Cuticular bristles and their innervation
Light microscopic examination of an antenna from a 1st instar (In1) locust reveals prominent bristles found at conserved locations on the cuticular surface of the six most apical annuli (A6, A5, A4, A3, A2, A1) (Fig. 1a, b). Immunolabeling against neuron-specific horseradish peroxidase (α-HRP) in sectioned antennae at the In1 stage reveals clusters of HRP-positive sensory cells in the epidermis of apical annuli such as A6 (Fig. 1c) and A4 (Fig. 1d). Note that as these are sectioned antennae the complete cell cluster comprising over 10 cells is not imaged (see Figs. 4, 5). Each cluster is associated with an HRP-positive neurite that terminates as a dendrite within the tip of the bristle, and an axon exits each cell cluster en route to the antennal nerve (not imaged here but see Fig. 5). The overall morphology and innervation pattern is consistent with basiconic-type sensilla (see Suppl. Figure 1; Slifer et al. 1957, 1959).
In this study we focus on basiconic-type bristles in apical annuli of the flagellum as a model for general sensory system development in the antenna. We identify mitotically active sense organ precursors for the associated sensory cell clusters, follow the subsequent differentiation of neuronal progeny in a cluster, and map the spatial organization of their axonal projections into antennal tracts running to the brain.
Ontogeny and development of sensory cell lineages in apical segments of the embryonic antenna
To identify sense organ precursors (SOPs) differentiating in the epithelium of apical annuli A1 – A4 and then generating sensory cell clusters we cultured early embryos with the S-phase label EdU (see Methods) and subsequently co-labeled with the nuclear stain DAPI (Fig. 2a). Confocal imaging at 30% of embryogenesis reveals differentiating SOPs are localized to defined bands in the epithelium each corresponding to an annulus. At 31%, triple labeling against Lachesin (α-Lach, a marker for differentiating epithelial cells), against the S-phase label EdU, and the nuclear stain DAPI reveals differentiating SOPs each with a column of lineage-related epithelial cells in A1 (Fig. 2b i,ii). Double-labeling against α-Lach and the proliferative cell marker phospho-histone 3 (α-PH3) demonstrates that at 33% of embryogenesis such SOPs are now mitotically active (Fig. 2c), and distributed circumferentially within the Lach-positive epithelial domains of these apical annuli (Fig. 2d).
Developing neuronal lineages (clones) associated with sensory organ precursors (SOPs) were identified by triple-labeling against the epithelial cell label Lachesin (α-Lach), the neuron-specific label horseradish peroxidase (α-HRP), and the proliferative cell label phospho-histone 3 (α-PH3) (Fig. 2d). At 38%, a representative Lach-positive proliferative SOP in annulus A2 has generated a clone of three Lach-/HRP-positive neuronal progeny (Fig. 2d, i). The SOP is typically connected to the cuticle by an epithelial foot (see Locke and Huie 1981). At 48% (Fig. 2d, ii), a typical mitotically active SOP has lost its epithelial foot and generated a clone of at least six Lach-/HRP-positive neuronal progeny. At 55% of embryogenesis (Fig. 2e), double-labeling against neuron-specific horseradish peroxidase (α-HRP) and phospho-histone 3 (α-PH3) reveals mitotically active SOPs each associated with a clone of HRP-positive progeny. These have now generated axonal processes that project centrally onto an antennal tract that runs via the scape to the brain (see Boyan et al. 2023). Dendritic processes from each cell cluster project peripherally to putatively innervate later developing cuticular sensilla.
Temporal differentiation of sensory cell clusters
Immunolabeling against neuron-specific α-HRP reveals that differentiation of sensory cell clusters proceeds in an apical to basal direction along the flagellum of the antenna during embryogenesis (Fig. 3). At 45% (Fig. 3a), sensory cell clusters have differentiated significantly only in A1, with just an initial small cluster present unilaterally in A4. Clusters belonging to Johnston´s organ are also visible in the pedicel. At 53% (Fig. 3b), differentiation of cell clusters advances in A1, A2 and A4 while isolated sensory cell clusters appear in A6, A8. At 56% (Fig. 3c), greater numbers of differentiated cell clusters are present throughout A1, A2, A4 and also appear in A3. Differentiation in more basal annuli A5 – A8 has not advanced significantly over 53%. At 65% (Fig. 3d), differentiation of cell clusters has progressed basally and established clusters now cover the epithelia of A1 – A8. Proliferative precursors are no longer found in the epithelium of these apical annuli after this age (data not shown), suggesting that embryonic differentiation of clusters in annuli A1 – A8 of the flagellum is complete.
We interpret these data as showing that the oldest neuronal population is found in cell clusters located near the antennal tip with progressively younger populations located more towards the base.
Innervation of basiconic-type bristles
With the sensory cell clusters largely differentiated in the epithelium of apical annuli at around 65% of embryogenesis, our next question was the time frame over which the basiconic bristles become innervated.
Labeling with neuron-specific α-HRP in apical segments A1 – A4 at 70% of embryogenesis reveals differentiated cell clusters that have generated axons projecting onto the tract system of the antenna (Fig. 4a). Dendritic processes from some cell clusters extend towards the cuticular edge, particularly in A1. Imaging at higher power from a further preparation at 70% (Fig. 4b) confirms that sensory cell clusters arrayed in the epithelium of A4, for example, have produced dendrites that extend to the cuticle edge but target basiconic bristles are not yet evident. At 85% of embryogenesis (Fig. 4c), such bristles are now present on the cuticle of A1 and A2 and are innervated by HRP-positive dendrites from epithelial cell clusters. Montaging optical stacks collected from different antennal depths demonstrates that cell clusters located dorsally or ventrally in the epithelium of A1, A2 direct axons symmetrically to the corresponding dorsal or ventral antennal tracts resulting in an overall topographic projection pattern.
Topographic projections from basiconic bristles into antennal tracts
We investigated the general organization of projections from cell clusters to the tract system of the antenna by first sectioning antennae longitudinally and immunolabeling with neuron-specific anti-horseradish peroxidase (α-HRP). Confocal imaging of flagellar segments A4, A5 at 95% of embryogenesis (Fig. 5a) reveals an array of sensory cell clusters in the epithelium. Sensory dendrites are seen innervating basiconic-type bristles on the cuticle and bundled axons from each cell cluster join a nerve tract in sequential order from apex to base.
We then examined whether cell cluster location, and hence bristle location on the cuticle, is also encoded topographically within the axon profile of the tract itself. To realize this we labeled cell clusters, their axon projections, together with the axonal organization of a tract, with α-HRP in sectioned antennae at the first instar (In1) stage and performed computer-based autotracings of axons from six representative HRP-positive sensory cell clusters in A5, A6 to this tract (Fig. 5b). These cell clusters we numbered sequentially from antennal apex towards the base. We found that autotraced axons stereotypically add laterally to the tract as one proceeds in a basal direction along the flagellum. Since axons join the tract strictly in order according to the location of the cell cluster in the epithelium this results in axons from apical clusters lying more medially in the tract than those from basal clusters, producing a topographic effect akin to tree rings (see Fig. 6).
Further, since cell clusters differentiate in an apical to basal direction along the antenna (see Fig. 3), the tract possesses a temporal topology so that axons from older neurons lie medially in the tract and those from younger neurons lie laterally. If this order is maintained further downstream it would allow spatially- and temporally-encoded sensory information to be transmitted along labeled lines from bristle to brain.