Neuner R , Cousin H , McCusker C , Coyne M .

DE016289 NIDCR NIH HHS, R01 DE016289-01A2 NIDCR NIH HHS, R01 DE016289-02 NIDCR NIH HHS, R01 DE016289-03 NIDCR NIH HHS, R01 DE016289-04 NIDCR NIH HHS, R01 DE016289 NIDCR NIH HHS

Xla Wt + adam19 MO (fig.5.a)
Xla Wt + adam19 MO (fig.5.b)
Xla Wt + adam19 MO (fig.6.a)
Xla Wt + adam19 MO (fig.6.c)
Xla Wt + adam19 MO (fig.7.a)
Xla Wt + adam19 MO (fig.8.d)
Xla Wt + adam19 MO (fig.8.e)
Xla Wt + adam19 MO (fig.8.f)

	Fig. 2. ADAM19 mRNA distribution. Whole mount in situ hybridization using a probe corresponding to Xenopus ADAM19. (A) Lateral view of a bisected gastrula with the dorsal side to the left and the animal pole at the top (ap). (B and G) Dorsal views of early tailbud stage embryos with the anterior at the top. (C–E, I) Lateral views of tailbud stage embryos with anterior to the left. (F) Ventral view of an early tailbud stage embryo. Photographs in (A, F and I) were taken with embryos in PBS to visualize superficial rather than internal staining. All other embryos were imaged in Murray’s clear. (A) ADAM19 is first expressed in the dorsal mesoderm of the gastrula, with the most intense signal in the dorsal blastopore lip (dbl). (B, F–H) At early tailbud stage, ADAM19 is found in the dorsal mesoderm including the notochord (n), somites (s), the cement gland (cg) and the neural tube (nt). (C, D and I) ADAM19 expression in these tissues persists during tailbud formation. In the anterior, ADAM19 is expressed in the brain (br), the otic (ot) and optic (o) vesicles as well as CNC segments and branchial arches (ba). (E) At stage 36, the expression in the pronephros (p) is also detected.
	Fig. 6. ADAM19 is critical for neural crest induction. (A) In situ hybridization of neurula (stage 15) embryos using Slug and Sox8 to detect neural crest cells. The injected side is to the left. Embryos injected with MO19 present a decrease expression of both neural crest markers (red arrowhead). (B) Real-time PCR analysis at stage 15. Level of gene expression was measured by the 2(-δCT) method and normalized to α-tubulin. Results are presented as the Log2 of the fold change compared to non injected embryos. Variation of Sox2, Sox8 and Slug are all significant (P < 0.01) in embryos injected with MO19 when compared to the control MO. C) lateral view of tailbud stage embryos (stage 24) visualizing GFP. Embryos were injected at the 16-cell stage in a dorso-lateral-animal blastomere to target neural crest cells. In the control, GFP is found in all CNC segments (white arrows). This is absent in 33% of the embryos injected with MO19.
	Fig. 7. Phenotypical analysis of ADAM19 KD at tailbud stage. (A) Whole-mount in situ hybridization of tailbud stage embryos (stages 22–24). Embryos were injected in one blastomere of the two-cell stage with 10 ng of MO19 or CMO. mRNA encoding β-galactosidase (β-Gal) was co-injected as a tracer. The embryos were processed by whole mount in situ hybridization with the markers myosin light chain (MLC), N-tubulin, NRP1, Sox2 and ADAM11. The injected side, as detected by β-Gal activity (light blue), is on the left side for all embryos. The percentages of embryos displaying these phenotypes are indicated and represent the average of three independent experiments. The black arrowheads point to the area of decreased gene expression. For Twist the embryo heads are presented as a side view. The injected side of each embryo is to the left. Decrease in twist expression is visible (arrowhead) in embryos injected with MO19. (B) Quantitative real-time PCR was performed on embryos injected at the one-cell stage with either the MO19 alone or with the ADAM19 rescue mRNA (MO19 + A19R) and allowed to develop until neurula (stage 17) and tailbud stage (stage 24). Alternatively, the MO19 was injected in 2 dorsal-animal blastomeres at the 8-cell stage (MO19 Ectoderm) to target dorsal ectoderm but not mesoderm derivatives. All genes were compared to non-injected control embryos. The relative abundance of MLC, N-tubulin, Sox2 and ADAM11 gene expression was measured by the 2(-δCT) method and normalized to α-tubulin. Results are presented as the log 2 of the fold change. Error bar correspond to the standard error. Asterisks represent a P < 0.05 using a standard t test assuming unequal variance.
	Fig. 5. Phenotypical analysis of ADAM19 KD at the gastrula stage. (A) Embryos were microinjected with 10 ng of either the MO19 or a control MO at the one-cell stage and allowed to develop until gastrula stage (stages 12 and 13). Whole mount in situ hybridization were performed using the markers Brachyury, Sox2 (Xbra Sox2, stage 12) and Chordin and Goosecoid (Chd and Gsc, stage 13). For all embryos, the dorsal view is presented with the anterior up. The red arrowheads point to the notochord. The number of embryos and the frequency of the phenotype depicted in the photographs are indicated in the text. (B) Quantitative real-time PCR was performed on embryos injected with either MO19 alone or together with ADAM19 Rescue mRNA (MO19 + A19R). The relative abundance of Chordin (Chd), Goosecoid (Gsc) and Brachyury (XBra) gene expression was measured by the 2(-) method using either embryos injected with the control MO or non-injected and normalized to α-tubulin. There was no effect of the control MO to gene expression. The asterisks represent statistical significance at P < 0.05 using a Student t test. There is a small decrease in Xbra (16%, not significant) and a robust increase in both Chd (187%) and Gsc (230%) which are rescued by the injection of the ADAM19 mRNA.
	ADAM metallopeptidase domain 19 (Adam19) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35-36, lateral view, anterior left, dorsal up.
