesis of one MB involves continuous proliferation of four equivalent Nbs through the embryonic, larval and pupal stages. During embryogenesis, four MB Nbs generate less than 50 neurons, which probably project their axons exclusively into the gamma lobe of adult flies. After larval hatching, each MB Nb produces about 500 more neurons. Based on their axonal projection patterns, there are three types of MB neurons. These three stereotypic types of axonal projections lead to formation of five distinct axonal lobes in the adult MB. Before the mid-3rd instar stage, all MB precursors generate gamma neurons. Between the mid-3rd instar stage and puparium formation, all MB precursors generate alpha'/beta' neurons. Both gamma and alpha'/beta' neurons acquire similar projection patterns before PF. After PF, gamma neurons undergo dramatic reorganizations in order to change the axonal projection pattern, while alpha'/beta' neurons remain relatively unchanged. Meanwhile, all newly born MB neurons after PF become alpha/beta neurons whose axonal processes form the adult-specific alpha/beta lobes (Lee, 1999 and references therein).

    What are the possible functions of different types of MB neurons? Studies by Lee (1999) provide new insights into the function of three distinct classes of neurons. Since the alpha/beta neurons are born starting from puparium formation, they obviously play adult specific roles. It is interesting that they are among the largest group of the neurons from an estimation of the volume contribution (42%) and their axons are the most densely packed. On the contrary, the gamma neurons probably play important roles in larval stages since they are likely born in embryos and certainly early stages of larval life. By the end of the third instar larva, the dendrites and axon projections into the lobes are highly elaborate. However, the gamma neurons undergo the most dramatic changes in the first day of pupal life so that their dendrites as well as their axons that branch into the larval vertical and medial lobes, are completely degenerated. Curiously, the adult projection pattern is medial lobe-specific, suggesting that the gamma neurons may change their network property altogether in larval and adult life (Lee, 1999).

    The alpha'/beta' neurons, although born at a later stage of larva life, may not be fully differentiated at the end of larval life, judging from the immature dendrites compared to the gamma neurons at the end of third instar, so their functions may also be largely adult-specific. However, they may play important roles in the transition between the larval mushroom body and adult mushroom body, both from the aspect of axon guidance and dendritic morphogenesis, and from the aspect of maintaining connections with input and output neurons. Since the gamma neurons completely change connection pattern during metamorphosis, the alpha'/beta' neurons, whose axon and dendrite projections may follow the routes established by the gamma neuron in larva, may prove instrumental to ensure that the adult-specific alpha/beta neurons project their axons correctly into both medial and dorsal lobes, and that their dendrites occupy the calyx region. Their function in axon guidance may be analogous to that of the subplate neurons in axon guidance of mammalian cerebral cortical neurons. Unlike the subplate neurons, the alpha'/beta' neurons are not themselves pioneer neurons. Their roles may be to transfer the routes established by the pioneering gamma neurons to the later-born alpha/beta neurons (Lee, 1999).

    It has been shown that Drosophila larvae are capable of olfactory learning and that memory can persist through metamorphosis. Although it has not been formally demonstrated that larval mushroom bodies are essential for larval learning, it is quite likely that they do play a role, to judge from the conserved organization of the MB neurons between larva and adult. For instance, both larval and adult mushroom bodies contain the calyx in analogous positions, and the axon projections consist of the dorsal and the medial lobes and are found in conserved relative positions. Moreover, many genes that are preferentially expressed in adult mushroom bodies are also preferentially expressed in larval mushroom bodies, including genes that are implicated in olfactory learning and memory. In light of these findings, alpha'/beta' neurons may play important roles in connecting the larval and adult functions of the mushroom bodies. It will be interesting to determine whether larvae younger than third instar, before the birth of the alpha'/beta' neurons, are capable of olfactory learning and to see if their memory can persist through metamorphosis (Lee, 1999).

    Origin of Drosophila mushroom body neuroblasts and generation of divergent embryonic lineages

    Key to understanding the mechanisms that underlie the specification of divergent cell types in the brain is knowledge about the neurectodermal origin and lineages of their stem cells. This study focused on the origin and embryonic development of the four neuroblasts (NBs) per hemisphere in Drosophila that give rise to the mushroom bodies (MBs), which are central brain structures essential for olfactory learning and memory. These MBNBs were shown to originate from a single field of proneural gene expression within a specific mitotic domain of procephalic neuroectoderm, and that Notch signaling is not needed for their formation. Subsequently, each MBNB occupies a distinct position in the developing MB cortex and expresses a specific combination of transcription factors by which they are individually identifiable in the brain NB map. During embryonic development each MBNB generates an individual cell lineage comprising different numbers of neurons, including intrinsic gamma-neurons and various types of non-intrinsic neurons that do not contribute to the MB neuropil. This contrasts with the postembryonic phase of MBNB development during which they have been shown to produce identical populations of intrinsic neurons. Different neuron types were shown to be produced in a lineage-specific temporal order, and neuron numbers are regulated by differential mitotic activity of the MBNBs. Finally, it was demonstrated that gamma-neuron axonal outgrowth and spatiotemporal innervation of the MB lobes follows a lineage-specific mode. The MBNBs are the first stem cells of the Drosophila CNS for which the origin and complete cell lineages have been determined (Kunz, 2012).

