A nervous system requires a precise network of connections between cells called neurons to work properly. Within the brain, the fiber-like connections between pairs of neurons are not running crisscross like a pile of spaghetti. Instead, connected partner neurons are organized into distinct layers and columns.

Many questions remain about how these partner neurons find each other and how the layers of fiber-like connections form. To answer these questions, scientists often study the part of the fruit fly nervous system that controls the insect’s vision. This brain-like structure is simple and can be easily manipulated with genetic engineering. Fruit fly studies have helped identify some molecules that play a role in helping partner cells find one another and connect. These studies have also shown that the timing of brain cell development appears to play a role. But the role that layer formation plays in the process is still a mystery.

Now, Kulkarni et al. show that the birthdate of neurons in the fruit fly visual system helps organize them into layers. These neurons are generated early in the development of the fly. Shortly after birth, a molecular clock under the control of a protein called Sequoia starts within each newly generated neuron. The Sequoia protein is a transcription factor and controls the activity of many genes, and the molecular clock provides the growing neuron fibers with information about where and when to look for its partner neurons.

By manipulating the amount and time that Sequoia is produced in the fly visual system, Kulkarni et al. show that this clock helps arrange the growing cells into layers. Cells with similar birthdates connect and are arranged into layers. How much and when Sequoia is produced dictates where each new layer begins. The next steps for the research will be to learn more about how the clock works and identify any intermediaries between the clock and cell growth patterns.

The identification of mechanisms that regulate the precise formation of neural circuits has been one of the major goals in developmental neurobiology. The Chemoaffinity hypothesis, formalized by Roger Sperry (Sperry, 1963), suggests that growing neurons must carry individual identification tags that allow the recognition between synaptic partners. Although many cell-type specific recognition molecules essential for neural circuit assembly have been identified in recent years (reviewed in Missaire and Hindges, 2015; Yogev and Shen, 2014), the precise developmental context in which these molecular tags control cell recognition and specify synaptic identity remain largely elusive.

The temporal pattern in which different types of neurons are generated and specified has been shown to influence their connectivity during further development (Kohwi and Doe, 2013; Osterhout et al., 2014; Pujol-Marti et al., 2012). In addition to cell type specification, all neurons undergo similar steps of cellular differentiation including the growth of specific processes with different molecular and functional properties (Rolls, 2011; Tahirovic and Bradke, 2009) accompanied by the expression of general neuronal molecules like N-Cadherin (Gärtner et al., 2015). These molecules common to most neurons also influence axon targeting (Brusés, 2011; Sakai et al., 2012) and synaptogenesis (Basu et al., 2015; Bekirov et al., 2008; Seong et al., 2015) but how their ubiquitous expression can support neuronal recognition is not well understood.

The Drosophila visual system, due to its highly stereotypic arrangement and genetic tractability, provides an excellent system to understand the mechanisms involved in neural circuit assembly (Clandinin and Zipursky, 2002). Each of the compound eyes is composed of approximately 800 units called ommatidia (Campos-Ortega, 1980) and each ommatidium contains eight photoreceptor or retinula cells (R1-R8). Axons of R1-R6 photoreceptors terminate in the outermost lamina neuropile (Fischbach and Dittrich, 1989). In contrast, R8 and R7 axons project topographically through the lamina and terminate in the medulla (Figure 1 A). This topographic projection leads to the formation of medulla columns that receive input from R7/R8 cells of the same ommatidium. Within the medulla column R8/R7 axons terminate in two different layers, M3 and M6 respectively (Fischbach and Dittrich, 1989), in which they contact their post-synaptic partner neurons (Fischbach and Dittrich, 1989; Gao et al., 2008; Karuppudurai et al., 2014; Melnattur and Lee, 2011; Ting et al., 2014).

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Figure 1—source data 1

R cell innervation quantification.

The data shows quantification of total number of R8 and R7 axons innervating medulla neuropile at three different developmental stages (6/12/24 hr APF) and axons labelled by 24B10 staining at each stage. The analysis shows the number of R7 and R8 axons for each brain as well as the average numbers for each stage at the bottom.

https://doi.org/10.7554/eLife.13715.004

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Figure 1—source data 2

Sequoia expression quantification.