	Fig. 1. ADAM19 expression. Quantitative real-time PCR expression of ADAM19 during early Xenopus development. Polyadenylated RNA from 2.5 embryos was used to make cDNA. PCR amplification was performed on a light cycler for 40 cycles. (A) The amplification product from both the control (−RT) and the experimental (+RT) were separated on a 2% agarose gel. (B) The relative abundance of the ADAM19 mRNA was measured by the 2(-δCT) method using stage 10 as a value of 1. Values are not adjusted to a control gene but to identical number of embryos. The CT values for each reaction are given below the graph. No CT values were obtained for the −RT control of each stage. ADAM19 mRNA is present as a maternal transcript at stage 2. Zygotic transcription starts during gastrulation and increases during neurulation (stage 16) continuing into early tailbud stage (stage 21).
	Fig. 3. Morpholino oligonucleotide knock-down of ADAM19. Western blot of protein extract from embryos injected with a control morpholino or a morpholino to either ADAM13 (MO13) or ADAM19 (MO19). The morpholinos (10 ng) were injected at the 1 cell-stage and embryos were extracted at stage 22. (A) Glycoproteins from 4 embryos equivalent were purified on Concanavalin A–agarose, separated by SDS–PAGE and probed with a rabbit polyclonal antibody to the ADAM19 cytoplasmic domain (ADAM19 cyt). A monoclonal antibody to integrin β1 (8C8) was used as a loading control. (B) The densitometric analysis of the blot shown in (A) shows that the ADAM19 protein is reduced by approximately 90% by the MO19 when normalized to the integrin β1 expression level. (C) The MO19 is capable of preventing translation of 1 ng of ADAM19 mRNA when co-injected in embryos (A19) but not a messenger with 4 mutations in the sequence corresponding to the MO19 (A19R). The ADAM13 morpholino does not affect ADAM19 translation.
	Fig. 4. ADAM19 is not required for blastopore closure. Time-lapse imaging of gastrula stage embryos injected with either a control MO (CMO) or the MO19. Embryos were microinjected with 10 ng of MO at the one-cell stage and allowed to develop through the end of gastrulation. The blastopore size in micrometers was measured using the OpenLab software at three different time points during gastrulation. The values represent the average of three different experiments using 8 embryos each for the control and the experimental. Error bars correspond to standard deviation from the mean. Representative images are shown below the histogram.
	Fig. 8. ADAM19 is essential for somite organization and muscle differentiation. Histological and molecular analysis of embryos with reduced ADAM19 protein. (A–F) Frozen sections of tailbud stage embryos. (A–D) Immunofluorescence using the affinity purified rabbit pAb to the ADAM19 cytoplasmic domain. (A) Frontal section, anterior is to the left. The ADAM19 protein is localized to the intersomitic boundary (arrowhead), the notochord (n), the neural tube (nt) and in the branchial arches (ba). (B) Transverse section. ADAM19 is in red, the muscle marker 12–101 in green and the nuclei (DAPI) in blue. ADAM19 staining appears associated with the ventral basal side of the neural tube (arrowhead) and the notochord (n). (C) Magnification of the somite from a frontal section, ADAM19 is in red, the arrowhead points to the intersomitic boundary. (D–F) Frontal sections of tailbud stage embryos injected at the two-cell stage in one cell with 5 ng of MO19. The injected side is labeled (yellow dots) and was detected either by using the ADAM19 antibody (D and E) or GFP-myc (F) that was co-injected with the MO19. The injection of MO19 completely abolished staining of the intersomitic boundary by the ADAM19 antibody. While the intersomitic boundary is still visible, the nuclei are not aligned on the injected side (white line). (E) ADAM19 is in green (left), 12–101 in red (center) and the merge presented with DAPI staining (right). There is a clear reduction of 12–101 staining on the injected side (MO19). (F) The MO19 was co-injected with GFP-myc to detect the cells that were targeted using the 9E10 mAb. Positive cells are visible in the right somites and some of the cells that contribute to the notochord (n). Fibronectin was detected using a rabbit pAb (32F). Some of the FN staining is absent in part of the intersomitic boundary (arrowhead). (G) Western blot using 12–101 on total protein extract. Stage 7 extract is used as a negative control. At stage 22, MO19 injected embryos but none of the other injected embryos have less 12–101 protein. (H) Whole mount immunostaining using mAb 4H2 to detect fibronectin. The injected side was detected prior to fixation using GFP fluorescence. The side injected with the MO19 shows an abnormal pattern of FN staining at some of the intersomitic boundary (arrowhead).
	Fig. 9. ADAM19 may interfere with EGF signaling. (A) Western blot using antibody to phosphorylated AKT, phosphorylated MAP kinase, and PACSIN2. Embryos were injected at the one-cell stage with either the control MO (CMO) or MO19. Total protein from 0.5 embryo equivalent were used for each blot. (B) Histogram representing the quantification of the Western blot from three independent injection experiments. All measures were normalized to PACSIN2. Variations in pMAPK were not significant while the decrease in pAKT was significant. (C) Real-time PCR analysis of Slug expression. Embryos were treated with 10 μM of Marimastat (Mar), 100 μM of AG1478 or DMSO (1/1000) as a control, between stages 10 and 12, based on the blastopore size (none of the treatment affected gastrulation timing in these conditions). Marimastat decreased Slug expression by an average of 34% while AG1478 decrease Slug by 60%.
	ADAM metallopeptidase domain 19 (Adam19) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anterior left, dorsal up.

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