    Applying the DiI labeling technique in combination with molecular markers and cell ablation experiments, the embryonic development was studied of a prominent central brain structure, the MBs. This approach allowed precise mapping of the origin of the MB in the procephalic NE, elucidation of the mode of MBNB formation, and tracing of the spatiotemporal development and composition of the entire embryonic lineages of the four MBNBs (see Origin of Drosophila mushroom body neuroblasts and generation of divergent embryonic lineages). Since cell numbers in the DiI-labeled MBNB clones (on average 29-45 cells at st17 and 36-48 cells in L1) differ drastically from those reported previously in flip-out clones (~14 cells at late st16 and ~8 cells in L1) (Larsen, 2009), the latter appear to encompass only part of an embryonic lineage. It is likely that this part represents the late-born cells, as recombination in an NB depends on a critical level of heat shock flippase, which does not become enriched before the early-born part of a lineage has developed (Larsen, 2009). Also, MARCM fails to disclose entire NB lineages in the late embryo, as clonal reporter expression additionally relies on the loss of the GAL80 repressor after recombination, which seems to persist throughout embryonic development (Kunz, 2012).

    MBNBs proliferate persistently throughout embryogenesis, in contrast to all other brain NBs (except one in the deutocerebrum), which enter a phase of mitotic quiescence in the late embryo. Accordingly, all four embryonic MBNB clones are significantly larger than those of other early NBs in the brain and VNC. Whereas the postembryonic MBNB lineages form identical subunits of the adult MBs, it was observed that during embryonic development each MBNB creates a distinct lineage with its own number and type of intrinsic and nonintrinsic interneurons, which are generated in a fixed temporal order. These findings imply genetic regulation of temporal cell fates in a lineage-specific manner. Further analyses will need to show whether the 'temporal gene cascade' hunchback-Krüppel-nubbin- castor-grainy head and/or Chinmo, a determinant for temporal neuronal identities during postembryonic MB development and/or other cues confer temporal cell fates to MBNB progenies during the embryonic period. Considering the higher diversity of neuronal cell types, the underlying regulatory mechanisms are presumably more complex compared with the postembryonic period, in which only three types of MB-intrinsic neurons are generated. Interestingly, as recently reported (Yu, 2010), the embryonic NB that generates the antero-dorsal projection neurons (adPN lineage) in the antennal lobe, in contrast to the larval period, alters temporal identity following each division (Kunz, 2012).

    Ey has been shown to regulate neuron numbers and Ey and Dac to control axonal differentiation and targeting of MB neurons. This study found that ey, dac and Rx are differentially expressed in MBNBs and daughter cells during embryonic neurogenesis, suggesting specific functions in certain neurons or neuron subtypes. It remains to be seen whether, for example, absence of Dac accounts for the delayed differentiation of γ- neurons in the MBNBd lineage. Moreover, Ey expression, which commences in the MBNBb/c lineages when γ-neurons are produced and becomes restricted to this cell type in the MBNBα/δ lineages (whereas it is downregulated quickly in ni-neurons), might be crucial for γ-neuron differentiation and its absence required for proper ni-neuron differentiation (Kunz, 2012).

    Clonal analysis reveals that ~95 γ-neurons develop in the embryo (~125 by 2-3 hours after larval hatching), which corresponds to the number of Kenyon cells (~100) observed in adult brain of MB-specific Gal4 strains after hydroxyurea ablation of MBNBs in the newly hatched larva. This suggests that most or all of the embryonic γ-neurons survive to adulthood, although they become dramatically reorganized during pupal development. Accordingly, γ-neurons do not undergo programmed cell death in the embryo, nor, presumably, during postembryonic stages. This is remarkable because ~30%-40% of embryonic brain neurons die around the time of larval hatching (Larsen, 2009). Interestingly, appetitive olfactory learning in L1 relies on a set of ~100 embryonic-born γ-neurons (included in the 201y-Gal4 and NP1131-Gal4 lines), indicating that the underlying neuronal circuits at the level of the MBs are established during embryogenesis (Pauls, 2010). As was observed a primordial calyx carrying dendritic arborizations of γ-neurons already in the embryo, it is likely that embryonic-born projection neurons (providing olfactory input from the antennal lobes) participate in these initial neuronal circuits (Marin, 2005; Raemakers, 2005; Yu, 2010) (Kunz, 2012).

    Although the four postembryonic MBNB clones consist of the same sets of Kenyon cells, which equally contribute to peduncle and lobes, it seems that they are not in fact identical as the dendrites of each MBNB clone occupy a specific calyx subregion. Projection neurons that terminate in distinct microglomeruli in the larval calyx convey olfactory information from specific antennal lobe glomeruli. Thus, each MBNB clone receives a different repertoire of olfactory information, which might be correlated with the unique properties of each of the four embryonic MBNBs. This raises the question as to which embryonic lineage (a-d) each postembryonic MBNB lineage evolves from. Interestingly, it was found that, for example, the most lateral subdivision of the MB cortex and calyx seems to be consistently occupied by the MBNBc lineage from the embryonic to the adult stage. This further suggests that aspects of embryonic calyx topology might be retained during postembryonic development. About 40 non-intrinsic interneurons are generated by the four MBNBs, most of which project into the contralateral brain hemisphere. Some of these interneurons form bouton-like structures at specific sites near the surface of the contralateral MB, reminiscent of MB-associated neurons, i.e.,'extrinsic' or 'non-Kenyon cell MB-intrinsic' neurons. Therefore, ni-neurons might be involved in processing information between the higher-order olfactory systems of the two hemispheres (Kunz, 2012).

