Novel polyclonal antibodies as a useful tool for expression studies in amphioxus embryos

Abstract

Cephalochordates, commonly called amphioxus or lancelets, are widely regarded as a useful proxy for the chordate ancestor. In recent decades, expression patterns of important developmental genes have been used extensively to infer homologies between cephalochordate and vertebrate embryos. Such comparisons provided important insight into cephalochordate biology and the origin of vertebrate traits. Most of the developmental expression data are collected using whole-mount in situ hybridization that allows the distributions of specific transcripts to be detected in fixed embryos. Here, we describe an experimental pipeline for production of small amounts of functional antibodies directed against amphioxus antigens for use in immunohistochemical labelling. In this pilot study, we generated antibodies against b-catenin and the transcription factors FoxA, Lhx1, Lhx3 and Pax6. We demonstrate the usefulness of antibodies by performing immunostainings on fixed specimens of B. lanceolatum and B. floridae. We anticipate that amphioxus-specific antibodies will provide a useful tool for high-resolution labelling of individual cells within the embryo and for determining the subcellular localization of the corresponding proteins. KEY WORDS: Branchiostoma, amphioxus, antibody, expression patternCephalochordates, commonly called amphioxus or lancelets, are regarded as a key animal group for understanding the origin of vertebrates, and a useful proxy to the ancestral chordate condition. This position has recently been affirmed especially thanks to the access to genome sequence data (Holland et al., 2008;Huang et al., 2014;Putnam et al., 2008), introduction of novel techniques (Acemel et al., 2016;Kozmikova and Kozmik, 2015;Li et al., 2017;Yue et al., 2016), and establishment of amphioxus as a model species for evolutionary developmental studies (for review see Bertrand and Escriva (2011)). Cephalochordates include three genera, namely Branchiostoma, Asymmetron and Epigonychtys. The phylogenetic relationships within the extant amphioxus lineage were recently investigated providing divergence time estimates and suggesting a rather recent diversification (Igawa et al., 2017). For example, the estimated divergence times among species within the Branchiostoma genus (22.6 +/-2.3 Mya for B.lanceolatum -B. floridae split) are comparable to those among rodents belonging to Muridae family (such as mouse and rat). Close phylogenetic relationship is mirrored by a high degree of coding sequence , which permits you to Share (copy and redistribute the material in any medium or format) and Adapt (remix, transform, and build upon the material for any purpose, even commercially), providing you give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. Printed in Spain Abbreviations used in this paper: TH, tyrosine hydroxylase; WMISH, whole mount in situ hybridization.identity (Holland et al., 2008;H...

Molecular Fingerprint of Amphioxus Frontal Eye Illuminates the Evolution of Homologous Cell Types in the Chordate Retina

“…Unfortunately, we were not able to detect c-opsins expression in putative photoreceptors of B. lanceolatum when using antibodies generated against B. floridae c-opsins ( Supplementary Figure 2 ). This was probably due to low amino acid sequence conservation of opsins between B. lanceolatum and B. floridae [( Pantzartzi et al, 2018 ); discussed also in Bozzo et al (2017) ]. Taken together, molecular fingerprint of B. lanceolatum and B. floridae frontal eye is spatially conserved.…”

“…Amphioxus-specific antibodies were prepared as described in details earlier in Bozzo et al (2017). Briefly, selected protein coding sequences were cloned into pET42a(+) vector (Novagen), sequenced and introduced into BL21 (DE3) RIPL bacteria (Stratagen).…”

“…The following "home-made" primary antibodies directed against amphioxus proteins were used (dilution of antibody is indicated): mouse anti-Otx (1:500) (Vopalensky et al, 2012), rabbit anti-Otx (1:800) (Vopalensky et al, 2012), mouse anti-Pax4/6 (1:500), rabbit anti-Pax4/6 (1:500) (Vopalensky et al, 2012), mouse anti-Pax2/5/8 (1:500), mouse anti-Brn3 (1:500), mouse anti-Brn1/2/4 (1:500), mouse anti-Mitf (1:500), rabbit anti-Mitf (1:500) (Vopalensky et al, 2012), mouse anti-Lhx1 (1:500) (Bozzo et al, 2017), mouse anti-Lhx3 (1:500) (Bozzo et al, 2017), mouse anti-c-opsin1 (1:250) (Vopalensky et al, 2012), mouse anti c-opsin3 (1:250) (Vopalensky et al, 2012). The specificity of "home-made" antibodies against Lhx1, Lhx3, Brn3, and Brn1/2/4 was validated as previously described (Vopalensky et al, 2012) by Western blotting (Supplementary Figure 8) and by immunostaining with antibody pre-adsorbed with antigen (Supplementary Figure 9).…”

Functional Conservation and Genetic Divergence of Chordate Glycinergic Neurotransmission: Insights from Amphioxus Glycine Transporters

“…3-N-X), GlyT transcripts were present in the most anterior part of the cerebral vesicle at the level of the frontal eye complex (Fig. 3N,O), which at this stage contains glutamatergic and glycinergic neurons [61,62], and in pairs of ventrolateral cells of the rhombospinal region of the nerve cord (Fig. 3Q-U).…”

Optical Clearing and Light Sheet Microscopy Imaging of Amphioxus

“…In this study, anti-acetyl-α-tubulin (1:500) and anti-melanopsin (1:250) were used. Mouse polyclonal antibody was generated against amphioxus melanopsin as described by Bozzo et al (2017) using a peptide derived from the C-terminus of B. floridae protein.…”