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J Dev Biol
2022 Jul 08;103:. doi: 10.3390/jdb10030029.
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The Core Splicing Factors EFTUD2, SNRPB and TXNL4A Are Essential for Neural Crest and Craniofacial Development.
Park BY
,
Tachi-Duprat M
,
Ihewulezi C
,
Devotta A
,
Saint-Jeannet JP
.
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Mandibulofacial dysostosis (MFD) is a human congenital disorder characterized by hypoplastic neural-crest-derived craniofacial bones often associated with outer and middle ear defects. There is growing evidence that mutations in components of the spliceosome are a major cause for MFD. Genetic variants affecting the function of several core splicing factors, namely SF3B4, SF3B2, EFTUD2, SNRPB and TXNL4A, are responsible for MFD in five related but distinct syndromes known as Nager and Rodriguez syndromes (NRS), craniofacial microsomia (CFM), mandibulofacial dysostosis with microcephaly (MFDM), cerebro-costo-mandibular syndrome (CCMS) and Burn-McKeown syndrome (BMKS), respectively. Animal models of NRS and MFDM indicate that MFD results from an early depletion of neural crest progenitors through a mechanism that involves apoptosis. Here we characterize the knockdown phenotype of Eftud2, Snrpb and Txnl4a in Xenopus embryos at different stages of neural crest and craniofacial development. Our results point to defects in cranial neural crest cell formation as the likely culprit for MFD associated with EFTUD2, SNRPB and TXNL4A haploinsufficiency, and suggest a commonality in the etiology of these craniofacial spliceosomopathies.
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35893124
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Figure 1. Developmental expression of eftud2, snrpb and txnl4a. (A) RT-PCR analysis indicates that eftud2, snrpb and txnl4a are expressed at all stages examined whereas sox10 is first activated at stage 15. odc is shown as a loading control. (B) At stage 15, eftud2 and txnl4a transcripts are enriched at the anterior neural plate (arrowheads) and the neural-crest-forming regions (arrows), whereas snrpb appears to be ubiquitously expressed. At stages 20 to 26, the three genes are enriched in the pharyngeal arches (brackets) and the eyes (arrows). At stage 29/30, the expression of eftud2, snrpb and txnl4a is maintained in the pharyngeal arches (brackets). Stages 15 and 20 are dorsal views, anterior to top. Stages 23, 25, 26 and 29/30 are lateral views, dorsal to top, anterior to right.
Figure 2. Eftud2 knockdown affects neural crest formation. (A) Increasing amounts of Eftud2MO, 10 ng (+), 100 ng (++) and 500 ng (+++), block translation directed by eftud2 mRNA. (-) Translation without Eftud2MO (B) Phenotype of Eftud2MO-injected embryos (30 ng) on neural crest (yellow arrows) and placode (red arrows) gene expression at stage 15. The gene analyzed in each panel is indicated in the lower left corner. D indicates dorsal view, anterior to top. L-cs indicates lateral view control side, anterior to left. L-is indicates lateral view injected side, anterior to right. (C) Quantification of the phenotypes. The numbers in each bar indicate the number of embryos analyzed. (D) sox10 expression in Eftud2MO-injected embryos is partially restored by expression of HuEFTUD2 resistant to the MO. The injected side is indicated by an asterisk. Dorsal views, anterior to top. (E) Quantification of the results. The numbers in each bar indicate the number of embryos analyzed. Statistically significant differences are indicated (*) p-value < 0.05 (Chi-squared test).
Figure 3. Snrpb knockdown affects neural crest formation. (A) Increasing amounts of SnrpbMO, 10 ng (+), 100 ng (++) and 500 ng (+++), block translation directed by snrpb mRNA. (-) Translation without SnrpbMO (B) Phenotype of SnrpbMO-injected embryos (5 ng) on neural crest (yellow arrows) and placode (red arrows) gene expression at stage 15. The gene analyzed in each panel is indicated in the lower left corner. D indicates dorsal view, anterior to top. L-cs indicates lateral view control side, anterior to left. L-is indicates lateral view injected side, anterior to right. (C) Quantification of the phenotypes. The numbers in each bar indicate the number of embryos analyzed. (D) sox10 expression in SnrpbMO-injected embryos is partially restored by expression of HuSNRPB resistant to the MO. The injected side is indicated by an asterisk. Dorsal views, anterior to top. (E) Quantification of the results. The numbers in each bar indicate the number of embryos analyzed. Statistically significant differences are indicated (*) p-value < 0.05 (Chi-squared test).
Figure 4. Txnl4a knockdown affects neural crest formation. (A) Increasing amounts of Txnl4MO, 10 ng (+), 100 ng (++) and 500 ng (+++), block translation directed by txnl4a mRNA. (-) Translation without Txnl4MO. (B) Phenotype of Txnl4MO-injected embryos (30 ng) on neural crest (yellow arrows) and placode (red arrows) gene expression at stage 15. The gene analyzed in each panel is indicated in the lower left corner. D indicates dorsal view, anterior to top. L-cs indicates lateral view control side, anterior to left. L-is indicates lateral view injected side, anterior to right. (C) Quantification of the phenotypes. The numbers in each bar indicate the number of embryos analyzed. (D) sox10 expression in Txnl4MO-injected embryos is partially restored by expression of Xenopus Txnl4 resistant to the MO. The injected side is indicated by an asterisk. Dorsal views, anterior to top. (E) Quantification of the results. The numbers in each bar indicate the number of embryos analyzed. Statistically significant differences are indicated (*) p-value < 0.05 (Chi-squared test).
Figure 5. Eftud2, Snrpb and Txnl4a knockdown increase cell death in the ectoderm. (A) TUNEL staining of representative morphant embryos at stage 15. The injected side is indicated by an asterisk. Dorsal view, anterior to top. (B) Quantification of the number of TUNEL-positive cells in control vs. injected sides of embryos injected with Eftud2MO (n = 33), SnrpbMO (n = 39) and Txnl4MO (n = 41). Values are presented as mean ± s.e.m.; (*) p-value < 0.0005 and (**) p-value < 0.0001 (Student’s t-test).
Figure 6. Eftud2, Snrpb and Txnl4a knockdown affect craniofacial cartilage formation. (A) Alcian blue staining of dissected craniofacial cartilages of control tadpoles and those injected with Eftud2MO (5 ng), SnrpbMO (1 ng) and Txnl4MO (30 ng), around stage 45. The injected side is indicted by an asterisk. The black lines indicate the midline. Note: Txnl4MO-injected tadpoles were collected at a slightly younger stage than their counterparts, which explains the overall size difference in craniofacial structures. (B) Quantification of the phenotypes. The numbers in each bar indicate the number of embryos analyzed. Control vs. MO-injected (*) p-value < 0.0001 (Chi-squared test).
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