    Ablation experiments suggest that γ-neurons from MBNBa are not instructive for axonal guidance of following γ-neurons from other MBNB clones. Interestingly, ni-neurons establish contralateral axonal projections before the onset of γ-neuron axogenesis. It has been shown that the Derailed receptor, which is expressed on contralateral axons of yet unidentified neurons, is necessary for proper growth of γ-neuron axons towards the midline, mediated through the Wnt5 ligand expressed in γ-neurons. Since contralateral fascicles of ni-neurons (non-intrinsic neurons that do not form part of the MB neuropils) are in close contact with developing γ-neuron axons they might correspond to those neurons that are instructive for γ-neuron axonal growth. It is therefore tempting to speculate that ni-neurons (non-intrinsic neurons that do not form part of the MB neuropils) interact with γ-neurons in two different ways: early in a developmental context and later in a functional context (Kunz, 2012).

    The MBNBs originate from adjacent γB cells that belong to the same proneural domain of the procephalic NE. This peculiar mode of neurogenesis parallels that reported for basal arthropods. In chelicerates, the MBs originate from a proneural gene (CsASH1)-expressing, invaginating NE in which all cells are destined to a neural fate. These similarities might suggest a common evolutionarily origin of a mode of neural progenitor formation that does not rely on lateral inhibition among cells of the NE. Recently, the early gene expression profile of two potential MBNBs, the Dac-positive Pcd8 and Pcd9, was used for a molecular comparison with the MB in the annelid Platynereis (Tomer, 2010). However, since dac and/or ey are not uniquely expressed in MBNBs, the identity of the MBNBs in the current NB map had been unclear. This study shows that Pcd9, but not Pcd8, belongs to the MBNBs. The gene expression profile in the other three MBNBs (Pcd2, Pcd4, Pcv9) is similar to that in Pcd9. Moreover, co-expression of Rx and dac/ey almost exclusively detected in the MB. Rx is also expressed in the annelid MB, which further supports homology between these brain structures in insects and annelids (Tomer, 2010). Remarkably, the molecular fingerprint of the MB in Platynereis was found to be conserved in a subregion of the vertebrate forebrain, the pallium (Tomer, 2010). It also seems to be largely conserved in Pcd9 and, albeit less strongly, in all other Drosophila MBNBs. This lends support to the hypothesis that the protostome (annelid, insect) MB and the deuterostome (vertebrate) pallium evolved from a common brain center present in the bilaterian ancestor (Tomer, 2010; Kunz, 2012 and references therein).

    Dynamics of glutamatergic signaling in the mushroom body of young adult Drosophila

    The mushroom bodies (MBs) are paired brain centers located in the insect protocerebrum involved in olfactory learning and memory and other associative functions. Processes from the Kenyon cells (KCs), their intrinsic neurons, form the bulk of the MB's calyx, pedunculus and lobes (see Mushroom body is a quadruple structure). In young adult Drosophila, the last-born KCs extend their processes in the alpha/beta lobes as a thin core (alpha/beta cores) that is embedded in the surrounding matrix of other mature KC processes. A high level of L-glutamate (Glu) immunoreactivity is present in the alpha/beta cores (alpha/betac) of recently eclosed adult flies. In a Drosophila model of fragile X syndrome, the main cause of inherited mental retardation, treatment with metabotropic Glu receptor (mGluR) antagonists can rescue memory deficits and MB structural defects. To address the role of Glu signaling in the development and maturation of the MB, the time course of Glu immunoreactivity was compared with the expression of various glutamatergic markers at various times, that is, 1 hour, 1 day and 10 days after adult eclosion. It was observed that last-born alpha/betac KCs in young adult as well as developing KCs in late larva and at various pupal stages transiently express high level of Glu immunoreactivity in Drosophila. One day after eclosion, the Glu level was already markedly reduced in the alpha/betac neurons. Glial cell processes expressing glutamine synthetase and the Glu transporter dEAAT1 were found to surround the Glu-expressing KCs in very young adults, subsequently enwrapping the alpha/beta lobes to become distributed equally over the entire MB neuropil. The vesicular Glu transporter DVGluT was detected by immunostaining in processes that project within the MB lobes and pedunculus, but this transporter is apparently never expressed by the KCs themselves. The NMDA receptor subunit dNR1 is widely expressed in the MB neuropil just after eclosion, but was not detected in the alpha/betac neurons. In contrast, evidence is provided that DmGluRA, the only Drosophila mGluR, is specifically expressed in Glu-accumulating cells of the MB alpha/betac immediately and for a short time after eclosion. The distribution and dynamics of glutamatergic markers indicate that newborn KCs transiently accumulate Glu at a high level in late pupal and young eclosed Drosophila, and may locally release this amino acid by a mechanism that would not involve DVGluT. At this stage, Glu can bind to intrinsic mGluRs abundant in the alpha/betac KCs, and to NMDA receptors in the rest of the MB neuropil, before being captured and metabolized in surrounding glial cells. This suggests that Glu acts as an autocrine or paracrine agent that contributes to the structural and functional maturation of the MB during the first hours of Drosophila adult life (Sinakevitch, 2010).

