Emilin3 is an essential component of the notochord sheath. (A) Lateral views of zebrafish embryos after immunofluorescence for Emilin3 and collagen II. Scale bars: 30 μm. (B) Lateral views of 24 hpf injected embryos. (C) BODIPY TR Ceramide staining of notochord cells in 24 hpf injected embryos. Scale bar: 50 μm. (D) Quantification of the mean number of vacuolated cells within the trunk region of control and treated notochord (not significantly different, n=10). Data are mean+s.e.m. (E) Transmission electron micrographs of the notochord sheath in transverse trunk sections of 30 hpf injected embryos. The arrow points at the medial layer of the notochord basement membrane, which is thinner and partially disorganized in Emilin3 double morphants. Scale bar: 500 nm. (F) Immunofluorescence for Emilin3 and collagen II in 24 hpf injected embryos. Scale bar: 10 μm. (G) Lateral views of 24 hpf injected embryos. col2a1 MO, col2a1 morpholino; Ctrl MO, control morpholino; e3a MO, emilin3a morpholino; e3b MO, emilin3b morpholino; i, inner layer; m, medial layer; no, notochord; o, outer layer.

Hh signaling is upregulated in Emilin3 morphant embryos. (A) Wild-type (WT) and Tg12x_Gli embryos were treated with 100 μM cyclopamine (cyc) and probed at 24 hpf by in situ hybridization for ptc1 or mCherry. (B) qRT-PCR for ptc1 and mCherry transcripts in Emilin3 morphant and control embryos (^*P<0.05; n=30). (C) In situ hybridization for nkx2.2a, tbx20 and myoD1 in 24 hpf injected embryos. All images are lateral views, except for bottom panels (dorsal views). (D) In situ hybridization for shh, ehh and ntl in 24 hpf injected embryos. (E) Immunofluorescence for engrailed in 24 hpf injected embryos. Scale bars: 150 μm. (F) Quantification of engrailed-positive cells at 24 hpf (^*P<0.05; n=10). Data are mean+s.e.m. Ctrl MO, control morpholino; e3a MO, emilin3a morpholino; e3b MO, emilin3b morpholino; e3a+e3b MO, emilin3a + emilin3b morpholinos; MPs, muscle pioneers; MFFs, medial fast fibers.

Overexpression of Emilin3 affects notochord patterning activity. (A) Lateral views of 24 hpf embryos probed for ptc1. Embryos were injected with the indicated morpholinos. (B) Wild-type embryos were injected with the indicated constructs at 50 ng/μl and photographed at 24 hpf (upper panels), or probed by in situ hybridization for myoD1 at eight somites and for ehh at 24 hpf (middle panels), or by immunofluorescence for engrailed and Emilin3 at 24 hpf (lower panels). Scale bar: 50 μm. (C) Quantification of engrailed-positive cells in 24 hpf embryos injected as in B (^*P<0.05; n=15). Data are mean+s.e.m. Ctrl MO, control morpholino; e3a+e3b MO, emilin3a + emilin3b morpholinos; MPs, muscle pioneers; MFFs, medial fast fibers.

Emilin3 functionally interacts with Scube2. (A-C) HEK293T were transiently co-transfected with the indicated constructs. Conditioned serum-free media and cell lysates were analyzed by western blot. (D) Cell lysates of HEK293T transfected with the indicated plasmids were analyzed by western blot or subjected to immunoprecipitation. (E-G) HEK293T were either co-transfected (E,G) or separately transfected (F) with the indicated plasmids. Media (E,F) and cell lysates (G) were analyzed by western blot or subjected to immunoprecipitation. (H) Transfected HEK293T were incubated for 24 hours with conditioned media derived from control or Scube2-transfected cells. (I) Stably transfected 293-Shh cells were incubated for 6 hours with fresh medium (DMEM) or with conditioned media derived from HEK293T co-transfected with the indicated plasmids. Media and cell lysates were analyzed by western blot. (J) Lateral views of 24 hpf embryos injected with the indicated morpholinos and immunostained for engrailed. Scale bar: 50 μm. (K) Quantification of engrailed-positive cells in injected embryos (^*P<0.05; n=6). Data are mean+s.e.m. CM, conditioned media; E3, murine full-length Emilin3; E3Δ1, murine Emilin3 lacking the EMI domain; E3Δ2, murine Emilin3 lacking the EMI domain and the coiled-coil region; ev, empty vector; IP, immunoprecipitation; PTC1, human FLAG-patched-1; WB, western blot; Ctrl MO, control morpholino; e3a+e3b MO, emilin3a + emilin3b morpholinos; Scube2 MO, Scube2 morpholino; MPs, muscle pioneers; MFFs, medial fast fibers.

Morpholino-mediated knockdown of zebrafish Emilin3 transcripts. (A) RT-PCR analysis of RNA extracted from 24 hpf non-injected embryos (WT) and embryos injected with the indicated morpholinos. β-actin was used as a loading control. (B) Whole mount immunofluorescence labeling for Emilin3 in 24 hpf embryos injected with 4 ng control morpholino (Ctrl MO), 2 ng/each of emilin3a and emilin3b splice-blocking morpholinos (e3a+e3b MO splice), or 2 ng/each of emilin3a and emilin3b translation-blocking morpholinos (e3a+e3b MO ATG). Scale bar, 50 μm. (C) Lateral views of 24 hpf embryos injected with 4 ng control morpholino (Ctrl MO) or 2 ng/each of emilin3a and emilin3b translation-blocking morpholinos (e3a+e3b MO ATG). Arrows point at notochord distortions in Emilin3 double morphants. (D) Lateral views of 24 hpf embryos injected with 10 ng control morpholino (Ctrl MO), 2 ng/each of emilin3a and emilin3b splice-blocking morpholinos (e3a+e3b MO), 6 ng p53 translation-blocking morpholino (p53 MO) and the combination of p53, emilin3a and emilin3b morpholinos (e3a+e3b+p53 MO). no, notochord.

