Research ArticleDEVELOPMENTAL BIOLOGY

The ancestral retinoic acid receptor was a low-affinity sensor triggering neuronal differentiation

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Science Advances  21 Feb 2018:
Vol. 4, no. 2, eaao1261
DOI: 10.1126/sciadv.aao1261
  • Fig. 1 Molecular components of retinoid metabolism and signaling.

    Schematic representation of retinoid metabolism, storage, transport, and signaling in vertebrates. Metabolic enzymes, binding proteins, and nuclear receptors are shown in blue. Names in bold letters indicate the presence of a gene encoding the ortholog of a given vertebrate protein in the P. dumerilii genome. Italic lettering indicates the absence of the complete corresponding gene family in P. dumerilii. Names in regular letters indicate the presence, in P. dumerilii, of members of a given gene family but the absence of a specific P. dumerilii ortholog of the corresponding vertebrate gene. Of note, the involvement of ADH enzymes in retinal synthesis in vertebrates has been challenged (3).

  • Fig. 2 Molecular characterization of PduRAR.

    (A) DNA recognition by the PduRAR/PduRXR heterodimer. Both receptors were synthesized in vitro, and the heterodimer was allowed to bind to a 32P-labeled DR2 RARE probe. Cold competitors correspond to 10- or 100-fold excess of unlabeled oligonucleotides (DR0 to DR5). A nonspecific element (NS) was used as a negative control. Unprogrammed reticulocytes were used as control (Ctrl). Protein and labeled probe complexes are indicated by an arrow (shown only in the PduRAR/PduRXR lane). (B) The ability of PduRAR to activate the transcription of the luciferase reporter gene was tested in transfected human embryonic kidney (HEK) 293T cells in the presence of increasing concentrations (0.1, 1, and 10 μM) of ATRA, 9cRA, and 13cRA. The Gal4 DNA binding domain construct alone was used as a negative control (Ctrl). Bars are means ± SD (n = 3). (C) The ability of PduRAR to bind different ligands was tested by LPA. The ligands (ATRA, 9cRA, and 13cRA) were used at increasing concentrations (0.1, 1, and 10 μM). Ethanol was used as a negative control (lane −), and protected bands are indicated by arrows. (D) Competition assay using increasing concentrations of the RAR antagonist BMS493 (0.1, 1, and 10 μM) in the presence of 10 μM ATRA. The Gal4 DNA binding domain construct alone was used as a negative control (Ctrl). Bars are means ± SD (n = 3). (E) Binding of PduRAR to increasing concentrations of BMS493 (0.1, 1, and 10 μM) as assessed by LPA. Ethanol was used as a negative control (lane −), and protected bands are indicated by arrows. (F) Titration of the fluorescein-labeled interaction domains (ID1 and ID2) of the corepressors NCOR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone receptors) by unliganded PduRAR monitored by fluorescence anisotropy. Assays were performed in three independent experiments, and data are expressed as means ± SEM. (G) Titration of the fluorescein-labeled interaction domain 1 (ID1) of the corepressor NCOR by PduRAR in the presence of different retinoids: ATRA, 9cRA, and 13cRA. Assays were performed in three independent experiments, and data are expressed as means ± SEM.

  • Fig. 3 Crystal structure of the PduRAR LBD.

    (A) Superimposition of the four PduRAR LBDs (Mol A to Mol D) contained in the asymmetric unit. α Helices (H1 to H12) are labeled. The two ATRA molecules present in subunits B and D are shown as yellow sticks. (B) Superimposition of the LBPs of human RARα bound to the agonist AM580 (yellow) and of the PduRAR (Mol B) bound to ATRA (blue). The ATRA present in Mol D (which was superimposed on Mol B and is not shown) is displayed as green sticks. Human RARα R276 and S287 that are engaged in polar interactions with the carboxylate moiety of AM580 are shown. (C) Close-up view of the LBP of PduRAR bound to ATRA. Residues in contact with the ligand are shown and labeled. (D) Superimposition of the LBPs of human RARα bound to the agonist AM580 (yellow) and of the PduRAR (Mol B) bound to ATRA (blue) showing how the V356F mutation affects ligand binding to the annelid receptor. (E) Transcriptional activity of the PduRAR V356F mutant, in transfected HEK 293T cells, in the presence of increasing concentrations of ATRA (1, 5, and 10 μM). The GAL4 DNA binding domain alone was used as a negative control (Ctrl). Bars are means ± SD (n = 3). (F) Superimposition of the LBPs of human RARα bound to the agonist AM580 (yellow) and of the PduRAR (Mol B) bound to ATRA (blue). The van der Waals interactions between AM580 and helix H12 residues I410 and L414 are indicated as dotted lines. (G) Superimposition of the LBPs of human RARα bound to the agonist AM580 (yellow) and of the PduRAR (Mol B) bound to ATRA (blue), highlighting the smaller distances between equivalent residue positions in helices H3 and H11 in the annelid receptor when compared to vertebrate receptors.

  • Fig. 4 Expression of retinoid receptors (rar and rxr) in P. dumerilii.

