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Exostosin 1 (EXT1) is a glycosyltransferase that contributes to the biosynthesis of heparan sulfate proteoglycans (HSPG). Loss of ext1 function leads to the human genetic disorder hereditary multiple exostoses (HME) and inhibits development in mouse, zebrafish and Drosophila. In Xenopus, loss of maternal EXT1 leads to impaired wnt11 signaling, resulting in a loss of dorsal embryonic development (Tao et al., 2005), but the functions of zygotic ext1 have not been elucidated. In this study, morpholino oligonucleotides were used to generate a zygotic partial loss of function for ext1, in order to evaluate the requirements for ext1 function in gastrulation and paracrine signaling. Transcriptional profiling was carried out by microarray. Validation and subsequent analyses of gene expression were performed using Q-RT-PCR and in situ hybridization. Western blots were used to assess paracrine signaling pathway activity. Introduction of ext1 MO led to gastrulation defects, which were partially rescued by co-injection of ext1 mRNA. Microarray-based comparisons of gene expression in control vs. Ext1 MO embryos identified several developmentally significant genes that are dependent upon Ext1 function, including brachyury (Xbra). In addition, decreased Ext1 was shown to reduce the level of Wnt8 and BMP4 signaling and disrupt ventral-specific gene expression. Ext1 function is required for maintenance of normal levels of BMP and wnt, as well as their target genes. In addition, expression of xbra and the establishment of ventralmesoderm depend upon normal levels of Ext1. These findings suggest that ext1-dependent synthesis of HSPG is critical for wnt and BMP signaling, mesodermal identity, and ventral pattern.
Fig. 1. Knockdown of ext1 results in gastrulation defects. (A) Embryos were injected with 20 ng of either the EXT1 MO or the mispair MO and lysed at st. 10.5 in preparation for immunoblotting to detect Ext1 and a-tubulin (loading control). Ext1 protein levels are decreased in EXT1-MO injected embryos. (B) Quantitative comparison of Ext1 protein accumulation. Relative Ext1 levels were normalized with a-tubulin. Bars represent mean ± S.D. N=3 independent experiments. (C) Midgastrula EXT1 MO-injected embryos exhibited incomplete blastopore formation and partial endoderm protrusion, whereas the mispair MO-injected embryos had a normal blastopore. (D,E) Embryos were injected with 20 ng of mispair or EXT1 MO at either the 2- cell stage or in either dorsal or ventral cells at the 4- cell stage; the phenotypes were compared at midgastrula (St. 10.5). A higher percentage of blastopore closure defects (D) or gastrulation arrest (E) were observed in EXT1 MO-injected embryos, irrespective of the stage or location of injection. N = 10 experiments, with a total of 400 embryos for each condition. (F) The phenotypes of embryos with head and tail defects in ext1 morphants. (G) The frequency of tail defects in ext1 morphants. Total number of tadpoles was 213. Bars present meant ± S.D. N=7 independent experiments. (H) The survival rates for EXT1 MO and EXT1 MIS embryos. Bars represent mean ± S.D. N=7 independent experiments (20-60 embryos/injection). (I) Blastopore closure defects and arrested gastrulation in ext1 morphants were partially rescued by coinjection with ext1 mRNA. Bars present meant ± S.D. N=5 independent experiments. (J) Embryos injected with 1ng of b-galactosidase or ext1 mRNA were collected and phenotypes were compared at gastrulation stage. The embryos overexpressing ext1 had higher frequencies of blastopore defects and gastrulation arrest compared to controls. Bars present meant ± S.D. N=5 independent experiments. * p < 0.05.
Fig. 2. Gene ontology (GO) of genes showing dysregulation in ext1 morphants. We characterized genes that showed a change of greater than two fold (A) or less than 0.5 fold (B) in ext1 morphant embryos. More than half of the genes up-regulated in response to ext1 knockdown had unknown functions. Among those with known functions, 16% were associated with metabolic processes, including transcriptional and translational regulation. Signaling-associated genes accounted for 6%, while 5% were involved in developmental processes. The remainder included establishment of localization (4%), cellular processes (3-4%), and others (6%). The “others” category includes multicellular organismal processes, response to stimulus, cell proliferation, cellular component organization, and reproductive processes. Similar proportions were observed for genes that were down-regulated following a reduction in Ext1 expression.
3. Validation of xbra and other genes affected by ext1 knockdown. (A) Selected genes identified by microarray were validated using Q-RT-PCR. Embryos injected with 20 ng of EXT1 MO or EXT1 MIS were collected at stage 10.5 for RNA extraction and Q-RT-PCR. (B) In situ hybridization shows differences in xbra expression in control vs ext1 morphant embryos. Results are representative of over 2/3 of embryos from 3 independent experiments. (C) Q-RT-PCR assays for expression of the xbra target genes eFGF and Wnt11. Relative fold enrichment of each target gene was calculated after normalization with the housekeeping gene ornithine decarboxylase (ODC) using the δδCt method. Bars represent mean ± S.D. N=3 independent experiments. *: p < 0.05.
Fig. 4. The effects of Ext1 knockdown on paracrine signaling pathways. Embryos were injected with EXT1 or mispair MO and lysed at stage 10.5. (A,C,E) Immunoblots showed levels of diphospho- Erk MAPK (dpMAPK), phospho-Smad3, and phospho-Smad1 in EXT1 or mispair MO-injected embryos. (B,D,F) Relative levels of phosphorylated forms of MAPK, Smad3, and Smad1 in EXT1 or mispair MO-injected embryos. Phospho-Smad1 is reduced in EXT1 morphant embryos, whereas levels of dpMAPK and phopho-Smad3 are unchanged. (G) Q-RT-PCR assays of BMP4 and the BMP4 target genes msx1, vent1, and vent2 in control or ext1 morphant embryos. (H) Q-RT-PCR assays of wnt8 and the wnt8 target genes HoxA1, HoxB1, HoxD1, and myoD. The relative -fold change of each target gene in EXT1 MO-injected embryos was calculated as described in Fig. 3. (B,D,F-H) Bars represent mean ± S.D. N=3 independent experiments. *: p < 0.05.
Fig. 5. Establishment of dorsal- or ventral-specific patterns of gene expression in ext1 morphant embryos. Embryos were injected with 20 ng of EXT1 or mispair MO. In situ hybridization of vent1 (A,B); vent2 (C,D); not1 (E,F); and otx2 (G,H). Expression patterns shown are representative of at least 2/3 of embryos from 3 independent experiments. (I) Q-RT-PCR assays of organizer-specific gene expression in ext1 MO or mispair MO embryos. Relative –fold changes were evaluated as described in Fig. 3. Bars represent mean ± S.D. N=3 independent experiments. *: p < 0.05.