Genealogical Correspondence of Mushroom Bodies Across Invertebrate Phyla

Home , Mushroom bodies

Current Biology 25, 38–44, January 5, 2015 ª2015 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2014.10.049 Report Genealogical Correspondence of Mushroom Bodies across Invertebrate Phyla

Gabriella H. Wolff1,* and Nicholas J. Strausfeld1,2 that are accorded the term ‘‘mushroom bodies’’ (insects) 1Department of Neuroscience, School of Mind, Brain, and and ‘‘hemiellipsoid bodies’’ (crustaceans). Four invariant Behavior, University of Arizona, Tucson, AZ 85721, USA characters define these centers’ ground pattern (Figure 1). 2Center for Insect Science, University of Arizona, Tucson, Clusters of basophilic globuli cells (Figures 1F and 1G) AZ 85721, USA giving rise to intrinsic fibers extending throughout the center provide approximately parallel arrangements (Figures 1H– 1K). Intrinsic fibers contribute to an orthogonal network with Summary arborizations of extrinsic (input/output) neurons (Figures 1H and 1J) and centrifugal cells that loop back to more distal Except in species that have undergone evolved loss, paired levels thereby forming feedback pathways (Figures 1D and lobed centers referred to as ‘‘mushroom bodies’’ occur 1E). The ground pattern is further defined by enriched expres- across invertebrate phyla [1–5]. Unresolved is the question sion of proteins essential for learning and memory functions in of whether these centers, which support learning and Drosophila (Figures 1F, 1G, and 2), namely, protein kinase memory in insects, correspond genealogically or whether A catalytic subunit alpha (DC0) [8]; Leonardo (Leo) [9], the their neuronal organization suggests convergent evolu- ortholog of mammalian 14-3-3z; and calcium/calmodulin- tion. Here, anatomical and immunohistological observations dependent protein kinase II (CaMKII) [10, 11]. Genealogical demonstrate that across phyla, mushroom body-like centers correspondence across phyla would be strongly supported share a neuroanatomical ground pattern and proteins if paired forebrain neuropils shared this composite ground required for memory formation. Paired lobed or dome-like pattern of structural organization and protein expression neuropils characterize the first brain segment (protocere- patterns. brum) of mandibulate and chelicerate arthropods and the nonganglionic brains of polychaete annelids, polyclad pla- Morphological Correspondence of the Mushroom Body narians, and nemerteans. Structural and cladistic analyses Ground Pattern across Arthropoda resolve an ancestral ground pattern common to all investi- Golgi impregnations and Bodian silver stains demonstrate gated taxa: chemosensory afferents supplying thousands in insects, myriapods, and chelicerates lobate centers, and of intrinsic neurons, the parallel processes of which estab- in decapod domed centers, surmounted by dense accumu- lish orthogonal networks with feedback loops, modulatory lations of globuli cells, each of which provides a long thin inputs, and efferents. Shared ground patterns and their axon-like fiber. Fibers contribute to successive layers of local selective labeling with antisera against proteins required circuits involving orthogonal arrangements of efferent and for normal mushroom body function in Drosophila are afferent arborizations (Figures 1F–1K; Figures S1A, S1C, and indicative of genealogical correspondence and thus an S1F–S1H available online) [2, 3]. Layers are supplied by ancestral presence predating arthropod and lophotrocho- interneurons originating in primary sensory neuropils as well zoan origins. Implications of this are considered in the as by afferents from other protocerebral areas. Golgi impreg- context of mushroom body function and early ecologies of nations reveal centrifugal processes linking different levels ancestral bilaterians. within these structures (Figure S1F). These arrangements all correspond to the mushroom body ground pattern (Figures Results and Discussion 1D and 1E) [2].

