Cell Reports, Volume 9

Supplemental Information

RGM Regulates BMP-Mediated Secondary Axis Formation

in the Sea Anemone Nematostella vectensis

Lucas Leclère and Fabian Rentzsch

Figure S1 – related to Figure 1

Figure S1, related to Figure 1. RGM is a conserved metazoan-specific protein.

(A) Predicted NvRGM protein structure; numbers below the sketch indicate amino-acid position

(top). Comparison of gene structures and intron positions between NvRGM and human RGM paralogs

(bottom). Thick boxes indicate open reading frame, coloured boxes correspond to predicted domains

shown in the protein sketch above.

(B) Metazoan phylogeny showing distribution and evolutionary origin of RGM, major BMP

signaling components and Neogenin. Dashed lines indicate controversial phylogenetic positions.

Filled and empty circles indicate presence and absence, respectively. Filled circles on branches

indicate evolutionary acquisition and crossed circles indicate evolutionary losses. The numbers

inserted in RGM circles indicate the number of paralogs found in each genome analysed. See Table

S1 for details about genome searches.

(C) Phylogenetic relationships between RGM sequences inferred by Maximum-likelihood

analyses. Maximum likelihood bootstrap replicates (200 replicates, left value) and Bayesian posterior

probabilities (right value) are indicated above each branch if superior to 50%. Nematostella RGM is

boxed in red. See Supplemental Experimental Procedures for details about the analyses, and Table

S1 for information about sequences. These phylogenetic analyses show that paralogous genes

present in mouse, human fish and chicken are the result of vertebrate specific duplications. The

topology outside vertebrates is not statistically supported and therefore not totally consistent with the

accepted metazoan phylogeny. Abbreviations for the two first letters of gene names: Ad: Acropora

digitifera, Am: Acropora millepora, Ap: Acyrthosiphon pisum, Bf: Branchiostoma floridae, Cbe:

Caenorhabditis brenneri, Cbi: Caenorhabditis briggsae, Ce: Caenorhabditis elegans, Cg: Crassostra

gigas, Ci: Ciona intestinalis, Cr: Caenorhabditis remanei, Ct: Capitella teleta, Dp: Daphnia pulex, Dr:

Danio rerio, Gg: Gallus gallus, Hm: Hydra magnipapillata, Hr: Helobdella robusta, Hs: Homo sapiens,

Is: Ixodes scapularis, Lg: Lottia gigantea, Ll: Loa loa, Mm: Mus musculus, Nv: Nematostella vectensis,

Oc: Oscarella carmela, Ph: Pediculus humanus, Pm: Petromyzon marinus, Sk: Saccoglossus

kowalevskii, Sm: Schmidtea mediterranea, Sp: Strongylocentrotus purpuratus, Ta: Trichoplax

adhaerens, Xt: Xenopus tropicalis.

Figure S2 – related to Figure 1

Figure S2, related to Figure 1. Supplementary information about NvRGM and NvNeogenin

morpholino injections.

(A, B) RT-PCR analysis of mRNA isolated from embryos injected with NvRGM and NvNeogenin

splice-blocking MOs. Both MOs target splice donor sites and result in intron retention and premature

stop codons. Gels on the left side show the results from the RT-PCR reactions (ctrl= non injected

embryos, MO= MO injected embryos). Diagrams on right indicate the location of primer and MO target

sequences relative to the transcript structure. Only the four 5’-most introns of NvNeogenin are

represented (21 introns in total). Abbreviations - SP: Signal Peptide, VWF-D: van Willebrand factor

type D domain, HR: Hydrophobic Region, GPI-a: GPI-anchor, IG: Immunoglobulin, FN: Fibronectin,

TM: Transmembrane domain, Neog-C: Neogenin Intracellular domain.