    In Drosophila and other arthropods, Glu is well characterized as the excitatory neurotransmitter of the neuromuscular junction. However, this amino acid has important signaling functions in the Drosophila brain as well. The Drosophila genome was predicted to encode 30 iGluR subtypes, including 3 AMPA- and 15 kainate-like, 2 NMDA-like, 4 δ-like and 6 divergent receptors. For now, the best characterized of these are the postsynaptic iGluRs expressed at the neuromuscular junction. Drosophila NMDA-like receptors are expressed in the central nervous system and have been implicated in learning and memory and locomotor control. The Drosophila genome encodes a single functional mGluR, DmGluRA, an ortholog of vertebrate group II mGluRs (Parmentier, 1996). This mGluR is presynaptic and expressed at the periphery of the active zones at the glutamatergic neuromuscular junctions, where it modulates both synapse excitability and fine structure (Bogdanik, 2004). DmGluRA is also expressed in the brain, in particular in lateral clock neurons, where it regulates circadian locomotor behavior (Hamasaka, 2007; Sinakevitch, 2010 and references therein).

    The mushroom bodies (MBs) are paired centers located in the protocerebrum of Drosophila and other dicondylic insects that play essential roles in olfactory learning and memory and other brain functions, such as the control of locomotor activity, courtship behavior, courtship conditioning, visual context generalization, and sleep. The intrinsic structure of the MB is provided by the Kenyon cells (KCs), which have their cell bodies in the brain cortex and their dendrites in the MB calyx, where they receive input from the antennal lobe projection neurons. Axon-like processes of KCs project anteriorly and ventrally in the peduncle to form the vertical and medial lobes, which are subdivided into discrete parallel entities, the vertical α, α' and the medial β, β' and γ lobes. In addition to the KCs, there are other MB intrinsic neurons and several classes of MB extrinsic neurons that connect the MB to other areas of the brain neuropil. Emerging evidence suggests that different subtypes of MB KCs may be involved in distinct mechanisms of memory formation due to their connections to different MB extrinsic neurons (Sinakevitch, 2010).

    Developmental studies have shown that the KCs are produced in each hemisphere of the brain by the division of four neuroblasts born early during the embryonic stage. The division of these neuroblasts sequentially produces the three morphologically and spatially distinct subtypes of KCs: γ, α'/β' and α/β. The γ neurons are generated up to the mid-third instar larval stage; they form the larval dorsal and medial lobe. The next KC subtype to be generated is the α'/β' neuron, which continues to be produced until puparium formation. Lastly, the α/β neurons are generated from the time of puparium formation until adult eclosion. In the α/β lobes, the KCs are organized in concentric layers. The youngest axon-like processes situated in the inner layer of the lobes are successively displaced outwards as they differentiate and newer α/β processes are added to the structure from the most recently born KCs (Kurusu, 2002). This volume of the α/β lobes into which grow the last-born axons contains densely packed and extremely thin fibers that are rich in actin filaments. This subset of processes has been named the α/β core (α/βc) (Sinakevitch, 2010).

    An increased response to mGluR activation may play a prominent role in the fragile X syndrome (FXS), the most common form of inherited mental retardation and the predominant cause of autism. Mutations in dFmr1, the Drosophila homologue of the gene implicated in FXS, lead both to learning deficits and altered development of the MB, of which the most common feature is a failure of β lobes to stop at the brain midline. These behavioral and developmental phenotypes can be successfully rescued in Drosophila by treatment with mGluR antagonists (McBride, 2005), implicating Glu in the pathology, as is the case in mammalian models. Recent studies showed that dFmr1 interacts with DmGluRA in the regulation of synaptic architecture and excitability at glutamatergic synapses (Gatto, 2008; Repicky, 2009). However, until now the precise role of Glu and mGluRs in FXS and MB development has remained obscure (Sinakevitch, 2010).

    This study presents evidence that Glu and its receptor DmGluRA are directly involved in construction of the MB neural circuits. Previous studies suggested that the Drosophila last-born α/βc KCs are immunoreactive to anti-Glu antibodies. The present study shows that these neurons express a high level of Glu-like immunoreactivity in newly eclosed adult flies. Interestingly, newborn KCs in late larval and pupal stages also appear to express as a rule a high level of Glu. To understand further the role and fate of Glu during KC maturation, the dynamics of Glu, DmGluRA and other Glu signaling-associated proteins were analyzed in the MB of young adult Drosophila from the time of their eclosion until 10 days post-eclosion. The results indicate that a transient Glu release likely regulates functional maturation of newborn KCs by a paracrine action during Drosophila post-embryonic development and the first hours after adult eclosion (Sinakevitch, 2010).

    One intriguing question in neuroscience is how newborn neurons establish a functional network during their period of growth and maturation. This work describes a study of the late maturation of a subset of the α/β intrinsic MB KCs, the α/βc neurons, during a short period after adult eclosion. Glu-like immunoreactivity has been observed in the ingrowth lamina of the cockroach MB, which contains axons of the youngest KCs. Similarly in Drosophila, Glu accumulates in the α/βc, which contains newly generated neurons, whereas taurine-expressing neurons were found in the outer α/βc and aspartate-expressing neurons in the rest of the α/β lobes. It has been shown in vertebrates that Glu can have a strong influence on cone motility and induce rapid filopodia protrusion from hippocampal neurites or cultured astrocytes. In the present study an extensive analysis was performed of the distribution of various glutamatergic markers in the MBs of young adult Drosophila. The results suggest that the α/βc neurons are not simply glutamatergic. Rather, the evidence provided in this study indicates that these newborn KCs may transiently use Glu as a paracrine agent to favor interactions with glial cell processes and become mature neurons forming functional circuits (Sinakevitch, 2010).