The file depicts the average Normalised Fluorescence Intensity (NFI) of Sequoia expression analysed using anti-Sequoia antibody. The average NFI for each row of R8 cells is shown. Average NFI for Sequoia expression in each row was calculated for 60 different 3^rd instar eye discs and was normalised against background to avoid individual differences in staining of each disc. The number represents the ratio of NFI for each R8 cell in a row against the background staining in each disc. To minimize the effect of variations in the staining of individual discs following modifications were made to imaging and analysis protocol- The discs were imaged as a single slice so that complete nuclei of all R8 cells are captured simultaneously to avoid fluorescence decay between slices. The fluorescence of each R8 nucleus was normalised against the background signal of exactly the same area. Further, to get rid of variation between samples, ratio of average R8 fluorescence against average fluorescence of background of each disc was considered while quantifying the NFI of Sequoia expression for each row.

https://doi.org/10.7554/eLife.13715.005

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The layer specific targeting of R7/R8 axons can be divided into two main developmental phases: First, targeting of R cell axons to distinct temporary layers in the early medulla and second, the selection of correct synaptic target layer within the mature medulla neuropile (Hadjieconomou et al., 2011; Ting and Lee, 2007; Ting et al., 2005). Multiple cell type specific molecules involved in layer specific targeting of R8 axons (Hakeda-Suzuki and Suzuki, 2014; Lee et al., 2003; Ohler et al., 2011; Pappu et al., 2011; Senti et al., 2003 Shinza-Kameda et al., 2006; Timofeev et al., 2012; Tomasi et al., 2008) and R7 axons (Astigarraga et al., 2010a; 2010b; Choe et al., 2006; Lee et al., 2001; Morey et al., 2008; Nern et al., 2005; Prakash et al., 2010; Ting et al., 2005) have been identified. Interestingly, most of these molecules function in the second phase of axon targeting and the molecular mechanisms governing the initial innervation of R8 and R7 axons as well as the importance of this temporary positioning for subsequent synaptic layer targeting remain elusive. The Zn finger transcription factor Sequoia and the cell adhesion molecule N-Cadherin are both expressed in R7 as well as R8 cells but are primarily required for the temporary layer positioning of R7 axons (Lee et al., 2001; Petrovic and Hummel, 2008; Ting et al., 2005). Additionally, the LRR molecule Capricious, expressed only in R8 cells, has been described to control R8 axon targeting during the second step (Shinza-Kameda et al., 2006) but its role in the initial targeting of R8 axons to temporary medulla position has not been addressed despite an early onset of expression.

Here we show that early self-patterning of R7/R8 afferents, mediated by relative difference in Sequoia levels, organizes an initial topographic map. This is achieved by a birth-order defined sequence of R7-R8 growth cone segregation leading to their differential positioning in the target field. Shortly afterwards, cell adhesion molecules, like Capricious in R8, consolidate these growth cone positions, which is critical for subsequent steps of synaptic partner recognition.

Here we demonstrate that the early, birth order dependent, segregation of R cell growth cones determines later synaptic layer identity in the Drosophila visual system. Small inter-ommatidial differences in Sequoia levels organize R cell growth cones within a layer whereas large intra-ommatidial differences segregate growth cones between layers. Changes in the positioning of growth cones directly correlate with changes in synaptic layer selection without affecting the expression of known cell-type specific targeting molecules. These results highlight the importance of initial afferent growth cone positioning for visual map formation prior to synaptic partner recognition (Figure 7).

Figure 7

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An early shift in the R8 growth cones to R7 position, induced by a short pulse of Sequoia expression, allows them to recognize the R7 target cell Dm8 as synaptic partner later during the development. Similarly, if R7 growth cones fail to segregate from R8 growth cones, they terminate together with R8 axons in the M3 synaptic layer independent of their intrinsic differentiation and targeting program. This extension of Frazzled-negative R7 axons (Timofeev et al., 2012) towards layer M3 could be explained by the default setting of R7 axons to tightly fasciculate and follow the R8 pioneer axons towards the target region (Fischbach and Heisinger, 2008; Meinertzhagen and Hanson, 1993).

R cell growth cone segregation can be controlled by axon-target interactions or axon-axon interactions or both. Although there is no experimental demonstration for direct R7-R8 afferent interactions, the following set of data indicate that such interactions occur during development. Support for direct interaction between R7-R8 afferents comes from Maurel-Zaffran et al., 2001, where the expression of LAR as membrane tethered ligand in R8 cells alone (using an R8-specific Gal4 driver line) could induce a response from R7 axons, indicating a direct signalling between R8-R7 axons. Further, induced Capricious expression in R8 and R7 cells, in Capricious null background (therefore resulting in a target region without any Capricious expression) is sufficient to mis-target R7 axons to layer M3, again indicating a direct R7-R8 afferent interaction (Berger-Müller et al., 2013). Here we show that final target layer of sequoia mutant R7 axons depends on the targeting of R8 axons, further suggesting that mis-targeting of sequoia mutant R7 axons to ectopic synaptic layer is the consequence of segregation defect rather than a change in target layer recognition.

