Neurogenesis and neuronal circuit formation in the Drosophila visual center

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1 The Japanese Society of Developmental Biologists Develop. Growth Differ. (2014) 56, doi: /dgd Review Article Neurogenesis and neuronal circuit formation in the Drosophila visual center Takumi Suzuki 1 and Makoto Sato 1,2 * 1 Laboratory of Developmental Neurobiology, Brain/Liver Interface Medicine Research Center, Kanazawa University, 13-1 Takaramachi, and 2 Graduate School of Medical Sciences, Kanazawa University, 13-1 Takaramachi, Kanazawa, Ishikawa, , Japan The Drosophila optic lobe is composed of a wide variety of neurons that form laminar structures and columnar units. The fly optic lobe shares structural features with the mammalian brain, and fly genetics allow precise genetic manipulations. Thus, the Drosophila visual center is an excellent model for studying the mechanisms underlying the establishment of a functional neuronal circuit during brain development. However, little is understood about the developmental mechanisms that produce neuronal diversity and establish neuronal circuits in the medulla, the largest component of the optic lobe. Our recent research revealed key features of medulla development, such as birth-order-dependent specification of neuronal types and the subdivision of the medulla primordium into concentric zones, which is characterized by the expression of four transcription factors. Here, we review recent investigations into the development of the medulla and discuss the mechanisms that establish functional neuronal circuits. Key words: Drosophila, medulla, neurogenesis, optic lobe, visual system. Introduction During brain development, a large number of various types of neurons are generated at the optimal time and location. The neurons are connected with their correct partners to establish precise neuronal circuits, and these complex and precise neuronal circuits of the brain are essential for correct brain function. However, the molecular mechanisms underlying neuronal diversity, spatio-temporal regulation of neurogenesis, specification of neuronal types, neuronal projection, and the formation of synapses with the correct partners remain obscure because the brain is too complex to elucidate such mechanisms. The Drosophila visual system provides a powerful model to investigate the molecular mechanisms of brain development because it shares structural characteristics with the vertebrate visual system, such as layer structure and columnar units, as described by *Author to whom all correspondence should be addressed. makotos@staff.kanazawa-u.ac.jp Received 12 May 2014; revised 23 June 2014; accepted 26 June Development, Growth & Differentiation ª 2014 Japanese Society of Developmental Biologists Ramon y Cajal (Cajal & Sanchez 1915). In addition, the medulla is genetically tractable and consists of a moderate number of various types of neurons that form a moderately complex neuronal circuit. The strongest point of this model is that Drosophila genetics follow high-resolution genetic analysis and artificial manipulation of neuronal activity in a spatially and temporally restricted manner (Kitamoto 2001; Pulver et al. 2009). The Drosophila retina consists of ommatidial units, each containing eight types of photoreceptor neurons (R1 8). The visual information received in the retina is transmitted to and processed by the optic lobe, which is composed of the lamina, medulla, lobula, and lobula plate. The second visual ganglion, the medulla, is the largest component of the optic lobe and contains neurons of at least 60 different types. The medulla may process all visual information received by the retina because R7 and R8 axons terminate in the medulla, and the visual information carried by R1 6 axons is sent to the medulla via lamina neurons. Currently, despite the importance of the medulla, the developmental mechanisms that generate the diverse types of medulla neurons and establish precise neuronal circuits are largely unknown. Recently, we and other groups reported key features of the developing medulla and unique phenomena that open the door to understand the molecular basis of

