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Figure 1. The X. laevis adsl.L gene encodes the adenylosuccinate lyase activity required in two non-sequential steps of the highly conserved purine synthesis pathways. (A) Schematic representation of the human and X. laevis purine biosynthesis pathways. Abbreviations: AMP, adenosine monophosphate; GMP, guanosine monophosphate; IMP, Inosine monophosphate; PRPP, Phosphorybosyl pyrophosphate; SAMP, Succinyl-AMP; SZMP, Succinyl-Amino Imidazole Carboxamide Ribonucleotide monophosphate; XMP, Xanthosine monophosphate; ZMP, Amino Imidazole CarboxAmide Ribonucleotide monophosphate. (B) Functional complementation of the growth defect of the yeast adenylosuccinate lyase deletion mutant via expression of the X. laevis adsl.L ortholog gene. Yeast wild-type and adsl knockout mutant (ade13 ade1) strains were either transformed with a plasmid, allowing for expression of the Saccharomyces cerevisiae (ADE13) or the X. laevis (adsl.L) adenylosuccinate lyase encoding genes, or with the empty vector (None). Serial dilutions (1/10) of transformants were dropped on SDcasaWA medium supplemented with either hypoxanthine or adenine as the sole external purine source. Plates were incubated for 48 h at 37 °C before imaging. Of note, the ade1 ade13 double deletion mutant was used in this experiment to avoid genetic instability associated with the accumulation of SZMP and/or its nucleoside derivatives observed in the single ade13 mutant [8].
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Figure 2. Spatiotemporal expression of adsl.L gene during X. laevis embryonic development. (A) Temporal expression profiles of adsl.L gene during embryogenesis. The expression profile was determined using RT-PCR from the cDNA of the fertilized oocyte (egg) and whole embryo at indicated stages covering the different phases of X. laevis embryogenesis. The ornithine decarboxylase gene odc1.L was used as a loading control and negative controls were performed by omitting either reverse transcriptase (-RT) or RNA (-RNA) in the reaction mix. Linearity was determined via dilutions of cDNA from stage 39 for adsl.L and 41 for odc1.L. Mid-blastula transition (MBT) is indicated. (B) Spatial expression profile of adsl.L gene during embryogenesis. Whole-mount in situ hybridization with adsl.L-specific DIG-labeled antisense or sense RNA probes was performed on embryos from stages (St.) 5 to 37–39. St. 5 and 9: animal (AP) and vegetal (VP) pole views, St. 12.5 and 19: dorsal (DV) and anterior (AV) views; later stages: lateral views, with dorsal up and anterior on the right, and dorsal view at stages 37–39. Transverse sections are dorsal up. Abbreviations: ba, branchial arches; e, eye; fmb, forebrain–midbrain boundary; h, heart; l, lens; mhb, midbrain–hindbrain boundary; n: nasal placode; ov, otic vesicle; ppt, pronephric proximal tubules; pit, pronephric intermediate tubules; s, somites. Bars: 0.5 mm.
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Figure 3. The adsl.L gene is required for somites and hypaxial muscle formation in X. laevis. (A) Representative images of adsl.L morphants following adsl.L MO1 injection at either 10 ng (weak and medium curvature) or 20 ng (medium and strong curvature). (B) Quantification and statistics of the curvature phenotype. (C–G) Immunostaining with the differentiated muscle-cell-specific 12-101 antibody revealed a strong alteration of somites and hypaxial muscle formation in adsl.L knockdown and adsl.L overexpressing embryos. Representative images (D,E) and quantification and statistics (C,F,G) of somites and hypaxial muscles phenotype at tailbud (C,D) and tadpole (E–G) stages. Injected side is indicated by asterisks. S, somites; HM, hypaxial muscles. Bars: 0.5 mm. White and red arrowheads point to typical somite chevron shapes and altered somites, respectively. The adsl.L RNA* refers to mutated RNA whose translation was not affected by the adsl.L specific MOs (Figure S3). Numbers above the bars of histograms correspond to p-values.
