DEV037820 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Real-time PCR analysis of six3b expression in response to increased levels of shha. Embryos injected with control mRNA (Tol2) or shha mRNA (1× ∼12.5 ng, 2× ∼25 ng) were harvested at 20s and subjected to real-time PCR analysis to quantify levels of shha (A) and six3b RNA (B). Results, normalized to gapdh, showed no significant increase in six3b transcript levels with increasing levels of shha (P=0.12). Each data-point is based on two technical replicates, each done in triplicate (i.e. six measurements), of a biological replicate comprising ten pooled embryos.

• Supplemental Figure S2 -

  Fig. S2. zic2a expression in the anterior CNS from 3s-12s. zic2a expression in the OS is first detectable at 3s-4s (A) and continues at 5s-6s (B) and 7s-8s (C) It is not detectable in the adjacent retina at these stages (A-C). Beginning at 11s-12s, zic2a is expressed both in the OS and the distal (dorsal) retina (D, asterisk). A-D are lateral views, anterior to the left. A′-D′ are dorsal views of the same embryos, anterior to the left.

• Supplemental Figure S3 -

  Fig. S3. Apoptosis does not contribute to OS patterning defects observed in Zic2a-depleted embryos. p53MO-injected embryos exhibit normal pax2a expression in the OS (A, 20/20 embryos, n=2). zic2a morphants exhibit strong pax2a expansion into the ventral retina (B, 20/23 embryos, n=2). Embryos co-injected with p53MO and zic2aMO show a similarly strong expansion of pax2a into the adjacent ventral retina (C, 21/21 embryos, n=2). A-C are lateral views, anterior to the left. A′-C′ are ventral views of the same embryos, anterior to the left. All embryos are at prim-5.

• Supplemental Figure S4 -

  Fig. S4. pax2a patterning defects are detected prior to fgf patterning defects in zic2a morphants. fgf8a expression is normal at 16s in control (A, 11/11 embryos) and zic2a morphants (B, 8/8 embryos). Similarly, spry4 is unaffected at 16s in control (C, 21/21 embryos, n=2) or zic2a morphants (D, 22/24 embryos, n=2). pax2a is expressed broadly in the OS and retinal primoridia at 16s (E, 15/15 embryos, n=2), and might be slightly expanded in zic2a morphants (F, 9/21 embryos, n=2). At 19s, fgf8a is weakly expressed in the presumptive OS (G, 12/12 embryos), and its expression is unaffected by knocking-down Zic2a (H, 9/9 embryos). spry4 is expressed in a broader domain than fgf8a (I, 20/20 embryos, n=2), and also appears unaffected in zic2a morphants (J, 21/25 embryos, n=2). pax2a expression has begun retracting from the retinal primordium by 19s in control embryos (K, 8/18, n=2), but not in zic2aMO-injected embryos (L, 9/15 embryos, n=2). Arrowheads mark the OS. All embryos are shown in ventral view, anterior to the left. Embryos in A-F are at 16s, while embryos in G-L are at 19s.

• Supplemental Figure S5 -

  Fig. S5. zic2a acts upstream of Fgf signaling and pax2a in OS development. (A-C) The expression of spry4, a direct target of Fgf signaling, after a 120-minute treatment with vehicle (A, normal in 13/13 embryos, n=2), a 30-minute treatment with SU5402 (B, strongly reduced in 10/10 embryos, n=2) and a 60-minute treatment with SU5402 (C, undetectable in 9/10 embryos, n=2). (D-F) zic2a expression after vehicle treatment for 120 minutes (D, normal in 16/16 embryos), after 30 minutes of SU5402 exposure (E, normal in 19/19 embryos) and after 60 minutes of SU5402 exposure (F, strongly reduced in the telencephalon, 2/17 embryos, but unaffected in the OS, 17/17 embryos). (G,H) zic2a expression at prim-5 after vehicle treatment starting at the tail bud (G, 25/25 embryos), or SU5402 treatment starting at the tail bud (H, absent in the telencephalon, 20/20 embryos, but normal in the OS). (I,J) zic2a is strongly expressed in the OS of both the wild-type (WT; I, 97/97 embryos, n=2) and in pax2a^−/− mutant siblings (J, 30/30 embryos, n=2). Arrows point to the telencephalon, arrowheads to the OS. Views are lateral, anterior to the left, except G′ and H′ that are ventral views, anterior to the left, of the embryos pictured in G and H. Embryos are shown at 16s (A-C), 19s (D-F) and prim-5 (G-H).