2 492 T. Suzuki and M. Sato medulla development. Here, we summarize recent advances in the developmental biology of the medulla primordium. Subdivision of the larval medulla primordium into concentric zones As mentioned above, the medulla is the primary component of the fly optic lobe, containing tens of thousands of neurons that form 10 layers (M1 10) and 800 columnar structures. Although at least 60 types of medulla neurons with different morphological features have been identified by Golgi staining (Fischbach & Dittrich 1989), the number of medulla neuronal types may be even higher. Thus, a highly complex neuronal circuit is established in the medulla to process various visual information, including motion, shape, and color. In the larval medulla, called the medulla primordium, the primary source of medulla neurons is thought to be neuroblasts (s), neuronal stem cell-like multipotent precursors, which are located on the cortical surface of the medulla primordium (Fig. 1). s produce ganglion mother cells (GMCs) that subsequently divide to produce medulla neurons (Egger et al. 2007). Many medulla neurons are produced from a single, with a linear and radial orientation toward the center of the developing medulla. To determine the genes that show unique expression patterns in the developing medulla and the patterning mechanisms of the medulla neurons, our group examined the expression patterns of highly conserved transcription factors. We identified concentric zones characterized by the expression of four conserved transcription factors: Drifter (Drf), a member of Brn family transcription factors; Runt (Run), a member of the Runx family; Homothorax (Hth), a member of the Meis family; and Brain-specific homeobox (Bsh), a member of the Bsx family (Fig. 1; Hasegawa et al. 2011; Sato et al. 2013). The concentric zones expressing Drf, Run, and Hth are adjacent to each other without mutual overlap. Bsh is expressed in the outer subdomain of the Hth expressing region. These transcription factors are collectively referred to as concentric transcription factors in this review. The concentric transcription factors are likely expressed stably and strongly during development. However, the concentric zones established in the larval medulla primordium are completely disorganized during pupal development. The concentric zones are essentially conserved during the early stage of pupal development until 8 h after puparium formation (APF). However, they begin to be disrupted at 12 h APF and subsequently become completely disorganized at 24 h APF. This disorganized pattern has also been observed in the adult medulla cortex. These observations imply that the cell bodies of medulla neurons migrate extensively in a radial orientation between 12 and 24 h APF (Fig. 1). When subsets of medulla neurons are constitutively labeled with green fluorescent protein (GFP), GFP-positive neurons are linearly arranged in the larval medulla primordium. This linear arrangement of GFP-positive neurons is kept constant at the beginning of the migration at 12 h APF. However, after the completion of the migration at 24 h APF, GFP-positive neurons are distributed stochastically (Hasegawa et al. 2011). Thus, medulla neurons migrate to change their relative locations during pupal development. In addition to the radial migration discussed above, medulla neurons may also migrate with (a) (b) (c) Neuroblast () Drf+ Runt+ Hth+ Bsh+ Hth+ Bsh- Radial migration Hth+ Bsh+ (Mi1) Lamina Hth+ Bsh- (Lawf2) Medulla A M1 M2 M3 M4 M5 M6 M7 V D Neuropil M8 M9 M10 Fig. 1. Schematics of developmental sequence of the medulla. (a) The developing medulla is subdivided into concentric zone that are characterized by expression of the four conserved transcription factors Hth, Bsh, Run and Drf. (b) The medulla neurons migrate to change their locations during the pupal period. Bsh/Hth double positive neurons radially migrate toward the outer surface of the medulla. (c) In the adult medulla, Hth positive neurons become four types of neurons including Mi1 and Lawf2. Bsh/Hth double positive neurons always differentiate into a single type of neuron, Mi1.

