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

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


INTRODUCTION
The Drosophila central complex is a composite of midline neuropils that include the protocerebral bridge, the fan-shaped body, the ellipsoid body (EB), the noduli and the lateral accessory lobes (Hanesch et al., 1989). These neuropils are interconnected in a modular way whereby columnar projection neurons leading to and from the central complex connect all its components that are themselves intersected by tangential layers of neural processes, which together form functional modules, each representing a segment of sensory space (Strausfeld, 2012). Functional studies have identified specific roles for the central complex in higher motor control, courtship and orientation behaviours, visual memory and place learning, as well as sleep, attention, arousal and decisionmaking (Strausfeld and Hirth, 2013;Pfeiffer and Homberg, 2014;Turner-Evans and Jayaraman, 2016).
Here we investigate the origin and formation of EB ring neurons R1-R4 in the developing and adult brain of Drosophila. We identify bilateral symmetric neuroblasts ppd5 in the embryonic procephalic neuroectoderm as founder cells of neuronal progeny that constitute R1-R4 subtypes of tangential ring neurons in the adult EB. Mutant analysis and targeted genetic manipulations reveal a lineagespecific requirement of engrailed (en) and Poxn activity that determines the number and identity of ppd5-derived progeny and their EB ring-specific connectivity pattern in the adult central complex of Drosophila.

EB ring neurons are lineage-derived progeny of embryonic neuroblasts ppd5
To gain insights into the origin and formation of the EB, we followed the expression of the Pax2/5/8 homologue Poxn which is expressed in the developing and adult EB as revealed by full enhancer analysis . In the embryonic protocerebrum, Poxn expression can be found at the protocerebral/deutocerebral neuromere boundary, which is also characterised by Engrailed-expressing cells . These Engrailed-expressing cells derive from neuroblasts ppd5 and ppd8 , which are distinguishable by dachshund (Dac) expression that is restricted to ppd8. Ppd5/8 neuroblasts can be visualised with en-Gal4 (Kumar et al., 2009) when combined with UAS-mCD8::GFP expression (Fig. 1), which reveals that neuroblasts ppd5/8 form bilaterally-symmetric lineages in the embryonic brain. The resulting neural progeny of ppd5/8 start to express Poxn, which can be visualised with en>mCD8::GFP ( Fig. 2A-D) but also with Poxn>mCD8::GFP, which reveals that Poxn-Gal4+ cells in the embryonic brain are labelled by Engrailed (Fig. 3A,B, arrowheads).
To test this hypothesis, we used a combination of Gal4/UAS and FLP/FRT cassettes  allowing the inheritance of a traceable, membrane-tethered marker (mCD8::GFP) which identifies progeny that share a common origin and are therefore clonally related. We first utilised the en-Gal4 driver line with Gal4 expression detectable from early embryogenesis in the procephalic neuroepithelium ( Fig. 1) and that remains active throughout development and in the adult (Fig. S2). Analysis of en>mCD8:: GFP flies co-labelled with anti-En revealed expression of endogenous Engrailed always within mCD8::GFP-labelled cells, including neuroblasts ppd5 ( Fig. 1C-E) and their progeny in the embryonic (Fig. 2C,D), larval ( Fig. S2A-E) and adult brain . These data establish that en>mCD8:: GFP recapitulates the spatio-temporal pattern of endogenous engrailed expression.
Anatomical and immunohistochemical analysis of en>mCD8:: GFP brains revealed that GFP-labelled En-expressing cells extend projections during larval development towards the midline of the central brain (Fig. S2A,B), which in the adult brain of en>mCD8:: GFP flies terminate in the superior protocerebrum (SP) (Fig. S2F). In addition to cell-specific labelling of en>mCD8::GFP projection patterns, cell-and lineage-specific labelling using en-mediated activation of the constitutively active tubulin driver tub-Gal4  Fig. S4; n=77 brains). Labelling of en>tub>mCD8::GFP-expressing cells in the posterior protocerebrum revealed neuronal projections that terminate in the SP (Fig. 4H, arrows) as well as in the ellipsoid neuropil (Fig. 4H, arrowheads). Labelling en>mCD8::GFP brains with anti-Poxn showed hardly any overlap between GFP and Poxn expression , which is detectable immediately adjacent to En-expressing cells (Fig. 4E). However, en>tub>mCD8::GFP brains immunolabelled with anti-Poxn revealed that protocerebral Poxn-expressing cells were co-labelled with mCD8::GFP and were located immediately adjacent to cells expressing compare to C to E). These data suggest that Poxn-expressing neurons labelled with en>tub>mCD8::GFP share a common lineage relationship with Engrailed-expressing cells.
