Early embryonic development of Johnston´s Organ in the antenna of the desert locust Schistocerca gregaria

doi:10.21203/rs.3.rs-1750012/v1

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Early embryonic development of Johnston´s Organ in the antenna of the desert locust Schistocerca gregaria

https://doi.org/10.21203/rs.3.rs-1750012/v1

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Johnston´s organ has been shown to act as an antennal auditory organ across a spectrum of insect species. In the hemimetabolous desert locust Schistocerca gregaria, Johnston´s organ must be functional on hatching and so develops in the pedicellar segment of the antenna during embryogenesis. Here we employ the epithelial cell marker Lachesin to identify the pedicellar domain of the early embryonic antenna, and then triple-label against Lachesin, the mitosis marker phosphohistone-3, and neuron-specific horseradish peroxidase to reveal the sense-organ precursors for Johnston´s organ and their lineages. Beginning with a single progenitor at approximately a third of embryogenesis, additional precursors subsequently appear in both the ventral and dorsal pedicellar domains, each generating a lineage or clone. Lineage locations are remarkably conserved across preparations and ages, consistent with the epithelium possessing an underlying topographic coordinate system which determines the cellular organization of Johnston´s organ. By mid-embryogenesis twelve lineages are arranged circumferentially in the pedicel as in the adult structure. Each sense-organ precursor is associated with a smaller mitotically active cell from which the neuronal complement of each clone may derive. Neuron numbers within a clone increase in discrete steps with age, are invariant between clones and across preparations of a given age. At mid-embryogenesis each clone comprises five cells consolidated into a tightly bound cartridge. A long scolopale extends apically from each cartridge to an insertion point in the epithelium, and bundled axons project basally towards the brain. We review data from Drosophila to suggest mechanisms which might regulate the developmental program of Johnston´s organ in the locust.

Locust

Embryo

Development

Antenna

Johnston´s organ

Johnston´s organ, first described as an auditory organ of the antenna of a mosquito (Johnston 1855), has since been shown to detect air displacement and so function as an antennal auditory organ in insect species as diverse as Drosophila (Göpfert and Robert 2001a, 2002; Göpfert et al. 2002; Todi et al. 2004; Eberl and Boekhoff-Falk 2007; Jarman 2014), cockroaches (Toh 1981; Toh and Yokohari 1985), mosquitos (Göpfert and Robert 2001b), bugs (Jeram and Pabst 1996), ants (Grob et al. 2020), and not least the desert locust Schistocerca gregaria (Gewecke 1972, 1979; Chapman 1982). Anatomical studies confirm a basic ground plan for Johnston´s organ which is conserved, but where the complement of cell clusters may still vary between species (see Lai and Orgogozo 2004; Jarman 2014; Grob et al. 2020).

From a developmental perspective, Johnston´s organ in Drosophila is constructed over the larval to pupal stages during metamorphosis (see Boekhoff-Falk 2005; Eberl and Boekhoff-Falk 2007; Jarman 2014) but only functions effectively as an auditory organ in the adult (see Göpfert and Robert 2001a, 2002). In orthopteroid insects with a hemimetabolous lifestyle, on the other hand, mechanosensory structures such as Johnston´s organ must be functional on hatching and so need to develop during embryogenesis. Given the significance of this structure for the behavioral repertoire of the locust (see Gewecke 1972, 1979), the absence of any modern developmental literature of which we are aware is surprising.

In this initial study, therefore, we employ immunolabeling coupled with anatomical reconstructions based on confocal microscopy to investigate the early cellular development of Johnston´s organ in the locust Schistocerca gregaria. We establish its epithelial domain of origin, identify putative sense-organ precursors and analyse the growth of the neuronal lineages these generate. We show that the organization of cell clusters comprising Johnston´s organ at mid-embryogenesis already reflects that in the adult. We then compare the pattern of development here with that reported for Johnston´s organ in other insects such as Drosophila.

Animals and preparation

Eggs were obtained from a crowded colony of Schistocerca gregaria maintained as previously described (Ehrhardt et al. 2015a,b; Ehrhardt et al. 2016) and embryos staged to the nearest 1% of developmental time according to Bentley et al. (1979).

Protocols for immunolabeling with primary and secondary antibodies, the composition of incubation media, incubation conditions, confocal, fluorescence and Nomarksi microscopy, and image processing were all as previously described (see Boyan and Williams 2004; Ehrhardt et al. 2015a,b; Ehrhardt et al. 2016).