    Gene expression is shown in blue. (A to E) P. dumerilii rar expression. (A to C) rar expression in forming neuroectoderm (ne), mesodermal precursor cells [dorsal longitudinal muscle (dlm) and mesodermal bands (mdb)], posterior growth zone (black arrowheads), and the brain region (br). (C) At 48 hpf, rar is expressed in neuroectoderm, in a region giving rise to motor neurons (yellow dashed circles), in the ventral midline (arrow, left) and subjacent cells (white arrowhead, right). (B to D) Stomodeal rar expression at 30 to 72 hpf (circles). (D) rar is expressed in two dorsally located domains of the brain at 72 hpf (white arrowheads). (E) At 6 dpf (days post-fertilization), rar is expressed in the midgut (mg) and posterior growth zone (black arrowhead). (F to I) P. dumerilii rxr expression. (F) At 24 hpf, rxr is expressed in the ventral midline (arrow) and two domains of the brain (black arrowheads). (G and H) In trochophore and nectochaete larvae, rxr is expressed in the brain (br) and the entire neuroectoderm (ne), including motor neuron domains (yellow dashed outline), and in the underlying mesodermal precursor cells (mdb) and stomodeum (circle). (I) By 5 dpf, rxr expression is restricted to two domains of the brain (arrowheads) and to tissues surrounding the midgut (mg). (J to M) Summary of the developmental expression in P. dumerilii of RA metabolism and signaling components. At 24 hpf (J), 48 hpf (K), and 72 hpf (L), aldh1a (green) genes are expressed in the blastoporal region and macromeres, whereas rar (purple) is in neuroectoderm and stomodeum. aldh1a and rar expression overlap in the forming mesodermal bands. (M) In the late nectochaete larva (5 to 6 dpf), aldh1a, cyp26, and rar expression overlap in the midgut (red). Ventral views of larvae are shown. Scale bars, 50 μm. * or circle, foregut; white dashed line, ciliated band; yellow dashed circles, motor neuron domain.

  • Fig. 5 Perturbation of RAR and RXR functions in P. dumerilii.

    (A to Q) Knockdown of rar and rxr by MO injections into P. dumerilii zygotes. (A, C, E, and G) rar and rxr MO injections cause a reduction of connectives (conn; white arrows) and misguidance of commissures (comm; yellow arrows) in the ventral nerve cord (VNC) as well as stomodeal defects including duplications (white dashed circle). (B, D, F, and H) Misplacement and reduction of larval serotonergic neurons (arrows), but not of neurons of the early nervous system (arrowheads), upon rar and rxr MO injections. (I) Histogram showing proportions of obtained knockdown phenotypes: normal development (“normal”), delayed development (“delay”), and VNC and stomodeum defects (“VNC & stomodeum defects”). (J and K) Box plots showing the number of serotonergic cells and neurons in the trunk of P. dumerilii larvae. Data distribution (circles), median values (bold line), and Tukey whiskers are shown. wt, wild type. Unpaired t test on the mean value was used for statistical analyses (n.s., nonsignificant). P = 0.3352 and P = 0.5842 in (J) and P = 0.0566 and P = 0.6064 in (K) for, respectively, rxr-5mm control MO versus rar MO and rxr-5mm control MO versus rxr MO. (L to O) Expression of the neuronal differentiation marker synaptotagmin (syt) (L and M) and of the motor neuron marker hb9 (N and O), showing that MO-induced rar knockdown disrupts differentiation in the larval nervous system (arrows), but not in the early larval nervous system (arrowheads). (P and Q) hox1 expression is unaffected by rar MO injections (arrowheads). (R to U) rar overexpression by mRNA injection into P. dumerilii zygotes. (R and S) Early larval nervous system is not affected by rar overexpression (arrows). (T and U) Injection of rar mRNA induces an increase of commissures in the larval nervous system (arrows). (V) Overview of embryonic and larval neurogenesis in P. dumerilii with respect to the differentiation (diff.) of functional neurons (29). Ventral views of larvae are shown. (A to H and R to U) Acetylated tubulin in green, serotonin in red, and nuclei in blue. (L to Q) Gene expression in blue. Number of affected over total number of assayed specimens is indicated. Scale bars, 50 μm.

  • Fig. 6 Effects of exogenous RA on developing P. dumerilii.