To corroborate these findings, we carried out mosaic analysis with a repressible cell marker (MARCM) (Lee and Luo, 1999) utilising a tubulin-Gal4 driver. Neuroblast lineage labelling was induced in early L1 and adult brains were screened for GFP expression in both Engrailed-expressing cells projecting to the SP and Poxn-expressing cells projecting to the EB. Following this protocol, we identified Engrailed and Poxn-expressing MARCM-labelled cells, both of which initially project together anterior-medially, before Engrailed-expressing cells branch off to the SP and Poxn-expressing cells project to the EB ring neuropil (Fig. S5). MARCM thus demonstrates that Engrailed and Poxn-expressing cells in the posterior protocerebrum are clonally related. Together with lineage tracing using en>tub>mCD8::GFP, our findings identify Poxn-expressing EB ring neurons and neighbouring SP-projecting Engrailed-expressing cells as clonally-related progeny that constitute two hemi-lineages derived from Engrailed-expressing neuroblasts ppd5. showing Engrailed (En) expression in the ectoderm (grey areas: hs, head spot; as, antennal stripe; is, intercalary stripe; md, mandibular stripe; mx, maxillary stripe) and (B) in the neuro-ectoderm from which brain neuroblasts delaminate (B, grey dots); these include neuroblasts ppd5 and ppd8 (B, green dots) that derive from the En head spot. Lateral views, anterior to the left. (C-H) At stage 11, en>mCD8::GFP (green) visualises expression patterns that mimic endogenous En expression, including the head spot (C,F, dashed areas) as well as neuroblasts ppd8 (D) and ppd5 (E) that both express mCD8::GFP (green) and En (magenta). (F) Dachshund (Dac, magenta) expression in the anterior head ectoderm is also found in the En head spot (F, dashed area) and in neuroblast ppd8 (G) but not in neuroblast ppd5 (H, arrowhead), both of which express en>mCD8::GFP (in H, ppd8 is highlighted with asterisk). D and E are enlargements of the dashed area in C at different focal planes; G and H are enlargements of the dashed area in F at different focal planes. C,F, projections of confocal sections; D,E,G, single sections; H, two confocal sections. n>20 for each condition. Scale bar: 25 μm.
ppd5 neuroblast-derived progeny form part of EB R1-R4 ring neuron circuitry We next wanted to know to which ring-neuron subtypes these Poxnexpressing EB-precursor cells give rise. Adult EB neurons are classified as large-field ring neurons based on their subtype-specific stereotypical pattern of synapse formation (Hanesch et al., 1989;Renn et al., 1999;Young and Armstrong, 2010). Previous reports identified and visualised R1-R4 neurons using subtype-specific Gal4 driver lines Wang et al., 2002;Martín-Peña et al., 2006;Young and Armstrong, 2010;Shaw et al., 2018), which combined with mCD8::GFP, reveal that axon terminals of R1-R3 neurons enter via the EB canal and synapse outwardly at different positions within the EB ring, whereas R4 projections reach the EB at the distal surface and synapse in the outer ring (Fig. S6). We made use of these Gal4 lines to investigate whether Poxn-expressing cells comprise different EB ring-neuron subclasses.

