The first vertebrate mineralized skeleton was an external bony armor coated with dental structures. The subsequent emergence of a mineralized endoskeleton and of teeth are considered key innovations in the diversification of vertebrates. Although time clouds our understanding of the initial evolution of these mineralized structures, recent re-emergences may shed light on the underlying processes. Loricarioid catfishes are a lineage that, much like the ancestral vertebrates, bear denticle-clad bony armor from head to tail. Loricarioid denticles (LDs) and oral teeth are very similar in superstructure. We show here that other extra-oral dental structures are found as ancestral characters only in lineages that are distantly related to loricarioids such as sharks or coelacanth, indicating that LDs have independently re-emerged in loricarioid catfishes. We investigate whether the similarities between LDs and teeth extend to their developmental and genetic context, and how their development compares to that of other vertebrate integument structures. Our detailed study of the development of LDs, and gene expression analyses through in situ hybridization confirm that all 12 genes from the tooth-forming gene regulatory network (oGRN) are expressed in developing LDs in a similar way as they are expressed in developing teeth. We then compare the developmental, structural, and genetic aspects of LD and teeth with that of other integument appendages such as fish scales, shark dermal denticles, feathers and hairs. We find that LDs share all developmental cues with teeth and, to a lesser extent, with the other vertebrate integument structures. Taken together, our results indicate that denticles have re-emerged on the trunk of loricarioid catfishes through the ectopic co-option of the oGRN rather than the resurrection of an ancestral trunk-specific denticle genetic pathway.

Sample fixation

All embryos were collected directly from the aquarium in which they were laid, at the desired age (2-6 dpf). Unhatched embryos (younger than 5 dpf) were dechorionated by hand, with forceps. All embryos were euthanized in a tank containing a lethal concentration (400g/ml) of tricaine methanesulfonate (MS-222), then fixed in 4% paraformaldehyde (PFA) pH 7.42 for at least 24 h at 4° C. Then, they were dehydrated in a series of methanol solutions and stored in 100% methanol at -20° C.

Sequencing and assembly of the embryonic transcriptome of A. triradiatus

Two embryos of 3, 4, and 5 dpf of Ancistrus triradistus were collected, pooled and their total RNA was extracted using the Trizol reagent. The mRNA was then purified using the Oligotex mRNA Mini Kit (Qiagen 70022). Library preparation, quality control and Illumina NGS sequencing was performed by the Genomics Platform of the University of Geneva (http://www.ige3.unige.ch/genomics-platform.php). Sequencing was performed on a HiSeq 2500 (Illumina) which generated 60 million single reads of 200bp.

Raw data still have Illumina adapters and low quality bases. A cleanup with Cutadapt (Martin and Wang 2011) is recommended.