rbm24a and rbm24b are expressed throughout somitogenesis.

ISH images of uninjected AB embryos using rbm24a or rbm24b riboprobes individually marking spatial and temporal RNA expression. Tailbud expression of bud stage embryos in lateral-slightly ventral facing (A, C) and dorsal (B, D) orientations. 8 somite embryos in dorsal facing orientations show all segmented somites (E, G) and PSM (F, H) marked by rbm24a and rbm24b riboprobes. Trunk images of embryos at 24 hpf show spatial expression of rbm24a in the somites concentrated at the posterior, while rbm24b is uniformly expressed in somites along the A-P axis (I-J). Inset of (I-J) highlights expression of rbm24a and rbm24b in the PSM. Embryos at 48 hpf show no somite rbm24a expression, while somite expression of rbm24b remains uniform thought the somites (K-L). som, somites; hpf, hours post fertilization; arrows, tailbud expression; A, anterior of embryo; P, posterior of embryo; bracket, PSM expression.

Rbm24a and Rbm24b are required for normal somitogenesis.

Bright field 8 somite embryos are shown in dorsal orientation (A-C). Spatial expression of myod in somites of 13 somite embryos was detected using anti-sense riboprobes (D-F). Measurements of individual chevron angle measurements of somites 6-10 of 13 somite embryos for uninjected, ctrlMO injected rbm24aMO and rbm24bMO embryos, n = 3-5 embryos each (G). Statistically significant differences in somite angle as compared to ctrlMO is shown using an asterisk. The trunks of 24 hpf embryos are shown in the lateral orientation stained via myod ISH (H-J). Average somite counts of uninjected, ctrlMO injected rbm24aMO and rbm24bMO 13 somite and 24 hpf embryos are shown (K). Number of embryos counted shown on bars. Error bars are standard error of the mean. Significance determined at 95% confidence with significance compared to ctrlMO shown as * = p≤0.05; ** = p≤0.01; *** = p≤0.001 and significance between respective MO only and MO rescue conditions shown as + = p≤0.05; ++ = p≤0.01; +++ = p≤0.001. Trunks of 24 hpf uninjected, ctrlMO injected rbm24aMO, rbm24bMO, rbm24aMO embryos with rbm24a mRNA and rbm24bMO embryos with rbm24b mRNA embryos are shown in the lateral orientation imaged in bright field (L-P), double immunohistochemistry for phalloidin and anti-pFAK (Q-U), phalloidin alone (V-Z).

Notch-signaling pathway transcripts are depleted in somites of rbm24aMO and rbm24bMO embryos.

ISH images of the PSM of dorsally oriented 13-15 somite and laterally oriented 24 hpf ctrlMO, rbm24aMO and rbm24bMO embryos. 13-15 somite embryos are shown in 2 different temporal stages of development. Spatial expression of dlc (A-F), dld (G-L), her1 (M-R) and her7 (S-X) and tbx16 (Y-DD). Asterisks highlight cycling expression. n = 8-12 embryos stained per time-point with no more than 1 deviating from the displayed expression. arrow, regions of interest for PSM expression.

Aberrant dlc and dld splice forms are detectable in cDNA from rbm24aMO and rbm24bMO embryos.

RT-PCR experiments to amplify dlc and dld mRNA transcripts using total cDNA generated from 13 somite uninjected, ctrlMO, rbm24aMO and rbm24bMO embryos (n = 50 embryos per condition). RT-PCR for dlc transcript yielded a fragment of correct length for all conditions and an additional short fragment for rbm24aMO and rbm24bMO cDNA (A). dlc pre-mRNA, dlc mRNA and dlc short 1 fragment sequences are depicted graphically to scale. RT-PCR for dld transcript yielded a fragment of correct length for all conditions and two additional short fragments for rbm24aMO and rbm24bMO cDNA (B). dld pre-mRNA, dld mRNA and dld short 1 and dld short 2 fragments are depicted graphically to scale. 13 somite and 24 hpf uninjected, ctrlMO, rbm24aMO and rbm24bMO embryos were used to generate cDNA used for qRT-PCR. All transcripts were assayed in triplicate per cDNA sample. Transcript levels were normalized to elfalpha transcript levels and fold change for each transcript is shown as compared to uninjected embryos. An asterisk denotes statistically significant fold changes compared to uninjected. * = p<0.05; ** = p<0.01; *** = p<0.001. black arrows, short dlc and dld fragments; boxes, exons; thick black lines, introns; thin black lines, wild type splicing, dashed lines, aberrant splicing.