In insects, mushroom bodies (Figures 1A and 1B) are paired DC0, pCaMKII, and Leo Immunoreactivity Resolve lobate centers. Each comprises a stalk (pedunculus) branch- Mushroom Body-like Centers across Arthropoda ing to 2–5 lobes made from thousands, sometimes hundreds Antibodies against the proteins DC0, CaMKII, and Leo also of thousands, of approximately parallel processes originating identify centers defined by the mushroom body neuronal from minute perikarya clustered above the dorsal surface ground pattern. Previous studies [3, 12] showing that insect of the forebrain [1]. Domed centers, also comprising many mushroom bodies and their crustacean homologs are ex- thousands of parallel fibers, occur at corresponding levels in ceptionally immunoreactive to antibodies raised against DC0 malacostracan crustacean forebrains (Figures 1C and 1G) suggested that DC0 expression at comparable levels should [2, 3, 6]. Paired lobate centers also occur in the asegmental resolve paired forebrain centers in other arthropod taxa brains of annelids [5, 6] and Platyhelminthes (flatworms) and as well (Figures 1F and 1G). Likewise, because Drosophila in the forebrain of Onychophora [7]. Here, we address whether DC0 protein and its mammalian ortholog, PKA-Ca are 82% these centers across Arthropoda and certain Lophotrochozoa conserved [13] and because the protein kinase A gene possess structural correspondences supporting homology sequence is retained across Metazoa [14, 15], the localization and whether these centers are likely to have existed in a bilat- of this enzyme should allow the detection of candidate homol- erian ancestor before the advent of the Cambrian explosion. ogous neuropils even across phyla. Another protein, CaMKII, To explore correspondences, we first identify unique neural is highly conserved throughout the Bilateria, with its regulatory arrangements in paired, lobed, or domed forebrain centers domains sharing nearly identical amino acid sequences in Drosophila and rat [16]. CaMKII impacts numerous neuron functions, including synaptic plasticity and induction of long- *Correspondence: [email protected] term potentiation [11]. Similarly, Leo, necessary for olfactory Genealogical Correspondence of Mushroom Bodies 39

Figure 1. Strategy for Identifying Mushroom Bodies across Invertebrate Phyla (A) Morphological ground pattern typifying basal insects. Small, basophilic globuli cells (gc) provide the intrinsic parallel fibers (pf) that comprise mushroom body (MB) lobes. Sensory afferents (aff) synapse onto intrinsic neurons while efferent neurons (eff) project from the lobes to other forebrain areas. Centrif- ugal neurons (c) extend back onto intrinsic neurons, providing feedback loops in the system. (B) Ground pattern elaborated in more derived taxa, such that discrete, parallel sets of globuli cells form layers of parallel fibers into the lobes, which are segmented into synaptic domains (ld), where efferent and afferent neurons are constrained to discrete levels. (C) In malacostracan crustaceans, the basal MB ground pattern has been modified to fit within the eyestalk, where its layered intrinsic neurons and other neurons retain the ground pattern circuitry. (D and E) Typically, centrifugal neurons (here, in the insects Apis mellifera in D and Musca domestica in E) project from deeper levels (shown here projecting from the vertical lobes [VL]) of the center to distal levels of intrinsic neurons. In insects, intrinsic neuron dendrites form a special structure called the calyces (CA). (F and G) Confocal laser scans from brain tissue sections of the cockroach, Periplaneta Americana, and the hermit crab, Coenobita clypeatus, respectively. The MBs of the cockroach and the layers of the hermit crab hemiellipsoid body (HB) are stained with relatively high intensity using the anti-DC0 antibody (magenta). a- tubulin immunoreactivity (cyan) and nucleic acid stain (green) provide structural reference. (H and I) Golgi impregnation (H) and reduced silver staining (I) of the cockroach