Differential Responses to Wnt and PCP Disruption Predict Expression and Developmental Function of Conserved and Novel Genes in a Cnidarian

Abstract

We have used Digital Gene Expression analysis to identify, without bilaterian bias, regulators of cnidarian embryonic patterning. Transcriptome comparison between un-manipulated Clytia early gastrula embryos and ones in which the key polarity regulator Wnt3 was inhibited using morpholino antisense oligonucleotides (Wnt3-MO) identified a set of significantly over and under-expressed transcripts. These code for candidate Wnt signaling modulators, orthologs of other transcription factors, secreted and transmembrane proteins known as developmental regulators in bilaterian models or previously uncharacterized, and also many cnidarian-restricted proteins. Comparisons between embryos injected with morpholinos targeting Wnt3 and its receptor Fz1 defined four transcript classes showing remarkable correlation with spatiotemporal expression profiles. Class 1 and 3 transcripts tended to show sustained expression at “oral” and “aboral” poles respectively of the developing planula larva, class 2 transcripts in cells ingressing into the endodermal region during gastrulation, while class 4 gene expression was repressed at the early gastrula stage. The preferential effect of Fz1-MO on expression of class 2 and 4 transcripts can be attributed to Planar Cell Polarity (PCP) disruption, since it was closely matched by morpholino knockdown of the specific PCP protein Strabismus. We conclude that endoderm and post gastrula-specific gene expression is particularly sensitive to PCP disruption while Wnt-/β-catenin signaling dominates gene regulation along the oral-aboral axis. Phenotype analysis using morpholinos targeting a subset of transcripts indicated developmental roles consistent with expression profiles for both conserved and cnidarian-restricted genes. Overall our unbiased screen allowed systematic identification of regionally expressed genes and provided functional support for a shared eumetazoan developmental regulatory gene set with both predicted and previously unexplored members, but also demonstrated that fundamental developmental processes including axial patterning and endoderm formation in cnidarians can involve newly evolved (or highly diverged) genes.

Diversity of Cnidarian Muscles: Function, Anatomy, Development and Regeneration

“…Those tools, in combination with “omics” and functional genomics approaches (Momose and Houliston, 2007; Rentzsch et al, 2008; Amiel et al, 2009; Chera et al, 2009; Genikhovich and Technau, 2009; Boehm et al, 2012; Röttinger et al, 2012; Layden et al, 2013; Lapébie et al, 2014; Bradshaw et al, 2015) as well as with the recently developed techniques for genome editing (Ikmi et al, 2014) are now opening new opportunities to functionally and thoroughly address the developmental and regenerative program of cnidarian muscles systems, but also the role(s) that epitheliomuscular cells, muscle fibers and muscle contraction can play on the regeneration process.…”

A G-protein-coupled receptor mediates neuropeptide-induced oocyte maturation in the jellyfish Clytia

“…From a comprehensive Clytia reference transcriptome (86,606 contigs) derived from mixed larva, polyp and jellyfish samples,we generated a list of predicted protein sequences from complete and incomplete ORFs using a homemade script [58]. This dataset was screened using TMHMM 2.0c to produce a list of Clytia complete protein sequences coding for 7 transmembrane domain proteins (7TMD) including putative incomplete sequences containing 2 to 7 transmembrane domains.…”

A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species

“…The protocol was adapted from (Lapébie et al, 2014) and from Takeda et al (in preparation) with regard to the chromogenic (CISH) and the fluorescent in situ hybridization (FISH), respectively.…”

“…The in situ hybridization technique is a well-established method for RNA detection in the hydrozoan Clytia hemisphaerica, and has proven valuable for assessing gene expression during embryonic development, oogenesis, and in adult structures such as in the tentacle bulb (Chevalier et al, 2006;Denker et al, 2008;Leclère et al, 2012;Lapébie et al, 2014;Kraus et al, 2015).…”