(C-I) NvRGM is required for asymmetric gene expression at gastrula stage (28hpf). In situ probes

are indicated above the images, with Morpholino on the left side. For each condition, a lateral view

with aboral pole to the left is shown next an oral view; Scale bar, 50 µm. (I) Proportion of embryos with

radial or asymmetric NvChd and NvBMP2/4 and NvRGM expression along the directive axis for each

experimental condition. Expression of NvBMP2/4, NvChd and NvRGM remains radial during

gastrulation in the case of NvRGM MO knockdown.

(J-AD) Indistinguishable effects of two different NvRGM Morpholinos and two different NvNeog

Morpholinos on gene expression patterns at the early planula stage (50 hpf). In situ probes are

indicated above the images, with Morpholino on the left side. For each condition, a lateral view with

aboral pole to the left is shown next to an oral view. Scale bar, 50 µm. (AD) Proportion of embryos with

radial or asymmetric NvBMP2/4 and NvRGM and NvChd expression along the directive axis for

control and NvNeog MO1 injected embryos.

(AE-AG) NvNeogenin in situ hybridizations in wild-type embryos at mid gastrula (AE, 26hpf),

early planula (AF, 50hpf) and late planula stages (AG, 5dpf) showing ubiquitous expression and slight

enrichment in the apical organ (arrow) and tentacle regions (arrow head) at late planula stages. Scale

bar, 50 µm

Figure S3 – Related to Figure 2

Figure S3, extended version of Figure 2. Establishment of bilateral symmetry during

Nematostella early development.

(A-H, K, L, N-S) confocal sections showing F-actin (phalloidin - green) and nuclear (DAPI - red)

stainings for mid gastrula (A, B – 24hpf), late gastrula (C, D – 36hpf), early planula (E-H – 48hpf), mid

planula (K, L, N-P – 72hpf) and metamorphosing primary polyp (Q-S – 5dpf). (B,D) Transverse

sections; (E, F, K, L) longitudinal sections along the directive axis, and (G, R) perpendicular to the

directive axis; (H, N, O, P, S) transverse sections. All transverse sections and longitudinal sections

along the directive axis are oriented with the 3-chambered/NvBMP2/4 side at the bottom. Mesenteries

are highlighted by arrow heads and arrows for primary and secondary mesenteries, respectively

throughout the figure. Grey triangles indicate the relative positions of the sections shown in the picture

corresponding to the letter inside the triangle. All confocal images are single sections except for (Q)

which is a maximum projection. Scale bar, 50 µm. (I) Rose diagram showing the orientation of the two

first mesenteries at 48hpf in relation to the directive axis. Confocal sections and optical sections of

NvBMP2/4 and NvHox6a in situ hybridization were used for assessing the position of primary

mesenteries. (I – 48hpf) NvHox6a, (J – 48hpf) NvBMP2/4, or (M – 72hpf) NvHox8 in situ hybridization

optical sections showing position of the primary and secondary mesenteries. Abbreviations: bp:

blastopore, ch: chamber, ec: ectoderm, e: endoderm, p.m: parietal muscle, ph. ec.: pharyngeal

ectoderm, ph. en.: pharyngeal endoderm, ph. l.: pharyngeal lobe, r.m: retractor muscle, t.b.: tentacle

bud. (T) Schematic drawings illustrating longitudinal views along the directive axis (top) and

transversal view (bottom) at different stages of development, oriented with the 3-chambered side at

the bottom for all cartoons. Grey triangles indicate the relative position of the transverse sections

shown in the bottom cartoons. Color code – light blue: ectoderm; orange: endoderm; green:

pharyngeal ectoderm; yellow: pharyngeal endoderm; purple: 8 parietal muscles; red: 8 retractor

muscles; grey: part of the pharynx not on the same plane as the rest of the cartoon; dark blue:

mesenteries.

Supplemental description of the results presented in Figure S3.