3 Neural development in fly visual center 493 a tangential orientation because radial migration cannot disrupt the linear arrangement of the neurons (Hasegawa et al. 2011; Morante et al. 2011). The changes in distribution of the medulla neurons appear to be stochastic. However, Hth and Bsh double-positive neurons show a clear pattern of migration. Bsh is expressed in a subset of Hth-positive neurons and therefore Bsh/Hth double-positive neurons are initially located in the inner area of the medulla primordium. They always migrate outward during metamorphosis and are consequently located at the outermost area of the adult medulla (Fig. 1). The final location of Drf-positive neurons in the adult medulla can also be classified according to neuronal type. These findings suggest that each type of medulla neuron exhibits a stereotyped migration pattern. In vertebrates, various types of cell migration play important roles during neurogenesis (Hatten 1999; Nadarajah & Parnavelas 2002). Although recent investigations advanced our understanding of the molecular mechanisms that regulate various types of neuronal migration, it is still unclear how neurons can migrate into the position where they should be located. Currently, few examples of active cell migration are known in Drosophila developmental neurobiology. Now, we can investigate the role of active cell migration during the establishment of a precise neuronal circuit using the developing medulla as a model. What is the role of the concentric genes in medulla neurons during development? Mosaic analyses using Gal4 drivers that mimic the expression of each concentric gene revealed that different types of medulla neurons are produced from each concentric zone. Four types of neurons are produced in the Hth expressing region, including Mi1 neurons, the most frequently observed neuronal type in classical Golgistaining (Fischbach & Dittrich 1989), and Lawf2 neurons, which were initially identified in our study (Hasegawa et al. 2011). In a subset of Hth-positive neurons, Bsh expression is observed throughout development. Hth and Bsh double-positive cells exclusively become Mi1 neurons in the medulla ganglion (Fig. 1). Indeed, mosaic analysis using bsh-gal4 specifically labels Mi1 neurons in the adult medulla (Figs 1, 5). Therefore, Bsh/Hth double-positive neurons always differentiate into a single type of neuron, Mi1. In contrast, nine types of neurons are produced in the Drf expressing region, which contains six lobula projection neurons and three medulla local interneurons (Hasegawa et al. 2011). Thus, concentric genes are expressed in a defined subset of medulla neurons throughout development, suggesting that medulla neuronal identities are pre-determined in the larval medulla primordium. Birth-order-dependent specification of neuronal types in the medulla What types of mechanisms regulate the formation of the concentric expression patterns of the transcription factors? As mentioned above, many neurons are produced from a single, with a linear and radial orientation toward the center of developing medulla (Fig. 1). Each clone of a neuron derived from a single forms a line that intersects the concentric zones and contains several types of medulla neurons. Because s produce neurons towards the center of the developing medulla, neurons produced early in development are located in the inner concentric zones, whereas neurons produced late in development are found in the outer concentric zones. The expression of concentric genes correlates with the birth order of medulla neurons, suggesting that medulla neurons are specified in a birth-order-dependent manner (Hasegawa et al. 2011). During central nervous system development, neural stem cells produce a variety of neuronal cells, depending on spatial and temporal information. In mammals, neural stem cells produce neurons and then glial cells in a stereotypical order determined by the temporal restriction of the precursor cell s fate (Cepko 1999). The transition from neurogenesis to gliogenesis is controlled by extrinsic and intrinsic factors. The cell-intrinsic mechanism that restricts the competence of stem cells has been well investigated in the developing embryonic central nervous system of Drosophila. s divide asymmetrically to produce a GMC and in a self-renewing manner (Wodarz & Huttner 2003). The GMC further divides into two neurons whose neuronal identities are specified at the birth of their mother cells. The birth-order identity of a GMC is determined by the expression of heterochronic transcription factors, including Hunchback (Hb), Kr uppel (Kr), Pdm1/Pdm2 (Pdm), Castor (Cas), and Granyhead (Grh). These transcription factors are expressed sequentially in each and maintained in their daughter GMCs to help specify the neuronal identity of their progeny (Isshiki et al. 2001). If such a mechanism also works in the developing medulla, genes that are transiently expressed may be involved in the temporal specification of medulla neurons. Although Hb, Kr, Pdm, Cas, and Grh are not expressed in the s of the developing medulla, different sets of transcription factors are transiently and sequentially expressed in the medulla s, as discussed below. Neuroblasts are differentiated from neuroepithelial cells (NEs) during the 3rd larval instar. The wave of differentiation, called the proneural wave, progresses in a medial-to-lateral orientation and induces the