MB resolves orthogonal arrangements of parallel fibers intersected by dendrites of efferent neurons. (J) Golgi impregnation of the HB reveals corresponding orthogonal arrangements of parallel fibers intersected by efferent dendrites. (K) Reduced silver staining of the HB demonstrates bundled parallel fibers characteristic of the MB ground pattern. The scale bar of (D) represents 50 mm; the scale bar of (E) represents 25 mm; scale bars of (F) and (G) represent 200 mm; scale bars of (H)–(K) represent 50 mm. See also Figure S1. learning and memory, is greatly enriched in Drosophila mush- track the occurrence of mushroom body characters within a room bodies, working in concert with CaMKII to activate neural cladogram. tyrosine hydroxylase [9, 17]. This 14-3-3 protein is highly DC0, phosphorylated CaMKII (pCAMKII), and Leo antisera conserved across the vertebrate and invertebrate species were applied to vibratome sections of brains of representative (with 88% conserved amino acid sequence between Leo and taxa from each major arthropod group, Insecta, Malacostraca, the mammalian 14-3-3z isoform [9]) and is also exceptionally and Myriapoda (Mandibulata), and to Scorpiones, Amblypygi, enriched in the hippocampi of mammals such as mice, where and Uropygi (Chelicerata). An earlier study showed the insect a deletion in 14-3-3z results in cognitive deficiencies [18]. mushroom bodies (exemplified by Periplaneta americana) Mammalian orthologs of PKA-Ca, 14-3-3z, and CaMKII are and crustacean hemiellipsoid bodies (exemplified by Coeno- implicated in a number of human neurological diseases and bita clypeatus) selectively resolved by DC0 immunoreactivity, disorders, such as autism, Alzheimer’s disease, and Angelman with their distalmost levels surmounted by basophilic globuli syndrome, which can cause learning and/or memory compli- cell clusters (Figures 1F and 1G; [3]). Mushroom bodies of cations [19–21]. Due to the conserved nature of these proteins, P. americana are also highly immunoreactive to antisera both in amino acid sequence and in their roles in learning against pCaMKII and Leo (Figures 2E and 2G). pCaMKII is en- and memory in insects and mammals, and their enriched riched in the hemiellipsoid body of C. clypeatus (Figure 2F); expression in mushroom bodies compared to other neuropils, however, Leo immunoreactivity could not be determined in they should prove useful as markers for putative mushroom this tissue. Paired lobate centers that are intensely immunore- body homologs in other phyla. Thus, a palette of neuroana- active to DC0 and originate from beneath dense clusters of tomical and immunohistochemistry methods can be been basophilic cell bodies are resolved in the protocerebrum of used to compare neural organization across species and the chilopod Scolopendra polymorpha and the diplopod Current Biology Vol 25 No 1 40

Figure 2. Anti-pCaMKII, DC0, and Leonardo Reveal Corresponding Immunoreactivity of Higher-Order Brain Centers across the Arthropoda (A–D) Confocal laser scans from brain tissue sections of the centipede, Scolopendra polymorpha (A), millipede, Narceus americanus (B), amblypygid, Phrynus marginemaculata (C), and scorpion, Hoffmannius spinigerus (D), labeled with antibodies against DC0 (magenta). MBs of the centipede, millipede, amblypigid, and scorpion are highly DC0 immunoreactive compared to surrounding tissue. (E, F, H, and I) pCAMKII antisera (magenta) applied to brain tissue of P. americana (E), C. clypeatus (F), S. polymorpha (H, left side), and vinegaroon, Mas- tigoproctus giganteus (I, left side). Anti-pCaMKII bound with the highest concentrations in MBs of the cockroach, centipede, and vinegaroon and the hermit crab HB. Antisera against Leonardo (Leo) resolved the MBs of P. americana (G), S. polymorpha (H, right side), and M. giganteus (I, right side). a-tubulin immunoreactivity (cyan) and nucleic acid stain (green) provide structural reference. Scale bars of (A) and (B) represent 100 mm; scale bars of (C)–(I) represent 200 mm. See also Figure S2.