    Although the last-born α/βc KCs show a high level of Glu immunoreactivity a few hours prior and after adult eclosion, Glu immunostaining is dramatically reduced in these cells 24 hours after eclosion and is entirely absent a few days later. Disappearance of this signal could result from the release or intracellular metabolism of this amino acid. Similarly, it was observed in cockroach MBs that newborn KCs loose Glu immunoreactivity when they become mature and establish contacts with extrinsic neurons. This study also presents the first evidence that Glu transiently accumulates at a high level in developing newborn KCs of Drosophila in late larva and during pupal stages. Therefore, transient Glu expression could correlate with KC growth and maturation not only in the α/βc around eclosion time but also in other lobes during earlier stages of MB development (Sinakevitch, 2010).

    Three subtypes of vesicular Glu transporters (VGluTs) have been identified in the mammalian nervous system with similar Glu transport functionality. Two of these (VGluT1 and VGluT2) present complementary distribution in central glutamatergic neurons. The third isoform, VGluT3, appears to be primarily expressed in neurons that release another transmitter (serotonin, dopamine, acetylcholine or GABA), where it may be required for efficient synaptic transmission. In the present study, neither the α/βc neurons nor any other intrinsic MB KCs were found to express the Drosophila vesicular transporter DVGluT. This may indicate that the Glu that is accumulated in the inner α/βc neurons is not stored in synaptic vesicles. However, the possibility cannot be excluded that these cells express another vesicular Glu transporter not yet identified in Drosophila. DVGluT immunoreactivity was observed in the MBs, particularly in the γ lobe and spur region and in the α lobes, but the punctuate labeling and localization suggest that this distribution corresponds to glutamatergic synapses belonging to extrinsic neurons (Sinakevitch, 2010).

    Can the Glu transiently stored in the newborn MB neurons be released into the extracellular space? In the absence of DVGluT or another similar transporter, this could involve a non-vesicular release of Glu. Non-conventional release of Glu from immature neurons has been demonstrated in the developing rat hippocampus where Glu release exerts a paracrine action that seems to particularly affect the migration of neighboring maturing neurons. To address this question indirectly, the presence in the MB of other proteins known to be involved in the recycling and degradation of Glu at glutamergic synapses was sought (Sinakevitch, 2010).

    An important role of glial cells is to capture Glu released from the synapse with specific transporters and then convert Glu to glutamine with GS. The only Drosophila high-affinity Glu transporter, dEAAT1, is expressed in subtypes of glial cells and is associated with Glu-release sites. GS2 is similarly expressed in glial cells in the Drosophila nervous system. This study shows that glial cells expressing dEAAT1 and GS surround the Drosophila MB lobe neuropiles, closely enwrapping the α/β lobes, thus isolating them from other lobes, and sending a mesh-like system of extensions inside these lobes. Enwrapping and invading of the MB β lobes by glia was also observed to occur in cockroach MBs, where glial cells are implicated in the removal of degenerating transient KC processes that occur during their establishment of mature connections with extrinsic cell dendrites. The high levels of glial dEAAT1 and GS within the Drosophila MB lobes suggest that this neuropil is tightly cordoned off from other parts of the brain and regulates the extracellular Glu level between the axons (Sinakevitch, 2010).

    These data show that GS expression is highly dynamic in the MB during the first day of adult life, suggesting that glial cells play a role in establishing the MB's functional network. During the first hour after eclosion, the meshwork of glial processes expressing Glu signaling-associated molecules (GS and dEAAT1) is not present in the inner α/βc region, but within 24 hours this area becomes covered by glial extensions. These glial elements are possibly guided towards the α/βc area by the gradient of Glu released by the last born KCs. Glia could be involved in reducing Glu concentration in this area and play a role in axonal guidance and final maturation of KCs. Evidence that Glu transporters are required for coordinated brain development has been previously reported for mice: the absence of two glial Glu transporters resulted in excess of extracellular Glu and abnormal formation of the neocortex (Sinakevitch, 2010).

    Assuming Glu is released by the newborn MB neurons, it has to interact with specific receptors. Therefore, the expression was sought of Glu receptors in MB neuropiles of young adult Drosophila, particularly those receptors that are likely to regulate neuronal growth and maturation through second-messenger pathways. Once activated by simultaneous Glu binding and membrane depolarization, the NMDAR channel allows calcium influx into the postsynaptic cell, where this ion triggers a cascade of biochemical events resulting in synaptic maturation and plasticity. Available antibodies against the constitutive dNR1 subunit of the Drosophila NMDAR were used. Immediately after eclosion, many processes in the MB neuropil were found to be dNR1-positive, with the exception of the α/βc neurons. The Glu released from either these α/βc neurons, or the surrounding glial cells, or extrinsic MB glutamatergic neurons may activate these NMDAR receptors. Thus, a widespread localization of NMDAR characterizes the MB immediately after eclosion, at the beginning of adult life when the MB is expected to receive the least inputs from sensory interneurons. Subsequently, with increasing sensory data being received and relayed to projection neurons, there is a dramatic and concomitant restructuring of NMDAR signaling: the majority of MB neurons no longer express these receptors. It is only those neurons that receive constant glutamatergic signaling that still address the dNR1 subunit in the vicinity of glutamatergic synapses expressing DVGluT. This occurs in particular within the spur region of the MB and the lateral horn. Such developmentally related regulation of NMDAR expression in the MBs of young adult flies may relate to adaptations of synaptic activity in response to sensory experience (Sinakevitch, 2010).

