Development of neural lineages derived from the sine oculis positive eye field of Drosophila

Arthropod Structure & Development 32 (2003) 303–317 www.elsevier.com/locate/asd

Development of neural lineages derived from the sine oculis positive eye field of Drosophila Ting Chang, Amelia Younossi-Hartenstein, Volker Hartenstein* Department of Molecular Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA Received 23 June 2003; accepted 10 September 2003

Abstract The Anlage of the Drosophila visual system, called eye field, comprises a domain in the dorso-medial neurectoderm of the embryonic head and is defined by the expression of the early eye gene sine oculis (so). Beside the eye and optic lobe, the eye field gives rise to several neuroblasts that contribute their lineages to the central brain. Since so expression is only very short lived, the later development of these neuroblasts has so far been elusive. Using the P-element replacement technique [Genetics, 151 (1999) 1093] we generated a so-Gal4 line driving the reporter gene LacZ that perdures in the eye field derived cells throughout embryogenesis and into the larval period. This allowed us to reconstruct the morphogenetic movements of the eye field derived lineages, as well as the projection pattern of their neurons. The eye field produces a dorsal (Pc1/2) and a ventral (Pp3) group of three to four neuroblasts each. In addition, the target neurons of the larval eye, the optic lobe pioneers (OLPs) are derived from the eye field. The embryonically born (primary) neurons of the Pp3 lineages spread out at the inner surface of the optic lobe. Together with the OLPs, their axons project to the dorsal neuropile of the protocerebrum. Pp3 neuroblasts reassume expression of so-Gal4 in the larval period and produce secondary neurons whose axonal projection coincides with the pattern formed by the primary Pp3 neurons. Several other small clusters of neurons that originate from outside the eye field, but have axonal connections to the dorsal protocerebrum, also express so and are labeled by so-Gal4 driven LacZ. We discuss the dynamic pattern of the sopositive lineages as a tool to reconstruct the morphogenesis of the larval brain. q 2003 Elsevier Ltd. All rights reserved. Keywords: Drosophila; Brain; Visual system; sine oculis; Neuroblasts

1. Introduction The Drosophila brain is formed by a set of stem cell-like neuroblasts each of which gives rise to lineages of neurons and glial cells. Approximately 85 neuroblasts produce the central brain, which is composed of the three neuromeres tritocerebrum, deuterocerebrum and medial protocerebrum (Younossi-Hartenstein et al., 1996). The lateral protocerebrum, or optic lobe, which represents the center processing input from the compound eyes, develops in a way that differs considerably from the stem cell-like proliferation pattern of neuroblasts (Meinertzhagen and Hanson, 1993). Thus, the optic lobe arises as a neuroepithelial placode that invaginates during mid-embryonic stages to become attached to the lateral surface of the central brain. From early on, the optic lobe placode is subdivided structurally * Corresponding author. Tel.: þ 1-22336635; fax: þ 1-3102063987. E-mail address: [email protected] (V. Hartenstein). 1467-8039/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2003.09.003

and molecularly into an anterior and posterior lip. Following invagination, the anterior lip attaches itself to the brain surface and is henceforth called inner optic Anlage (IOA), and the posterior lip settles at the outside, becoming the outer optic Anlage (OOA). During larval development, both optic Anlagen proliferate and give rise to the lobula, lobula plate, medulla, and lamina. Several regulatory genes have been identified that are expressed in the early embryo in parts of the neurectoderm and that are essential for the development of the neural structures derived from these parts. The homeobox gene sine oculis (so) is expressed in a contiguous domain in the dorso-posterior head of the blastoderm stage embryo (Cheyette et al., 1994). From the domain characterized by so expression, called the eye field (Chang et al., 2001), develops the optic lobe placode, as well as a number of neuroblasts of the central brain and part of the head epidermis. The larval eye (Bolwig’s organ) forms from the optic lobe placode and is therefore also a derivative of the

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eye field (Green et al., 1993). It has been proposed that the Anlage of eye antenna disc that will give rise to the adult compound eye is also part of the eye field (Cheyette et al., 1994); however, this notion has not been clearly demonstrated, mainly based on the unfortunate fact that early markers for the eye antenna disc have not yet been discovered. Expression of so gradually disappears as morphogenesis of the eye field takes place. This down regulation of so follows a temporal gradient, with cells located more anteriorly and dorsally losing expression of this gene before cells more posteriorly and laterally (Cheyette et al., 1994). In mid-stage embryos, only the primordium of the larval eye maintains so. As a result of this early down-regulation of so, it has so far not been possible to analyze the later fate of cells derived from the eye field, in particular the neuroblasts that delaminate from the eye field at an early stage and become part of the central brain. In this paper we have generated a Gal4 line driven by the so promoter. Reporter genes such as lacZ or GFP driven by so-Gal4 persist much longer than so itself, and we were therefore able to follow the fate of eye field derived neural lineages into the larval stage. One of the questions we wanted to address concerned the morphogenetic movements of neural lineages that take place during embryonic and larval development. When they first appear neuroblasts are laid out in a two-dimensional array, roughly in the shape of an oval, underneath the surface of the embryonic head (Younossi-Hartenstein et al., 1996; Urbach et al., 2003). During neuroblast proliferation, neurons are given off towards the interior of the embryo, resulting in a thickening of the neural primordium. The brain of the late embryo is a three-dimensional, spherical structure. Neuronal cell bodies form the outer layer, or cortex, of the brain hemisphere; neuronal processes (neurites) project radially towards the center where they form the neuropile. It is not known where neuronal pericarya born at a defined position within the twodimensional neural primordium of the early embryo end up in the late embryonic brain, nor whether during subsequent stages of development neuronal cell bodies further change in position. It is also unknown how the position of a neuronal cell body relates to the projection pattern of its neurites within the neuropile. Is the projection highly topographic, with the position of the cell body determining where dendrites and/or axons project in the neuropile, or does cell body position have nothing to do with axonal/dendritic projection? Markers expressed continuously over time in neuroblasts, neurons and their projections allow one to address these questions, and we have made use in this study of so-Gal4 driven reporter genes to characterize the so-positive lineages derived from the eye field. Another aspect that makes the eye field derived lineages a significant object of curiosity is their relationship, or lack thereof, to the visual system. The central target neurons of the photoreceptors of the compound eye form the optic

ganglia, lamina, medulla and lobula complex (reviewed in Strausfeld, 1976; Fischbach and Dittrich, 1989; Bausenwein et al., 1992), all of which are derived from the optic lobe placode of the embryo. The output of the optic lobe is mediated mainly by neurons of the lobula complex whose axons form the massive optic tracts of the adult brain. The optic tracts terminate on interneurons of the so-called optic foci, the lateral horn, and other compartments which are all part of the central brain (Strausfeld, 1976). The origin and development of these brain parts is unknown. We asked whether neuroblasts that give rise to neurons that contribute their neurites to the optic foci or lateral horn could be derived from the eye field. A related matter is the question of the targets of the larval eye. These neurons, called the optic lobe pioneers (OLPs) were studied in several previous papers (Tix et al., 1989; Campos et al., 1995; HelfrichFoerster, 1997; Malpel et al., 2002). They have recently received a lot of attention because they express the Per protein, and seem to represent a key element in the central circuit that mediates circadian activity rhythm in both adult and larval brain. The origin of the OLPs in the early embryo has not been established, and the question arises whether these cells, like the larval eye itself, are derivatives of the eye field. Our so-Gal4 driven reporter gene expression allowed us to address these questions, and thereby contributes to our understanding of essential apsects of visual system development in Drosophila.