4 494 T. Suzuki and M. Sato transition of NEs to medulla s, which is triggered by the expression of lethal of scute (l sc), a member of the bhlh proneural gene family (Fig. 2; Yasugi et al. 2008; Sato et al. 2013). As a result, early born s are located medially on the surface of the larval medulla primordium, while later-born s, which tend to be larger than the medial ones, are located laterally (Sato et al. 2013). The s located between the lateral and medial s are designated intermediate s. In the s of the developing medulla, five transcription factors have been identified as candidate heterochronic factors (Fig. 3; Suzuki et al. 2013). In addition to medulla neurons, Hth is also expressed in NEs and lateral s but not in intermediate and medial s. Klumpfuss (Klu) is strongly expressed in s located in the lateral region of the medulla cortical surface adjacent to differentiating NEs and is gradually weakened in s located in more medial regions. Eyeless (Ey) expression is detected in the Klu-expressing s, except for the most lateral cells. Sloppy paired (Slp) expression occurs in intermediate s, in which Klu is weakly expressed. Ey and Slp expression overlaps, but the Slp domain is medial to the Ey domain. As observed in the expression pattern of Klu, both Ey and Slp expression levels gradually decrease from lateral to medial s. These observations suggest that Hth and Klu are strongly expressed in the newer s; however, Ey and Slp are expressed in middle-aged s. In contrast to these four factors, Dichaete (D) is expressed strongly in s located in the medial region, indicating that D is expressed in older s. The expression Medial Older Late-born neuron Lateral Newer NE Early-born neuron L sc proneural wave Early born Late born Hth+ Bsh Hth+ Bsh+ Run+ Drf+ Fig. 2. Sequential neuroblast () production in the developing medulla. The proneural wave progresses in a medial-to-lateral orientation and induces the transition of NEs to medulla s, which is triggered by the expression of Lsc. s are numbered in a numerical order: the 1 is firstly differentiated from NE and the 6 is the last. NE7 is the NE cell that is just differentiating to. s sequentially produce medulla neurons in the order of cells labeled in light green (Hth +, Bsh ), dark green (Hth +, Bsh + ), purple (Run + ), blue (Drf + ) and grey (unidentified cell type). D Slp Older Ey Klu Newer Newer Homothorax (Hth) Hth Meis family homeobox pattern of these five transcription factors suggests that the expression of Hth, Klu, Ey, Slp, and D is sequentially initiated and decreases according to the age of medulla s (Fig. 3). In addition, Tailless (Tll) is expressed in the oldest s but not in D positive s (Li et al. 2013). A regulatory mechanism that controls the sequential transition of these transcription factors has been investigated as follows (Fig. 4). In medulla s, Ey and Slp are co-expressed in medial s, and their expression levels are inversely related. Slp is weakly expressed in s that strongly express Ey, while Slp is strongly expressed in those that weakly express Ey. This implies a mutual regulation between Ey and Slp. Similarly, Slp and D expression levels are also inversely related, suggesting a mutual regulation between Slp and D. Such mutual regulatory mechanisms are clearly revealed by loss-of-function and gain-of-function experiments (Li et al. 2013; Suzuki et al. 2013). Slp NE Klumpfuss (Klu) EGR family Zn-finger Eyeless (Ey) Pax family paired box Sloppy paired (Slp) Fox family forkhead box Dichaete (D) Sox family HMG box Older Fig. 3. The expression domains of the transcription factors that are expressed sequentially and transiently in medulla neuroblasts (s). Hth and Klu are strongly expressed in newer s; Ey and Slp are expressed in middle-aged s; D is expressed in older s. The expression of these transcription factors is sequentially initiated and decreased according to the ages of the medulla s. Newer Neuroblast () Neuron Early born neuron Hth Klu Ey Slp D Bsh Run Drf X Y Older Late born neuron Fig. 4. Schematic model of the molecular mechanism that regulates specification of the neuronal type in a birth-order dependent manner. The temporal window of Hth-, Klu-, and Ey-positive neuroblasts (s) corresponds to the production of Hth/Bsh-, Run-, and Drf-positive neurons, respectively. The temporal windows of Slp- and D-positive s corresponds X and Y, unidentified types of neurons.

