Meis/Hth genes are not essential for pax6/ey expression. (A,B) pax6b, detected at 20 hpf by in situ hybridization, is expressed at similar levels in both control-MO (A) and meis1-MO (B) injected embryos, despite the already noticeable reduction of the eye primordium in meis1-morphants. We have noticed, though, a consistent reduction of pax6b expression in the lens of meis1 morphants. (C) RT-PCR of two independent experiments (#1 and #2) for pax6b and actin, as internal control. Total RNA, from dissected eyes or remaining bodies of 19-hpf injected embryos (control-MO and meis1-MO), was isolated using an RNeasy Kit (Qiagen) and cDNA prepared with standard methods. PCR was performed with specific primers for pax6b, forward 5′-GATTTTGCAGGTGTCGAATG-3′ and reverse 5′-TTCACCACCGTTGTCCTGT-3&prime& β-actin primers as in (Duffy et al., 2005). PCR protocol used: denaturation of 95°C for 2 minutes, followed by 28 cycles of 95°C for 1 minutes, 60°C for 30 seconds, and 72°C for 1 minute, and a final elongation of 72°C for 5 minutes. (D) The image processing and pixel intensity values of ethidium bromide-stained agarose gels were obtained with Typhoon 9410 Variable Mode Imager. Results from two independent experiments (each performed in duplicate), are expressed as mean±STDEVP (standard deviation using the entire data population as argument). (E,F) Drosophila late third larval stage eye disc containing clones of cells mutant for a strong hth hypomorphic allele, hthP2. The clones are marked by the absence of CD2 (clones are outlined in F). ey expression remains unaltered in hth mutant cells. Hth clones were induced between 48 and 72 hours after egg laying by a 20-minute 37°C heat shock on larvae resulting from the cross of FRT82B hthP2/TM2 males to yw hsFLP 122; FTR82B hsCD2 y+ M/TM2 females (e.g. Pichaud and Casares, 2000 20-minute 37°C heat shock on larvae resulting from the cross of FRT82B hthP2/<29. Expression of CD2 was induced by a 30-minute heat shock at 37°C, 30 minutes prior to dissecting wandering third instar larvae. Mouse anti-CD2 is from Serotec, and rabbit anti-Ey is a gift from Patrick Callaerts. Secondary antibodies were from Molecular Probes. Imag
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