2. Materials and methods 2.1. Fly stocks Flies were cultured on standard yeast-apple juice-agar medium. Oregon R flies were used as the wild-type stock. An so-GAL4 stock was generated by P-element replacement (Sepp and Auld, 1999) using P[Gal4] to hop into the LacZ position of so-lacZ (Cheyette et al., 1994). 2.2. Immunohistochemistry Embryos were dechorionated and fixed in 4% formaldehyde containing PT (1% PBS, 0.3% Triton X-100) with heptane. Embryos were then devitellinized in methanol and stored in ethanol before labeling with antibody, following the standard procedure (Ashburner, 1989). Expression of bgalactosidase in soGAL4; UAS-lacZ was detected with a polyclonal anti-b-galactosidase antibody (Upstate Biotechnology) at a dilution of 1:500. Monoclonal antibody antiFasII (Grenningloh et al., 1991) was used at a 1:1000 dilution to detect FasII. Whole mount stained embryos where dehydrated in graded ethanol and acetone, left overnight in a 1:1 mixture of acetone and Epon, and then 4 h in Epon. Embryos were mounted as wholemounts and analyzed with a Zeiss Axiophot microscope. Selected embryos were placed in moulds, oriented, and placed for

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16 h at 608 to enable Epon polymerization. An LKB ultratome was used to obtain 2 mm sections. Confocal images were taken on a Biorad MRC1024ES microscope using Laser sharp version 3.2 software. Confocal images were analyzed using the ImageJ software. 2.3. In situ hybridization Plasmid pBS-pF3k (Cheyette et al., 1994) linearized with Bam HI was used as template to synthesize the so RNA probe. pBSK-svp (Mlodzik et al., 1990; kindly provided by Dr J. Lengyel) was linearized with BamHI and used as template to generate the svp RNA probe. Embryos were dechorionated and fixed in phosphate-buffered saline (PBS) containing 5% formaldehyde and 50 mM EGTA and stored in ethanol. They were then treated with xylene and fixed for a second time in PBS containing 0.1% Tween-20 and 5% formaldehyde. The embryos were then hybridized with probes synthesized using digoxigenin-labeled UTP (Boehringer) according to standard protocol (Ashburner, 1989). Anti-digoxigenin antibody (Boehringer) was used according to the manufacturer’s instructions to detect hybridized probe, after which the embryos were dehydrated in ethanol and embedded in Epon.

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fragments that are taken up by macrophages (Fig. 2B, arrow). During stage 12 the optic placode invaginates and forms the inner and outer Anlagen of the optic lobe that become attached to the central brain (Fig. 2K and L). As the optic lobe invaginates, a small group of cells that give rise to the Bolwig’s organ stay in the surface ectoderm and are pulled away from the optic lobe during the course of later development (Fig. 3H and J). The so-Gal4 driven lacZ reporter is expressed in two groups of neuroblasts (Fig. 1E – G) that delaminate from the eye field. One group, located dorsally of the optic placode proper, arises during stages 9 and 10. It comprises three to five contiguous neuroblasts which we identify as members of the posterior central protocerebral groups, Pc1 and Pc2. As described by Younossi-Hartenstein et al. (2003), the Pc1 neuroblast is marked by its expression of the svp gene. This neuroblast is located posteriorly in the dorsal cluster of sopositive neuroblasts (Fig. 1F). The remainder of the dorsal so-positive cluster then represents the svp-negative Pc2 group of neuroblasts. The second group of so-positive neuroblasts appears slightly later than the first one during stage 11. It encompasses three neuroblasts which delaminate from the anterior part of the optic lobe placode which gives rise to the inner optic Anlage. These neuroblasts express the marker svp and thereby can be identified as the posterior protocerebral group, Pp3 (Fig. 1G).

3. Results 3.1. The so-Gal4 construct is expressed in the Drosophila embryonic eye field and its derivatives The expression of the early eye gene sine oculis (so; Cheyette et al., 1994) defines the eye field, an unpaired Anlage in the dorso-medial head ectoderm of the early embryo (Cheyette et al., 1994; Chang et al., 2001). Starting around gastrulation (stage7), the eye field undergoes dramatic morphogenetic changes during which it gives rise to several different tissues, including the larval eye/ Bolwig’s organ (BO), the inner (IOA) and outer optic lobe (OOA), the dorsal head epidermis, and postero-medial protocerebrum (see Hartenstein and Reh, 2002, for recent review). Expression of so is only short-lived and disappears in the derivatives of the eye field before they differentiate, with the exception of the larval eye which expresses so until mid-stages of development. By inserting Gal4 into the so promoter via P-element replacement (Sepp and Auld, 1999) we were able to drive the lacZ reporter in the so pattern. The so-GAL4 driver reproduces the expression pattern of the endogenous so mRNA (Fig. 1A –D). Due to the inherent nature of the GAL4-UAS system, the reporter was slightly delayed, starting expression during stage 8 embryogenesis in a small dorso-median field of cells (Fig. 1C). During stages 9 – 11 the eye field splits into the bilaterally symmetric optic placodes. Cells in the dorsal midline flatten and become head epidermis. Many of these cells undergo apoptotic cell death, which can be seen by lacZ positive cell

3.2. Embryonic morphogenesis of neural lineages descended from the eye field After delaminating the Pc1/2 and Pp3 neuroblasts proliferate and form clusters of progeny that occupy positions dorso-medially and laterally in the primordium of the protocerebrum (Fig. 2). With respect to the optic lobe, the Pc1/2 cluster is located medially, the Pp3 cluster ventroanteriorly. As part of the global posterior rotation of the brain (Boyan et al., 1995; Younossi-Hartenstein et al., 1996) the clusters move posteriorly and ventrally. At the same time, the clusters increase in size due to continued neuroblast proliferation. Both clusters adopt a wedge shape, with the tips of the wedges pointing anteriorly (Pc1/2; Figs. 2G and O, and 3H and N) and antero-medially (Pp3; Figs. 2G and O, and 3H and M). At their tips, which contain the oldest neurons, both clusters come into close contact. It is here that axon outgrowth starts during stage 13 (see below). At the base, the two clusters are separated by a domain of so-negative neurons. We assume that these are the neurons produced by the Pp2 and Pp5 neuroblasts, which are located in between Pc1/3 and Pp3, and which do not express so-Gal4 (arrow in Fig. 3H and N). In the late embryo, some neurons of the eye field derived lineages differentiate. Located at the tips of their respective wedge-shaped lineages, approximately 10 neurons project axons dorsomedially. Axons from the Pp3 neurons form a loose bundle, joining axons of the FasII-positive P4l cluster which pioneer the posterior transverse tract (Fig. 3C, G, J,