Interactions among afferent axons have been implicated in the assembly of visual and olfactory circuits in vertebrates as well as invertebrates (Brown et al., 2000; Clandinin and Zipursky, 2002; Ebrahimi and Chess, 2000; Feinstein and Mombaerts, 2004; Komiyama et al., 2007; Petrovic and Schmucker, 2015; Sweeney et al., 2007). It has recently been shown that Eph-Ephrin signalling mediates local sorting of RGC axons in mammalian visual system (Brown et al., 2000; Suetterlin and Drescher, 2014). Notch signalling was demonstrated to play a role in spacing of DCN cluster neuron axons via neighbour axon interactions (Langen et al., 2013). But, whether these afferent interactions influence synaptic partner recognition is not known.

Here we show that in the Drosophila visual system, relative levels of Sequoia determine the segregation of afferent R7/R8 growth cones within or between layers. By creating Seq^high-Seq^low R cell combinations using Sequoia gain-of-function R7 mosaics we observed that difference in Sequoia levels among neighbouring cells could induce growth cone segregation. The endogenous differences in Sequoia levels most likely arise as a result of the temporal sequence of R cell specification, suggesting a self-patterning mechanism in early visual circuit assembly (Hassan and Hiesinger, 2015; Roignant and Treisman, 2009, Tomlinson and Ready, 1987). How the relative differences in Sequoia levels in the nuclei of R cells translate into growth cone segregation remains elusive. We have tested candidate signalling pathways including Semaphorin/Plexin (Cafferty et al., 2006; Hsieh et al., 2014; Pecot et al., 2013; Yu et al., 2010), TGF-beta ligand Activin and its receptor Baboon (Ting et al., 2007) and Notch (Langen et al, 2013) but did not find evidence for a critical role in initial growth cone segregation (data not shown). This suggests a so far unknown molecular mechanism in which the growth state of an axon is directly coupled to differential growth cone adhesion. As we could demonstrate a cell-autonomous function of Sequoia in R8 for columnar segregation as well as in R7 for layer segregation, we envision a mechanistic model related to the concept of cell competition, in which strong cell-cell interactions induce cell-autonomous responses (Rhiner et al., 2010).

The initial segregation of afferent growth cones into distinct positions is then consolidated by expression of Capricious in R8 axons in the same posterior-to-anterior pattern in which they arrive in the medulla. We speculate that Capricious mediated growth cone consolidation serves two purposes: 1. It removes the temporal difference in the arrival of R8 axons and 2. It maintains R8 axons in the position where they are responsive to subsequent NetrinB signal provided by L3 neurites (Pecot et al., 2014; Timofeev et al., 2012). This is supported by two different sets of results: First, as we show in this study, the displacement of R8 growth cones to deeper medulla position leads to their mis-targeting to layer M6 in spite of normal Frazzled expression in these R8 cells. Second, the ectopic expression of Frazzled in R7 cells cannot re-direct them to M3 layer in response to localized NetrinB signal present in the superficial position but R8 axons can be re-directed to a different layer (M1/M2) by ectopic expression of localized NetrinB in a position deeper to the superficial R8 medulla position (Nern et al., 2008; Pecot et al., 2013; Timofeev et al., 2012). Taken together, these observations suggest that M3 layer targeting via L3-mediated NetrinB signalling requires R8 axons to be positioned superficially in the medulla further underscoring the importance of R8 growth cone consolidation in this position (Figure 7).

Previous studies have identified several molecules necessary for M6 targeting of R7 axons (Clandinin et al., 2001; Hofmeyer and Treisman, 2009; Hofmeyer et al., 2006; Maurel-Zaffran et al., 2001; Ting et al., 2005; Tong et al., 2011), including Liprin-alpha/beta/gamma, PTP69D and D-Lar. The loss of these molecules specifically affects the stabilization of R7 growth cones during the second step of targeting. Additionally, these molecules along with N-Cadherin have been shown to be critical for establishment of synaptic contacts between pre and post-synaptic neurons (Arikkath and Reichardt, 2008; Astigarraga et al., 2010b; Garrity et al., 1999; Hummel and Zipursky, 2004; Lee and Godenschwege, 2015; Nagaoka et al., 2014; Prakash et al., 2010; Reines et al., 2012). We confirm previous observations, that R7 growth cones are in close proximity with their primary post-synaptic target neurons, Dm8, at the end of growth cone segregation (Figure 6—figure supplement 1A–B', Ting et al., 2014). This raises the possibility that targeting of R7 axons to M6 layer, later in the development, could be the direct result of R7->Dm8 contacts mediated by N-Cadherin. Recently it was shown that N-Cadherin function is necessary for stabilizing R7 growth cones in the deeper medulla position but not for targeting and subsequent extension of R7 axons to M6 layer seems to be a result of passive dislocation (Özel et al., 2015). Additional support for the role of N-Cadherin in the formation and maintenance of R7->Dm8 contacts, following their initial segregation from R8 growth cones, comes from our observation that early expression of Sequoia in CadN mutant R7 cells under weak elav-Gal4 driver can rescue the mis-targeting of R7 growth cones in the superficial medulla position along with R8 growth cones at 24 hr APF (Figure 6—figure supplement 2A,A’,C,C’), but fails to rescue the later mis-targeting to layer M3 eventually resulting in a mis-targeting phenotype identical to CadN mutant R7 axons (Figure 6—figure supplement 2B,B’,D,D’). Interestingly, the R8 growth cones initially mis-positioned in the deeper medulla eventually mis-target to layer M6 and form synaptic contacts with Dm8. In addition, these R8 cells, with axons mis-targeted to layer M6, do not show changes in any of their known cell-type specific molecules including early specifier of cell identity (Senseless), guidance receptors (Frazzled, Capricious) and sensory receptors (Rh6). In addition, no expression of R7 specific molecules (Prospero, R3, Rh4) can be detected. Thus, the R8 cells interact with Dm8 neurons most likely via ubiquitously expressed molecules such as N-Cadherin expressed in both, R7 as well as R8, cells. This is supported by our observation that N-Cadherin is required for stabilization of R8 axons at the layer M6 (Figure 6—figure supplement 2E–F’).