5 Neural development in fly visual center 495 and D are downregulated in s by knocking-down Ey, whereas they are expressed precociously in the younger s by ectopic expression of Ey. These results indicate that Ey positively regulates the expression of Slp and D. In slp mutant clones, Ey expression is not repressed but is prolonged in medial s. Although ectopic Slp expression does not affect the expression of Ey in s, Slp is necessary to repress Ey expression in medial s. In contrast to Ey, D expression is abolished in slp mutant s and precociously induced in s expressing Slp. Conversely, Slp expression is not repressed but is prolonged in D mutant medial s and is abolished in s expressing D. These facts indicate that Slp is necessary and sufficient for inducing D expression, while D is necessary and sufficient for suppressing Slp expression in s. This regulatory relationship may be responsible for the temporal transition of transcription factor expression in the order Ey, Slp, and D in medulla s (Fig. 4; Suzuki et al. 2013). Similar results have been demonstrated by Li et al. (2013). What are the roles of the above genes that are expressed transiently in medulla s? Green fluorescent protein-labeling of s and their progeny revealed a correlation between the expression of Hth, Klu, Ey, Slp, and D and the neuronal types that are produced from the s (Suzuki et al. 2013). Hth-positive s located adjacent to NEs have a small number of Hthpositive progeny that do not contain Bsh-positive neurons, whereas those located more medially contain Bsh-positive neurons. This type of correlation has also been observed between the progeny of Klu-positive s and Run-positive neurons and between those of Ey-positive s and Drf-positive neurons (Suzuki et al. 2013). These findings suggest that Hth-, Klu-, and Ey-positive s produce Bsh-, Run-, and Drf-positive neurons, respectively. This hypothesis is strongly supported by the loss-of-function and gain-of-function results for each transcription factor. The temporal window of Hth-positive s corresponds to the production of Hth- and Bsh-positive neurons. Hth is continuously expressed through s to neurons, and the Hth-domain contains Bsh-positive neurons. Bsh expression is completely lost in hth mutant clones and induced by the ectopic expression of Hth in neurons (Hasegawa et al. 2011), suggesting that Hth-positive s produce Bsh-positive neurons through inherited Hth expression in medulla neurons. The temporal window of Klu- and Ey-positive s corresponds to the production of Run- and Drf-positive neurons, respectively. Klu is at least sufficient to induce Run expression instead of Drf expression in the outer domain. When ey is knocked down, Drf expression is abolished, whereas Run expression is derepressed and expands to the outer domain. In contrast, when Ey is ectopically expressed, Drf expression is ectopically induced in the inner domain, whereas Run expression is abolished. In addition, ectopic Ey expression also represses Hth and Bsh expression in the inner concentric area. These results suggest that Klu is sufficient to induce the production of Run-positive neurons and conversely, to suppress Drf-positive neuron production. Ey is necessary and sufficient to induce the production of Drf-positive neurons and suppress that of Run-positive neurons. The temporal windows of Slp and D-positive s appear to correspond to the production of unidentified types of neurons that are produced after the production of Drf-positive neurons. In slp mutant clones, Run and Drf expression are derepressed and expand to the outer domain. In contrast, both Run and Drf expression are lost in clones expressing Slp. The expression of Hth, Bsh, and Drf are lost in clones expressing D, although the expression of all concentric genes is not affected in D mutant clones. These results suggest that Slp and D suppress the production of Drfpositive neurons. The temporal window of Tll corresponds to the production of medulla neuropile glia (Li et al. 2013). Taken together, Hth, Klu, and Ey are responsible for the production of Hth/Bsh-, Run-, and Drf-positive neurons, respectively (Fig. 4). Slp and D negatively regulate the production of neurons that occupy the inner concentric zones and may positively regulate the production of neurons located in the outer concentric zones. Thus, the identities