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Fig. 1. Expression of so-Gal4 in the eye field. A, B: Dorsal view of stage 6 (A) and stage 9 (B) embryo labeled with so cDNA probe. The eye field initially forms an unpaired dorsomedial domain (arrow in A) that later splits into a left and right optic lobe placode (arrows in B). Expression of so rapidly decreases dorsomedially. C, D: Dorsal view of stage 7 (C) and stage 10 (D) embryos expressing the lacZ reporter gene driven by so-Gal4 in the dorsomedial eye field at stage 7. As the eye field splits (D), lacZ expression remains high dorso-medially. Three domains can be distinguished within the eye field: the primordium of the dorsal head epidermis (he), the dorsal central protocerebral neurectoderm (pcnd), and the ventral posterior protocerebral neurectoderm (ppnv) that overlaps with the optic lobe placode (opl). E: Schematic lateral view of brain neuroblast map of stage 11 embryo (after Younossi-Hartenstein et al., 1996). The eye field (magenta) gives rise to two clusters of neuroblasts. Pc1/2 neuroblasts delaminate from the dorsal central protocerebral neurectoderm, Pp3 neuroblasts from the ventral posterior protocerebral neuerectoderm. F, G: Two focal planes of stage 11 embryo (lateral view) double labeled with the neuroblast marker svp (cDNA in situ probe, blue) and so-Gal4;UAS-lacZ (brown). The svp marker is expressed in Pc3 and Pc1 (F, deep focal plane), as well as in the Pp3 group (G, superficial focal plane). Pc1 and Pp3 are included within the so-Gal4 positive eye field. Additional so-Gal4-positive, svp-negative neuroblasts are located in between the Pc1 and Pc3. They represent the Pc2 group (G). Other abbreviations: Da, Dc, Dp anterior, central and posterior deuterocerebral domain, respectively; Pa, Pc, Pp anterior, central, posterior protocerebral domain, respectively; T tritocerebral domain.

Fig. 2. Shape and location of so-Gal4 positive, eye field derived structures during stages 11 (A –H) and 12 (I –P) of embryonic development. Each stage is represented by six cross-sections of embryonic heads (two rows with three panels each on the left of figure) and two focal planes of a wholemounted embryo (right column; upper panel shows deep focal plane, with images of neuroblasts and neurons; lower panel represents superficial focal plane, showing surface of embryo). In cross-sections only the right half of the head is shown (embryonic midline to the left of each panel; dorsal up). Anti-beta-Gal (brown) visualizes expression of so-Gal4;UAS-lacZ. Sections were counterstained with methylene blue/toluidine blue/borax. Position of sections along the antero-posterior axis is indicated by lettered bars in panels G and O, respectively. By stage 11 so-Gal4 positive neuroblasts have delaminated from the eye field and form two separate clusters underneath the surface ectoderm. The Pc1/2 group (B, C, G) lies underneath the dorsal central protocerebral neurectoderm (pcnd; C, D, H). The Pp3 group (B, C, G) is attached to the inner surface of the ventral posterior protocerebral neurectoderm (ppnv; D, H) that forms part of the optic lobe placode (opl; C, D, H). By stage 12 (I –P) eye field derived neuroblasts have undergone three to four rounds of mitosis each. Their progeny form coherent clusters. The Pp3 cluster (I–K, O) is located medial and anterior of the anterior lip of the invaginating optic lobe (ola; L, P). The Pc1/2 cluster borders Pp3 medio-dorsally (K –M, O). Cells of the so-Gal4-positive protocerebral neurectoderm that remain at the surface differentiate into dorsal and posterior head epidermis (hed and hep, respectively; M, N, O, P). Arrow points at so-Gal4 positive former cells that have undergone apoptotic cell death and are internalized to later be taken up by macrophages. Other abbreviations: es esophagus.

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Fig. 3. Pattern of eye field derived cells at stage 14. Panels A –F show cross-sections as specified in legend of Fig. 2. G–I represent focal planes of wholemount (dorsal view; anterior to the left, lateral to the top; G superficial plane near dorsal head epidermis; H 20 mm beneath dorsal epidermis; I 40 mm beneath dorsal epidermis). J–O: Confocal sections of head of embryo (dorsal view; anterior to the left; lateral up, midline at bottom of each panel) double labeled with antiFasII (red; labels brain pioneer tracts and larval visual system) and so-Gal4; UAS-GFP (green). Confocal sections are ordered from dorsal (J) to ventral (O). The optic lobe (ola anterior lip; olp posterior lip) has invaginated and resides as a vesicle with an inner lumen at the ventro-lateral surface of the brain (br; B–E; I; M–O). The larval eye (Bo) has separated from the optic lobe. It moves anteriorly, trailing the optic nerve (Bn) behind it (A, H, J). The Pp3 neuroblast derived lineages are located antero-dorsally of the optic lobe (B, C, H, L, M). Some neurons, located at the central tip of the Pp3 cluster, extend axons (ax in C, G, L). These axons fasciculate with the pioneer tract formed by the fasII-positive P4 l and P3l clusters (J, K) and project towards the brain commissure (sec in C, J). The Pc1/2 derived group of neurons has moved posteriorly and forms an elongated cluster dorso-medially of the optic lobe (E, F, H, N). A group of surface glial