Primary antibodies

anti-Horseradish peroxidase (α-HRP, polyclonal rabbit, Dianova) recognizes a neuron-specific epitope in insects (see Jan and Jan 1982). anti-Lachesin (Mab 1C10, mouse, gift of M. Bastiani) recognizes a GPI-linked cell surface molecule belonging to the Ig superfamily (see Karlstrom et al. 1993). Expression occurs initially on all differentiating epithelial cells but only cells involved in neurogenesis such as precursors continue to express the molecule later. anti-Lazarillo (Mab 10E6, mouse, gift of D. Sánchez) recognizes a glycosylphosphatidylinositol (GPI)-linked cell surface lipocalin expressed by sensory and pioneer neurons in the grasshopper embryo (Sánchez et al. 1995; Ganfornina et al. 1995). anti-phospho-Histone H3 (Ser10, rabbit, Millipore) binds the phosphorylated form of the amine terminal of Histone 3 so that staining is only possible when the chromatin lies dissociated from the nucleosome complex, as occurs during mitotic chromosome condensation, and is strongest in the metaphase of the cell cycle (see Hendzel et al. 1997).

Secondary antibodies

Single staining involved: Alexa® 488 (goat anti-rabbit, Invitrogen) or Cy3 (goat anti-rabbit, Dianova) for fluorescence-based α-HRP; peroxidase (PO)-conjugated goat anti-rabbit (Jackson ImmunoResearch) for Nomarski-based α-HRP followed by counterstaining with diaminobenzidine (DAB) using Sigma Fast DAB tablets; Cy3 (goat anti-mouse, Dianova) for α-Lachesin and α-Lazarillo; Cy3 (goat anti-rabbit, Dianova) for α-PH3. Double-staining (α-HRP/α-Lazarillo) involved Alexa® 488 (goat anti-rabbit, Invitrogen) for α-HRP and Cy3 (goat anti-mouse, Dianova) for α-Lazarillo. Triple-labeling (α-HRP/α-Lach/α-PH3) involved Cy5 (donkey anti-goat, Dianova) for α-HRP, Alexa® 488 (donkey anti-mouse, Invitrogen) for α-Lach, and Cy3 (donkey anti-rabbit, Dianova) for α-PH3.

Controls for the specificity of all secondary antibodies involved (a) the lack of a staining pattern in the absence of the primary antibody, and (b), in all cases a staining pattern consistent with previously published data (see above).

Sensory apparatus of the antennal base

The locust antenna is a true appendage (c.f. leg: Gibson and Gehring 1988; Casares and Mann 1998) comprising three articulations: a basal scape which links the antenna to the head capsule, a short intermediary pedicel, and a distal elongated flagellum which is not actively motile (Fig. 1a; see Gewecke 1972, 1979; Chapman 1982). The antennal flagellum is also subdivided into articulations but these do not represent true segments and so have been termed meristal annuli (Chapman 2002). The major nerve root of the antenna is the antennal nerve which conducts sensory fibers originating from mechanosensory hairs, propioceptive chordotonal organs, campaniform sensilla and olfactory sensilla from all three segments to the deutocerebrum of the brain (for a full description see Chapman and Greenwood 1986; Ochieng et al. 1998; Chapman 2002). In addition, the scape possesses a musculature to move the pedicel (Fig. 1a) and this musculature is innervated by axons from motoneurons in the brain (see Gewecke 1972, 1979).

Johnston´s organ is located in the pedicellar segment and in the adult locust the organ comprises symmetrical ventral and dorsal cell clusters each of which are then subdivided into medial and lateral fields. An adult cluster comprises six to seven scolopales which insert into a membrane at the pedicellar/flagellar junction (Fig. 1a; inset, i). The cell clusters project axons into pedicellar nerves that run basally to fasciculate with respective medial or lateral nerves in the scape and then join the antennal nerve (Fig. 1a; see Gewecke 1972, 1979 for details). In the hemimetabolous locust, Johnston´s organ must be functional on hatching and so develops during embryogenesis. Labeling with neuron-specific α-HRP reveals that the innervation pattern, distribution of sensory organs, and organization of clusters of sensilla in Johnston´s organ existing in the antennal base at mid-embryogenesis (Fig. 1b) already bears a great similarity to that of the adult (c.f. Figure 1a). Our findings suggest that major developmental steps building Johnston´s organ occur prior to mid-embryogenesis. We therefore decided to limit the scope of our present study, which involved screening a total of 235 preparations, to this same time span.