of medulla neurons are specified by transcription factors that are transiently and sequentially expressed in medulla s (Suzuki et al. 2013). Li et al. (2013) proposed a similar regulatory mechanism using a different set of markers for medulla neuronal types. In addition to this birth-order-dependent specification of neuronal types, asymmetric division of GMCs also contributes to neuronal diversity in the medulla (Li et al. 2013). Clonal analysis implies that GMCs of medulla divide asymmetrically to produce two different types of neurons, Apterous (Ap) positive and negative neurons. This asymmetric division of medulla GMCs involves Notch signaling. When Notch signaling is suppressed, GMCs divide to produce two Ap positive neurons. These results suggest that inactivation of Notch signaling causes symmetric division of GMCs (Li et al. 2013). Specification of neuronal types by a combination of transcription factors The Hth-positive domain in the larval medulla primordium is differentiated into four types of neurons: Mi1, Lawf1, Lawf2, and Pm3 (Hasegawa et al. 2011). The

6 496 T. Suzuki and M. Sato neurons located in the outer half of the Hth-positive domain are Bsh-positive in the developing medulla, and such Hth/Bsh double-positive neurons differentiate into Mi1 neurons, which are most likely cholinergic (Figs 1, 5). Mi1 is a local interneuron because it shows small arborizations at the M1, M5, and M9 10 layers of the medulla but no projections to the lamina or lobula complex (Figs 1, 5). Hth and Bsh are constantly expressed in Mi1 neurons from the larval to the adult stage. What are the roles of Hth and Bsh in the differentiation and maturation of Mi1 neurons? In hth mutant clones, normal Mi1 neurons are never observed but are transformed into neurons that are Hth-negative in a wild-type background, such as Tmtype lobula projection neurons. This phenotype is partially rescued by introducing exogenous hth. Thus, loss of hth transforms Mi1 neurons into lobula projection neurons. Such transformation from Mi1 to Tm-type neurons also occurs in bsh mutant clones. This transformation phenotype of the bsh mutant is partially rescued by exogenous bsh. Therefore, both hth and bsh are necessary for the specification of Mi1 neuronal identities. Because Bsh expression is positively regulated by Hth in the developing medulla, the transformation phenotype of an hth mutant can be explained by the loss of Bsh expression. However, the transformation phenotype of an hth mutant is not rescued by introducing exogenous bsh. In addition, Mi1-like abnormal neurons in hth mutant clones are still Bshpositive (Hasegawa et al. 2011). These observations indicate that hth mutant neurons never differentiate into normal Mi1 neurons, even if Bsh is expressed. In contrast, Hth expression is not altered in bsh mutant clones. bsh mutant neurons never differentiate into normal Mi1 neurons, even if Hth is normally expressed. Thus, the contribution of both of hth and bsh are necessary for the specification and successful development of Mi1 neurons (Fig. 5). Drf-positive neurons differentiate into several types of Tm-type lobula projection neurons. Ectopic expression of Hth or Bsh in Drf-positive neurons under the control of drf-gal4 results in the transformation from Tm-type to Mi1-like neurons (Hasegawa et al. 2013). Approximately 40% of Drf-positive neurons are transformed into Mi1-like neurons by the ectopic expression of Bsh alone, although few Mi1-like neurons are induced by the ectopic expression of Hth alone in Drfpositive neurons. However, the morphology of these ectopic Mi1-like neurons is abnormal, with its wide arborization in the M5 layer and loss of fish-hook-like shape in the M9 10 layers. In contrast, when both Hth and Bsh were ectopically expressed simultaneously, Mi1-like neurons were induced without this abnormal morphology. These observations indicate that Bsh Neuron Hth Hth Ncad Bsh Mi1 Ncad Mi1 (Hth+ Bsh+) bsh-gal4 UAS-CD8GFP UAS-sytHA alone can induce some features of Mi1 neurons, but together with Hth, it can make Mi1-like neurons that are more morphologically similar to wild-type Mi1. Thus, the cooperative action of Hth and Bsh is important for the specification and normal morphogenesis of Mi1 neurons (Fig. 5). Both Hth and Bsh are transcription factors and therefore they regulate the expression of downstream