Narceus americanus (Figures 2A and 2B). These centers are mushroom bodies regardless, indicating that this protein’s likewise highly immunoreactive to antisera against CaMKII expression in mushroom bodies is independent of sensory and Leo (Figure 2H), thus demonstrating that Myriapoda modality (Figures S2A and S2B). and Pancrustacea (Hexapoda + Crustacea sensu lato) share corresponding mushroom body protein expression. Lophotrochozoa The second major arthropod subphylum is Chelicerata, Bilaterians are generally divided into taxa that follow a represented by marine and terrestrial taxa but best known deuterostome or protostome pattern of development. Proto- for arachnids, such as spiders, scorpions, amblypygids, stomes are further divided into Ecdysozoa, to which arthro- and uropygids. Here, anti-DC0 was applied to the brains of pods belong, and Lophotrochozoa, which includes annelids, the African amblypygid, Phrynus marginemaculata, and the planarians, and nemerteans [24]. striped-tail scorpion, Hoffmannius spinigerus (Figures 2C Polychaetes, Annelida and 2D), both exemplifying the diversity of lobed neuropils The supraesophageal but asegmental brains (‘‘acron’’) of poly- typical of chelicerate protocerebra [6]. Their lobed centers chaete annelids possess centers characterized by thousands are intensely DC0 immunoreactive. Sister group to the ambly- of minute processes originating from dense clusters of small pygi are Uropygi (vinegaroons), whose enfolded mushroom basophilic somata. The processes extend in parallel to the body lobes are resolved with antisera against pCaMKII main lobe and then adopt an orthogonal organization inter- and Leo (Figure 2I). With the inclusion of these taxa, DC0, sected by afferent and efferent arborizations as well as centrif- pCaMKII, and Leo are shown localized to mushroom bodies ugal elements between their different levels (Figure S3). These in Mandibulata and Chelicerata. features correspond to the mushroom body ground pattern. In As evidenced by comparable immunoreactivity in the brain sabellid and polynoid polychaetes, chemosensory receptor of the ﬁg beetle, Cotinis mutabilis, which possesses the sense neuron axons from the head appendages terminate in discrete of olfaction [22], and in the anosmic whirligig beetle, Dineutus glomeruli occupying lobes immediately caudal to the mush- sublineatus [23], DC0 immunoreactivity is elevated in the room bodies [5, 6]. However, in certain other polychaetes, Genealogical Correspondence of Mushroom Bodies 41

Figure 3. MB Homologs in the Lophotrochozoa (A) This platyhelminth, Notoplana sanguinea, possesses a discrete brain (inset) equipped with paired MBs (lo, lobe; gc, globuli cells). (B–D) Sections from brain tissue of N. sanguinea showing MB lobes and a central neuropil (cc) expressing DC0 (magenta) at a highly elevated level compared with surrounding brain tissue. (E) Confocal laser scans from brain tissue sections of the roundworm, Lineus viridis, stained with antisera against DC0 (magenta) reveal enrichment of this protein in the cerebral organ (co) and, to a lesser extent, in the dorsal lobes (dl). (F) Confocal laser scans of the polychaete annelid, Nereis virens, brain reveal intense DC0 immunoreactivity (magenta) in the MB lobes. a-tubulin immunoreactivity (cyan) and nucleic acid stain (green) provide structural reference. The scale bar of (A) represents 1 mm; the scale bar of the inset of (A) represents 100 mm; scale bars of (B)–(D) represent 100 mm; scale bars of (E) and (F) represent 200 mm. See also Figure S3.

In Lineus viridis, a structure called the cerebral organ, which contains tightly packed somata corresponding to globuli cells, is attached to each dorsal lobe posteriorly. Applications of DC0, Leo, and pCaMKII antisera to sectioned neural tissue of L. viridis resolve the dorsal lobes and cerebral organs, together possessing the highest concentration of these proteins (Figures 3E, S3F, and including Nereis virens, corresponding glomeruli flank the S3G), with DC0 most concentrated in the cerebral organ, which mushroom body pedunculus and lobes from which intrinsic may correspond to the calycal layer of insect mushroom fiber processes extend out to grasp them (Figures S3A–S3C). bodies. Leo and pCaMKII are most concentrated in the Using N. virens