Epithelial domains

The pedicel, along with the other articulations of the antenna can be identified at the end of embryogenesis via epifluorescent illumination which causes the septal-like cuticular bands to autofluoresce (Fig. 1c). However, this method only functions with cuticularization (after mid-embryogenesis), and does not reveal early epithelial domains. Immunolabeling against the GPI-linked cell surface antigen Lachesin, however, can be shown to identify the epithelial domain of the pedicel at very early embryonic stages, even prior to the formation of Johnston´s organ (Fig. 1d). At mid-embryogenesis (Fig. 1e), Lachesin expression in the scape and pedicel clearly matches the segmentation of the antennal base revealed later by epifluorescent illumination (c.f. Figure 1c) and so allows an unambigous identification of these domains. In combination with neuron-specific labels such as α-HRP (see Figs. 2, 4 below), the α-Lach label allows the cellular development of Johnston´s organ to be followed from its earliest origins within a molecularly identifiable region of the antenna.

Sensory precursors and initial lineages of Johnston´s organ

We identified the sense-organ precursors (SOPs) generating the neurons of Johnston´s organ (JO) in the epithelial domain of the pedicel by triple-immunolabeling against the epithelial cell marker Lachesin (α-Lach), the mitosis marker phosphohistone-3 (α-PH3), and the neuron-specific marker horseradish peroxidase (α-HRP) (Fig. 2). Our analysis here initially focusses on the ventral epithelium but applies equally to the dorsal region (see Fig. 5 below).

Beginning at about 36% of embryogenesis (Fig. 2a), a single large Lach-positive/PH3-positive mitotically active cell is present in the region of the pedicellar domain where the clusters of Johnston´s organ will form. The precursor is itself HRP-negative, and at this age has generated a small lineage of Lach-positive progeny, two of which are also HRP-positive and so represent the initial neurons of this cell cluster of the JO. At 42% (Fig. 2b), three PH3-positive mitotically active cells are generating lineages of the JO in the ventral Lach-positive domain of the pedicel. Towards mid-embryogenesis (48%, Fig. 2c), six PH3-positive progenitors are seen distributed throughout the ventral Lach-positive domain of the pedicel, the dendritic projections of their progeny project apically towards the border with the flagellum. Comparisons across a range of preparations of this age show that the number of active progenitors and lineages in the ventral Lach-positive pedicellar domain already matches the compliment of cell clusters belonging to the mature Johnston´s organ (see Fig. 6, c.f. Figure 1).

Along with the SOP, a smaller mitotically active cell can be seen in lineages at both 42% (Fig. 2b) and 48% (Fig. 2c) of embryogenesis. A time series imaged at higher resolution (Fig. 2d: 38%, 40%, 42%) allows SOPs and their lineages to be analysed more precisely. The data establish that there is only a single large SOP associated with each progressively expanding lineage which therefore constitutes a single clone. At each age there is a smaller cell associated with the clone and in the same location with respect to the SOP. At two of these ages (38%, 42%) the cell in this location is mitotically active, while at 40% it is between cell cycles. We can clearly not be sure that this smaller cell is the same cell each time, and it could equally represent a series of similar second order precursors, each generated asymmetrically by the SOP before dividing.

3D confocal reconstructions following α-HRP labeling (Fig. 2e) document the age-dependent increase in neuronal numbers contributing to a lineage (36%:2 progeny, 38%:3 progeny, 41%:5 progeny), and reveal that these neuronal progeny are consolidated into tightly bound cartridges.

Topographic organization of the pedicellar epithelium

Our evidence up to this point is that a subset of Lach-positive/PH3-positive proliferative cells we have identified in the pedicellar domain generate the neuronal progeny of Johnston´s organ. We reasoned that if the locations of these SOPs were fixed, then this should be reflected in a conserved distribution of their initial lineages in the epithelium, which in turn would argue for a topographic organization of the Lach-positive pedicellar domain.