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K, and L; Nassif et al., 1998, 2003). This tract crosses the midline as the dorsal fascicle of the supraesophageal commissure. Several so-positive axons extend across the midline as part of this commissure. Pc1/2 neurons form shorter axon stumps that grow anteriorly and intermingle with the Pp3 fibers. The eye field derived lineages give rise to several glial cells. Notably, a thin sheath of surface glia extends out of the postero-lateral edge of the Pc1/2 cluster and spreads out around the posterior surface of the protocerebrum during stages 14 –16 (Fig. 3F, G, and O). These glial cells represent the dorsal protocerebral cluster of surface glia (DPSG) identified in previous work in which the anti-Repo antibody was utilized to map the origin of brain glial cells (Hartenstein et al., 1998). The DPSG group contains 4 –6 cells when it first becomes positive during stage 12, and increases to about twice that number in late (stage 16) embryos. The DPSG group forms the glia sheath surrounding the posterior half of the protocerebrum; a similarly sized cluster, VPSG, produces the surface glia covering the anterior protocerebrum (Hartenstein et al., 1998). Although it is difficult to count individual cells in the so-positive clusters (in which labeling is cytoplasmic), we estimate that the number of so-positive surface glial cells is in the order of 5 –10. This suggests that the Pc1/2 lineages give rise to the entire DPSG group of glial cells. 3.3. Pattern of so-positive neurons in the late embryonic brain So-Gal4 driven reporter gene expression in the Pc1/2 and Pp3 lineages gradually decreases towards later stages, but remains detectable even into larval stages. Within the Pp3 lineage, a few cells remain strongly labeled. One pair is located at the tip of the Pp3 cluster, suggesting its cells are among the first-born Pp3 neurons (Fig. 4A4). These cells (abbreviated as Pp3c cells in the following) form massive dendritic and axonal arborizations in the dorsal protocerebrum during the larval period (see below). In addition, groups of smaller cells, containing 4 –6 cells each, can be seen at the base of the Pp3 cluster. These cells (called Pp3v; Fig. 4A5) do not form neurites until the first instar larva; by this stage, their axons fasciculate with the Pp3c axons (Fig. 4B3 – 5). Finally, a tight group of neurons closely attached to the optic lobe remain strongly so-positive. Based on their location and projection pattern, these cells represent the optic lobe pioneer neurons (OLP), the central targets of the larval optic nerve (Tix et al., 1989; Fig. 4A6). We assume that the OLPs form part of the Pp3 lineages, although it is also possible that these cells

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differentiate directly from the optic lobe placode. Axons of the OLPs follow the dorsally directed trajectory of the Pp3v and Pp3c neurites and along with these form abundant arborizations in the dorsal protocerebrum (Helfrich-Foerster, 1997; see below). Beside the eye field derived lineages, another subset of neurons expresses so in the late embryo. De novo so expression in these cells [which was not reported in our previous analysis (Cheyette et al., 1994) of this gene] is also reproduced by the so-Gal4 driver line. We distinguish six small clusters: the anterior and posterior dorso-lateral protocerebral so-positive cluster (pdla, pdlp); the anterior and posterior dorso-medial cluster (pdma, pdmp), the lateral and medial baso-anterior clusters (bal, bam; Fig. 4A1). The dorsolateral clusters form neurits that, like the Pp3 neurons located further basally, join the posterior transverse tract. The remaining clusters establish separate projection patterns that will be described below. 3.4. Loss of so function results in the absence of OLPs and other eye field derived lineages Previous studies had shown that in embryos homozygous for a null allele of so the larval eye and outer optic Anlage does not form (Cheyette et al., 1994). Labeling with antiFasII demonstrates that the so mutation also results in at least partial loss of the neural lineages derived from the eye field (Fig. 5). Most conspicuously, the OLPs and their axons, which form the LOT tract, are absent. Brain hemispheres of so homozygous embryos are flattened in the dorso-ventral axis, indicating that beside the OLPs, a substantial number of neurons located in the basal brain hemisphere is lacking. We assume that the missing neurons are those of the Pp3 and Pc1/2 lineages, which are derivatives of the so-expressing eye field. 3.5. Pattern of so-positive neurons in the larval brain The fact that lacZ reporter gene expression persisted until the second instar allowed us to establish where the Pc1/2 and Pp3 lineages end up in the larval brain, and to analyze in detail their neuritic arborization pattern. As an anatomical framework we use the system of neuropile compartments and long axon tracts defined in previous papers (Nassif et al., 2003; Younossi-Hartenstein et al., 2003; Dumstrei et al., 2003). The Pp3 cluster is located in between the inner Anlage (IOA) of the optic lobe and the baso-posterior lateral compartment. Growth of the optic lobe has a major effect on the location of the neurons derived from the Pp3 lineage, i.e., Pp3c, Pp3v, and OLP. The cell bodies of Pp3c are

cells grows out of the Pc1/2 cluster (DPSG in F, G, M). Eye field derived head epidermal cells form a thin epithelium covering the brain dorsally (hed in A– D). Arrows in panels H and N point at domain of so-negative neurons separating Pc1/2 and Pp3. Other abbreviations: es, esophagus; P2l, P4m fasII positive positive brain neuropile founder clusters; sbc, subesophageal commissure.

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pushed dorsally, those of Pp3v ventrally, and the OLPs remain in the center of the optic lobe, wedged in between the outer and inner optic Anlage (Figs. 4A4, B4 and C –E3, and 6A and C). As described in Section 3.4 for late embryos, the axons of Pp3c project dorso-medially and arborize profusely in the centro-posterior lateral (CPL), dorsoposterior (DP), and dorso-anterior (DA) neuropile compartments (Figs. 4C –E3, and 6B and F). Pp3v cell bodies are pushed in the opposite direction than Pp3c, towards ventrally (Fig. 4A5, B5, C6, and E4, and 6A and C). During the third instar, the small group of primary Pp3v neurons (those born during embryogenesis) is joined by a large cluster of secondary neurons (Pp3sec; Fig. 6D3 and E3). We suggest that the same Pp3 neuroblasts that generated the primary Pp3 lineages in the embryo are responsible for the secondary neurons. Neurites of the secondary Pp3 lineages, along with the primary Pp3v fibers, travel straight dorsally, following the inner surface of the inner optic Anlage. At the dorsal edge of the inner optic Anlage, the axons join the Pp3c axons. Secondary lineages with this kind of position (ventral of optic lobe) and neurite projection (dorsad along IOA) were defined as the basolateral ventral group (BLV; Dumstrei et al., 2003). We therefore conclude that the secondary Pp3 lineages are members of the BLV group. The cell bodies of the OLPs remain in the vicinity of the outer optic Anlage where they were located in the embryo (Figs. 4A – C6 and D4, and 6A, C, and E). As shown by previous authors (Tix et al., 1989; Campos et al., 1995; Helfrich-Foerster, 1997), these neurons become part of the medulla primordium that forms in between the outer and inner optic Anlage during late larval development. OLP neurites, along with the larval optic nerve, project medially, passing the posterior edge of the inner optic Anlage. As the

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Fig. 5. Loss of so function results in the absence of eye field derived lineages. A: Lateral view of late embryonic wild-type brain labeled with anti-FasII (lateral view, dorsal up, anterior to the right). The optic lobe (ol), optic lobe pioneer cluster (P5m ¼ olp) and other neuropile founder clusters (P4l, P3l) are labeled. Arrow points at axon tract formed by P5m. B: Corresponding view of so mutant brain. Note absence of optic lobe and P5m. A large ventral domain of the brain cortex (arrowhead) is missing. Other abbreviations: crc, cervical connective.