Quantification of the mean number of notochord cells. (A) Notochord cell nuclei of the same trunk region from 24 hpf control and Emilin3 double morphant embryos were stained with Hoechst. Scale bar, 50 μm. (B) Nuclei, stained as in (A), were counted and reported as mean number of nuclei/somite (not significant; n=15). Ctrl MO, control morpholino; e3a+e3b MO, emilin3a + emilin3b morpholinos.

Notch activity is not grossly affected in the notochord of Emilin3 depleted embryos. (A) Notchresponsive reporter zebrafish (Tg_Hbb:EGFP) were injected with the indicated morpholinos and the fluorescence analyzed at 24 hpf. Scale bar, 50 μm. (B) Quantification of the mean number of GFP-positive notochord cells in the trunk region (250 μm in length) (not significant; n=10). Ctrl MO, control morpholino; e3a+e3b MO, emilin3a + emilin3b morpholinos.

Analysis of collagen II and laminin expression in Emilin3 morphant embryos. (A, B) Zebrafish embryos were injected with the indicated morpholinos and probed for col2a1 expression at 24 and 48 hpf, respectively. The fraction of embryos displaying the corresponding phenotype is provided in each panel. Note that at 24 hpf, even embryos injected with emilin3a or emilin3b morpholino alone displayed a slight increase in col2a1 expression. (C) Lateral views of controls and Emilin3 morphant embryos labeled by immunohistochemistry for laminin at 24 hpf. The panels display projection of confocal sections around somite 10, showing the notochord. Scale bar, 25 μm. Ctrl MO, control morpholino; e3a MO, emilin3a morpholino; e3b MO, emilin3b morpholino; e3a+e3b MO, emilin3a + emilin3b morpholinos.

Hedgehog upregulation in Emilin3 morphant embryos is dependent on Emilin3 protein depletion. (A, B) Embryos were injected with the indicated splice- or translation-blocking morpholinos (ATG) and probed for ptc1 expression at 24 hpf. Lateral view of the trunk, head is on the left. (C) Control and Emilin3 double morphant embryos were probed for ptc1 expression at 8-somite stage. (D) Dorsal view of wild-type embryos immunostained for Emilin3 and β-catenin at at 8-somite stage. Note the absence of Emilin3 in the notochord (marked by the asterisks) at this stage of development. Scale bar, 100 μm. The fraction of embryos displaying the corresponding phenotype is provided in each panel. Ctrl MO, control morpholino; e3a+e3b MO, emilin3a + emilin3b morpholinos; p53 MO, p53 morpholino.

Upregulation of Hedgehog target genes in Emilin3 morphant embryos. (A) Lateral views of 24 hpf embryos injected with the indicated morpholinos and probed for olig2, vegfa and eng2a expression. (B) Expression of fgf8 is similar between control and Emilin3 double morphant embryos, thus excluding a generalized developmental delay. Head is on the left. The fraction of embryos displaying the corresponding phenotype is provided in each panel. Ctrl MO, control morpholino; e3a+e3b MO, emilin3a + emilin3b morpholinos.

Hedgehog upregulation in Emilin3 morphant embryos is not dependent on BMP or TGF-β signaling. (A) Lateral views of 24 hpf embryos injected with the indicated morpholinos and treated at 8 hpf with 5 μM cyclopamine (lower panels) or with the corresponding volume of ethanol as a control (upper panels). Embryos were then stained with the monoclonal 4D9 antibody (green) and the polyclonal pSMAD1/5/8 antibody (purple). The panels show magnifications around somite 10. Scale bar, 50 μm. (B) Wild-type embryos were injected with the indicated morpholinos. Embryos were then left untreated or treated at 8 hpf with 30 μM of the selective TGF-β receptor inhibitor LY364947 from 8 hpf and probed for ptc1 expression at 24 hpf. The fraction of embryos displaying the corresponding phenotype is provided in each panel. Ctrl MO, control morpholino; e3a+e3b MO, emilin3a + emilin3b morpholinos.

Injection of Emilin3 cDNA rescues the phenotype of Emilin3 morphants. (A) Lateral views of 24 hpf embryos co-injected with the indicated morpholinos (4 ng/embryo) and with the indicated cDNA constructs (25 ng/μl). Arrows point at notochord distortions in Emilin3 double morphants, which are largely rescued by co-injection with the pCS-twhh-mEmilin3 construct. The fraction of embryos displaying the corresponding phenotype is provided in each panel. (B) Whole mount immunofluorescence labeling for engrailed and Emilin3 in 24 hpf embryos injected as in (A). Scale bar, 25 μm. (C) Quantification of engrailed-positive cells in 24 hpf co-injected embryos (*, P < 0.05; n.s., not significant; n = 10). Ctrl MO, control morpholino; e3a+e3b MO, emilin3a + emilin3b morpholinos; Trunk MPs, trunk muscle pioneers; Trunk MFFs, trunk medial fast fibers.

Morpholino-mediated knockdown of zebrafish Emilin3 and Scube2. (A) Lateral views of 24 hpf embryos injected with 6 ng of control morpholino (Ctrl MO) or 2 ng/each of the indicated morpholinos. (B) qRT-PCR for scube2 transcript in control and Emilin3 double morphant embryos (not significant; n = 30). Ctrl MO, control morpholino; e3a+e3b MO, emilin3a + emilin3b morpholinos; Scube2 MO, Scube2 morpholino.