    mGluRs are neuromodulatory G-protein-coupled receptors that are involved in many aspects of brain physiology, including neuronal development, synaptic plasticity, and neurological diseases. Whereas eight distinct mGluRs are present in the mammalian genome, a single functional mGluR is expressed in Drosophila, DmGluRA. The fly mGluR is structurally and pharmacologically closer to the mammalian group II mGluRs, which are mainly presynaptic receptors negatively coupled to adenylate cyclase. Attempts to locate DmGluRA with the commercially available monoclonal antibody 7G11 were not successful because the antibody produced by the hybridoma clone recently lost its binding specificity. To monitor DmGluRA distribution, a new GAL4 line was used that carries an enhancer trap insertion close to the mGluR start site of transcription, keeping in mind that expression of this GAL4 reporter may, in part, differ from the mGluR pattern. Strikingly, the DmGluRA-GAL4 line was found to express GFP selectively in the Glu-accumulating α/βc KCs of newly eclosed adult flies. This is in contrast to commonly used MB GAL4 driver lines (17d-, c739- and 201Y-GAL4) that do not express GFP in these neurons immediately after eclosion. Ten days later, the GFP staining in the DmGluRA-GAL4 line appeared strongly reduced in the α/βc; in contrast, the MB drivers now expressed GFP in these neurons (Sinakevitch, 2010).

    Because the GAL4 reporter method reveals whole neurons, it could not be determined where the receptor is addressed restrictively in cell bodies, dendrites or axons. A previous study performed with an active lot of 7G11 antibody indicated that DmGluRA is present in nearly all neuropiles of the mature adult fly brain, including the MB calyces, but not in the MB lobes (Devaud, 2008). However, thas study did not report on the localization of DmGluRA in newly eclosed Drosophila. Further work is required to precisely locate the subcellular localization of DmGluRA in the newborn α/βc neurons, either with a new antibody or a DmGluRA-GFP fusion gene. The source of Glu binding to this mGluR receptor may be the neighboring glial cells or newborn KCs themselves, or both. Through activation of these receptors, Glu is likely to have a transient paracrine action on the α/βc neurons during the first day after eclosion that could be required for dendrite growth or synaptic maturation (Sinakevitch, 2010).

    Although the α/βc KCs represent a minor part of the α/β lobe neurons, the maturation of these cells appears to be essential for proper MB functioning. Selective expression of the rutabaga (rut)-encoded adenylate cyclase in the α/βc neurons with 17d-GAL4 was shown to partially restore olfactory learning and memory in 2- to 5-day-old rut mutant flies. In contrast, no rescue of the rut defect was observed with c739-GAL4, which expresses in more peripheral α/β neurons at this stage. Therefore, the network involved in olfactory learning and memory apparently requires the α/βc neurons and is already functional in 2- to 5-day-old flies. Furthermore, treatment with mGluR antagonists restored courtship behavior, memory deficits and MB structural defects in DFmr1 mutants, a Drosophila model of FXS. These positive effects are even stronger when the pharmacological treatment is applied both during larval development and after eclosion. This suggests that these behavioral defects relate to an abnormally high level or prolonged duration of DmGluRA expression in the α/βc neurons of DFmr1 mutants. Further study should determine the distribution of Glu and DmGluRA during MB development in Drosophila FXS models (Sinakevitch, 2010).

    The ubiquitin-proteasome system is one of the major conserved cellular pathways controlling protein turnover in eukaryotic cells. Substrate protein ubiquitination plays important roles in neuronal differentiation, axonal targeting, synapse formation and plasticity. In addition to strong Glu immunolabeling in the inner α/βc KCs, a high level of anti-ubiquitin immunoreactivity was also observed in these neurons immediately after eclosion. Such a high staining level was no longer detected in 10-day-old flies. In contrast, the spur region of the MB showed a constant high ubiquitin immunoreactivity that did not change with the age of the animal. This could suggest that synaptic plasticity is particularly active in this MB area (Sinakevitch, 2010).

    Similarly, labeling of the cockroach MB β lobe with anti-ubiquitin showed, at specific stages in each developmental instar, as well as at an early adult stage, consistent staining of newly generated KC axons. Anti-ubiquitin also labeled the extending transiently Glu-immunoreactive collateral processes from developing KCs in the ingrowth zone, the hemimetabolous homologue of Drosophila's core KCs. This study showed that ubiquitin expression precedes degeneration of these collaterals and their subsequent removal by scavenging glial cells. Glu receptors can be endocytosed by an ubiquitin-dependent mechanism. The down-regulation of Glu and its receptor protein, possibly mediated by ubiquitin, thus appear to be important steps in the maturation and differentiation of the α/βc KCs (Sinakevitch, 2010).

    In conclusion the present study suggests that the Glu accumulated in the α/βc KCs of young adult Drosophila is used for cell growth and maturation rather than for neurotransmission. The distribution and dynamics of glutamatergic markers indicates that Glu released from newborn KCs can bind to intrinsic mGluRs in the α/β cores and to NMDARs in the rest of the MB neuropil before being captured and metabolized by surrounding glial cells. As an autocrine or paracrine agent, Glu is likely to play a role in pathway finding within the lobe, namely, interactions between maturing KCs and extrinsic neuron dendrites, guidance of glial cell outgrowth and glial process targets into and around the relevant lobes, and maturation of synaptic networks required for a functional MB. Further study of the paracrine function of Glu in wild-type flies and in the Drosophila FXS model may shed light on similar actions of this neurotransmitter in the developing human brain in normal and pathological conditions (Sinakevitch, 2010).

