Characterization of plexinA and two distinct semaphorin1a transcripts in the developing and adult cricket Gryllus bimaculatus

Guidance cues act during development to guide growth cones to their proper targets in both the central and peripheral nervous systems. Experiments in many species indicate that guidance molecules also play important roles after development, though less is understood about their functions in the adult. The Semaphorin family of guidance cues, signaling through Plexin receptors, influences the development of both axons and dendrites in invertebrates. Semaphorin functions have been extensively explored in Drosophila melanogaster and some other Dipteran species, but little is known about their function in hemimetabolous insects. Here, we characterize sema1a and plexA in the cricket Gryllus bimaculatus. In fact, we found two distinct predicted Sema1a proteins in this species, Sema1a.1 and Sema1a.2, which shared only 48% identity at the amino acid level. We include a phylogenetic analysis that predicted that many other insect species, both holometabolous and hemimetabolous, express two Sema1a proteins as well. Finally, we used in situ hybridization to show that sema1a.1 and sema1a.2 expression patterns were spatially distinct in the embryo, and both roughly overlap with plexA. All three transcripts were also expressed in the adult brain, mainly in the mushroom bodies, though sema1a.2 was expressed most robustly. sema1a.2 was also expressed strongly in the adult thoracic ganglia while sema1a.1 was only weakly expressed and plexA was undetectable

<i>De novo</i> assembly of a transcriptome for the cricket <i>Gryllus bimaculatus</i> prothoracic ganglion: An invertebrate model for investigating adult central nervous system compensatory plasticity

<div><p>The auditory system of the cricket, <i>Gryllus bimaculatus</i>, demonstrates an unusual amount of anatomical plasticity in response to injury, even in adults. Unilateral removal of the ear causes deafferented auditory neurons in the prothoracic ganglion to sprout dendrites across the midline, a boundary they typically respect, and become synaptically connected to the auditory afferents of the contralateral ear. The molecular basis of this sprouting and novel synaptogenesis in the adult is not understood. We hypothesize that well-conserved developmental guidance cues may recapitulate their guidance functions in the adult in order to facilitate this compensatory growth. As a first step in testing this hypothesis, we have generated a <i>de novo</i> assembly of a prothoracic ganglion transcriptome derived from control and deafferented adult individuals. We have mined this transcriptome for orthologues of guidance molecules from four well-conserved signaling families: Slit, Netrin, Ephrin, and Semaphorin. Here we report that transcripts encoding putative orthologues of most of the candidate developmental ligands and receptors from these signaling families were present in the assembly, indicating expression in the adult <i>G</i>. <i>bimaculatus</i> prothoracic ganglion.</p></div

The central auditory system of adult <i>G</i>. <i>bimaculatus</i> shows large-scale compensatory anatomical plasticity after injury.

<p>A) Auditory afferents (red “claw”) terminate in the prothoracic ganglia, and synapse with ascending neurons (AN-2 shown in green and gray). AN-2 neurons have lateral dendrites (L) and medial dendrites (M) that grow up to but not across the midline (dashed line). AN-2 soma (S) are on the opposite side of the midline from the dendrites, and its axons (Ax) projects up to the brain. B) After unilateral loss of an ear, afferents degrade (X), leaving the ipsilateral ANs deafferented. AN-2 dendrites sprout across the midline and become synaptically connected to the contralateral afferents, compensating for the loss of the ear. Fig adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199070#pone.0199070.ref013" target="_blank">13</a>].</p

Netrin and Frazzled proteins in <i>Drosophila</i> and <i>G</i>. <i>bimaculatus</i> share most domains.

<p>A) The type, number, and order of protein domains was assessed by using <i>Drosophila</i> Netrin A, isoform C (AHN59713) and the putative <i>G</i>. <i>bimaculatus</i> Netrin A (TRINITY_DN160784_c2_g1_i2) as queries in a conserved domain search. <i>Drosophila</i> Netrin A, isoform C possessed a laminin N-terminal domain (yellow rectangle), three laminin-type EGF-like domains (blue ovals), and a netrin-like domain (red rectangle). The angled lines on the <i>G</i>. <i>bimaculatus</i> protein representation indicate missing N-terminal sequence, but the remaining sequence matches that of <i>Drosophila</i>, with three laminin-type EGF-like domains (blue ovals), and a netrin-like domain (red rectangle). B) The type, number and order of protein domains in <i>Drosophila</i> Frazzled, isoform A (AAF58493) and the putative <i>G</i>. <i>bimaculatus</i> Frazzled (TRINITY_DN172587_c0_g1_i7) was assessed using a conserved domain search. Frazzled had at-least two Ig-like domains (purple) and 6 Fibronectin Type III domains. The predicted <i>G</i>. <i>bimaculatus</i> protein was similar, though some sequence was missing from the N-terminal end (angled lines).</p

Summary of <i>G</i>. <i>bimaculatus</i> prothoracic ganglia samples and their Illumina sequencing.

