Transcriptional profiling of identified neurons in leech

Elizabeth Heath-Heckman,Diana Bautista,Veronica B Lammardo,James Angstadt,Francisco F De-Miguel,Shinja Yoo,Maurizio Pellegrino,David Weisblat,Christopher Winchell

doi:10.1186/s12864-021-07526-0

Abstract

BackgroundWhile leeches in the genus Hirudo have long been models for neurobiology, the molecular underpinnings of nervous system structure and function in this group remain largely unknown. To begin to bridge this gap, we performed RNASeq on pools of identified neurons of the central nervous system (CNS): sensory T (touch), P (pressure) and N (nociception) neurons; neurosecretory Retzius cells; and ganglia from which these four cell types had been removed.ResultsBioinformatic analyses identified 3565 putative genes whose expression differed significantly among the samples. These genes clustered into 9 groups which could be associated with one or more of the identified cell types. We verified predicted expression patterns through in situ hybridization on whole CNS ganglia, and found that orthologous genes were for the most part similarly expressed in a divergent leech genus, suggesting evolutionarily conserved roles for these genes. Transcriptional profiling allowed us to identify candidate phenotype-defining genes from expanded gene families. Thus, we identified one of eight hyperpolarization-activated cyclic-nucleotide gated (HCN) channels as a candidate for mediating the prominent sag current in P neurons, and found that one of five inositol triphosphate receptors (IP3Rs), representing a sub-family of IP3Rs absent from vertebrate genomes, is expressed with high specificity in T cells. We also identified one of two piezo genes, two of ~ 65 deg/enac genes, and one of at least 16 transient receptor potential (trp) genes as prime candidates for involvement in sensory transduction in the three distinct classes of leech mechanosensory neurons.ConclusionsOur study defines distinct transcriptional profiles for four different neuronal types within the leech CNS, in addition to providing a second ganglionic transcriptome for the species. From these data we identified five gene families that may facilitate the sensory capabilities of these neurons, thus laying the basis for future work leveraging the strengths of the leech system to investigate the molecular processes underlying and linking mechanosensation, cell type specification, and behavior.

Highlights

While leeches in the genus Hirudo have long been models for neurobiology, the molecular underpinnings of nervous system structure and function in this group remain largely unknown
Each neuronal phenotype exhibits a distinctive transcriptional profile To determine the transcriptional profile of the four cell types, we first created a reference transcriptome by combining the RNA-Seq libraries made from pools of identified T, P, N, and Rz neurons, and libraries made from the remainder of the ganglion after dissection of the four cell types, hereinafter referred to as ganglion-minus (Gm)
To explore the extent to which the neuronal markers identified in Hirudo may be applicable to other leech species, we identified Helobdella orthologs for several of the differentially expressed Hirudo genes described above, and performed in situ hybridization (ISH) for their transcripts on Helobdella embryos at stage 10–11 of development, by which time the nervous system is fairly well differentiated and yet ISH can be carried out on intact embryos without dissection (Fig. S2)

Summary

Introduction

While leeches in the genus Hirudo have long been models for neurobiology, the molecular underpinnings of nervous system structure and function in this group remain largely unknown. The broad outlines of how this is achieved through cascading interactions of unequal cell divisions and inherited determinants, intercellular signaling, and transcriptional networks is understood, but numerous questions remain. Nowhere is this more evident than in the nervous system, where the diversity of morphologically defined cell types is further complicated by molecular and physiological distinctions [1,2,3]. Large scale transcriptional profiling at the single cell level (scRNAseq) is a powerful approach to this problem for complex vertebrate nervous systems, but at present this approach suffers from major limitations. While spatially resolved transcription profiling techniques are emerging [4], these approaches would be expected to reduce sensitivity to transcriptional differences even further than standard single-cell approaches

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