as a representative taxon, brains embedded dorsal lobes. for immunocytology were treated with antisera raised against DC0, pCaMKII, and Leo. Each of these proteins was enriched Resolution of Mushroom Bodies by Anti-Leo, pCaMKII, in the mushroom bodies, corresponding to the arthropod and DC0 pattern of expression (Figures 3F, S3D, and S3E). Although 14-3-3 proteins are involved in a wide range of Platyhelminthes, Polycladida molecular pathways, studies suggest that high levels of Leo These triploblastic acoelomates lack segmentation or special- expression in the mushroom bodies are acutely necessary ized sensory appendages but have well-defined anterior for olfactory learning and memory, whereas lower expression brains surrounded by a sheath (Figure 3A). The brains possess levels are sufficient for the normal function of other cell types prominent paired centers that are DC0 positive (Figures 3B– [26]. However, CaMKII and DC0 are ubiquitous among cell 3D). In Notoplana sanguinea, each center originates from a and tissue types. Why these proteins are resolved at such cluster of minute basophilic somata situated outside the sur- exceptionally higher levels, differentiating them from the rounding sheath. About 300 thin processes extend from these surrounding neuropils, is still open to interpretation. cell bodies into the brain, reaching the brain’s midline, with CaMKII has a multitude of functions in various cell types, but some processes crossing the brain’s midline. A second lobe important for learning and memory is the necessity for auto- expands ipsilaterally into a broader terminus. Reduced silver phosphorylation of this protein at the postsynaptic density in reveals an organization like that of a small mushroom body: order to achieve long-term potentiation of neurons [27]. It is parallel fibers intersected by other neuronal processes. How- possible that antisera used in this study against autophos- ever, there is no evidence that sensory interneurons supply phorylated CaMKII localize at high levels in the mushroom the lobes. Rather, the arrangement of globuli cells and the bodies because these structures are involved in learning and initial segments of parallel fibers outside what is interpreted memory and their intrinsic cells must have constitutively phos- as the blood-brain barrier suggest that the distal parts of the phorylated CaMKII to achieve synaptic plasticity. intrinsic neurons directly receive information about the chem- In its inactive form, PKA exists as a holoenzyme consisting ical composition of the immediate environment. of two catalytic subunits negatively modulated by two regula- Nemerteans, Heteronemertea tory subunits. Upon binding cyclic AMP (cAMP), the holoen- Nemerteans (roundworms) are unsegmented, predatory ani- zyme dissociates, leaving the catalytic subunits free to phos- mals possessing complex brains, comprising of a pair of phorylate various substrates, including transcription factors, dorsal and ventral lobes that lie flat in the head capsule [25]. synaptic vesicle proteins, and voltage-gated or ligand-gated Current Biology Vol 25 No 1 42

Figure 4. Neurophylogeny of the Euarthropoda with Lophotrochozoan Outgroups Phylogeny of the protostoma based on neural cladistic (2011) [24]. The polyclad flatworm, Notoplana sanguinea, serves as a proxy for the ancestral protostome MB ground pattern (upper left). This MB is characterized by small basophilic globuli cells, giving rise to parallel, intrinsic fibers forming the lobe. The polychaete MB (N. virens) is lobed and comprises parallel intrinsic fibers from globuli cells, distal efferent inputs, and efferent, afferent, and centrifugal neurons in its lobe. These features comprise the MB ground pattern. Evolved modifications of this ground pattern, exemplified by Insecta, are shown to the right. In insects whose MBs serve a single modality, such as the case in the mayfly, Potamathus luteus, the MBs receive primarily olfactory sensory afferents distally. Centrifugal neurons provide feedback from parallel fibers to this distal level. Elsewhere, organization is like that of the ancestral type (lower left). Certain taxa, exemplified by the diving beetle, Thermonectus marmoratus, have evolved a secondary loss of olfactory receptor neurons and a consequent loss of