Fig. 4. Pattern of so-Gal4 positive neurons from late embryonic to late larval development. Panels show confocal sections of brain hemispheres in which soGal4; UAS-GFP positive structures are shown in green. Panels of one column belong to one brain. Column A shows brain of embryonic stage 17, B first larval instar, C second larval instar; D early third instar; E late third instar. Brains are double labeled with anti-FasII (red; labels long axon tracts; columns A–D) or anti-Syntaxin (red; labels neuropile compartments; column E). Brains in columns A–C are presented in a dorsal view (anterior up, lateral to the right) and are ordered from dorsal (1) to ventral (7). Intervals between focal planes are approximately 8 mm. Third instar brains in D and E are in posterior view (dorsal up, lateral to the right) and are ordered from posterior (1) to anterior (7). Intervals between sections are 12 mm. Brains of different stages are presented at different magnifications. Scalebars in the first row correspond to 20 mm. The dorsal group of so-positive neurons (pdma/p, pdla/p) is placed around the calyx of the mushroom body (A1-E1). Pdla/p form profuse dendritic arborizations (dorso-posterior optic arborization; DPOA) in the dorsal neuropile compartments (CPL, DP, DA in A2-E2, A3-C3). Although these arborizations span several compartments, there are sudden changes in branch density or gaps at compartment boundaries (arrowheads in C2 and E3). One or two large, eye field derived neurons (Pp3c) flank the dorsal margin of the inner optic Anlage (IOA; A4-C4; D3E3). These neurons project dorso-medially through the CPL, DP and DA compartments, joining the arborizations of the (non-eye field derived) pdl neurons (D3). Pp3 derivatives located ventrally and posteriorly of the IOA (Pp3v; A5-B5, C6, E5) also join the Pp3c/pdl arborizations in the dorsal neuropile. One large secondary lineage, Pp3sec, expresses so-Gal4 from early third instar onward (D3, E3). The axon bundle formed by this lineage projects straight dorsally, passing the inner surface of the IOA, and then curves medially, to terminate in the center of the CPL compartment, right underneath the arborizations formed by the so-positive primary neurons (E3). The optic lobe pioneers (olp) are eye field derived neurons that represent the central target of the larval optic nerve (Bn). These neurons are nudged in the space between inner and outer Anlage and remain so-positive until early third instar (A6-C6, D4). Their axons project dorsally, forming the posterior optic tract (POT), and terminate in the CPL (A4-6; B5-6; C4-6). So-positive neurons not participating in the dorsal neuropile projection are the pdm and ba neurons. Pdm axons project straight ventrally in the dorsal posterior protocerebral tract (DPPT), passing the calyx on its medial surface (B16; C1-6; D1-4; E1-5). Axons of the pdma leave the DPPT and form the secondary corpora cardiaca nerve (nccII; D1, E2). Pdmp axons project straight down to the ventral nerve cord. Their form dense arborizations in the BPM compartment (C6-7; D3-5; E3-5). Prominent collaterals of pdm axons leave the DPPT and project dorso-laterally to the BPL and CPL (arrowhead in D3). The basal anterior so-positive clusters are located laterally (bal) and medially (bam) of the BA neuropile compartment (antennal lobe; B7, C7, D6-7; E6-7). Bal neurons are probably olfactory interneurons. They have dendritic arborizations in the BA (D67; E6-7) and axons that project via the antenno-cerebral tract. Bam neurons arborize in the tritocerebral neuropile (TR; D6-7, E6-7).

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Fig. 6. 3D digital model of so-positive neurons in first instar larval brain. Panels depict different views of one brain hemisphere in which each group of sopositive neurons described in the text is shown in its own color. Only strongly so-Gal4 positive, individualized neurons are included in the model; clusters of weakly stained neurons (such as the entire Pc1/2 derived group which loses so-Gal4 expression in the larva) are omitted. Landmark structures (mushroom body, optic lobe, long axon tracts) are shown in gray. A, B: posterior view (dorsal up, lateral right). C, D: lateral view (dorsal up, posterior right). E, F: dorsal view (anterior up, lateral right). In panels of the upper row neuropile compartment boundaries are omitted to allow clearer view of axons inside the neuropile. Domains of axonal and dendritic arborizations of so-positive neurons are depicted in uniform light brown color. In panels of lower row (B, D, F) neuropile boundaries are shown as semi-transparent gray surfaces. In these models domains of terminal arborizations of so-positive neurons appear in the same color as the belonging cell bodies and axons. For further details see text and legend to Fig. 4. Abbreviations: So-positive neurons (abbreviations given in upper row of models): bal/m, basal anterior lateral and medial cluster; olp, optic lobe pioneers; pdla/p, anterior and posterior dorso-lateral cluster; pdma/p, anterior and posterior dorsomedial cluster; Pp3c/v, central and ventral Pp3 derived neurons eye field derived primary neurons. So-positive axon tracts (abbreviations given as violet letters in lower row of models): DPC, dorso-posterior commissure; DPPT, dorso-posterior protocerebral tract; DPPTr, recurrent collateral of DPPT; NccII, secondary corpora cardiaca nerve; POT, posterior optic tract; PTT, posterior transverse tract. Landmark structures (abbreviations given as white and grey letters in upper row of models): cx, calyx; dl, dorsal lobe of mushroom body; LCT, lateral cervical tract; MCT, medial cervical tract; ml, medial lobe of mushroom body; PCT, posterior cervical tract; ped, peduncle. Neuropile compartments (abbreviations given as black letters in lower row of models): BA, basoanterior compartment; an antennal nerve; BC, baso-central compartment; BC, baso-central compartment; Bcv, baso-cervical compartment; BPL, basoposterior lateral compartment; BPM, baso-posterior medial compartment; CA, centro-anterior compartment; CPI, centro-posterior intermediate compartment; CPL, centro-posterior lateral compartment; CPLv, ventral domain of centro-posterior lateral compartment; CPM, centro-posterior medial compartment; CX, calyx; DA, dorso-anterior compartment; DP, dorso-posterior compartment.

optic Anlagen bend posteriorly to adopt the shape of a letter C, with the opening of the C pointing postero-dorsally, the OLP tract comes to lie in the central hole of the C-shaped IOA. After passing through the IOA, OLP neurites turn dorsally and extend towards the centro-posterior lateral (CPL) neuropile compartment where they form terminal arborizations intermingled with those ones of the Pp3v and Pp3c neurons (Figs. 4C3 – 6, and 6B and D). This projection corresponds to the dorsally directed axon bundle that can be

distinguished already in the late embryo (see above). Despite the fact that Pp3c/v axons and OLP axons establish terminal arborizations in the same neuropile compartments, the two bundles formed by these axons become increasingly separated from each other. OLP axons follow a more posterior course (Fig. 6B). They fasciculate with the FasII positive larval optic commissural tract (LOCT; Nassif et al., 2003). Pp3v and Ppc3c axons are located further anteriorly and follow a straight dorsally directed course.