During gastrula stage, ectoderm, endoderm, pharyngeal endoderm and pharyngeal ectoderm are

generated (A-D). The embryos display cylindrical symmetry, characterized by a single oral-aboral axis

of polarity. Immediately following gastrulation, the aboral part of the pharynx tissue starts to elongate

in two lobes and the pharyngeal cavity becomes slit-shaped on the transverse plane, oriented along

the directive axis (E-H). The “pharyngeal lobes” are bilayered, being composed both of pharyngeal

ectoderm and pharyngeal endoderm. The more oral part of the pharynx can be slit-shaped, lozenge-

shaped or round (e.g. B, D).

Bilateral symmetry, i.e. the presence of two axes of polarity (the oral-aboral and directive axes),

becomes apparent at early planula stage by the asymmetric development of the first two mesenteries:

they form closer to one end of the directive axis and thereby divide the transverse plane of the body

column into a smaller and a larger part (H, I - mean value of the angle between the directive axis and

the first two mesenteries: 99.5°).

At mid-planula stage, the remaining six mesenteries are formed, reinforcing the bilateral organization

of the endoderm (“secondary mesenteries”): two of them are added in the smaller part and four are

added in the larger part of the transverse plane. Thus, due to the unequal addition of the secondary

mesenteries, the “smaller part” contains three chambers delimited by mesenteries (3-chambered side),

whereas the “larger part” contains five chambers (5-chambered side). The spacing between the

mesenteries is also bilateral, with the two secondary mesenteries on the 3-chambered side further

apart from each other than any other adjacent mesenteries (N, O). At this stage the two pharyngeal

lobes grow aborally along the first two mesenteries, generating a marked bilateral symmetry of the

aboral part of the pharynx (K, L, O).

At late planula stage the retractor muscles develop on one side of each mesentery in a bilateral

symmetric manner (S, T), as previously reported (Frank and Bleakney, 1976). Using tubulin antibody

staining we could not detect a siphonoglyph (ciliated groove) on one side of the pharynx, neither at

late planula or primary polyp stages. This structure has been described in mature polyp and therefore

probably only appears at later stages. By combining our data about the pattern of mesentery

formation, the position of retractor muscles, and the previous description of the relative position of

siphonoglyph and retractor muscles (Frank and Bleakney, 1976), it can be deduced that the

siphonoglyph appears in polyps on the 3-chambered side, the same side where NvBMP and NvChd

are expressed at planula stage.

Figure S4. Related to Figure 3

Figure S4, related to Figure 3. Controls for pSmad1/5/8 antibody and additional phenotypes

of NvRGM and NvBMP knockdown.

(A-G) Smad1/5/8 expression pattern and knockdown at gastrula and planula stage. In situ

hybridization using anti-sense (A, C) and sense (B, D) NvSmad1/5/8 probes at mid gastrula (A, B) and

early planula (C, D) stages. Anti pSmad1/5/8 antibody stainings, transverse sections (all maximum

projections) at early planula stages after injection of NvSmad1/5/8 MO (E), NvSmad1/5/8 5-mismatch

MO (F) and NvSmad2/3 MO (G). (E-G) Lower panels show high magnifications of the stainings shown

above. Note the loss of ectodermal and endodermal staining in the case of NvSmad1/5/8 MO injection

but not in the case of NvSmad1/5/8 5-mismatch MO (F) and NvSmad2/3 MO (G) injections (as

examples, 4 ectodermal and 4 endodermal stained nuclei are shown by white and yellow arrows,

respectively in the lower F and G panels). Scale bar, 50 µm.

(H-U) Effect of NvRGM, NvBMP2/4 and NvBMP5/8 knockdowns at early planula stage and

“primary polyp” stages. Confocal section of early planula embryos stained for F-actin (phalloidin)

and DAPI (nuclear) in the case of (H, I) control, (J, K) NvRGM, (L, M) NvBMP2/4 or (N, O) NvBMP5/8

morpholino injection. (H, J, L, N) longitudinal sections; (I, K, M, O) transversal sections. At early

planula stage, NvRGM, NvBMP2/4 and NvBMP5/8 MO injected embryos do not develop primary

mesenteries. (P-S) At 6d post fertilization NvRGM, NvBMP2/4 and NvBMP5/8 MO injected embryos

have elongated dramatically, possess a cylindrical pharynx (everted in most embryos) and do not have

mesenteries. (T, U) These elongated embryos possess an epithelial endoderm, including transverse

muscle processes, but no parietal or longitudinal muscles (in T, arrows indicate parietal muscle fibers).