We observed that R8 axons form functional synapses with Dm8, a known R7 target neuron, in the layer M6. This raises the fundamental question of how synaptic layer selection influences synaptic partner recognition. The cellular complexity of potential post-synaptic target layer encountered by ingrowing R cell axons has not been fully determined, leaving room for selective recognition for synaptogenesis within a layer. In fact, it has been shown that within M6, R7 axons form synapses with Dm8 but not with Tm5c (Karuppudurai et al., 2014) which also arborize the M6 layer. In addition, we have identified various medulla columnar neurons within M6 that are not contacted by R7 axons (unpublished results). Similarly in layer M3 R8 and L3 select distinct post-synaptic partners (Takemura et al., 2013).

The types of neurons present in the medulla at the time of R8 and R7 axon innervation have not been fully identified. Based on the data from Hasegawa et al., 2011; Li et al., 2013; Suzuki et al., 2013, the medulla neurons are generated in temporal fashion (reviewed in Sato et al., 2013; Suzuki and Sato, 2014) and therefore they likely innervate the medulla at different time points. Experiments presented here support a developmental scenario in which the medulla context for arriving R cell axons reduces the complexity of synaptic partner selection. For example R8 and L3 have different arrival times at M3 (Nern et al., 2005; Pecot et al., 2013; 2014), thereby would encounter a different local environment of potential post-synaptic partners competent for synaptogenesis. It is plausible that some form of temporal co-ordination of afferent axons and their post-synaptic partner cell neurites would actually simplify the synaptic partner matching. The concept of temporal identity would argue that R7 and R8 axons arriving at the same medulla position approximately the same time, as shown in the Sequoia gain-of-function background, will pick the same synaptic partners exemplified by Dm8. Support for such proximity-based axon-target interaction for synaptogenesis comes from earlier analysis of ectopic axons in Drosophila as well as Zebrafish (Edwards and Meinertzhagen, 2009; Berger-Müller et al., 2013; Pujol-Martí et al., 2014).

From an evolutionary perspective, such proximity-induced synapse formation has several advantages over mechanisms that require regulation and expression of distinct sets of cell recognition molecules. Considering R7 as the most recently added cell to the precursor ommatidium (Mavromatakis and Tomlinson, 2012): During development, R7 is recruited using mechanisms similar to R8 and therefore possesses default R8 specification program. However, this default R8 program is suppressed to facilitate R7 specification (Cook et al., 2003; Morey et al., 2008). Thus, a temporally separated, novel R7 cell is generated with basic neuronal differentiation similar to that of an R8 cell (Brennan and Moses, 2000; Friedrich et al., 2011; Roignant and Treisman, 2009). Interestingly the temporal difference in the R8/R7 differentiation is then translated into Sequoia mediated layer segregation of their growth cones (Petrovic and Hummel, 2008), with Sequoia expression being part of common differentiation program. Thus, the evolutionary recent R7 cell seems to recognize its synaptic targets via pan neuronal molecules like N-Cadherin as part of the default neuronal differentiation program, instead of the invention of an additional recognition code.

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Author details

1. Abhishek Kulkarni

   Department of Neurobiology, University of Vienna, Vienna, Austria

   Contribution

   AK, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Deniz Ertekin

   Department of Neurobiology, University of Vienna, Vienna, Austria

   Contribution

   DE, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-9480-8622

3. Chi-Hon Lee

   Section on Neuronal Connectivity, Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, United States

   Contribution

   C-HL, Conception and design, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

4. Thomas Hummel

   Department of Neurobiology, University of Vienna, Vienna, Austria

   Contribution

   TH, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   thomas.hummel@univie.ac.at

   Competing interests

   The authors declare that no competing interests exist.

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