Rbm24a and Rbm24b are required for craniofacial development. Bright field, ISH, and Alcian blue images of ctrlMO, rbm24aMO and rbm24bMO embryos. 72 hpf lateral oriented embryos show expression of rbm24a and rbm24b in presumptive optic muscles, pharyngeal arch muscles, mandibular muscles, and fin bud. rbm24a shows additional expression in the otic vesicle (A). The anterior region of 72 hpf and 96 hpf embryos are shown oriented laterally for visualization of eye and mandible phenotypes (B). Bright field and ISH images of myod expression in the anterior region of dorsally oriented 72 hpf ctrlMO, rbm24aMO and rbm24bMO embryos (B rows 1 and 2). myod expression is detected in the find bud, optic muscles, pharyngeal arch muscles and mandibular muscles of uninjected embryos. Normal myod expression is diminished in rbm24aMO and rbm24bMO embryos. Bright field and Alcian blue cartilage staining of ctrlMO, rbm24aMO and rbm24bMO embryos in lateral orientation at 96 hpf (B rows 2 and 3). Normal cartilage staining is observed in the fin buds, ethmoid plate, palatoquadrate, hyomandibular and Meckel′s cartilage of uninjected embryos. Cartilage formation of these structures is ablated in rbm24aMO embryos and severely reduced in rbm24bMO embryos. Bright-field craniofacial images of RNA rescue rbm24aMO and rbm24bMO phenotypes at 96 hpf (C). rbm24aMO rescue, by co-injection of 5 ngrbm24aMO with 800 pg of capped poly-A rbm24a mRNA and rbm24bMO rescue, by co-injection of 8 ngrbm24bMO with 200 pg of capped poly-A rbm24b mRNA, show rescue of somite and craniofacial rbm24aMO and rbm24bMO phenotypes. fb, fin bud; om, optic muscles; ov, otic vesicle; ph, pharyngeal muscles; mm, mandibular muscles. white arrow, eye; black arrow, mandible; m, Meckel′s cartilage; ep, ethmoid plate; pq, palatoquadrate; hm, hyomandibular cartilage.

notch1a and notch3 transcripts do not shown reduced tailbud expression in rbm24aMO and rbm24bMO embryos. ISH of Notch pathway receptors transcripts notch1a (A-C) and notch3 (D-F) in the somites of 24 hpf ctrlMO, rbm24aMO and rbm24bMO embryos.

rbm24a and rbm24b are expressed before and after maternal-zygotic transition. ISH of uninjected embryos with anti-sense and sense rbm24a and rbm24b riboprobes. Imaging of 16-cell (cleavage ~1.5 hpf) (A-D), high (blastula ~3.3 hpf) (E-H), shield (early gastrula ~6 hpf) (I-L) and 75% epiboly (late gastrula ~8 hpf) (M-P) show both rbm24a and rbm24b are expressed both before and after the maternal-to-zygotic transition.

rbm24aMO and rbm24bMO embryos do not display gastrulation defects. ctrlMO, rbm24aMO and rbm24bMO embryos during gastrulation. Bright field imaging of 75% epiboly embryos (A-C). Bright field imaging of bud stage (D-F). ISH of bud stage embryos with tbx16 (G-I) and ntla (J-L) riboprobes.