DEV01572 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Effects of interference with Prdm1 activity on embryonic morphology, slow muscle and pectoral fins. (A-C,F-M) Lateral view, anterior towards the right. (D,E) Frontal view, dorsal towards the top. (N,O) Dorsal view, anterior towards the top, left side shown. (A,D,F,H,J,L,N) Untreated controls (ctrl). (B,C,E,G,I,K,M,O) Embryos microinjected with 1 ng of MO^prdm1 oligonucleotides (MO). (A-C) Overall morphology at 24 hpf; embryo in B scored as strong phenotype with kinked tail (kt); embryo in C scored as severe phenotype exhibiting degeneration in head and trunk (dg; embryos of that class were not subjected to molecular anlyses). (D,E) Head morphology at 24 hpf. (F,G) Morphology of three somitic segments at 24 hpf; somites are U-shaped and lack horizontal myoseptum (hm, broken line) in morphants morphants as seen in ubo^tp39 mutant embryo. (H,I) emx1 expression in diencephalon (de) (Morita et al., 1995), but not krox20 expression in hindbrain rhombomeres 3 and 5 (r3, r5), is slightly reduced at 24 hpf. (J,K) eng2a expression at the mid/hindbrain boundary (mhb) (Ekker et al., 1992) is normal and reduced in muscle pioneers (mp) in 18 hpf morphants. tbx5 expression in prospective pectoral fin buds is missing (fb) (Begemann and and Ingham, 2000). (L,M) Slow muscle fiber formation, detected by a-sMHC antibody (mAB F59) (Crow and Stockdale, 1986), is severely affected in morphants (white arrows), as observed in ubo^tp39 mutant embryo (Baxendale et al., 2004), and the level of sMHC protein expression is low (image overexposed); broken line highlights somite boundaries; Cy2-conjugated secondary antibodies by Jackson ImmunoResearch were used, photographed using a Zeiss LSM510 inverted confocal laser scanning microscope. Seventy-five percent of morphants (n=51) displayed 0-50% of labeled slow muscle fibres compared with 17% (n=60) when co-injected with 50 pg of prdm1^mut RNA. (N,O) Loss of pectoral fins (pf) and failure of swim bladder (sb) inflation. Scale bar: 200 µm in A,I,P,Q,T; 100 µm in D,O; 50 µm in F,M.

• Supplemental Figure 2 -

  Fig. S2. Additional organogenesis defects resulting from downregulation of Prdm1 activity in branchial arches, eyes and cloaca. (A,B,H,I,K,M,R,S) Lateral view, anterior towards the left. (C,D) Ventral view, anterior towards the left. (E-G) Dorsal view, anterior towards the left. (J) Dorsal view, anterior towards the top. (L) Ventral view, anterior towards the top. (N-Q) Ventral view, anterior towards top right, left eye. (J,K and L,M) Different views of the same embryos. (A,C,E,F,H,J,K,N,P,R) Untreated controls (ctrl). (B,D,G,I,L,M,O,Q,S) Embryos microinjected with 1 ng of MO^prdm1 oligonucleotides (MO). (A,B) Morphants exhibit jaw (jw) and gill arch defects (ga), and lack inflated swim bladders (sb); eye (ey). (C,D) Development of pharyngeal skeleton, visualized by Alcian Blue cartilage staining in 5 dpf morphants (Piotrowski et al. 1996); hyoid (hy) is abnormal, ceratobranchials 1-3 (cb) are shortened and ceratobranchials 4-5 are missing (class III, 24%; class II morphants lack cb 3-5 and class I lack cb1/2-5; 63% and 13%, respectively, n=215). (E,F) prdm1 and aldh1a2 share expression domains in the branchial arch (ba) and pectoral fin bud (fb) primordia (Grandel et al., 2002). (F-I) Morphant embryos exhibit reduced aldh1a2 expression in branchial arch and pectoral fin primordia; and eyes (ey) and somites (sm). (J-M) Eye size and rho expression in photoreceptor cell layer (pl) (Vihtelic et al., 1999) is reduced in morphants; lens (le) and retina lamination (rt) appear normal, whereas rho expression is generally reduced and missing regionally (arrows). (N,O) Reduced number of green-sensitive Opsin-expressing cells in PCL of morphants, detected by a-grOpn antibody (Vihtelic et al., 1999); single focal level obtained by confocal microscopy. (P,Q) Strongly reduced number of Rhodopsin-expressing cells in PCL of morphants, detected by a-Rho antibody (Zpr3/Fret11, Vihtelic et al., 1999); images obtained by 3D reconstruction. Morphant eye dimensions are smaller. Cy3-conjugated secondary antibodies by Jackson ImmunoResearch were used. (R,S) Cloaca (cl) morphology is abnormal in morphants. Scale bar: 200 µm in A,C,I; 100 µm in G,J,K; 50 µm in O,U.

  References

  Begemann, G. and Ingham, P. W. (2000). Developmental regulation of Tbx5 in zebrafish embryogenesis. Mech. Dev. 90, 299-304.

  Crow, M. T. and Stockdale, F. E. (1986). Myosin expression and specialization among the earliest muscle fibers of the developing avian limb. Dev. Biol. 113, 238-254.

  Ekker, M., Wegner, J., Akimenko, M. A. and Westerfield, M. (1992). Coordinate embryonic expression of three zebrafish engrailed genes. Development 116, 1001-1010.

  Grandel, H., Lun, K., Rauch, G.J., Rhinn, M., Piotrowski, T., Houart, C., Sordino, P., Kuchler, A. M., Schulte-Merker, S., Geisler, R. et al. (2002). Retinoic acid signalling in the zebrafish embryo is necessary during pre-segmentation stages to pattern the anterior-posterior axis of the CNS and to induce a pectoral fin bud. Development 129, 2851-2865.

  Morita, T., Nitta, H., Kiyama, Y., Mori, H. and Mishina, M. (1995). Differential expression of two zebrafish emx homeoprotein mRNAs in the developing brain. Neurosci. Lett. 198, 131-134.

  Piotrowski, T., Schilling, T. F., Brand, M., Jiang, Y. J., Heisenberg, C. P., Beuchle, D., Grandel, H., van Eeden, F. J., Furutani-Seiki, M., Granato, M. et al. (1996). Jaw and branchial arch mutants in zebrafish II: anterior arches and cartilage differentiation. Development 123, 345-356.

  Vihtelic, T. S., Doro, C. J. and Hyde, D. R. (1999). Cloning and characterization of six zebrafish photoreceptor opsin cDNAs and immunolocalization of their corresponding proteins. Vis. Neurosci. 16, 571-585.