• Supplemental Figure S6 -

  Fig. S6. The epistatic relationships between zic2a, pax2a and fgf8a. (A-H) conMO-injected WT siblings exhibit normal fgf8a expression in the OS and MHB (A, 58/60 embryos, n=2). pax2^−/− mutant embryos injected with conMO express fgf8a in the OS, but not in the MHB (B, 20/20 embryos, n=2). WT siblings injected with zic2aMO show expanded fgf8a (C, green arrow, 55/80 embryos, n=2). pax2^−/− mutants injected with zic2aMO also have expanded fgf8a in the ventral retina (D, 13/23 embryos, n=2), but lack MHB expression. Vehicle-treated embryos injected with conMO display normal pax2a expression (E, 35/36 embryos, n=4), whereas conMO-injected embryos treated with SU5402 show reduced pax2a (F, 34/37 embryos, n=4). Vehicle-treated zic2aMO-injected embryos have expanded pax2a expression (G, 28/32 embryos, n=4), but zic2aMO-injected and SU5402-treated embryos have reduced pax2a expression (H, 33/36 embryos, n=4). (I) The proposed regulatory relationship between pax2a and fgf8a downstream of zic2a. Arrowheads point to the OS. A-H are lateral views, anterior to the left. A′-H′ are ventral views of the same embryos, anterior to the left. Embryos in A-D are at prim-5 and embryos in E-H are at ∼prim-1.

• Supplemental Figure S7 -

  Fig. S7. Gross retinal patterning and retinal neurogenesis appear unaffected in Zic2a knockdown embryos. (A-H) foxg1 expression in the anterior retina in conMO (A, 20/20 embryos) and Zic2a-depleted embryos (B, 21/21 embryos). efna5a expression in the anterior retina in conMO (C, 32/32 embryos, n=2) and zic2a morphants (D, 30/34 embryos, n=2). atoh7 expression marks the ventral retina in conMO-injected embryos (E, 40/46 embryos, n=2). Expression of atoh7 is absent in zic2aMO-injected embryos (F, red arrow, 49/68 embryos, n=2). Tg(pou4f3:gap43-GFP)^s356t embryos allow direct visualization of retinal ganglion cells (RGCs) and their axons in living embryos. RGC differentiation and axon guidance (white arrows) are largely normal in control (G) and Zic2a-depleted embryos (H, 7/7 embryos, n=2). A-D show embryos in dorsal view, anterior at the top. E-F show dissected retina at prim-5, anterior to the left. G and H depict live transgenic embryos at 5 dpf imaged by confocal microscopy, ventral view, anterior up.

• Supplemental Figure S8 -

  Fig. S8. The combined effects of Zic2a depletion and Hh pathway hyperactivation on pax6a, fgf8a and spry4 expression. (A-L) Zic2a knockdown does not affect pax6a expression in the anterior retina (B, normal in 10/10 embryos). By contrast, overexpression of shha inhibits pax6a expression throughout the retina, most noticeably in the nasal retina (C, red arrow, 9/13 embryos). Embryos co-injected with shha mRNA and zic2aMO exhibit an overall reduction of pax6a in the retina, with a more pronounced clearing in the nasal retina (D, orange arrow, 12/12 embryos). fgf8a expression is expanded in zic2aMO-injected embryos (F, green arrow, 11/12 embryos). Similarly, ectopic fgf8a expression is observed in shha-mRNA-injected embryos (G, green arrow, 3/11 embryos). In shha mRNA plus zic2aMO injected embryos, dramatically increased ectopic fgf8a expression is observed (H, orange arrows, 4/12 embryos). spry4 expression is also expanded in zic2aMO-injected embryos (J, green arrow, 10/10 embryos). shha mRNA induces ectopic spry4 (K, green arrow, 11/13 embryos). Co-injection of shha mRNA and zic2aMO leads to a stronger expansion of spry4 expression than either single injection (L, orange arrow, 7/11 embryos). A-D are lateral views of isolated retina, anterior forward. E-L are lateral view, anterior to the left. E′-L′ are ventral views of the same embryos, anterior to the left. All embryos shown at prim-5.

• Supplemental Figure S9 -

  Fig. S9. Six3b depletion does not rescue the retinal patterning defect in Zic2a-depleted embryos. (A) Uninjected embryos show normal pax2a expression in the OS and ventral retina (33/33 embryos, n=2). (B) zic2a morphants exhibit ectopic expression of pax2a in the ventral retina (15/18 embryos, n=2). (C) six3b morphants show normal pax2a patterning (25/25 embryos, n=2). (D) Embryos co-injected with six3bMO and zic2aMO show ectopic pax2a expression in the retina similar to that observed in zic2a morphants (38/46 embryos, n=2). (E) A graphic summary of the pax2a rescue experiments. Red, expanded pax2a; blue, normal pax2a. Arrowheads point to the posterior limit of retinal pax2a expression. Lateral views with anterior to the left shown at prim-5.