target genes to control the morphogenesis of Mi1 neurons. What are the downstream targets of Hth and Bsh that in turn regulate Mi1 neuron development? In the larval medulla primordium, N-cadherin (Ncad), a type of cell adhesion molecule, is strongly expressed in the Hth/Bsh-positive concentric zone. Ncad is upregulated in Hth-expressing clones and cell-autonomously reduced in hth mutant clones, indicating that strong expression of Ncad is controlled by hth in the larval medulla primordium (Hasegawa et al. 2011). Ncad is also upregulated in Bsh-expressing clones without Hth upregulation. However, Ncad expression was not altered in bsh mutant clones. In bsh mutant clones, Hth expression is normal, which may be sufficient to sustain Ncad levels. Therefore, Hth but not Bsh is necessary for maintaining normal Ncad expression levels (Hasegawa et al. 2013). Because Ncad is a homophilic cell adhesion molecule, neurites of Mi1 neurons with high Ncad expression may innervate targets that also express high levels of Ncad. In fact, Mi1 neurons form two arborization sites (proximal and distal to the Ncad domain) along the neuropile structure that strongly express Ncad during early pupal development (Hasegawa et al. 2011). In Ncad mutant M1 M5 M9-10 Fig. 5. Specification of neuronal identity by the combination of concentric transcription factors. Hth/Bsh neurons are produced from Hth+ neuroblasts (s). In the neruons, Hth and Bsh act cooperatively to regulate expression of their downstream genes, including Ncad, to control the differentiation, arborization and migration of Mi1 neurons.

7 Neural development in fly visual center 497 Mi1 neurons, proximal arborizations are eliminated during the early pupal period, indicating that Ncad upregulation in Mi1 influences the arborization site and its morphology. Thus, the cooperative action of Hth and Bsh regulates Ncad expression, and Ncad partially controls the morphogenesis of Mi1 neurons (Fig. 5). Acknowledgments This work was supported by Program for Improvement of Research Environment for Young Researchers and PRESTO from JST, Grant-in-Aid for Scientific Research on Innovative Areas and Grant-in-Aid for Scientific Research (B) from MEXT (to M.S.). Conclusion Recent studies have revealed key features of medulla development, such as the concentric subdivision of the developing medulla, birth-order-dependent specification of neuronal types, and regulation of neuronal morphogenesis by cooperative action of transcription factors. However, the developmental mechanisms of the medulla remain largely unknown. Development of the medulla ganglion shares characteristics with various forms of neurogenesis observed in the mammalian central nervous system. For example, the concentric zones established in the larval medulla primordium resemble the dorsoventral subdivisions of the spinal cord (Jessell 2000). The locations of cell bodies are organized according to their neuronal types, and the distribution may be similar to the cortical organization of the cerebral cortex. Therefore, the medulla can be used as a suitable model for the development of the central nervous system, and understanding the developmental mechanisms of the medulla may help elucidate the whole picture of brain development. When the genes that specify each type of neuron are found, the functions of defined types of neurons can be examined. As mentioned above, bsh-gal4 is solely expressed in Mi1 neurons in the medulla, which is predicted to be involved in motion detection in the course of visual processing (Hasegawa et al. 2011). Although Bsh is also expressed in other regions of the optic lobe, intersectional strategies may enable specific manipulation of Mi1 neuron activity by inducing expression of neurogenetic tools. In addition to such neuronal type-specific Gal4 lines (Morante & Desplan 2008; Hasegawa et al. 2011), a large collection of Gal4-driver lines is available to manipulate neuronal activity (Pfeiffer et al. 2008; Jenett et al. 2012). In fact, the function of 12 types of lamina neurons was analyzed using this collection (Tuthill et al. 2013). Furthermore, the connectivity of medulla neurons was also comprehensively analyzed by electron microscopy reconstruction to identify a candidate motion detection circuit (Takemura et al. 2013). 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