(I, P) Mesenteries are highlighted by arrow heads for primary mesenteries and arrows for secondary

mesenteries. (H, J, L, N) Dotted lines indicate the shape of the aboral extremity of the pharynx. e.ph:

everted pharynx; ph.l: pharyngeal lobe; ph: pharynx. Scale bar, (H-O, T, U) 50 µm or (P-S) 500 µm.

Figure S5. Related to Figure 5

Figure S5, related to Figure 5. Only the 3-chambered (low pSmad1/5/8 activity) side is affected

by partial knockdown of NvBMP activity.

(A-D) At mid planula stage, the position of the secondary mesenteries located on the 5-chambered

side is not significantly affected in the case of injection of NvRGM MO ((C) m1: p = 0,352; (D) m2: p =

0,138).

(E-H) The position of the primary mesenteries (see Figure 5), secondary mesenteries located on the 3-

chambered side (E) (m4: p = 0,00038), and the area of expression of NvBMP2/4 (p = 5,27 e-12) are

significantly shifted away from the area of NvRGM expression in NvRGM MO injected embryos,

compared to the control. All images are oral views with the 5-chambered side to the top. Scale bar, 50

µm. (C,D,E,H) Box-plot of the angle shown in the images on the left. Boxes indicate quartiles; and

whiskers extend to extreme values. Number of embryos analyzed for mesentery position: 26 for

NvRGM MO and 16 for control MO; and for NvBMP2/4 expression: 29 for NvRGM MO and 23 for

control MO. Statistical test used: two-tailed Student’s t-test assuming unequal variance – n.s: p > 0,05;

**: p < 0,01.

(I-M) Plot of the relative nuclear pSmad1/5/8 staining intensity along the directive axis from the intense

pSmad1/5/8 staining side to the opposite side. Consistently, pSmad1/5/8 staining is reduced in the low

BMP activity side of the embryo (right half) upon injection of NvRGM MO and low dose NvBMP2/4 MO

compared to the control but not significantly in the high BMP activity half (left half) for most sample

comparisons. Results from the statistical tests for sample comparisons are shown in Table S2.

Table S1, related to Figure 1; Sequence and database information for genome survey and

phylogenetic analysis of RGM (TableS1.xls).

Table S2, related to Figure 5. Results of the two-tailed Student’s t-test assuming unequal variance for comparison of the pSmad1/5/8 intensity values between samples – Four random samples for Control MO and NvRGM MO injected embryos (A, B, C and D) and three samples for NvBMP2/4 MO injected embryos (A, B and C) were analyzed. For the three conditions, plots of the relative nuclei pSmad1/5/8 staining intensity along the directive axis are shown in Figure 5 (T, U) for samples A and in Figure S5 (I-M) for samples B, C and D. Bold numbers indicate statistical significance at p<0.001. For comparisons within the same experimental condition the values along the entire directive axis were compared. Note that control MO embryo C appears to be an outlier that differs also from other control MO embryos.