antennal lobes and thus projections to the MBs. They have retained parallel fibers, efferents and afferents to the lobes and centrifugal cells. Multi- sensory elaboration is exemplified by Hymenoptera, such as the wasp, Polistes flavus. Additional modalities supplying distal levels of the MB are served by additional parallel fibers, resulting in great increases in volume. In contrast, certain taxa, such as the whirligig beetle, Dineutus sublineatus, reveal an evolved modality switch where the MBs serve the integration of exclusively visual information. As indicated by the color coding, all these modifications are derived from the ancestral ground pattern. Filled black circles in front of species names indicate taxa in which an evolved loss of MBs or HBs has occurred, as in spiders, branchiopod crustaceans, and Archaeognathan insects. ion channels [28]. One possible explanation for the relatively high DC0 immunoreactivity. This shared trait and the fact high immunoreactivity of the DC0 antibody in learning and that such centers share neuroanatomical characters imply memory centers is that the epitope might only be available in that a last common ancestor of all protostomes possessed the dissociated, active form of the catalytic subunit and that such centers. PKA is persistently active to a large extent in these structures. In insects, a persistently high ratio of PKA catalytic to regu- For example, persistent PKA activity may be achieved by latory subunits may allow for the integration of sensory infor- increased production of cAMP during synaptic plasticity as mation to form associations. If even the simplest mushroom well as during ubiquitination of regulatory subunits, leading bodies, such as in planarians, likewise support learning and to degradation in the proteasome and an overall higher ratio memory, then we might conclude that this functional property of catalytic to regulatory subunits [29]. is likely to be ancient. The polyclad mushroom body can use- Although high levels of DC0 have previously been reported fully serve as a proxy for such a structure in the brain of the in the mushroom bodies of D. melanogaster [8] and PKA has lophotrochozoan-ecdysozoan ancestor, the ground pattern long been implicated in a role for synaptic facilitation across of which has been conserved across invertebrate phyla, irre- the Metazoa [28, 30], it is significant that all the protostome spective of evolved elaborations (Figure 4). Such elaborations groups studied here share the character of having paired include the absence, presence, or multiplication of levels, such lobed centers in the protocerebrum that show extremely as the calyces, to receive unimodal or multimodal inputs. In Genealogical Correspondence of Mushroom Bodies 43

certain taxa, directly relayed sensory input to the mushroom Received: June 27, 2014 bodies has been lost [1] or, as in whirligig beetles, has Revised: September 12, 2014 switched from one modality to another [23]. Accepted: October 16, 2014 Published: December 18, 2014 Based on the conserved pattern across invertebrate phyla of DC0, pCaMKII, and Leo expression in centers having a corre- References sponding neural ground pattern, we conclude that the last common ancestor of the ecdysozoans and lophotrochozoans 1. Strausfeld, N.J., Sinakevitch, I., Brown, S.M., and Farris, S.M. (2009). also possessed that ground pattern. Relatively high expres- Ground plan of the insect mushroom body: functional and evolutionary sion of learning and memory proteins suggest that, when implications. J. Comp. Neurol. 513, 265–291. 2. Brown, S., and Wolff, G. (2012). Fine structural organization of the hemi- present, mushroom body-like centers support learning and ellipsoid body of the land hermit crab, Coenobita clypeatus. J. Comp. memory as they do in insects and that these centers enabled Neurol. 520, 2847–2863. early bilaterians to form multisensory associations, particularly 3. Wolff, G., Harzsch, S., Hansson, B.S., Brown, S., and Strausfeld, N. in the more complex, turbid and evolving ecologies typifying (2012). Neuronal organization of the hemiellipsoid body of the land her- the end Ediacaran and early Cambrian [31]. Indeed, the novel mit crab, Coenobita clypeatus: correspondence with the mushroom ability to form and act on sensory associations likely contrib- body ground pattern. J. Comp. Neurol. 