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Besides terminating in the dorsal protocerebrum, OLP axons form short collaterals that contribute to a small neuropile compartment located in between the inner and outer optic Anlagen. This compartment represents the larval optic neuropile (LON on Fig. 4E4; Campos et al., 1995; Helfrich-Foerster, 1997). Axons of the larval eye (Bolwig’s organ) terminate in the larval optic neuropile, which later will become part of the accessory medulla neuropile of the adult brain. The LON is also the site of termination of the larval optic nerve; only the OLP axons continue dorsally and arborize in central and dorsal protocerebral compartments. The Pc1/2 cluster remains weakly labeled until mid second instar. No strongly expressing cells develop in this cluster, nor are there any secondary neurons that form adjacent to Pc1/2 during the third instar. Pc1/2 neurons are situated at the posterior apex of the protocerebrum, flanking the centro-posterior lateral compartment (Fig. 4A –C5). Immediately dorsal of the Pc1/2 cluster are the Kenyon cells of the mushroom body. Pc1/2 axons project anteriorly and penetrate into the CPL compartment, lateral of the peduncle of the mushroom body (arrow in Fig. 4B5). With respect of to cell body location and axon trajectory, Pc1/2 resembles the centro-posterior (CP) group of lineages (Dumstrei et al., 2003). All of the non-eye field derived, so-positive neurons delineated in the previous section for the late embryonic brain can be followed throughout larval life. With the exception of the lateral baso-anterior one (bal), no secondary neurons are added to these clusters. However, their axonal projection and arborization becomes more elaborate. The two dorso-lateral pairs of neurons (pdla, pdlp) are located laterally adjacent to the calyx of the mushroom body (Figs. 4A – E1, and 6A, C, and E) and project straight ventrally towards the CPL compartment, where they join the axons of Pp3 neurons and form arborizations in the CPL, DP and DA compartments (Fig. 6B). Secondary lineages with this characteristic location and axonal trajectory represent the dorso-posterior lateral group (DPL; Dumstrei et al., 2003). The dorso-medial so-positive neuronal clusters (pdma, pdmp) are located medially adjacent to the calyx of the mushroom body (Figs. 4A – E1, and 6A and E). Axons of these clusters are directed ventrally, following the dorsoposterior protocerebral axon tract (DPPT) that penetrates through the central posterior intermediate (CPI) into the baso-posterior medial (BPM) neuropile compartment (Figs. 4B – E3, and 6A and B). Neurons with these characteristics were defined as dorso-posterior medial (DPM) lineages in our reconstruction of the larval brain (Dumstrei et al., 2003). The pdma and pdmp axonal projections have different targets. The pdma axons turn medial and leave the brain at its medial surface, forming part of the secondary nerve to the corpora cardiaca (NccII; Figs. 4D1, E2, and 6A and B). This nerve carries neurosecretory axons terminating in the corpora cardiaca, a neurohemal gland surrounding the aorta

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of the larva (Siegmund and Korge, 2001). We conclude that the pdma neurons are part of the population of neurosecretory cells. Neurons of the pdmp cluster do not contribute to the NccII. Their axons branch widely and form arborizations in several compartments. Strong labeling (interpreted as high density of axon terminals) is seen in two foci in the basoposterior medial compartment (Figs. 4D3 –5, E3-5 and 6B; see Section 3.6). These arborizations increase in size and density throughout larval development. In the early first instar larva, only a minute terminal arbor, located in the dorsal BPM, is seen. During late larval stages, dpmp axons continue to grow into the ventral nerve cord where they have not been followed in detail. A prominent collateral tract branches off the dpmp bundle and extends dorsolaterally, reaching the BPL and CPL compartments (Fig. 4D3). Correspondingly, in both BPL and ventral CPL, a low intensity signal can be discerned that diffusely fills out most, if not all, of the volume occupied by these compartments (Fig. 4D3 –5, E5). An additional dpmp collateral with terminal arborization seems to be elaborated during late larval stages in the BA (antennal) compartment. 3.6. Pattern of so-positive axon arborization in relationship to neuropile compartmental boundaries Neuropile compartments of the larval brain are surrounded by glial sheaths that in principle may act as a barrier for axons to freely pass from one compartment to the other. Thus, for example, the bundles of axons of secondary lineages (cell body fiber tracts; Dumstrei et al., 2003) grow radially towards the neuropile where they either terminate, or turn to make a ‘detour’ that brings them to one of the openings in the neuropile glia sheath (glial portal). Axons enter through these portals and grow into the neuropile, mostly following glial sheaths in between compartments (Pereanu et al., in prep). In the following we will describe how the pattern of axonal arborization of so-positive neurons relates to compartment boundaries. The most conspicuous system of terminal neurites is formed in the dorsal protocerebral neuropile by the various subsets of Pp3 neurons (Pp3c, Pp3v, OLP) and the pdla/p neurons. Due to the high number and density of neurites we could not distinguish whether any one of these neuronal subsets differ in regard to their terminal arborization; we will therefore consider the entire arborization as one entity, for which we will use the term dorsal protocerebral optic arborization (DPOA; Fig. 4B2, C2, D2-3 and E2-3), given that the OLPs, the central targets of the larval visual system, contribute to this arborization. The DPOA spans part of three neuropile compartments, the DA, DP and CPL (Fig. 6D and F). Conspicuously, the main axons pass over the outer surface of the neuropile, reaching first the lateral aspect of the CPL, then passing dorso-antero-medially over the DP and DA compartments and then extending through the DPC commissure to the contralateral hemisphere

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(Fig. 6B, D, and F). Highly branched terminal fibers branch off the main axons and penetrate the neuropile. Given the external location of the main axons, the density of terminals is highest near the outer surface of the compartments and decreases towards the interior (Fig. 4E2). In the DA and DP terminal fibers branch throughout the entire volume occupied by these compartments, although, due to the external-to-internal gradient in axon density, fibers are very scarce in the deeper portions of DA and DP. In the CPL terminal fibers are restricted to a small superficial focus. The volumes occupied by neuropile compartments are very small in the late embryo and increase in size continuously in the larva; terminal arbors grow correspondingly, thereby maintaining their spatial relationships within the compartments. In the third instar, as ingrowth of axons of secondary lineages lead to a rapid increase in overall neuropile volume, DPOA terminal arbors do not keep up with this growth and become restricted to superficial slabs within the dorsal compartments. An interesting spatial relationship exists between these late larval terminal arbors and the incoming axon bundle formed by the secondary Pp3 neurons. Thus, rather than fasciculating with the superficially located primary Pp3 tract, they penetrate the CPL and dorsal compartments, thereby subdividing these compartments into a superficial and a deep stratum. The DPOA arborizations, formed by primary Pp3 and pdla/p neurons, are almost completely restricted to the superficial strata (Fig. 4E3). Although the DPOA spans several compartments, boundaries in between neighboring compartments are still visible either because terminal axons become scarce at these positions (e.g. boundary between CPL and DP in Fig. 4E3), or because the overall density and/or pattern of terminal axons differs in one compartment from that one of the adjacent compartment (e.g. DA versus DP in Fig. 4C2). This suggests that many terminal axons do indeed respect intercompartmental boundaries as barriers that they do not penetrate freely. The same holds true for the terminal arbors of the dpmp, bal and bam neurons, which are located in the BPM, BPL, BA and tritocerebral compartments.