    (A to Q) Effects of ATRA and 13cRA on larval development (48 to 80 hpf). (A to E) Reduction of differentiation within the VNC (in green, acetylated tubulin), particularly in longitudinal connectives (conn; white arrows) and less in commissural projections (comm; yellow arrows), was observed with increasing RA concentrations. (F to J) Treatments reduce the number of differentiating serotonergic neurons (in red, serotonin) along the VNC (arrows). (K to O) Number of proliferating cells (in purple, 4-hour EdU incubation) in larval neuroectoderm (circles) is reduced by ATRA and 13cRA, whereas cell proliferation in the larval brain is less affected (dashed circles). (A, F, and K) The first (I), second (II), and third (III) larval segments are indicated. (P and Q) Box plots of VNC length and width and of serotonergic neuron numbers in dimethyl sulfoxide (DMSO) controls and after treatments with 4 μM ATRA or 4 μM 13cRA. Data distribution (circles), median values (bold line), and Tukey whiskers are shown. Unpaired t test on the mean value was used for statistical analyses (**P < 0.01). (P) Ratio of VNC length and total body length, as shown in (A), and comparison of VNC width in the first two body segments, indicated as width I and II in (A). Left box plot: P = 0.4048 for DMSO versus ATRA and P = 0.0006 for DMSO versus 13cRA. Right box plot: P = 0.002 and P = 0.0001 (width I) and P = 0.0017 and P = 0.0001 (width II) for, respectively, DMSO versus ATRA and DMSO versus 13cRA. (Q) Serotonergic neuron numbers in DMSO-, ATRA-, and 13cRA-treated embryos. P = 0.6504 for DMSO versus ATRA and P = 0.0001 for DMSO versus 13cRA. (R to Y) Treatments reduce nk6 (R to T) and pax6 (V to X) expression in the VNC during larval development (34 to 40 hpf). Gene expression in pink. (U and Y) Orthogonal optical sections showing the absence of nk6 and pax6 from proliferating neuroectoderm (superficial layer, white outline) and decrease of nk6 and pax6 expression in the postmitotic layers below the neuroectoderm (middle and deep layers, purple and yellow outlines, respectively). Ventral views of developing P. dumerilii are shown. Number of affected over total number of assayed specimens is indicated. Scale bars, 50 μm [except (U) and (Y), 20 μm].

  • Fig. 7 A simplified phylogeny of metazoan animals illustrating major events of RA signaling evolution.

    The color used for each taxon highlights the RA binding capacity of the RAR: light blue for low-affinity sensors and dark blue for high-affinity receptors. Red circles highlight hypothesized events of RAR evolution, and green boxes highlight the likely concomitant appearance of the two main developmental roles of RA signaling: in neuronal differentiation at the base of bilaterians and in anteroposterior (AP) regional patterning via hox gene regulation at the base of chordates. Red stars indicate secondary RAR loss. Deutero, deuterostomes; Proto, protostomes.

  • Table 1 Endogenous retinoids in P. dumerilii.

    Values are means ± SD. Serum values are pmol/g tissue. n.d., not detected. Developmental stages correspond to gastrulation at 18 hpf, the early metatrochophore larva at 51 hpf, and the early nectochaete larva at 74 hpf.

    StageATRA9cRA13cRARetinolRetinyl esters
    Unfertilized egg
    (n = 4)
    0.92 ± 0.74n.d.0.73 ± 0.4150 ± 31.25180 ± 68.66
    Gastrulating protrochophore
    (n = 2)
    4.58 ± 0.16n.d.6.34 ± 2.21420 ± 2001000 ± 417.81
    Early metatrochophore larva
    (n = 9)
    2.74 ± 1.52n.d.7.47 ± 4.63830 ± 489.341880 ± 1004.92
    Early nectochaete larva
    (n = 6)
    3.39 ± 1.10n.d.2.53 ± 1.17300 ± 125.65900 ± 567.46
    Adult worm
    (n = 4)
    38.3 ± 4.03n.d.46.8 ± 3.89878.5 ± 102.322170 ± 4581

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/2/eaao1261/DC1

    fig. S1. Phylogenetic analyses of the molecular components of retinoid metabolism and signaling in P. dumerilii.

    fig. S2. Alignment of RAR sequences.

    fig. S3. Transactivation assays using the PduRXR.

    fig. S4. Structural analysis of the LBD of the PduRAR.

    fig. S5. Expression of RA metabolic enzymes (aldh1a and cyp26) in P. dumerilii.

    fig. S6. Validation of MOs for microinjection into P. dumerilii zygotes.

    fig. S7. Effects of ATRA and 13cRA treatments on P. dumerilii larval development.

    table S1. Data collection and refinement statistics for the PduRAR LBD–ATRA crystal structure complex.

    movie S1. Locomotion of P. dumerilii larvae upon application of exogenous RA.

    movie S2. Locomotion of P. dumerilii prelarvae upon application of exogenous RA.

    References (61, 62)

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Phylogenetic analyses of the molecular components of retinoid metabolism and signaling in P. dumerilii.
    • fig. S2. Alignment of RAR sequences.
    • fig. S3. Transactivation assays using the PduRXR.
    • fig. S4. Structural analysis of the LBD of the PduRAR.
    • fig. S5. Expression of RA metabolic enzymes (aldh1a and cyp26) in P. dumerilii.
    • fig. S6. Validation of MOs for microinjection into P. dumerilii zygotes.
    • fig. S7. Effects of ATRA and 13cRA treatments on P. dumerilii larval development.
    • table S1. Data collection and refinement statistics for the PduRAR LBD–ATRA crystal structure complex.
    • Legends for movies S1 and S2
    • References (61, 62)

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.avi format). Locomotion of P. dumerilii larvae upon application of exogenous RA.
    • movie S2 (.avi format). Locomotion of P. dumerilii prelarvae upon application of exogenous RA.

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