<p>Summary of <i>G</i>. <i>bimaculatus</i> prothoracic ganglia samples and their Illumina sequencing.</p

Plexin proteins predicted from the <i>G</i>. <i>bimaculatus</i> transcriptome share most of the functional domains found in <i>Drosophila</i> Plexin proteins.

<p>A) The type, number, and order of protein domains was assessed using <i>Drosophila</i> Plexin A, isoform A (AAF59394) and the putative <i>G</i>. <i>bimaculatus</i> Plexin A (TRINITY_DN159247_c0_g1_i2) as queries in a conserved domain search. Both proteins possessed a sema domain (red rectangle), three plexin repeats (blue rectangle), four IPT domains (Ig, Plexin Transcription factor domains; green circles), and a rasGAP domain (purple rectangle). B) The type, number, and order of protein domains was assessed by using <i>Drosophila</i> Plexin B, isoform B (AFH06760) and the putative <i>G</i>. <i>bimaculatus</i> Plexin B (TRINITY_DN161683_c0_g1_i2) as queries in a conserved domain search. Both proteins possessed a Sema domain (red rectangle) two or three plexin repeats (blue rectangles), three or four IPT domains (Ig, Plexin Transcription factor domains; green circles), and a rasGAP domain (purple rectangle). Diagonal lines indicate sequence missing from the N-terminal end of the <i>G</i>. <i>bimaculatus</i> protein.</p

Semaphorin proteins predicted from the <i>G</i>. <i>bimaculatus</i> transcriptome share most of the functional domains found in <i>Drosophila</i> Sema proteins.

<p>A) The type, number, and order of protein domains was assessed using <i>Drosophila</i> Sema1a, isoform G (AGB92801) and the putative <i>G</i>. <i>bimaculatus</i> Sema1a (TRINITY_DN169697_c2_g1_i3) as queries in a conserved domain search. Both proteins possessed a sema domain (red rectangle) and a plexin repeat (blue rectangle). Diagonal lines indicate sequence missing from the N- and C-terminal ends of the predicted <i>G</i>. <i>bimaculatus</i> protein. B) The type, number, and order of protein domains was assessed using <i>Drosophila</i> Sema2a, isoform E (AAS64837) and the putative <i>G</i>. <i>bimaculatus</i> Sema2a (TRINITY_DN173773_c2_g2_i6) as queries in a conserved domain search. Both proteins possessed a sema domain (red rectangle) and an Ig domain (purple). <i>Drosophila</i> Sema2a, isoform E possessed a plexin repeat (blue rectangle) which was missing in <i>G</i>. <i>bimaculatus</i>. C) The type, number, and order of protein domains was assessed using <i>Drosophila</i> Sema5C, isoform B (ACZ94701) and the putative <i>G</i>. <i>bimaculatus</i> Sema5 (TRINITY_DN165105_c1_g2_i8) as queries in a conserved domain search. Both proteins possessed a sema domain (red rectangle) and a plexin repeat (blue rectangle). <i>Drosophila</i> Sema5c isoform B had six thrombospondin type 1 domains while the predicted Sema5 in <i>G</i>. <i>bimaculatus</i> had just four (light blue ovals).</p

<i>G</i>. <i>bimaculatus</i> prothoracic ganglion transcriptome assembly statistics.

<p><i>G</i>. <i>bimaculatus</i> prothoracic ganglion transcriptome assembly statistics.</p

Developmental guidance molecules mined from adult prothoracic transcriptome.

<p>Developmental guidance molecules mined from adult prothoracic transcriptome.</p

Summary of the results of mapping RNA-Seq reads to the complete <i>G</i>. <i>bimaculatus</i> prothoracic ganglion assembly.

<p>Summary of the results of mapping RNA-Seq reads to the complete <i>G</i>. <i>bimaculatus</i> prothoracic ganglion assembly.</p