Embryonic formation of Poxn-expressing lineages requires engrailed function
Our lineage analysis identified Poxn-expressing ring neurons as progeny of Engrailed expressing neuroblasts ppd5, suggesting that engrailed might be required for their development and/or specification. To investigate these hypotheses, we first analysed two different alleles affecting engrailed function. en CX1 affects embryonic patterning but does not completely remove the engrailed orthologues en and invected (inv) (Heemskerk et al., 1991). Df(2R)en E is a deficiency removing the entire en locus and the majority of the inv locus, resulting in the absence of en and inv gene products, which is therefore considered to be a null allele of engrailed (Tabata et al., 1995).
Analysis of the embryonic brain and ventral nervous system of Df(2R)en E -homozygous mutants revealed severe patterning defects including absent or fused commissures, fused or broken connectives and a disrupted peripheral nerve pattern. Anti-Poxn immunolabelling of these mutant brains revealed a complete absence of Poxn-labelled neurons in 94.7% (n=19) of all cases examined that developed beyond stage 13 ( Fig. 2H,I). These data suggest that engrailed is required for the formation of Poxnexpressing progeny in the embryonic protocerebrum.

Determination of ring-neuron identity depends on lineagespecific Poxn function
The extended post-embryonic phase of EB lineage development made it necessary to bypass embryonic lethality associated with recessive lethal mutations, as seen for Df(2R)en E homozygous mutants. Moreover, previous studies had shown that Poxn mutants are adult viable but present with an affected EB neuropil Minocha et al., 2017). We therefore used lineagespecific genetic manipulations to gain insights into the mechanisms of engrailed-and Poxn-mediated EB development. To this end, we used UAS-mediated overexpression and RNA interference (RNAi) targeted by en-Gal4 and co-expressed Dicer-2 (Dcr2) to enhance RNAi efficiency (Dietzl et al., 2007). We first tested whether on its own, en-Gal4-mediated UAS-Dcr2 expression interfered with lineage formation and EB development. For this we analysed adult brains of en>mCD8::GFP controls and en>mCD8::GFP, Dcr2 co-immunolabelled with anti-Poxn to visualise Poxnexpressing ring neurons, and with anti-En to visualise adjacent We next studied whether overexpression of engrailed and Poxn might interfere with lineage formation and EB development. Analysis of en>mCD8::GFP,en brains revealed projection patterns and anti-Poxn and anti-Engrailed immunolabelling  indistinguishable from controls ( Fig. 4A-E). In contrast, we were not able to analyse adult brains of en-Gal4-mediated, lineage-specific overexpression of UAS-Poxn due to early developmental lethality of en>mCD8::GFP,Poxn flies. We then analysed the brain phenotypes of RNAi-mediated knockdown of engrailed and Poxn. Again, we were not able to analyse adult brains of en>mCD8::GFP,Dcr2,en-IR animals due to early developmental lethality.

Lineage-specific formation of EB ring-neuron circuitry
Previous studies suggested the Drosophila EBas part of the central complexdevelops from precursor cells that differentiate during larval development and during pupal stages generate the EB neuropil (Hanesch et al., 1989;Renn et al., 1999;Ito and Awasaki, 2008;Yu et al., 2009a,b;Bayraktar et al., 2010;Young and Armstrong, 2010;Omoto et al., 2017). Our lineage analysis demonstrates that at least part of its origin can be traced back to the embryonic procephalic neuroectoderm. We identified Engrailedexpressing neuroblasts ppd5 as embryonic stem cells that give rise to Poxn-expressing progeny, which ultimately differentiate into EB ring neurons. Genetic tracing with en-Gal4 identified R1-R4 ring neurons, suggesting that embryonic neuroblasts ppd5 are the major source of Poxn-expressing progeny leading to EB ring neurons detected in our study. Based on their position, morphology, gene expression patterns and axonal fasciculation, our findings suggest that ppd5-derived larval lineages (Fig. 3) correspond to previously described larval lineages variously called 'EB-A1/P1' Ito et al., 2013;Yu et al., 2013;Yang et al., 2013), 'DALv2/3' (Spindler and Hartenstein, 2011;Lovick et al., 2013;Omoto et al., 2017), 'MC1' (Kumar et al., 2009) or 'DM' (Bayraktar and Doe, 2013;Yang et al., 2013). We previously demonstrated that these larval lineages express Poxn and give rise to gamma-amino butyric acid (GABA)-ergic ring neurons in the central complex of the adult brain . We therefore propose to (re-) name them according to their embryonic origin.