    Fate mapping of brain progenitors using photoactivated gene expression

    The Drosophila brain is generated by a complex series of morphogenetic movements. To better understand brain development and to provide a guide for experimental manipulation of brain progenitors, a fate map was derived using photoactivated gene expression to mark cells originating within specific mitotic domains and time-lapse microscopy to dynamically monitor their progeny. Mitotic domains 1, 5, and 9 give rise to discrete cell populations within specific regions of the brain. Two novel observations are reported: the antennal sensory system, composed of four disparate cell clusters, arises from mitotic domain 5 and that mitotic domain B produces glial cells, while neurons are produced from mitotic domains 1, 5, and 9. Time-lapse analysis of marked cells show complex mitotic and migratory patterns for cells derived from these mitotic domains. Photoactivated gene expression was also used either to kill, to induce ectopic divisions, or to alter cell fate. This revealed that deficits were not repopulated, while ectopic cells were removed and extra glia were tolerated (Robertson, 2003).

    To enable the marking of cells in a spatially and temporally restricted manner, and alter their behavior, a method for activating gene expression using a microbeam of light was used. This method, which is referred to as photoactivated gene expression, is based on the GAL4-expression method. Instead of supplying GAL4 genetically, chemically 'caged' GAL4VP16 is injected into syncytial stage embryos that carry a UAS-transgene. Expression of the UAS-transgene is activated by briefly irradiating the cell, or cells, of choice with a long-wavelength UV microbeam, thus uncaging the GAL4VP16 protein. This method was used to activate the expression of benign markers, such as LacZ and GFP, and to alter cell behavior with agents such as Cyclin E, Reaper (Rpr), and Head involution defective (Hid). Time-lapse microscopy and whole-mount embryo preparations were used to track the normal and altered behavior of marked cells (Robertson, 2003).

    The procephalic region of the embryo is made up of 13 mitotic domains (individual mitotic domains are abbreviated as deltaN). Seven procephalic mitotic domains (delta2, delta3, delta8, delta10, delta15, delta18 and delta20 have been described. All of these mitotic domains produce nonoverlapping sets of distinctly fated cells. These observations led to the conclusion that mitotic domains represent fate domains. Only one of these mitotic domains, delta20, gives rise to brain tissue, the optic lobe. Mitotic domains 1, 5, 9, and B form the embryonic brain. It was of interest to determine the morphogenetic movements of brain-forming cells. How are these cells internalized? Do they form discrete brain regions? Do they differentiate into neurons and glia? What other cell types are generated by these mitotic domains? To map the fates of cells within selected mitotic domains, photoactivated gene expression was used to initially mark cells and their development was monitored either by three-dimensional, time-lapse microscopy, or postfixation immunohistochemical staining (Robertson, 2003).

    The most difficult aspect of fate mapping the head region of the Drosophila embryo is its complex morphogenesis. The majority of mitotic domains within the Drosophila procephalic blastoderm was fate mapped by using the photoactivated gene expression system; it was determined that the embryonic brain develops from five mitotic domains: the posterior-dorsal part of delta1 and delta5, delta9, deltaB, and delta20. The final position of the mitotic domain progeny within the brain does not reflect their relative blastoderm positions. Thus, the mitotic domains follow specific morphogenetic trajectories. Several different mechanisms are employed to internalize brain progenitors: the posterior-dorsal part of delta1 and deltaB invaginate en mass; delta5 and delta20 likely invaginate together, and delta9 uses oriented mitosis and possibly delamination. Together, these mitotic domains constitute nonoverlapping regions of the brain. This fate map will provide an avenue for performing region-specific experiments. The discrete behavior of the brain-forming mitotic domains raises several interesting questions about the ancestral origin of the brain. One such question is, did the various brain compartments evolve from a common group of cells and later specialize or did the compartments evolve independently and later coalesce to form the brain (Robertson, 2003)?

    How does the mitotic domain-based fate map compare to previous studies? [Expression of the neurogenic gene; lethal of scute (l'sc) has been reported to correlate with the location of mitotic domains, delta5, delta9, and the posterior-dorsal part of delta1. A region of the blastoderm has been described that invaginates en mass and gives rise to neurons and glia; this corresponds to the posterior-dorsal part of delta1 and deltaB. It has been suggested that the mushroom body neuroblasts arise from deltaB. However, l'sc is only transiently expressed in deltaB, and photoactivation-based fate mapping clearly shows that deltaB produces glia, not neurons. delta1 produces neurons that populate the region of the brain that forms the mushroom body. Finally, the anatomical classification of the deutero-, proto-, and tritocerbrum does not correlate with separate mitotic domains; delta9 spans these three regions. The mitotic domain fate map correlates well with previous studies and provides a new level of precision and experimental options (Robertson, 2003).

    There is a tremendous amount of cell and tissue movement in the head region of the developing embryo. These movements are very difficult to describe without a context. Mitotic domains provide such a context by dividing the embryo into discrete regions with distinct boundaries. The following is a synopsis of the morphogenesis of mitotic domains 1, 5, 9, and B. Mitotic domain 1 first appears as a two-lobed structure with the posterior-dorsal lobe invaginating to form the anterior-medial part of the protocerebrum. The anterior-ventral portion of delta1 remains on the embryos surface forming the epithelial surface of the clypeolabrum. Thus, there is a margin within delta1 that forms a furrow boundary between the anterior-ventral and posterior-dorsal regions. The opposite side of the furrow is derived from delta9. The zippering up of this furrow joins delta1 to delta9; this causes the internalization of the posterior-dorsal portion of delta1 and deltaB. This, coupled to the posterior elongation of delta3 and delta18, draws delta9 dorsally and pulls delta5 and delta20 ventrally and anteriorly, creating a vortex centered on delta9. The initial division of delta9 is perpendicular to the embryo surface. Later delta9 divisions may also be perpendicular to the surface or cells may delaminate to generate a population of migratory cells. As delta5 and delta20 move ventrally along the cephalic furrow, a large portion of each mitotic domain forms a placode that is involuted to form the antennal and visual systems, respectively (Robertson, 2003).