Control MO RGM MO

A B C A B C

D 0,034 0,726 1,5E-04 D 0,091 0,0021 0,331

Control MO C 7,1E-11 7,4E-05

RGM MO C 0,429 0,027

A 0,0033

A 0,174

BMP2/4 MO

A B

BMP2/4 MO C 0,836 0,010

B 0,0068

Control MO

A B C D

A 0,127 0,838 3,8E-05 0,693

BMP2/4 MO B 0,084 0,053 5,2E-11 8,0E-04

C 0,001 0,002 0,212 0,136

Control MO High pSmad1/5/8 side

A B C D

RGM MO

A 0,778 0,035 3,5E-09 0,029

B 0,121 0,008 1,3E-10 0,071

C 0,467 0,008 1,8E-10 0,008

D 0,102 4,1E-04 1,9E-12 6,2E-04

Control MO

A B C D

A 5,6E-14 6,5E-13 2,8E-27 3,3E-12

BMP2/4 MO B 3,7E-18 2,2E-08 7,9E-31 4,1E-16

C 2,4E-38 1,2E-34 4,8E-37 7,2E-24

Control MO Low pSmad1/5/8 side

A B C D

RGM MO

A 4,3E-10 2,1E-16 5,3E-23 1,1E-07

B 4,1E-16 5,0E-19 7,0E-12 0,399

C 1,6E-12 5,0E-19 2,2E-23 3,0E-08

D 5,7E-26 5,8E-30 1,7E-28 2,8E-14

Supplemental Experimental Procedures

NvRGM and NvNeogenin Sequence Identification and Phylogenetic Analyses

RGM and Neogenin sequences searches were performed by tBLASTn on the Nematostella genome

database (Joint Genome Institute). NvRGM and NvNeogenin were cloned using specific primers

designed from JGI gene predictions and confirmed by RACE (SMART RACE cDNA Amplification Kit,

BD Biosciences). NCBI accession numbers are KM975939 (NvRGM) and KM975940 (NvNeogenin).

Presence of orthologs of RGM in publicly available opistokont genomes was investigated using BLAST

searches (blastp and tblastn, see Table S1). In addition, we searched RGM conserved domains in the

PFAM domain database (http://pfam.sanger.ac.uk/; domain references: RGM_N, PF06535; RGM_C,

PF06534). RGM amino acid sequences were aligned using the software MUSCLE (Edgar, 2004) using

default parameters. Poorly aligned positions were excluded using the program Gblocks 0. 91b

(Castresana, 2000) (default settings used, except “allowed gap positions” set to half) as well as

positions with more than 50 % gaps. Maximum Likelihood (ML) analyses were performed using

PhyML3.0 (Guindon et al., 2010), with a LG + G (10 categories) model of amino acid substitution and

200 bootstrap replicates; Bayesian analyses were performed using MrBayes 3.2.1 with WAG + G (5

categories) model of amino acid substitution running for 1,000,000 generations sampled every 100

generations (burnin: 50%). Phylogenetic analyses of Neogenin proteins can be found in Leclère and

Rentzsch (2012).

Immunostaining protocol

Embryos were fixed for two minutes in cold 3,7 % paraformaldehyde + 0,25 % glutaraldehyde in PBS-

0.2 % triton-X100 (PBTr) followed by one hour in cold 3,7 % formaldehyde in PBTr, rinsed quickly with

100 % methanol (except when coupled with phalloidin staining), rinsed five times in PBTr, incubated in

PBTr – normal goat serum (NGS) 5% - BSA 1% for two hours, and then incubated overnight at +4°C

with the primary antibody (anti-phospho-Smad1/5/8 Cell Signaling, #9511, 1:100 and/or acetylated

tubulin 1:200) diluted in PBTr-NGS-BSA. Embryos were washed five times with PBTr, incubated two

hours with PBTr-NGS-BSA and then incubated overnight at +4°C with the secondary antibody diluted

in PBTr-NGS-BSA, in addition to Alexa Fluor 488-conjugated phalloidin (1:50, Molecular Probes) for

detection of filamentous actin. The secondary antibody was washed five times with PBTr, incubated

half-an-hour with 1:1000 DAPI (4',6-diamidino-2-phenylindole) for nuclear stain and finally washed two

times with PBTr. Embryos were mounted in ProLong Gold antifade reagent (Molecular Probes).

