Fig 1.
Maximum likelihood (ML) tree of Alx proteins.
The tree was constructed using conserved amino acid sequences. The numbers on the nodes indicate the bootstrap values (1,000 pseudoreplicates). Values greater than 50% are shown. Ak: Amphipholis kochii (brittle star); Ap: Asterina pectinifera (starfish); Bf: Branchiostoma floridae (amphioxus); Dr: Dario rerio (zebrafish); Hl: Holothuria leucospilota (sea cucumber); Hp: Hemicentrotus pulcherrimus (sea urchin); Hs Homo sapiens (Human); Mr: Metacrinus rotundus (sea lily); Pm: Patiria (Asterina) miniata (starfish); Sk: Saccoglossus kowalevskii (acorn worm); Sp: Strongylocentrotus purpuratus (sea urchin).
Fig 2.
The expression patterns of alx genes in ambulacraria.
(A–D) The expression pattern of starfish alx1 (Apalx1). (A–C) Expression was barely evident prior to the bipinnaria stage. (D) Expression was evident in mesenchyme cells of the posterior region of late bipinnarial larvae. (E-H) The expression pattern of brittle star alx1 (Akalx1). (E) Expression was evident in the putative vegetal plate of the blastula (F) and in mesenchyme cells that ingressed from the vegetal plate. (G) At the gastrula stage, expression was evident in mesenchyme cells located bilaterally to the archenteron. (H) In plutei, expression was evident in mesenchyme cells located along the arms. (I-K) The expression pattern of sea cucumber alx1 (Hlalx1). (I) Expression was not evident prior to the gastrula stage. (J) In mesenchyme of the gastrula, expression was evident in a few cells located in the posterior larval region. (K) In auricularia larvae, expression was evident in mesenchyme cells. (L-N) The expression pattern of starfish Calx (ApCalx). (L) Expression was evident in the vegetal plate of the blastula. (M) Expression was noted at the tip of the archenteron of the gastrula. (N) In early bipinnariae, expression persisted in the coelom and de novo expression was evident in the posterior enterocoel that invaginated from the left side of the gut. (O-Q) The expression pattern of sea urchin Calx (HpCalx). Red fluorescent signals indicate HpCalx transcripts and blue signals denote nuclei stained by DAPI. (O) Expression was evident in ingressed primary mesenchyme cells (PMCs) of the blastula mesenchyme. (P) In late-stage gastrulae, signals disappeared from PMCs but were observed in secondary mesenchyme cells (SMCs) located around the tip of the archenteron. (Q) Expression was restricted to the coelomic pouches of prism larvae. (R, S) The expression pattern of acorn worm alx (Bsimalx). (R) In the mid-gastrula, expression was evident in cells at developing protocoel. (S) At a later stage of gastrula, expression was observed in the proximal part of the protocoel. Scale bars: 50 μm.
Fig 3.
Differentially expressed (DE) genes after the injection of alx1 mRNA into starfish.
(A) MAplot of DE genes identified by RNAseq. Data represent individual putative gene in a log2 ratio versus log2 average plot. Red plots indicate DE genes with q value (FDR) < 0.001. (B) Relative expression levels in 24-h-old embryos were calculated via real-time PCR. The data show–ΔΔCt values relative to the embryos in which EGFP mRNA was overexpressed. The expression levels were normalized to that of ApEF1a. Data from three trials using different batches of eggs are shown. Black points indicate experimental data derived using sea urchin Hpalx1, and gray points show data derived using starfish Apalx1. The horizontal bars indicate mean values. Asterisks indicate significant differences between ΔCt values of sample pairs (paired t-test with Bonferroni correction, p < 0.05).
Fig 4.
Expression patterns of putative skeletogenic genes in embryos expressing alx1.
Solutions 0.5 mg/ml in mRNA for Hpalx1 (A-D), 1 mg/ml in mRNA for Apalx1 (E-G) or EGFP (I-L) were injected into starfish eggs, which were next reared for 15 h (to the early gastrula stage) or 24 h (to the mid-gastrula stage). (A) App19 was detected in the presumptive mesoderm. (B) App19 expression was retained by mesoderm cells at the tip of the archenteron. (C) ApCA1 mRNA was also detected in the presumptive mesoderm. (D) ApCA1 expression was retained in the mesoderm of gastrulae. (E-H) The similar expression patterns to the sea urchin Hpalx1 overexpression were observed in the starfish Apalx1 overexpressed embryos. (I-L) Neither App19 nor ApCA1 was detectable in control embryos. Scale bars: 50μm. (M) Changes in the expression levels of skeletogenic gene orthologs upon injection of Hpalx1 mRNA into starfish. Relative expression levels in the 24-h-old embryos were measured via real-time PCR. The data show -ΔΔCt values relative to those of (control) EGFP-expressing embryos. The expression level of each gene was normalized to that of the ApEF1a gene. Three distinct trials with different batches of eggs were run. Asterisks indicate significant difference between ΔCt values of control and Hpalx1 overexpression (paired Student’s t-test, p < 0.05). Endogenous Apalx1 was highly upregulated upon ectopic Hpalx1 expression.
Table 1.
DE genes in Alx1 over-expressed embryos.
Fig 5.
The expression patterns of App19 and App16.
(A) Changes in the expression of App19 and App16 during larval development of starfish. The expression levels shown are relative to those of ApEF1a. (B–D) Spatial App19 expression patterns. (B) No expression was evident in the gastrula. (C) Signals were emitted from two rows of mesenchyme cells located in the posterior regions of the bipinnaria (an aboral view). (E) Signals were emitted from mesenchyme cells that were dispersed radially at the brachiolarial stage (an aboral view of the adult rudiment). Scale bars: 100μm.