4. Discussion 4.1. Dynamic pattern of neural lineages during embryonic brain morphogenesis Previous studies of insect brain development had concluded that neural lineages of the brain undergo a dramatic spatial reorganization between the time they first appear and the late embryonic stage at which the spherical brain takes shape (Boyan et al., 1995; Younossi-Hartenstein et al., 1996). The brain primordium rotates posteriorly around an imaginary transverse axis penetrating the center of the protocerebrum (Fig. 7A). As a result of this posterior rotation, neuroblasts starting out at a given position will end

up further posteriorly in the late embryonic brain. For example, the Pc3 neuroblasts giving rise to the mushroom body are located at a mid-dorsal position within the neuroblast map of a stage 11 embryo. Late in development, these neuroblasts form the posterior apex of the brain (Noveen et al., 2000). Lineages that give rise to the pars intercerebralis originate at an antero-medial position and end up at the dorsal pole of the brain (Zacharias et al., 1993; Younossi-Hartenstein et al., 1996). The morphogenetic shifts of the Pc1/2 and Pp3 lineages described in the present paper can be seen as part of the posterior brain rotation summarized above. Pc1/2 lineages, whose neuroblasts in the stage 10 embryo appear at a dorsolateral position within the protocerebral neurectoderm, end up at the posterior pole of the late embryonic/larval brain (Fig. 7B –D). Pp3 neuroblasts start out on the lateral pole of the protocerebral neurectoderm, a position close to the imaginary axis around which the sagittal rotation occurs. As a result, Pp3 lineages are not affected as much by the sagittal rotation. Reconstructing the position of the so-positive lineages during embryonic development reveals a second global movement that occurs concurrently with the posterior rotation. This second movement can be best described as a ‘curved stretching’ of the posterior part of the brain primordium in the transverse axis (Fig. 7A). As part of this movement, neuroblasts located along the dorsal edge of the neuroblast map of a stage 11 embryo move medially and ventrally. In this manner, the disc shaped neural primordium of an early embryo is transformed into the almost spherical cortex of the late embryonic brain. The morphogenetic shift of the so-positive Pc1/2 lineages illustrate the curved stretching of the brain primordium in an impressive manner. These lineages first appear near the dorsal apex of the neuroblast map and are located at a ventro-medial position in the late brain. One should note in this context that the name ‘hemisphere’ to describe one half of the larval brain is a misnomer, since each hemisphere is represented by a full sphere, rather than a half-sphere. During larval development, the growth of the optic lobe has a profound influence on the pattern of central brain lineages. Without the growing optic lobe (a situation generated artificially driving a cell-death inducing hid-rpr construct in the optic lobe placode; Wang et al., in preparation) the central brain would retain its spherical shape throughout larval development. Neuroblasts and their lineages would be located all around the sphere. However, in normal larval development, the optic lobe forms an expanding disc covering the lateral surface of the brain. Central brain lineages originally situated laterally are pushed in all directions away from the optic lobe, dorsally, ventrally, posteriorly, and anteriorly (see Fig. 5 in Dumstrei et al., 2003). This movement is dramatically exemplified by the so-positive Pp3 lineages. In the late embryo, these lineages form a compact group of neuronal cell bodies located medially adjacent to the small optic lobe placode.

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This group stretches in the dorso-ventral axis so that, in the first instar larva, some cell bodies (Pp3v) are near the ventral edge of the IOA, others at the dorsal edge (Pp3c). As the optic lobe grows, Pp3c and Pp3v are further pushed away from each other. Although additional markers for other neural lineages will be required to reconstruct in detail the overall morphogenetic movements shaping the brain primordium between early embryonic and late larval stage, we can present a tentative (and very low-resolution) ‘genealogy’ of the larval neuroblast lineages (Fig. 7E– G). The dorsoanterior medial (DAM), dorso-posterior medial (DPM), and centro-medial (CM) lineages, occupying a position within the medial wall of the larval brain, are derived from the dorso-medial edge of the embryonic brain primordium which is formed by the Pa groups of neuroblasts (Younossi-Hartenstein et al., 1996; Fig. 7E). Pc neuroblasts give rise to the dorso-anterior lateral (DAL), dorso-posterior lateral (DPL), and centro-posterior (CP) lineages, and to the mushroom body. Pp neuroblasts form the basolateral lineages (BLA, BLD, BLP, BLV) which are grouped around the optic lobe. Deuterocerebral neuroblasts (Da and Dc) give rise to the baso-anterior (BA) lineages.

Fig. 7. A– D: Morphogenetic movement of eye field derived structures during embryonic brain development. A: Schematic brain neuroblast map of stage 11 embryo (anterior to the right, dorsal up). Eye field derived neuroblasts (Pc1/2; Pp3) and optic lobe placode are colored violet. Mushroom body neuroblasts are dark gray. Blue arrows indicate directions of global neuroblast shift. B–D: 3D digital models of early first instar brain hemisphere in lateral view (B; dorsal up, anterior to the left), dorsal view (C; anterior up, lateral to the right) and posterior view (D; dorsal up, lateral to the right). Outline of mushroom body (MB; gray) and eye field derived lineages (violet; OL optic lobe; Pc1/2 and Pp3 lineages) is indicated. Note that the relative positions of mushroom body, optic lobe, Pp3 and Pc1/2 is maintained as the brain primordium undergoes a posteriorly and medially directed rotation (blue arrows in A). The mushroom body ends up occupying the dorso-posterior pole; the Pc1/2 cluster is located ventroposteriorly of the mushroom body; the optic lobe is ventro-laterally of the mushroom body; the Pp3 cluster is splayed out at the medial surface of the optic lobe. The gray hatched line shows in each panel the course taken by cervical connective (cc) and brain commissure (com). E–G: Proposed relationship of embryonic brain neuroblast map (A) to the pattern of larval neuroblasts (B, lateral view; C, dorsal view; after Dumstrei et al., 2003). Each topologically defined group of larval neuroblasts is shown in its own color (see color key at upper right). Neuroblast group-identifying colors are superimposed on the embryonic neuroblast map to indicate in a tentative manner where in the embryo a given group is derived from. Hatched lines in A demarcate the major topological subdivisions of the brain neuroblast map (Da/c/p anterior/central/posterior deuterocerebrum; Pa/c/p anterior/central/posterior protocerebrum; T tritocerebrum). Abbreviations of neuroblast groups and landmark structures: BA, baso-anterior group; BLA, basolateral anterior group; BLD, baso-lateral dorsal group; BLP, baso-lateral posterior group; BLV, baso-lateral ventral group; CM, centro-medial group; CP, centro-posterior group; DAL, dorso-anterior lateral group; DAM, dorso-anterior medial group; DPL, dorso-posterior lateral group; DPM, dorso-posterior medial group; MBnb, mushroom body neuroblasts; mb, mushroom body; OL, optic lobe. H: Schematic cross-section of third larval instar brain, depicting the spatial relationship between elements of the larval (green) and adult (blue) visual system. For details, see Section 4.

