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Figure S3. Effects of HUA Treatment on Retinal Histogenesis
Retinal sections of st. 42 embryos, treated with HUA (150 μM hydroxiurea, 20 μM aphidicoline) from st. 30, compared to control. In situ hybridization of mRNAs (neurotubulin, hermes, prox1, and IRBP) are detected with Fast Red, and antibodies (anti-R5, amacrine antibodies panel) are immunodeteced with Oregon green–conjugated secondary antibody. According to Harris et al. [30], HUA blocks cell proliferation in 4 h from the beginning to the end of treatment, as detected by BrdU-incorporation assay and immunodetection of mitotic cells with the phosphorylated form of Histone3 (not shown). The treatment reduces retinal size but does not impede terminal cell differentiation, as shown by the expression of the Müller glial marker R5 [31] and neurotubulin, the staining of which in treated embryos is comparable to control. Immunostaining of the neuronal marker acetylated tubulin (Sigma T6793; 1:1,000) confirmed the observation obtained by in situ hybridization with a neurotubulin probe (not shown). The pattern of neurotubulin and Müller glial staining indicates that retinal layering is compromised. This happens, even more severely, when treating from earlier stages (not shown, compare to Harris et al. [30]). The expression of markers for ganglion cells (hermes) and horizontal cells (prox1) is not affected by treatment. The expression of markers for amacrine cells (amacrine antibodies panel as in Figure 1: anti-5-HT, anti-GABA, and anti-tyrosine hydroxilase) and photoreceptors (IRBP) is often reduced but is still detectable with a pattern similar to that of control in all the examined embryos. Treatment from st. 25 strongly reduces IRBP and amacrine markers, but allows the expression of hermes and prox1 (not shown).
https://doi.org/10.1371/journal.pbio.0040272.sg003 |