Subclass-specific Gal4 lines together with Poxn expression identifies these lineage-related, ppd5-derived sister cells as R1-R4 ring neurons. Moreover, brain-specific Poxn-Gal4 mediated labelling identifies ring neurons and their axonal projections covering all layers of the EB neuropil, thus suggesting neuroblasts ppd5 give rise to the majority, if not all, of ring neuron subtypes. The ontogenetic relationship between Engrailed-expressing neuroblasts ppd5 and Poxn-expressing EB ring neurons is affirmed by the fact that en-Gal4 and Poxn-Gal4-targeted RNAimediated knockdown of Poxn causes similar EB neuropil-specific phenotypes. Together, these data establish that ppd5-derived progeny are clonal units contributing to the EB ring neuron circuitry in the central complex in Drosophila.

Lineage-related Poxn and engrailed function specifies EB ring neurons
How are these units specified? In both insects and mammals, the patterning and specification of neural lineages is regulated by genetic programs from neurogenesis to neuronal differentiation (e.g. Skeath and Thor, 2003;Guillemot, 2005;Gao et al., 2013;Allan and Thor, 2015). Our study in Drosophila shows that the development and specification of EB-specific circuit elements is likewise dependent on the lineage-specific activity of developmental regulatory genes. Early formation and maintenance of Poxn-expressing ppd5 lineages requires engrailed function as revealed with a deficiency removing both engrailed orthologues, en and invected (Fig. 2H,I). Previous studies showed that, engrailed/ invected are required for the specification of neuroblast identity in the developing nervous system (Bhat and Schedl, 1997), suggesting that engrailed is also required for the specification of ppd5. We also found a later, lineage-specific function of engrailed in the specification of ring neuron numbers (Fig. 6), which is consistent with its transient expression in Poxn+ lineages in the embryonic brain (Fig. 3A,B) but not at later developmental stages nor in adult ring neurons (Figs 3M and 4A-E). engrailed codes for a homeodomain transcription factor mediating the activation and suppression of target genes, regulatory interactions that are required for neural lineage formation and specification in the procephalic neuroectoderm (McDonald and Doe, 1997;Gallitano-Mendel and Finkelstein, 1997;Seibert and Urbach, 2010). In contrast, no function for Poxn in embryonic brain development has been  Table S1 for details. Scale bar: 10 μm.
reported Kimura, 1997, 2001;Boll and Noll, 2002;Minocha et al., 2017), suggesting that Poxn is only during later stages of development required for lineage and/or neuronal specification in the central brain.
Indeed, our experiments identify a postembryonic requirement of Poxn in the specification of ppd5-derived progeny. Previous studies showed that zygotic mutations of Poxn perturb EB neuropil formation, in that presumptive ring neurons are unable to project their axons across the midline and as a consequence, the EB ring neuropil is not formed Minocha et al., 2017). In the present study, en-Gal4-targeted knockdown of Poxn reveals Engrailed-expressing cells that project across the midline and form a ring-like neuropil instead of their normal ipsilateral projections to the SP. Significantly, we did not observe any ppd5-derived GFPlabelled cells that project ipsilaterally towards the SP, neurons that are normally detectable with en-Gal4 targeted GFP expression in the adult brain (Fig. 5B, asterisks). Furthermore, en>Poxn-IRtargeted, EB neuron-like projections do not form a torroidal ring but are rather characterised by a ventral cleft. These en>Poxn-IR cells aberrantly retain Engrailed expression even though their axonal projection and connectivity pattern clearly identify them as ring neurons that are normally devoid of Engrailed but instead express Poxn (Fig. 4C-E). Together these data suggest that, based on their morphology, Engrailed expression, axogenesis and ring-specific projection patterns, en>GFP cells normally projecting to the SP have been transformed into EB ring neurons in en>mCD8::GFP,Dcr2, Poxn-IR flies.