Support for this hypothesis takes the form of a series of confocal images showing the initial cell clusters in the ventral epithelium of the pedicel in five repeat preparations of the same age (39%) after α-HRP labeling (Fig. 3). The data clearly show neuronal clusters of Johnston´s organ as well as campaniform sensilla at remarkably conserved locations in the epithelium across these preparations. This is also the case for a range of other ages investigated (Suppl. Figure 1) from which we infer that the distribution of PH3-positive proliferative precursors generating these lineages is equally conserved and leads us to propose that the future cluster organization of Johnston´s organ is based on a topographic organization of the epithelium.

Developing neuronal clusters of Johnston´s organ

We investigated the developing pattern of cell clusters (clones) making up Johnston´s organ in the pedicellar domain by labeling with epithelial cell-specific α-Lachesin and neuron-specific α-HRP. At 40% of embryogenesis (Fig. 4a), two major clusters of Lach-positive cells are present in the ventral epithelial domain. Each cluster contains a lineage of differentiating neurons co-labeled by α-HRP, some of which are sprouting initial dendritic and axonal processes. By 42% of embryogenesis (Fig. 4b), five clusters of Lach-positive cells are present in the ventral epithelium, only three of which contain HRP-positive neurons at this stage. At 48% of embryogenesis (Fig. 4c), six Lach-positive cell clusters are now present all of which contain HRP-positive neurons with significant axonal and dendritic processes. At 55% of embryogenesis (Fig. 4d), six clusters of cells are still present in the ventral Lach domain, all contain HRP-positive differentiated neurons. These neurons have generated extensive dendritic processes projecting apically towards the flagellum, and axons projecting basally to the scape where they fasciculate with the antennal nerve to the brain (see Fig. 1b).

The cluster number we see at each developmental stage reflects the number of active progenitor cells present, and by mid-embryogenesis matches that for the ventral subregion of the adult JO (c.f. Figure 1a). Subsequent development does not, therefore, appear to involve the generation of additional lineages.

Our data above refer to events in the ventral epithelium, but there is a parallel, symmetrical, development dorsally. In order to map the complete developmental pattern, we labeled cell clusters of the JO with α-HRP and then optically reconstructed these in 3D (Fig. 5). A transverse optical slice through the pedicel at 41% of embryogenesis shows three cell clusters with dendrites extending towards the ventral epithelial surface, and a mirror symmetrical group of three cell clusters with dendrites extending towards the dorsal epithelial surface (Fig. 5a). At 55% of embryogenesis (Fig. 5b), the number of ventral cell clusters has increased to six (c.f. Figure 4d), and a transverse view shows an equal number of dorsal clusters so that the complete JO is now organized circumferentially in the pedicellar epithelium. A 3D confocal reconstruction from a further preparation at 55% of embryogenesis (Fig. 5c) shows that scolopales from all six ventral and six dorsal cell clusters in the pedicel extend in parallel for over 50µm towards the flagellum (c.f. Wolfrum 1990). We were able to visualize the scolopale insertion points in cap cells via Nomarski optics following α-HRP/PO labeling and DAB counterstaining (Fig. 5d).

The developing pattern of cell clusters, and the number of neurons contributing to each cluster of Johnston´s organ up to mid-embryogenesis, are summarized graphically in Fig. 6. Our data reveal that for both ventral and dorsal pedicellar domains: (a) cluster numbers increase in a stepwise manner each 5% of development; (b) cluster numbers are remarkably constant across all preparations, and between antennae, at each given age; (c) the spatial distribution of clusters within a domain changes in a binary fashion with age (Fig. 6, i);

The number of HRP-positive cells/cluster (Fig. 6) also increases with age up to 41% of embryogenesis at which it saturates. The very small variance in neuronal number per cluster between preparations at a given age may be a function of our method. We staged preparations to the nearest 1% (5 hours) of developmental time so that some embryos could have been slightly advanced or retarded temporally compared to others. There was also no significant difference in neuronal numbers comprising the ventral and dorsal cell clusters for a given age (data not shown). It should be emphasized that our data for cell numbers here are based exclusively on HRP-positive, differentiated, neurons. Double-immunolabeling against neuron-specific HRP and the sensory cell marker Lazarillo, however, reveals the presence of one or more extrinsic cells associated with each cluster (Suppl. Figure 2). These extrinsic cells are exclusively HRP-negative/Laz-positive over the age spectrum we tested and their identity must be established in a later study.