520, 2824–2846. 4. Kenyon, F.C. (1896). The brain of the bee. A preliminary contribution to uted to the rapid diversification of animal evolution, which is the morphology of the nervous system of the arthropoda. J. Comp. known as the ‘‘Cambrian explosion.’’ Neurol. 6, 133–210. If protostome bilaterian forebrains are denoted by paired 5. Heuer, C.M., and Loesel, R. (2009). Three-dimensional reconstruction of mushroom bodies, are the forebrains of deuterostomes like- mushroom body neuropils in the polychaete species Nereis diversicolor wise endowed? That the ancestral bilaterian brain possessed and Harmothoë areolata (Phyllodocida, Annelida). Zoomorphology 128, a tripartite organization is supported by molecular evidence 219–226. showing that orthologous genes required for fore-, mid-, and 6. Strausfeld, N.J. (2012). Arthropod Brains: Evolution, Functional Elegance, and Historical Significance (Boston: Harvard Belknap Press). hindbrain are present in both protostomes and deuterostomes 7. Strausfeld, N.J., Strausfeld, C.M., Stowe, S., Rowell, D., and Loesel, R. [32]. Cellular organization in mammalian hippocampus and in- (2006). The organization and evolutionary implications of neuropils and sect mushroom bodies suggests forebrain correspondence their neurons in the brain of the onychophoran Euperipatoides rowelli. [6]. Homologous transcription factor gene expression in the Arthropod Struct. Dev. 35, 169–196. developing mushroom bodies of the polychaete Platynereis 8. Skoulakis, E.M., Kalderon, D., and Davis, R.L. (1993). Preferential dumerilii and the developing mouse pallium, and to some de- expression in mushroom bodies of the catalytic subunit of protein kinase A and its role in learning and memory. Neuron 11, 197–208. gree in the mushroom bodies of D. melanogaster [33], resolves 9. Skoulakis, E.M., and Davis, R.L. (1996). Olfactory learning deficits in mu- conserved tissue patterning related to corresponding gene tants for leonardo, a Drosophila gene encoding a 14-3-3 protein. Neuron expression and the differentiation of comparable neurons. 17, 931–944. High levels of proteins expressed in mushroom bodies also 10. Kamikouchi, A., Takeuchi, H., Sawata, M., Natori, S., and Kubo, T. denote the mammalian hippocampus (G.H.W. and N.J.S., (2000). Concentrated expression of Ca2+/ calmodulin-dependent pro- 2014, Society for Neuroscience Annual Meeting, abstract). tein kinase II and protein kinase C in the mushroom bodies of the brain Our current research now focuses on whether this chordate of the honeybee Apis mellifera L. J. Comp. Neurol. 417, 501–510. 11. Pasch, E., Muenz, T.S., and Ro¨ ssler, W. (2011). CaMKII is differentially forebrain center might share sufficient correspondence with localized in synaptic regions of Kenyon cells within the mushroom mushroom bodies to suggest homology. bodies of the honeybee brain. J. Comp. Neurol. 519, 3700–3712. 12. Farris, S.M. (2005). Developmental organization of the mushroom Experimental Procedures bodies of Thermobia domestica (Zygentoma, Lepismatidae): insights into mushroom body evolution from a basal insect. Evol. Dev. 7, Please refer to the Supplemental Information for detailed experimental 150–159. procedures. 13. Kalderon, D., and Rubin, G.M. (1988). Isolation and characterization of Drosophila cAMP-dependent protein kinase genes. Genes Dev. 2 (12A), 1539–1556. Supplemental Information 14. Perino, A., Ghigo, A., Scott, J.D., and Hirsch, E. (2012). Anchoring proteins as regulators of signaling pathways. Circ. Res. 111, 482–492. Supplemental Information includes Supplemental Experimental Proce- 15. Li, H.L., Song, L.S., and Qian, P.Y. (2008). Cyclic AMP concentration and dures, three figures, and one table and can be found with this article online protein kinase A (PKA) gene expression at different developmental at http://dx.doi.org/10.1016/j.cub.2014.10.049. stages of the polychaete Hydroides elegans. J. Exp. Zoolog. B Mol. Dev. Evol. 310, 417–427. Author Contributions 16. Wang, Z., Palmer, G., and Griffith, L.C. (1998). Regulation of Drosophila Ca2+/calmodulin-dependent protein kinase II by autophosphorylation G.H.W. and N.J.S. designed the research, performed the research, analyzed analyzed by site-directed mutagenesis. J. Neurochem. 