The resulting additional ring neurons in en>mCD8::GFP,Dcr2, Poxn-IR flies are accompanied with a ventrally open EB ring neuropil. A comparable phenotype is seen in brains of Poxn (757) >Poxn-IR flies which are characterised by an increased number of Poxn (757) -Gal4-targeted ring neurons, suggesting that increasing numbers of EB ring neurons lead to an arch-like neuropil reminiscent of the arch-like EB seen in the majority of arthropods (Strausfeld, 2012). In support of this notion, we previously demonstrated that in vivo amplification of ppd5-derived progenitor cells can lead to fully differentiated supernumerary GABAergic ring neurons that form functional connections often characterised by a ventrally open EB ring neuropil . Together, these data identify differential roles of Poxn activity during neuroblast lineage formation, in that Poxn is required for cell identity determination of ppd5-derived progeny, as well as for the specification of cell numbers and terminal neuronal projections of EB ring neurons (Fig. 7).
These Poxn functions in ppd5-derived brain lineages are reminiscent of Poxn activity in the peripheral nervous system (PNS) which mediates the specification of sensory organ precursor (SOP) cell lineages giving rise to external sense organs, the tactile and gustatory bristles, respectively (Ghysen and Dambly-Chaudiere, 2000). In these SOP lineages, differential Poxn activity determines progeny fate between chemosensory (gustatory) or mechanosensory (tactile) neuronal identities Awasaki and Kimura, 1997;Layalle et al., 2004). Furthermore, SOP lineagespecific Poxn function specifies the number of these neurons and their connectivity pattern (Nottebohm et al., 1992(Nottebohm et al., , 1994Awasaki and Kimura, 2001). The apparent functional commonalities between Poxn-mediated specification of ppd5 neuroblast-derived lineages in the brain and SOP lineages in the PNS, suggest that evolutionarilyconserved mechanisms (Alberch, 1991;Hirth and Reichert, 1999) underlie the development and specification of clonal units as cellular substrates for neural circuit and sensory organ formation.

Clonal units as cellular substrates for neural circuit evolution
The cytoarchitecture of both the insect and mammalian brain are characterised by neural lineages generated during development by repeated asymmetric divisions of neural stem and progenitor cells (Shen et al., 1998;Kim and Hirth, 2009;Sousa-Nunes and Hirth, 2016). These ontogenetic clones are thought to constitute building blocks of the insect and mammalian brain Rakic, 2009). In support of this notion, lineage-related progeny constitutes sets of circuit elements of the mushroom bodies (Ito et al., 1997) and antennal lobes in Drosophila (Lai et al., 2008). Clonal relationship also characterises the lineage-dependent circuit assembly in the mammalian brain, where stem cell-like radial glia give rise to clonally-related neurons that synapse onto each other, as has been shown for cortical columns and GABAergic interneurons in the neocortex (Noctor et al., 2001;Yu et al., 2009a,b;Brown et al., 2011;Xu et al., 2014;Shi et al., 2017) and for striatal Fig. 7. Poxn-expressing EB ring neurons R1-R4 descend from engrailed-expressing neuroblasts ppd5. During embryogenesis, engrailed-expressing neuroblasts ppd5 and ppd8 (large blue circles) derive from the procephalic neuroepithelium; they can be distinguished by Dachshund expression (Dac+) restricted to ppd8. At stage 11, ppd5/8 have produced a small number of Engrailed-expressing progeny (small blue circles). At stage 14, two classes of ppd5/ 8-derived neuron are visible: En + /Poxn − (small blue circles) and En − /Poxn + (small red circles). At this stage, cells are already sending axons towards the interhemispheric commissure. The lineages continue to expand during larval and pupal development and acquire their adult morphology during metamorphosis. Genetic tracing and mosaic analysis with a repressible cell marker identify En + /Poxn − (small blue circles) and En − /Poxn + (small red circles) as hemi-lineages derived from bilateral symmetric neuroblasts ppd5. Poxn neuro expression identifies R1-R4 ring neurons of the adult EB.
compartments of the basal ganglia (Kelly et al., 2018). Our study in Drosophila shows that a pair of bilateral symmetric, engrailedexpressing embryonic stem cells, neuroblasts ppd5, give rise to R1-R4 subtypes of tangential ring neurons that contribute to the layered EB neuropil. Thus, ppd5 neuroblast lineages constitute complete sets of circuit elements intrinsic to the adult central complex in Drosophila (Fig. 7).
