Lee SH , Lee HK , Kim C , Kim YK , Ismail T , Jeong Y , Park K , Park JW , Kwon OS , Kang BS , Lee DS , Park MJ , Park DS , Lee HS .

	Fig. 1. Spatial-temporal expression profiles of SRSF1 during Xenopus embryogenesis. (A) Spatial expression of SRSF1 was determined by whole-mount in situ hybridization of Xenopus embryos from stage 0–32. SRSF1 is mainly localized in the animal hemisphere in an early embryo (∼stage 6) and sequentially widens to the vegetal hemisphere through gastrulation (∼stage 12). At the late neurula stage, SRSF1 is expressed in the neural plate- (∼stage 18) and then is strongly expressed in the central nervous system (CNS), including the front head, eye, and branchial arches (∼stage 22 and stage 32). (B) Reverse transcriptase-PCR (RT-PCR) results to show temporal expression patterns of SRSF1. SRSF1 is continuously expressed from stage 0 embryos to stage 40 tadpoles and expression levels reduced from stage 0–8 were restored at gastrula stage (stage 17). This restored expression again decreased after the neurula stage but also increased and peaked at stage 35. Ornithine decarboxylase (ODC), was used as a loading control.
	Fig. 2. Loss of SRSF1 induces microcephaly by abnormal formation of brain cartilage. Alcian blue staining was performed to show cartilage formation. SRSF1 MO-injected embryos showed abnormal cartilage shapes, such as cb, ch, ir, and mc (red arrows) compared to those of control MO-injected embryos; cb = ceratobranchial, ch = ceratohyal, ir = infrarostral, mc = Meckel’s cartilage. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
	Fig. 3. SRSF1 regulates heart development through the regulation of Nkx-2.5 expression. (A) Expression of Nkx-2.5 was detected by whole-mount in situ hybridization. Stage 32 tadpoles were fixed in MEMFA for this experiment. SRSF1 morphants showed significant downregulation of Nkx-2.5 expression. (B) The graph showed the expression level of Nkx-2.5 in each experiment. Expression of Nkx-2.5 in stage 32 embryos, detected by whole-mount in situ hybridization, was slightly reduced (approximately 30%) in SRSF1 MO-injected groups. ***p < 0.001 compared with control MO-injected. (C) Expression of Nkx-2.5 was detected by RT-PCR in stage 16 whole embryos or isolated animal caps injected with DNBR. (D) The graph exhibited only DNBR-induced Nkx-2.5 expression in stage 16 animal caps. In stage 16 embryos, expression of Nkx-2.5, detected by RT-PCR, was dramatically reduced in SRSF1-injected embryos (approximately 70%). ***p < 0.001 compared with control MO-injected and SRSF1*-injected.
	Fig. 4. Loss of SRSF1 dramatically reduces the transcription of chordin. We performed RT-PCR to show the expression of BMP-4 signaling-related molecules in SRSF1 MO-injected embryos. In the SRSF1 MO-injected groups, only chordin RNA was down-regulated, while other BMP-4 signaling-related molecules such as BMP-4 and follistatin were not changed.
	Fig. S1. SRSF1-depleted embryos show abnormal organ development, especially in head formation, gut looping, and edema in the heart. (A) SRSF1-depleted embryos did not show significant malformation of organs until the tail bud stages. The first traits of SRSF1 morphants were detected at stage 33 tadpoles, in the period of eye development (left). At stage 45, SRSF1-depleted tadpoles showed abnormal phenotypes such as edema in the head and heart and miscoiled intestines. (B) Graphs show the percentage of tadpoles that showed abnormal development in the test groups. More than 80% of tadpoles showed loss-of-SRSF1 phenotypes at stages 33 and 45.
	Fig. S2. Exogenous Xenopus SRSF1 is expressed in nuclear speckles. Exogenously expressed Xenopus SRSF1 proteins were localized in the nucleus, especially in the nuclear speckles in HeLa cells. NSrp70 was used as a control because it is a known nuclear speckle protein and interacts with SRSF1 physiologically. (Blue: DAPI, Green: SRSF1, Red: NSrp70). Immunohistochemistry was performed to show localization.
	Fig. S3. SRSF1 MO-induced abnormal phenotypes are specific and rescued by knock-in of exogenously expressed RNAs. (A) In order to rescue the defects of SRSF1 MO, SRSF1* RNA (SRSF1 Res), which is resistant to SRSF1 MO, was co-injected with SRSF1 MO. SRSF1* RNA (400 pg) was injected and did not affect embryogenesis (third row panels). Furthermore, embryos injected with 400 pg of SRSF1* RNA with SRSF1 MO showed partially rescued phenotypes at stage 45 tadpoles (bottom panels). (B) The histogram shows the percentage of tadpoles with abnormal phenotypes among SRSF1 MO and rescued tadpoles. Approximately 50% of the failed development in tadpoles was restored in the rescued groups.
	srsf1 (serine/arginine-rich splicing factor 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.