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4.2. The eye field forms the larval visual system and part of the adult visual system Holometabolous insects, including dipterans, lepidopterans and coleopterans, have a larval visual system that arises separately from the adult visual system. Larval eyes, called stemmata (sing.: stemma) are formed by arrays of photoreceptors and pigment cells which do not form the highly regular ommatidia typical for the adult complex eye (for review, see Paulus, 1989). The larval (stemmatal) optic nerve targets a small neuropile, the larval optic neuropile. Both the larval eye and its central target neurons arise in close proximity to their adult counterparts. Typically, the larval eye forms posteriorly adjacent to the adult eye, and the larval optic neuropile is enclosed within the outer and inner optic Anlagen which form the adult optic lobe. In Drosophila (and probably other holometabolans as well) the larval visual system, after a major remodeling process occurring during metamorphosis, persists into the adult. The larval eye (Bolwig’s organ) gives rise to an internalized cluster of cells, the eyelet (Hofbauer and Buchner, 1989; Yasuyama and Meinertzhagen, 1999), and the larval optic neuropile forms the accessory medulla. Recent studies have provided a considerable amount of detail regarding the structure and function of the larval visual system in Drosophila. An important discovery was that the target neurons of the larval eye (OLPs) are among the period expressing neurons and play a role in controlling the circadian rhythm in both larvae and adults (HelfrichFoerster, 1997). The OLPs, also called ‘small PDFMe neurons’ because of their expression of pigment dispersing factor (PDF), form in the embryo and are closely associated with the optic lobe. We show here that these neurons express so. They either form directly from the optic lobe placode, or are among the early born neurons of one of the Pp3 lineages which delaminate from the optic lobe placode. OLP neurites form dendritic arborizations close to the cell bodies, in the cleft that develops between the inner and outer optic Anlage. Dendrites are joined by the terminal arborizations of the larval optic nerve (Bolwig’s nerve) and of other protocerebral neurons, in particular a cluster of serotonergic neurons (Mukhopadhyay and Campos, 1995). Together, OLP dendrites, Bolwig’s nerve terminals and serotonergic terminals make up the minute larval optic neuropile (LON; Fig. 7H). The LON represents the larval counterpart of the large optic lobe (lobula complex, medulla, lamina) of the adult. As briefly described in Section 1, the adult optic lobe targets parts of the central brain, notably the optic foci and the lateral horn that are located in the lateral protocerebrum. Can one identify an equivalent of these fourth order visual neuropiles in the central brain of the larva? And furthermore, are (parts of) the adult and/or larval central visual neuropiles derived from the eye field? In the larva, besides forming dendritic arborizations in the LON, the OLP neurons send axons into the dorso-lateral

protocerebrum (Fig. 7H). This projection pattern was described already by Helfrich-Foerster (1997). Our results identify the neuropile compartment that receives the OLP axons as the dorsal part of the centro-posterior lateral (CPLd) compartment. Based on location and connectivity, it has been suggested that the larval CPL will turn into the lateral horn of the adult brain (Younossi-Hartenstein et al., 2003). The adult lateral horn receives input of different sensory modalities, among them collaterals of the antennocerebral axons that carry olfactory information from the antennal lobe (Strausfeld, 1976), but also prominently tertiary visual axons from the lobula complex. This connectivity suggests that the lateral horn of the adult brain represents a multimodal sensory association center with important visual function. The same may be true for the larval CPLd, which also receives visual input from the OLP neurons. Many central brain neurons derived from the sine oculis expressing eye field of the embryo also have axonal and/or dendritic arborizations in the CPLd. Among these neurons are the primary neurons Pp3c, as well as large clusters of secondary neurons (Pp3sec). The common origin from a coherent eye field and the shared expression of sine oculis by photoreceptors and central interneurons targeted by them may constitute an example of a neural circuit defined by the expression of a common regulatory gene. As shown schematically in Fig. 7H, we can summarize the relationship between elements of the larval and adult visual system discussed so far as follows: Sense organ Peripheral neuropile Central neuropile

Larval Larval Larval Larval

eye optic neuropile (LON) CPLd optic focus (LOF)

Adult compound eye Adult optic lobe neuropile Adult lateral horn Adult optic foci

We will conclude the discussion by briefly considering the origin of the optic foci which constitute the major visual neuropile of the adult central brain. The neuropile of the optic foci is formed by terminal arborizations of axons of the lobula complex, as well as central brain interneurons. Both populations of neurons differentiate from the late third larval instar onward. In the late larval brain, a small rudimentary larval optic focus (LOF) becomes visible at the lateral surface of the protocerebral neuropile (YounossiHartenstein et al., 2003). During pupal development, the LOF becomes incorporated into the BPL and CPL compartments (V.H., unpublished) and leads to an enormous increase in size of these compartments. The neural lineages whose neurites form the BPL and CPL have not been defined in much detail yet. Our published data on the pattern of secondary lineages (Dumstrei et al., 2003) indicate that the basolateral groups of lineages (BLA, BLD, BLP) form tracts of neurites that become ‘accreted’ to the lateral surface of the protocerebral neuropile, and whose arborizations are therefore the most likely candidates to contribute to both to the CPL/BPL and the LOF compartments. The BLA/BLD/BLP lineages are not derived from the eye field. Continuously expressed markers will be required to chart the exact origin of BLA/BLD/BLP in the

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embryo; as shown tentatively in Fig. 7E, they should occupy a position anteriorly adjacent to the eye field.

Acknowledgements We thank Dr J. Lengyel for providing probes. This work was supported by USPHS National Research Service Award GM07185 to T.C. and NSF Grant IBN-0110715 to V.H.

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