Johnston´s organ is a prominent sensory structure of the antenna across a wide spectrum of insect species and has been universally found to function in audition (Toh 1981; Toh and Yokohari 1985; Jeram and Pabst 1996; Göpfert and Robert 2001a,b; 2002; Göpfert et al. 2002; Todi et al. 2004; Boekhoff-Falk 2005; Eberl and Boekhoff-Falk 2007; Jarman 2014). In the locust Schistocerca gregaria, Johnston´s organ is located in the pedicellar segment of the antenna (Gewecke 1972, 1979) and, as with other sensory stuctures in hemimetabolous insects (see Anderson 1973; Chapman 1982), must develop during embryogenesis in order to be functional on hatching. We show here that the cellular organization of Johnston´s organ at mid-embryogenesis already strongly resembles that of the adult (Figs. 1, 4, 5) and is consistent with the timeframe over which the sensory complement of the tympanal ear forms in orthopteroid insects (Meier and Reichert 1990; Klose 1991). It is likely that maturation of Johnston´s organ will take place during late embryonic and postembryonic development and involve changes in mechano-sensitivity as reported for the tympanal ear of orthopteroid insects (Ball and Young 1974; Ball and Hill 1978; Ball 1979; Michel and Petersen 1982). Such changes in transduction are effected in Drosophila by a spectrum of transcriptional and motor proteins (see Boekhoff-Falk 2005; Eberl and Boekhoff-Falk 2007) with the proneural gene atonal playing a key regulatory role as it does in both insects and vertebrates (Jarman et al. 1993, 1995; Sun et al. 1998; Göpfert and Robert 2001a; Göpfert et al. 2002; Wang et al. 2002; Todi et al. 2004; Todi et al. 2005).

Topographic organization of the pedicellar epithelial domain

The scape, pedicel and flagellum of the locust antenna represent true segments and their development is regulated by molecular mechanisms homologous to those forming other head and body appendages (see Gibson and Gehring 1988; Casares and Mann 1998). Accordingly their epithelia express conserved expression patterns of cell surface factors such as Annulin (Bastiani et al. 1992; Boyan et al. 2018) and Lachesin (Karlstrom et al. 1993; Boyan and Ehrhardt 2020). Lachesin expression in the embryonic pedicel takes the form of a single stripe (Fig. 1) within which putative sense-organ precursors (SOPs) generating the cell clusters of Johnston´s organ are located (Fig. 2). The initial neuronal progeny of these SOPs are seen to occupy remarkably conserved locations within the pedicellar domain (Fig. 3; Suppl. Figure 1) consistent with their having a predetermined position and suggesting a topographic organization of the Lach-positive epithelial domain of the pedicel. Our data correlate with the position-specific coordinate system for neuroectodermal progenitors of the central nervous system of the locust and Drosophila (Doe et al. 1991; Doe 1992), as well as for sensory epithelia such as the retina of Drosophila (see Fischbach and Hiesinger 2008). Molecular mechanisms involving seven up, prospero and gooseberry-distal (a Pax gene homolog) have been shown to regulate the position of such progenitors (Skeath et al. 1995) and we speculate that they also function in the pedicel of the locust antenna.

Neuronal lineages

As the sense-organ precursors (SOPs) generate their lineages, their conserved locations translate into the subsequent organization of cell clusters making up Johnston´s organ (Figs. 3, 4, 6). We found the number of clusters contributing to Johnston´s organ at any given age to be remarkably constant, with no variance at all across 18 preparations at 40% of embryogenesis for example (Fig. 6). We detected only one large SOP associated with each lineage indicating that the progeny represent a single clone, as in the cockroach (Blöchl and Selzer 1988) and Drosophila (Campos-Ortega and Hofbauer 1977; Lawrence and Green 1979). Selection of a single SOP has been shown in Drosophila to result from lateral inhibition involving Delta-Notch signalling (see Artavanis-Tsakonas and Simpson 1991), while specification of the SOP itself is under the control of achaete (Ruiz-Gomez and Ghysen 1993), and as these factors are conserved (see Singhania and Grueber 2014), they probably determine SOP identity in Johnston´s organ of the locust as well.