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Figure 4. The adsl.L gene is required for the expression of tcf15 and mespa genes involved in early somitogenesis. Expression of tcf15 (A,B) and mespa (C,D) genes at late neurula stage is altered by the knockdown of adsl.L gene. Representative images of trf15 (A) and mespa (C) RNA expression revealed via in situ hybridization. Statistics are shown in (B) and (D) for trf15 and mespa RNA, respectively. White arrows point to the anterior domain of the somitomere S-II on both sides of the embryos. Injected side is indicated by asterisks. Numbers above the bars of histograms correspond to p-values. Bars: 0.5 mm.
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Figure 5. Expression of the myogenic regulatory factors myod1 and myf5 in paraxial mesoderm was strongly affected by the knockdown of the adsl.L gene. (A) Expression of myod1 gene at the early neurula stage was altered by the knockdown of adsl.L gene and rescued by adsl.L RNA* and human ADSL RNA. (C) Representative images of the effect on myf5 RNA expression by adsl.L knockdown at stage 12.5 revealed via in situ hybridization. (B,D) Quantification and statistics of the myod1 and myf5 expression phenotypes are presented in (A,C), respectively. (E) Knockdown of adsl.L did not alter the mesoderm formation, as revealed by the absence of change in tbxt (xbra) RNA expression domain at stage 11. Injected side is indicated by asterisks. The amounts of MO and RNA presented in this figure are in ng. Bars: 0.5 mm. Numbers above the bars of histograms correspond to p-values. White arrows point to the domain where expression of either myod1 or myf5 is altered.
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Figure 6. Knockdown of adsl.L gene caused an impaired expression of myod1 and myf5 at late tailbud and tadpole embryonic stages, leading to somites and craniofacial muscle formation defects. (A–D) Representative images of the adsl.L-dependent altered expression of myod1 (A) and myf5 (C) domains in either somite (S), dorsal somite border (DB), ventral somite border (VB) or craniofacial muscles (CM), as revealed via in situ hybridization in late tailbud embryos. Blue, red, green and purple arrowheads point to myod1 altered expression in somites, ventral border, craniofacial muscles and hypaxial muscles, respectively. (E,G) Representative images of the adsl.L-dependent altered expression of myod1 (E) and myf5 (G) domains in the unsegmented mesoderm in the most posterior tail (T), somites (S), ventral somite border (VB) and craniofacial muscles (CM) in tadpole embryos. Black and pink arrowheads point to myf5 altered expression in pharyngeal arch muscle enlagen intermandibularis muscle enlagen respectively. (B,D,F,H) Quantification and statistics of the myod1 and myf5 expression phenotypes presented in (A,C,E,G), respectively. Craniofacial muscles: ih, interhyoïedus anlage; im, intermandibularis anlage; lm, levatores mandibulae anlage; pam, pharyngeal archmuscle anlagen; q/oh, common orbitohyoïdeus and quadrato-hyoangularis precursors. Injected side is indicated by asterisks. Bars: 0.5 mm. Numbers above the bars of histograms correspond to p-values.
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Figure 7. The adsl.L gene is required for hypaxial muscle migration. (A,B) The hypaxial muscle defect associated with adsl.L knockdown was rescued by the MO non-targeted adsl.L RNA*. Representative images (A) and quantifications (number in bars) and statistics (B) of the adsl.L-dependent alteration of myogenin gene expression monitored via in situ hybridization. (C–F) Migration of myoblasts in the hyoid region was found to be severely affected in adsl.L morphants, as shown by lbx1 (C,D) and pax3 (E,F) gene expression pattern alterations. Injected side is indicated by asterisks. Bars: 0.5 mm. HM and H stand for hypaxial muscles and hyoid region, respectively. Numbers above the bars of histograms correspond to p-values.
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