    The antennal sensory and visual systems are derived from adjacent mitotic domains. There are three main sensory organs at the anterior tip of the embryonic brain: the antennal sensory/dorsal organ, Bolwig's organ, and the maxillary sense/terminal organ, each of which generates an axonal tract that terminate in the brain. Delta20 gives rise to Bolwig's organ and the maxillary sense/terminal organ. The morphology and position of the anterior delta5-derived cells indicates that they are the antennal sensory organ. Moreover, the axonal tract generated by delta5-derived cells follows the path of the antennal nerve. There are many parallels between mitotic domains 5 and 20: they follow similar morphogenetic pathways; they produce brain neurons, distally located sense organs, and specific epithelial contact sites. The middle group of cells are likely nonneuronal guidance cells associated with the antennal sensory system. It has yet to be determined whether the antennal system epithelia ultimately form imaginal disc tissue, as is the case for the visual system. The way in which these sensory systems develop ensures that the correct axonal connections are maintained from the very beginning of the structure's development. These similarities suggest a common ancestral sense organ (Robertson, 2003).

    The development of the antennal sensory system and antennal lobe from delta5 largely correlates with the engrailed expression patterns of the antennal stripe, antennal spot, and head spot at all stages of development. The engrailed head and antennal spots correspond, respectively, to the location of the antennal lobe and middle group from delta5. The antennal stripe correlates with migration patterns of delta5 throughout development, for example, at stage 9 the antennal stripe is ventral-lateral, as are delta5-derived cells. Time-lapse analysis of photoactivated cells provides evidence for the dynamic connection between the antennal sense organ and the antennal lobe, as well as associated cells: this connection could not be inferred from fixed preparations (Robertson, 2003).

    Neurons and glia in the embryonic brain do not arise from the same stem cells, but rather glial cells are found to be almost exclusively associated with deltaB. This surprising result is in contrast to findings in the ventral nerve cord. However, this neuron/glia specification is more reminiscent of vertebrate brain development. The data taken together with recent fate maps in the chicken and data which show remarkable similarities between the olfactory systems of Drosophila and mice, suggest that the development of the Drosophila embryonic brain may be more similar to the embryonic vertebrate brain than previously thought (Robertson, 2003 and references therein).

    DeltaB contributes subperineural glial cells to the embryonic brain, not neuropil glia. Clusters of GFP-positive/Repo-negative cells were observed. The ultimate fate of these marked cells was not determined. They may represent as yet, undifferentiated cells; Repo labels postmitotic cells. The origin of all brain glia was not determined. All of deltaB was not photoactivated, and procephalic mitotic domains delta23 and delta24 have yet to be mapped. A cluster of neuropil glia progenitors found at the deutero-tritocerebral boundary has been mapped to a ventral-lateral region by Repo expression at stage 11. It is not clear how these cells arrive at this position. Further photoactivation studies are needed to completely map the origin of brain glia (Robertson, 2003).

    Embryogenesis is dependent on the establishment and maintenance of morphology, which requires balancing cell proliferation and death. Embryos of several species have been shown to have a remarkable ability to repair patterning defects as a consequence of cell ablation, increased cell density, or alterations of the anterior-posterior fate map. However, all of these studies affect large areas of the embryo. The ability of the embryo to repair defects induced during the development of specific regions of the brain was tested. The most commonly used method for function mapping has been cell ablation. The correlation between ablation mapping and photoactivated gene expression fate mapping was examined, and whether ablated regions were repaired by repopulating cell divisions was determined. Cell ablation of brain progenitors leads to a reduction in brain size, but the precise location of the defect is obscured by the tissue deforming to accommodate the missing volume. It is clear that the deficit is not repaired at the cellular level. These results agree with studies of mispatterned embryos where compressed regions of the fate map are not repaired by new cell growth. The functional consequences of localized ablation will require further investigation (Robertson, 2003).

    Ectopic cells are efficiently removed. It is interesting that many ectopic cells are found outside of the brain, something never observed in control embryos. Time-lapse analysis shows that these extra cells fail to be incorporated into the brain, indicating that the selection, or counting process occurs during cell migration, before incorporation into the final brain structure. This selection process is not dependent on differentiation, since differentiation at the level of ELAV expression is often delayed. This observation is contrary to a common belief that cell death is a finishing step and occurs at the end of a developmental process (Robertson, 2003).

    Another common notion is that inappropriately differentiated cells are removed by apoptosis. To test this point, glial cell differentiation was induced in neurogenic delta1. The embryo appears to accommodate this fate switch and incorporate these cells into the brain, in spite of their being in excess. Perhaps the excess glia are not removed because they are normally associated with neurons. Why aren't glia counted when neuronal precursors are? It will be worthwhile to see the effect of switching neurons to a more unusual cell type, such as muscle (Robertson, 2003).

    Embryos need to continuously monitor cell density and eliminate excess or malfunctioning cells. This is the purpose of pattern repair. By further studying pattern repair in different tissues, more about the specific limits of pattern repair will be learned as well as the types of defects that lead to malformation and disease (Robertson, 2003).


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