Morpholino injection

Morpholinos (GeneTools) were tested for appropriate working concentrations and then injected at a

concentration of 200 μM (NvChd MO, NvBMP2/4 MO and NvBMP5/8 MO) or 500 μM (all other MOs),

together with Alexa dye coupled dextran (final concentration 50 ng/μl). We used two control

morpholinos: NvRGM 5-mismatch MO1 and a generic control MO, used previously (Nakanishi et al.,

2012; Sinigaglia et al., 2013) both having no detectable effect on gene expression and morphology.

The control MO and NvRGM MO presented in all our analyses and figures are the generic MO and

NvRGM MO1, respectively. Sequences of the morpholinos:

NvRGM MO1: 5’-AGCTACAAACACGTACCTTGTATGA-3’;

NvRGM control MO1 (5-mismatch): 5’-AGGTAGAAACAGGTACGTTCTATGA-3’;

NvRGM MO2: 5’-AAATTGTCCCGGTGTTGCTAACCAT-3’;

NvNeog MO1: 5’-GTTTTCAAACTTACAGTTCACTTTG-3’;

NvNeog MO2: 5’-ATATAAAGACATCCTATCACAGGCC-3’.

NvSmad1/5/8: 5’-TGTGAAAGAAAACAGGGACGCCATT-3’.

NvSmad1/5/8 5mm: 5’-TGTCAAACAAAAGAGGCACCCCATT-3’.

NvSmad2/3: 5’-AGGCAACAGGGAAGTCATCTCACTT-3’.

Morpholinos against NvBMP2/4, NvBMP5/8 and NvChd are as in Saina et al. (2009).

Angle and pSmad1/5/8 staining intensity measurements.

To calculate angles of mesentery positions and gene expression in transversal sections (shown in

Figure 5 and Figure S5), images were oriented along the directive axis based on the positions of the

pharyngeal lobes and mesenteries; the geometrical centre of the embryo and the bisection of the

middle chamber on the 5-chambered side were used as reference points.

For generating the pSmad1/5/8 plots we used pSmad1/5/8 immunostainings performed in parallel on

embryos from the same batch. Image stacks of lateral views (oriented along the directive axis) were

generated using identical settings for all recordings on a Leica SP5 confocal miscrocope. Images were

processed using ImageJ. Artifactual bright spots were removed from the unprocessed images using

the Despeckle function. Maximum intensity projections of the endoderm were generated. For each

body wall endodermal nucleus (300±50 per embryo) the maximum fluorescence intensity and the

position along the directive axis were recorded.

Data were then processed in Excel. Comparing DAPI staining between samples (irrespective of the

condition analysed), we could observe that the overall intensity was different between embryos even if

all the recording settings were the same (depending on the thickness of the preparation and the

position of the embryos within the preparation). Therefore, to normalize the staining, (1) the 2%

highest intensity data points were removed (to avoid that few anomalously bright nuclei affect the

normalization) and (2) the relative intensity values were recalculated using the most strongly stained

nucleus of each embryo (after 2% data subtraction) as reference point. To avoid distortion of the

intensity distribution, no background normalization was performed. The relative position for each

nucleus was calculated with the two most distant nuclei along the directive axis as reference points.

Finally, for each sample, normalized intensities and relative positions data were plotted on graphs (see

Figure 5 and S5) or used for statistical student t-tests (see Table S2).

Supplemental References

Edgar RC (2004) MUSCLE: a multiple sequence alignment method with reduced time and space

complexity. BMC Bioinformatics 5: 113.

Castresana J (2000) Selection of conserved blocks from multiple alignments for their use in

phylogenetic analysis. Mol Biol Evol 17: 540-552.

Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, et al. (2010) New algorithms and methods

to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol

59: 307-321.

Nakanishi N, Renfer E, Technau U, Rentzsch F (2012) Nervous systems of the sea anemone

Nematostella vectensis are generated by ectoderm and endoderm and shaped by distinct

mechanisms. Development 139: 347-357.

Sinigaglia C, Busengdal H, Leclère L, Technau U, Rentzsch F (2013) The bilaterian head patterning

gene six3/6 controls aboral domain development in a cnidarian. PLoS Biology 11:e1001488.