Each clone might then derive from successive asymmetric cell cycles of an SOP, as has been proposed for antennal pioneers in the locust (Boyan and Ehrhardt 2017), Johnston´s organ in Drosophila (Jarman 2014), and visual neurons of the medulla in Drosophila (Apitz and Salecker 2014). While we do not have the means to identify precursors individually (c.f. Pearson and Doe 2003), we speculate that the neuronal complement of the clone may be traced to a precursor such as the smaller cell we see associated with each SOP (Fig. 2b–d) and so represent constituents of the pIIb/pIIIb path reported for neurons in the homologous structure in Drosophila (Jarman 2014). Taken together, our data are consistent with Johnston´s organ in the locust possessing a type I sensory organization in common with other internal chordotonal organs for stretch or vibration (see Yack 2004; Singhania and Grueber 2014). Indeed, comparative data reveal a remarkably similar circumferential organization of cell clusters in both the JO of the embryonic locust (Fig. 5b) and the phylogenetically distant adult ant (Grob et al. 2021, their Fig. 4), consistent with a conserved structural requirement for detecting and encoding these environmental signals.

Cartridge composition

Our confocal reconstructions show that the neurons of each lineage of Johnston´s organ are organized into cartridges (Fig. 5; Suppl. Figure 1) whose organization is reminiscent of that of retinal photoreceptors in the fly (see Strausfeld 1976; Meinertzhagen and Hanson 1993; Jarman et al. 1995). Indeed, the fact that in Drosophila atonal functions in the initial development of Johnston´s organ, stretch receptors, and the eye, has led to the suggestion that these organs derive from an ancestral atonal-dependent protosensory organ (Niwa et al. 2004).

The final number of HRP-positive cells contributing to a cartridge of Johnston´s organ in the locust is five (Fig. 6), as in the adult scolopidium of the connective chordotonal organ in the pedicel of the cockroach (Blöchl and Selzer 1988) and the mosquito (Schmidt 1967), or olfactory sensilla on moth antennae (Keil and Steiner 1990; Keil 1997). Consistent with this, comparative data suggest the sensory organ precursor (SOP) generates the clonal cartridge comprising cap cell, attachment cell, scolopale cell, ligament cell and neurons according to a ground plan for sensillum development which is conserved across species (see Lai and Orgogozo 2004; Jarman 2014). The mechanisms for determining cell fates in sensory cartridges of Drosophila involve homothorax expression induced by the iroquois complex and wingless pathway (see Fischbach and Hiesinger 2008), while assembling the postsynaptic cell complement involves Hedgehog together with the epidermal growth factor receptor (EGFR) ligand Spitz (see Huang et al. 1998). Given the shared organization of sensory cartridges across species these mechanisms are also likely to be conserved.

When we examined the sensory clusters of Johnston´s organ in the locust via double-labeling against Lazarillo, a GPI-linked cell surface lipocalin expressed by sensory cells in both the locust and Drosophila (Ganfornina et al. 1995; Sánchez et al. 1995), and neuron-specific HRP (Jan and Jan 1982), we found additional Lazarillo-positive/HRP-negative cells associated with each cartridge (Suppl. Figure 2). Such non-neuronal cells could represent the attachment or ligament cells found in the JO of Drosophila (Jarman 2014) or glia-like accessory cells common to sensory units associated with mechano- and olfactory sensilla of insect antennae (Blöchl and Selzer 1988; Keil and Steiner 1990; Keil 1997). Their identity in the locust is still unclear as previous experiments with the glia-specific homeobox gene repo failed to find an expression compatible with the developing cell clusters of Johnston´s organ (Boyan and Williams 2004, 2007). Additional labeling, for example with α-Tubulin which in the cockroach specifically stains the attachment cell, or fluorescent phalloidin which exclusively stains the scolopale (see Wolfrum 1990), may prove insights into their identity as would a future ultrastructural analysis.

Central projections

At mid-embryogenesis we see axons from cell clusters of Johnston´s organ projecting basally into pedicellar nerves which then fasciculate with their respective medial or lateral nerves in the scape before joining the antennal nerve (Fig. 1b). The resulting overall pattern of tracts within the pedicel and scape of the antenna at this developmental stage already strongly resembles that described for the adult locust (Gewecke 1972). Gewecke (1979) additionally described cobalt-stained fibers from JO projecting via the so-called tractus pedicello protocerebrum posterior-medianus through the deutocerebrum and towards the protocerebrum of the adult locust. He hypothesized that this tract might be homologous to bundle B4₁ in the bee Apis mellifera (Pareto 1972) and to a tract referred to as T6I in Apis mellifera by Suzuki (1975). More recent findings on the adult desert ant Cataglyphis (Grob et al. 2020) show subsets of fibers from JO projecting to the protocerebrum via a branch these authors also refer to as T6I. The gross projection patterns in the adult locust, bee and ant are indeed so similar as to suggest that they might be conserved (see also Kamikouchi et al. 2006; Brockmann and Robinson 2007).

