<i>m1061</i> mutants show increased <i>th</i> expression and increase in number of hypothalamic DA neurons from 3 dpf onwards.

<p>Analysis of <i>th</i> gene expression in <i>cnot8<sup>m1061</sup></i> mutants and WT siblings as indicated in header. (A–F) <i>th</i> transcript levels are not altered in <i>m1061/m1061</i> mutants at 1 and 2 dpf. Embryos were genotyped by PCR. (G–N) <i>m1061</i> mutants at 3 dpf display stronger WISH signal indicating increased <i>th</i> mRNA levels in DA groups 1 to 7 (arrowhead) and NA sympathetic cells (asterisk). (K, L) NA locus coeruleus (LC) and DA pretectal cells (Pre) do not display altered <i>th</i> expression levels in <i>m1061</i> mutant embryos. (M, N) The number of DA retinal amacrine cells (rAC) is reduced in <i>m1061</i> mutant embryos. Additionally <i>m1061</i> mutants display a lens defect (arrowheads). Genotypes were inferred by <i>th</i> WISH analysis. (O, P) anti-TH immunohistochemistry of <i>m1061</i> mutant embryo and WT sibling at 4 dpf document DA neurons in the ventral diencephalon. Data confirm a higher cell number of DC7 DA neurons in the caudal hypothalamus of <i>m1061</i> mutants. Dorsal view z-projections of partial confocal stacks representing the ventral diencephalon are shown. (A, B, E, F, G, H, M, N) lateral views, anterior at left; (C, D, G, H, K.L, O, P) dorsal views, anterior at left. Scale bars 100 µm in A for A, B; in C for C–F; in G for G–N; in P for O, P. Abbreviations: DC - early diencephalic DA group (DC2, DC4), 1 - ventral thalamic DA group, 2,4,5,6 posterior tubercular and hypothalamic Orthopedia-dependent DA groups, 3 - medial hypothalamic DA group, 7 - caudal hypothalamic DA group, VT - ventral thalamus, PT/H - posterior tuberculum/hypothalamus, PO - preoptic area, AAC - arch associated catecholaminergic neurons/carotid body, sym - sympathetic NA neurons, Pr - pretectum, TC - telencephalon, CH - caudal hypothalamus, LC locus coeruleus, MO - medulla oblongata, rAC - retinal amacrine cells. (Q) Quantification of CA cell numbers in <i>m1061</i> embryos at 4 dpf. Cell counts of DC1-7 and LC TH-expressing cells. Bars show the average number of CA neurons in five <i>m1061</i> mutants and five WT sibling embryos. Error bars indicate standard deviation. Significance was evaluated using Mann-Whitney test (see text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113829#pone.0113829.s002" target="_blank">Table S1</a>).</p>