71, 378–387. the data, and wrote the paper. Both authors read and approved the final 17. Ichimura, T., Isobe, T., Okuyama, T., Takahashi, N., Araki, K., Kuwano, manuscript. R., and Takahashi, Y. (1988). Molecular cloning of cDNA coding for brain-specific 14-3-3 protein, a protein kinase-dependent activator of Acknowledgments tyrosine and tryptophan hydroxylases. Proc. Natl. Acad. Sci. USA 85, 7084–7088. We thank Nick Gibson, University of Arizona, for providing valuable help 18. Cheah, P.S., Ramshaw, H.S., Thomas, P.Q., Toyo-Oka, K., Xu, X., with western blot analyses. We would like to once again thank Daniel Kal- Martin, S., Coyle, P., Guthridge, M.A., Stomski, F., van den Buuse, M., deron, Columbia University, for his generous donation of anti-DC0 antibody et al. (2012). Neurodevelopmental and neuropsychiatric behaviour and Ronald Davis for his generation of antisera against Leonardo. N.J.S. defects arise from 14-3-3z deficiency. Mol. Psychiatry 17, 451–466. thanks Martin Heisenberg for many discussions about insect mushroom 19. Ji, L., Chauhan, V., Flory, M.J., and Chauhan, A. (2011). Brain region- bodies. Work was supported by an Alexander von Humboldt Foundation specific decrease in the activity and expression of protein kinase A in Senior Research Prize to N.J.S. G.H.W. was funded by a National Science the frontal cortex of regressive autism. PLoS ONE 6, e23751. Foundation Graduate Research Fellowship Grant (DGE-1143953, 7/15/ 20. Steinkellner, T., Yang, J.W., Montgomery, T.R., Chen, W.Q., Winkler, 2011-6/30/2016). Other support came from the Center for Insect Science M.T., Sucic, S., Lubec, G., Freissmuth, M., Elgersma, Y., Sitte, H.H., and grant AFRL FA86511010001 to N.J.S. and Kudlacek, O. (2012). Ca(2+)/calmodulin-dependent protein kinase Current Biology Vol 25 No 1 44

IIa (aCaMKII) controls the activity of the dopamine transporter: implications for Angelman syndrome. J. Biol. Chem. 287, 29627–29635. 21. Umahara, T., Uchihara, T., Tsuchiya, K., Nakamura, A., Iwamoto, T., Ikeda, K., and Takasaki, M. (2004). 14-3-3 proteins and zeta isoform con- taining neurofibrillary tangles in patients with Alzheimer’s disease. Acta Neuropathol. 108, 279–286. 22. Larsson, M.C., Hansson, B.S., and Strausfeld, N.J. (2004). A simple mushroom body in an African scarabid beetle. J. Comp. Neurol. 478, 219–232. 23. Lin, C., and Strausfeld, N.J. (2012). Visual inputs to the mushroom body calyces of the whirligig beetle Dineutus sublineatus: modality switching in an insect. J. Comp. Neurol. 520, 2562–2574. 24. Andrew, D.R. (2011). A new view of insect-crustacean relationships II. Inferences from expressed sequence tags and comparisons with neural cladistics. Arthropod Struct. Dev. 40, 289–302. 25. Beckers, P., Faller, S., and Loesel, R. (2011). Lophotrochozoan neuro- anatomy: An analysis of the brain and nervous system of Lineus viridis (Nemertea) using different staining techniques. Front. Zool. 8, 17. 26. Philip, N., Acevedo, S.F., and Skoulakis, E.M. (2001). Conditional rescue of olfactory learning and memory defects in mutants of the 14-3-3 zeta gene leonardo. J. Neurosci. 21, 8417–8425. 27. Yamauchi, T. (2005). Neuronal Ca2+/calmodulin-dependent protein kinase II—discovery, progress in a quarter of a century, and perspective: implication for learning and memory. Biol. Pharm. Bull. 28, 1342–1354. 28. Abel, T., and Nguyen, P.V. (2008). Regulation of hippocampus-dependent memory by cyclic AMP-dependent protein kinase. Prog. Brain Res. 169, 97–115. 29. Nguyen, P.V., and Woo, N.H. (2003). Regulation of hippocampal synaptic plasticity by cyclic AMP-dependent protein kinases. Prog. Neurobiol. 71, 401–437. 30. Burrell, B.D., and Sahley, C.L. (2001). Learning in simple systems. Curr. Opin. Neurobiol. 11, 757–764. 31. Erwin, D.H., and Tweedt, S. (2012). Ecological drivers of the Ediacaran- Cambrian diversification of Metazoa. Evol. Ecol. 26, 417–433. 32. Hirth, F., Kammermeier, L., Frei, E., Walldorf, U., Noll, M., and Reichert, H. (2003). An urbilaterian origin of the tripartite brain: developmental genetic insights from Drosophila. Development 130, 2365–2373. 33. Tomer, R., Denes, A.S., Tessmar-Raible, K., and Arendt, D. (2010). Profiling by image registration reveals common origin of annelid mushroom bodies and vertebrate pallium. Cell 142, 800–809.