It has been suggested that clonal expansion of neural lineages contributed to the evolution of complex brains and behaviours (Fish et al., 2008;Enard, 2011;Nielsen, 2015). Key to this hypothetical scenario are ancestral circuit elements in the form of genetically encoded stem cell-derived clonal units, like the ones described in our study here. In such a scenario, lineage-related ancestral circuit elements might have been multiplied and co-opted or diversified during the course of evolution. Multiplication and co-option have been suggested for the evolution of the multiple-loop architecture of the basal ganglia that allows processing of cognitive, emotional and motor information (Stephenson-Jones et al., 2011;Enard, 2011). In line with this hypothesis, quantitative control of the transcription factor Prospero is sufficient to cause clonal expansion of ring-neuron circuitry in Drosophila , which has been implicated in cognitive and motor information processing (e.g. Fiore et al., 2015;Fiore et al., 2017;Kottler et al., 2019) and resembles extensive correspondences to vertebrate basal ganglia, ranging from comparable developmental genetics to behavioural manifestations and diseaserelated dysfunctions (Strausfeld and Hirth, 2013).
In contrast to multiplication and co-option, the diversification of stem cell lineages can equally contribute to neural circuit evolution. Our results presented here identify differential and tightly regulated spatio-temporal functions of engrailed and Poxn that lead to the differentiation of ppd5 progeny into hemi-lineage specific identities in the adult brain. Loss of engrailed affects the formation of precursors cells, whereas its lineage-specific knockdown affects the number of Poxn expressing ring neurons. Correspondingly, en-Gal4-driven lineage-specific knockdown of Poxn results in an identity transformation of Engrailed-expressing neurons in the adult brain in that they no longer project to the SP, but instead reveal an EB ring-neuron identity. These data indicate a binary switch of hemilineage identities as the result of a feed-forward mechanism between engrailed and Poxn. engrailed may activate transcription (directly or indirectly) of Poxn, which in turn represses engrailed to permit differentiation of R1-R4 neurons, thereby regulating the specification of neuronal identities in ppd5 hemi-lineages. This hypothesis is consistent with lineage tracing (Fig. 4) and MARCM experiments (Fig. S5), as well as the transient expression of engrailed in embryonic ppd5 lineages but not in adult EB ring neurons. However, further studies are required to elucidate the nature and extend of these putative regulatory interactions between Engrailed and Poxn.
In summary, our findings presented here establish a causal relationship between a pair of bilateral symmetric embryonic stem cells, neuroblasts ppd5 and the lineage-related assembly of their EB ring neuron progeny as structural units of the central complex in Drosophila. Based on these observations we propose that amplification and diversification of ontogenetic clones together with the repurposed use or exaptation (Gould and Vrba, 1982) of resulting circuitries, is a likely mechanism for the evolution of complex brains and behaviours.

Drosophila genetics
All lines were obtained from the Bloomington Stock Center and raised at 25°C with a 12 h/12 h light/dark cycle. Embryonic and larval gene expression Z-projections were created and analysed using FIJI. Neurons expressing UAS-mCD8::GFP were counted using the ImageJ Cell Counter Plugin (http://rsbweb.nih.gov/ij/plugins/cell-counter.html). Images were processed using Adobe Photoshop and figures constructed in Adobe Illustrator.

Statistics
Statistical analysis was carried out using GraphPad prism 6. Comparison of means from multiple experimental conditions (>2) with one independent variable was performed using the one-way analysis of variance (ANOVA), followed by Bonferroni's multiple comparisons post-hoc test. The alpha level for all tests was 0.05, for details see Table S1.