In situ hybridization of neuropeptide-encoding transcripts afp-1, afp-3, and afp-4 in neurons of the nematode Ascaris suum

Abstract

The gene transcripts encoding both the AF8 and AF2 neuropeptides of the nematode Ascaris suum have been identified, cloned, and sequenced. The AF8 transcript (afp-3) encodes five identical copies of AF8; each peptide-encoding region is flanked by the appropriate dibasic or monobasic cleavage processing sites. The AF2 transcript (afp-4) encodes three identical copies of AF2 along with the appropriate cleavage sites. In contrast, the afp-1 transcript (Edison et al. [1997] Peptides 18:929-935) encodes six different AF peptides (AF3, 4, 10, 13, 14, 20) which all share a -PGVLRFamide C-terminus but have different N-terminal sequences. By using in situ hybridization, gene transcript expression patterns of afp-1, afp-3, and afp-4 (As-flp-18, As-flp-6, and As-flp-14, respectively, in the naming convention proposed by Blaxter et al. [1997] Parasitol Today 13:416-417) were determined in the adult A. suum anterior nervous system. Each gene transcript can be localized to a different subset of neurons. These subsets of neurons are different from the subsets of Caenorhabditis elegans neurons that were shown to express identical or similar peptides by the use of promoter GFP constructs (Kim and Li [2004] J Comp Neurol 475:540-550).

Figure 1

The nucleotide sequence and the deduced amino acid sequence of the afp-3, afp-4, and afp-1 cDNAs. The riboprobe’s targeted regions are indicated by the bold nucleotides. The putative start sites are in parentheses. The peptide-encoding amino acid sequences are bold and underlined. The peptide-forming cleavage sites are in italics. The bold forward slash indicates the predicted signal peptide cleavage site. An SL1 spliced leader nucleotide sequence is present and underlined in afp-4 and afp-1. A dash signifies a stop codon. Numbering of sequences is bold for amino acids, and unbold for nucleotides. A: (top panel) AF8-encoding cDNA (GenBank Access. no. AY386833). B: (middle panel) AF2-encoding cDNA (GenBank Access. no. AY386834). C: (bottom panel) afp-1 cDNA (Edison et al., 1997; GenBank Access. no. U15279).

Figure 2

Diagrams of the head ganglia of A. suum, modified from Goldschmidt (1908). The preparation has been split near the dorsal axis and opened flat. Neuronal cell bodies and commissural processes are shown. The nerve ring (NR), ventral ganglion (VG), dorsal ganglion (DG), lateral ganglia, and retrovesicular ganglion (RVG) are indicated. LLL: left lateral line; RLL: right lateral line; VC ventral nerve cord; DC: dorsal nerve cord; DeC: deirid commissures; AC: amphidial commissures. The central diagram is an overall view of the head ganglia. Surrounding boxes are magnifications of individual ganglia showing the location of identified neurons. The DG contains ALA and RID (RMED is in the nerve ring). The VG contains the identified neurons AVK, RIS, AIY and AIM (not yet distinguished from each other), RIR, AVL, and RIH (RMEV is in the nerve ring). The RVG contains two AVFs, two RIFs, RIG, and interneuron B (INTB). The lateral ganglia contain the “large” neuron and ADE, the anterior deirid.

Figure 3

ISH with afp-1 antisense probe. A: A single neuron, RID, is stained in the dorsal ganglion (DG). In the left lateral ganglia (LG) the “large” neuron (long arrow) is heavily stained and a medium neuron near the amphidial commissure (medium arrow) is lightly stained. In this preparation a spherical neuron near the nerve ring is lightly stained (arrowhead). B: The same neurons are stained in the right lateral ganglion, as are five neurons in the ventral ganglion (VG). A and B are from the same preparation. C: The RIG neuron at the posterior end of the RVG is heavily stained and four other neurons are more lightly stained. D: The second RIG neuron, located posterior to the RVG in the ventral cord, is also heavily stained. E,F: Control ISH with afp-1 sense strand probe. VG and LG: E; RVG: F. No neurons are stained. Scale bars = 100 μm.

Figure 4

ISH with afp-4 (AF2) antisense probe. A: The RID neuron in the dorsal ganglion (DG) is heavily stained. The ventral ganglion (VG) has two lightly stained neurons. The lateral ganglia (LG) have staining in the “large” neuron (short arrow) and in a small neuron near the amphidial commissure (long arrow). B: In a different preparation, the VG and one LG, containing the same stained neurons as in A, but with additional lightly stained neurons in the VG and LG. C: Retrovesicular ganglion (RVG), showing intense staining of the pair of RIF neurons and light staining of four other neurons. D: Control ISH with AF2 sense probe. No neuronal staining is seen. Scale bars = 100 μm.

Figure 5

ISH with afp-3 (AF8) antisense probe. A: The ALA neuron is heavily stained in the dorsal ganglion (DG). B: A pair of small cells in the anterior part of the ventral ganglion (VG) is lightly stained (arrows), as was a pair of neurons in the lateral ganglion (LG) near the nerve ring (arrow; only one LG is shown). Scale bars = 100 μm.

Figure 6

ISH with both afp-3 and afp-4 (AF2 and AF8) antisense probes. The RID and the ALA neurons are both heavily stained in the dorsal ganglion (DG). Scale bar = 100 μm.