Functional divergence of paralogous transcription factors supported the evolution of biomineralization in echinoderms
- Carnegie Mellon University, United States
Figures
Figure 1
Comparison of the predicted amino acid sequences of Alx1 across the echinoderms.
(A) Clustal Omega alignment of the Alx1 sequences from Lytechinus variegatus (LvAlx1), Strongylocentrotus purpuratus (SpAlx1), Eucidaris tribuloides (EtAlx1), Parastichopus parvimensis (PpAlx1), Pati…
Figure 2 with 2 supplements
Expression of MO-resistant LvAlx1 rescues PMC specification in LvAlx1 morphants.
(A) Six silent mutations were introduced into the MO target site to generate a MO-resistant Lv-alx1 mRNA (LvAlx1.WT.GFP). Boxed nucleotides indicate changes introduced. A mutant form of …
Figure 2—figure supplement 1
Quantitative analysis of MO rescue experiments.
Each vertical bar indicates the mean percentage of 6a9-positive cells relative to uninjected control embryos, calculated from 80 to 100 embryos. Vertical lines represent standard errors. An average …
Figure 2—figure supplement 2
LvAlx1 with premature stop codons, LvAlx1 C region mutants, LvAlx4 and PmAlx1 are expressed at levels similar to that of LvAlx1.WT.GFP.
(A) Fluorescent images showing expression of LvAlx1.GFP proteins in living embryos at 4 hpf. The dotted line indicates the outline of embryo injected with LvAlx1.3STOPS.GFP, which cannot be …
Figure 3 with 1 supplement
The N-terminus is dispensable for LvAlx1 function in PMC specification and patterning.
(A) Constructs generated with different N-terminal deletions and truncations. (B–G) Embryos fixed at the mid-late gastrula stage and labelled with 6a9 antibody. (B’–G’) DIC/fluorescence composite …
Figure 3—figure supplement 1
The putative MAPK phosphorylation site T28 is not essential for PMC specification or patterning.
(A) Alignment of part of the N-terminal region of Alx1 from L. variegatus (LvAlx1), S. purpuratus (SpAlx1) and E. tribuloides (EtAlx1). Boxed region indicates protein sequences that conform to the …
Figure 4
The C-terminal region contains motifs essential for Alx1 function.
(A) Constructs generated with different C-terminal deletions and truncations. (B–G) Embryos fixed at the mid-late gastrula stage and labelled with 6a9 antibody. (B’–G’) DIC/fluorescence composite …
Figure 5
Domain 2 is essential for Alx1 function.
(A) Constructs with deletions within the C-terminal region. (B–I) Embryos fixed at the mid-late gastrula stage and labelled with 6a9 antibody. (B’–I’) DIC/fluorescence composite images of the same …
Figure 6 with 2 supplements
Determination of a minimal construct sufficient to rescue LvAlx1 morphants.
(A) Constructs tested. All mutant constructs contained the homeodomain and Domain 2, both of which were required for PMC specification. (B–G) Embryos fixed at the mid-late gastrula stage and …
Figure 6—figure supplement 1
Basic residues flanking the LvAlx1 homeodomain function as nuclear localization signals.
(A) Top: Clustal Omega alignment of Alx1 homeodomains from L. variegatus (LvAlx1), S. purpuratus (SpAlx1), E. tribuloides (EtAlx1), P. miniata (PmAlx1), P. parvamensis (PpAlx1), H. sapiens (HsAlx1), …
Figure 6—figure supplement 2
Rescued embryos show normal skeleton development at late embryonic stages.
Embryos were visualized using DIC optics. (A–F) Prism stage embryos (ventral views). Arrowheads indicate spicule primordia. (H–M) Early pluteus stage embryos (posterior views). Arrowheads indicate …
Figure 7
LvAlx1 and LvAlx4 are not functionally redundant; however, insertion of LvAlx1 Domain 2 into LvAlx4 is sufficient to confer skeletogenic function.
(A) Clustal Omega alignment of the predicted amino acid sequences of LvAlx4 and LvAlx1. (C–I) Embryos fixed at the mid-late gastrula stage and labelled with 6a9 antibody. (C’–I’) DIC/fluorescence …
Figure 8
Alx1 proteins from closely related echinoderm species, but not from distantly related species, are functionally interchangeable with LvAlx1.
(A) Three silent mutations (solid boxes) were introduced into the S. purpuratus alx1 translational start site to generate a form of Sp-alx1 mRNA (SpAlx1.WT.GFP) that was resistant to the LvAlx1 MO. …
Figure 9
Evolution of echinoderm alx gene structure.
Left side: molecular phylogeny of echinoderm Alx1 and Alx4 proteins (based on Koga et al., 2016), with a hemichordate as an outgroup. Branch lengths are arbitrary. Right side: intron-exon …
Figure 10
Inclusion of Domain 2 is regulated by alternative splicing.
(A) Agarose gel showing different LvAlx1 splice forms. Wild-type LvAlx1 is the predominant splice form throughout embryonic development. MB: mesenchyme blastula; EG: early gastrula; LG: late …
Figure 11
Model representing the evolution of L. variegatus alx genes.
Following gene duplication, the ancestral Lv-alx1 underwent rapid evolution through multiple intron gains and more importantly, acquired Domain 2 through exonization of previously non-coding …
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.32728.018
