DOI: 10.5061/dryad.rr4xgxdc4
title: Cephalopod retinal development shows vertebrate-like mechanisms of neurogenesis: Multiple sequence alignments and phylogenetic trees

description: Coleoid cephalopods, including squid, cuttlefish and octopus, have large
 and complex nervous systems and camera-type eyes that are comparable only
 to features that have independently evolved in the vertebrate lineage. The
 changes in development that result in the evolution of nervous system size
 and diversity of neural cell-types are not well understood. Here, we have
 pioneered live-imaging techniques and performed functional interrogation
 to show the squid, Doryteuthis pealeii, utilizes mechanisms during retinal
 neurogenesis that are hallmarks of vertebrate processes. Given the
 convergent evolution of elaborate visual systems in cephalopods and
 vertebrates, these results reveal common mechanisms that underlie the
 growth of highly proliferative neurogenic primordia that may alter
 ontogenetic allometry and contribute to the evolution of complex nervous
 systems.
description: Genes were first identified by using annotated sequences from
 model organisms from major lineages for BLAST (Altschul et al., 1990) into
 a custom local database of the <em>D</em>.
 <em>pealeii</em> transcriptome in Geneious. For top hits the
 entire sequence in the <em>D</em>.
 <em>pealeii</em> transcriptome was retrieved, the longest ORF
 was extracted and translated, then the amino acid sequence was trimmed for
 coding sequence. To find related sequences, BLASTp was used, searching
 both the Uniprot database in NCBI and retrieving only select vertebrate
 and <em>D</em>. <em>melanogaster</em> hits. BLASTp
 was performed again using the non-redundant protein database, and
 searching specifically for cephalopods, select mollusks, and
 <em>Limulus</em>. Trees that were not well resolved after
 these steps required an additional round of BLASTp, this time including
 more spiralian and ecdysozoan hits. Full sequences (or as long as is
 available) were aligned with our <em>D</em>.
 <em>pealeii</em> sequences for each tree using MAFFT v.7.450
 in Geneious (Katoh, 2013). The only exception was our Sox tree where we
 used the alignment from (Schnitzler et al., 2014), which only included the
 HMG box of Sox proteins. This alignment focused on early metazoan species,
 so we added select vertebrates, mollusks, and ecdysozoans as described
 above, but trimmed sequences to include the HMG box for all. For all
 alignments we checked sequence redundancy and proper outgroups Fast Trees
 were made using FastTree2 v.2.1.11 (Price et al., 2010). We constructed
 maximum-likelihood trees on the FASRC Cannon cluster supported by the FAS
 Division of Science Research Computing Groat Harvard University. We
 exported relaxed Phylip formatted alignment files and used IQ-TREE 2
 v.2.1.0 with the following settings: iqtree2 -s ALIGNMENT.phy -st AA -nt
 AUTO -v -m TEST -bb 1000 -alrt 1000 (Minh et al., 2020). Unrooted trees
 were visualized as rooted by known outgroups and labeled by known
 annotated orthologues.
description: <strong>Supplemental Data Files</strong> <strong>MAFT Multiple Sequence Alignments:</strong> Delta_jagged.phy EGFR_EphR.phy ELAV.phy Fringe.phy HES.phy NeuroD_G.phy RAR.phy Retinochrome.phy Sox.phy VEGFR.phy   <strong>IQ Phylogenetic Trees:</strong> 211019_Retinochrome.phy.treefile 211020_Delta_jagged.phy.treefile 211027_Fringe.phy.treefile 211027_VEFGR.phy.treefile 211105_EGFR_EphR.phy.treefile 211105_ELAV.phy.treefile 211105_HES.phy.treefile 211105_NeuroD_G.phy.treefile 211112_RAR.phy.treefile 211112_Sox.phy.treefile