An examination of the central projections from afferents of the early embryonic JO proved beyond the scope of our current study, so we are currently unable to expand on Gewecke´s (1979) findings for the adult locust. However, we have previously shown that by mid-embryogenesis neurons pioneering the two major axon tracts of the locust antenna do project to the protocerebrum where they target the pioneers for the primary axon scaffold of the brain, including commissural tracts of the developing central complex (Boyan and Ehrhardt 2015; Boyan et al. 2017). All the neurons contributing to this pathway express the cell surface lipocalin Lazarillo (Ganfornina et al. 1995; Sánchez et al. 1995), an epitope which is shared by the neurons of Johnston´s organ (Suppl. Figure 2). This feature could represent a molecular basis for a topographic projection of afferents from Johnston´s organ to the locust brain similar to that in the honeybee (Ai et al. 2007).

Acknowledgments

We thank Karin Fischer for excellent technical assistance.

Compliance with ethical standards

All experiments were performed in accordance with the guidelines for animal welfare as laid down by the Deutsche Forschungsgemeinschaft (DFG).

Funding declaration

During the course of this study both authors received funding and other support directly from the Graduate School of Systemic Neuroscience, University of Munich.

Competing interest

The authors declare that they have no competing interests.

Author contributions

G.B. and E.E. both wrote and checked the text of the manuscript. G.B. and E.E. both conceived, created, and checked all the figures. G.B. and E.E. agree with the content and format of the final manuscript.

Data availability

Core data supporting this study are available on request from Dr. E.E. Ehrhardt, AG Ito,

Institute of Zoology, Universität Köln, Zülpicher Str 47b, 50674 Cologne, Germany.

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Suppl.Fig.1.tif
Supplementary Fig. 1Confocal images show neuronal lineages belonging to Johnston´s organ (JO) as well as campaniform sensilla (cs) and chordotonal organs (white arrowheads) in the ventral epithelium of the pedicel following a-HRP labeling in three repeat preparations (P1–P3) at each of 36%, 38%, 41% of embryogenesis. Note the conserved locations of the cell clusters across preparations (vertical white dashed lines; c.f. Fig. 3) consistent with there being an underlying topographic coordinate system to the epithelium. Scale bar represents 50µm throughout
Suppl.Fig.2.tif
Supplementary Fig. 2Extrinsic cells associated with sensory clusters of Johnston´s organ (JO). Confocal images of a sensory cluster in transverse view from two preparations (a–c, d–f) both at 40% of embryogenesis following double-labeling against neuron-specific HRP (a, d; a-HRP, green) and sensory-cell specific Lazarillo (b, e; a-Laz, red). In each case the cluster has a cartridge-like form and comprises four neuronal profiles (white stars) which are co-labeled by a-HRP and a-Laz (c, f; merge, yellow) and so represent differentiated sensory neurons. Associated with the cluster are the profiles of further cells (open white arrowheads) which are HRP-negative/Laz-positive. The identities of such non-neuronal cells remain to be determined. g. 3D reconstruction of a cell cluster at 40% in side view following double-labeling against a-HRP (green) and a-Laz (red) and subsequent superposition of channels (merge, yellow). An HRP-negative/Laz-positive extrinsic cell (red, open white arrowhead) is associated with four HRP-positive/Laz-positive sensory neurons (yellow, white stars). Coordinates point to the dorsal and ventral epithelial surfaces. Scale bar represents 25µm in a-f; 8µm in g

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Early embryonic development of Johnston´s Organ in the antenna of the desert locust Schistocerca gregaria

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Abstract

Figures

Introduction

Materials And Methods

Animals and preparation

Primary antibodies

Secondary antibodies

Results

Sensory apparatus of the antennal base

Epithelial domains

Sensory precursors and initial lineages of Johnston´s organ

Topographic organization of the pedicellar epithelium

Developing neuronal clusters of Johnston´s organ

Discussion

Topographic organization of the pedicellar epithelial domain

Neuronal lineages

Cartridge composition

Central projections

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1