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Figure 1. Serotonin Signaling Is Required for the Development of Leftward Flow (A and B) Altered Pitx2c mRNA expression patterns and quantification of results in stage (st) 28-33 xHtr3 morphants and LBD-injected specimens. The following abbreviations are used: wt, left-sided; bi, bilateral. (C) Leftward flow in dorsal explants of stage 17 embryos. Quantification of flow quality (rho) is shown, as calculated from time-lapse movies. (D) GRP analysis. (D) SEM photographs of GRP areas in wild-type embryo (D), MO1 morphant (E), MO1 + xFL mRNA (F), and xLBD-injected specimen (G). Coloration indicates nonciliated cells (red), cells with normal posteriorly polarized cilia (green), and cells with cilia in other locations (blue). Insets show cilia in higher magnification (arrowheads). Scale bar represents 25 mm. (H) Quantification of ciliation rate (H), relative cilia length (I), and cilia polarization (J) of GRP cilia in defined areas, indicated by white squares in (D)G). x in box plots represents mean values. The following abbreviations are used: a, anterior; l, left; p, posterior; r, right; *, significant; **, highly significant; ***, very highly significant; n.s., not significant. Numbers in parentheses indicate number of embryos or dorsal explants analyzed. Statistical significance was calculated using Pearson chi-square test (B) or Student t test (C and H; [43]). See also Figure S1. |
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Figure S1, Serotonin Receptors: Expression, Structure and Phenotypes, Related to Figure 1 (A) Expression of xHtr3 (left) and xHtr4 (right) by RT-PCR analysis. Absence of maternal transcripts and onset of zygotic xHtr3 transcription post mid-blastula transition. No detectable xHtr4 mRNA transcription before tadpole stages. (B) 5'-UTR and translational start sequence of xHTR3. Binding sites for MOs are indicated. (C) Serotonin receptor type 3. Schematic representation of domain structure of full- length frog (xFL) and human (hFL) receptor subunits (x5-Ht3, h5-HT3A), consisting of signal peptide (blue), ligand binding domain (LBD; orange) and C-terminal region (green). Six protein loops in the LBD involved in serotonin binding (A-F; red), and four transmembrane domains (TM, dark green) are characteristic features of h5- HT3A [33]. A human splice variant (hsv) lacks essential loops F and C and contains additional amino acids (black) due to read-through into intronic sequences (5-HT3AT in [33]). The truncated constructs encoding xLBD and hLBD lack the C-terminus. The domain structure of xFL was deduced from amino acid alignments with the hFL and a hydrophobicity plot. (D, E) Serotonin signaling on the dorsal side is required for asymmetric marker gene expression. (D) No significant alteration of Pitx2c expression upon ventral injection of co-MO, MO1, and xLBD or hLBD. (E) Highly significant reduction of wildtype left- sided expression of Xnr1 in morphants following dorsal MO1 injection. (F) Embryonic malformations in embryos injected with higher MO or LBD doses. Embryos were injected at the 4-cell stage into the prospective DMZ of dorsal blastomeres and analyzed for Pitx2c expression at stage 28-33. Knockdown of serotonin signaling resulted in absence of Pitx2c expression, reduced head size, dorsal bending and neural tube closure defects (NTD). (G-J) Expression of cilia and GRP marker gene Tekt2 (G, J) was reduced in MO1 morphants (H, J) and rescued upon xFL co-injection (I, J). Quantification of expression levels (J). Dorsal explants are displayed in ventral view (anterior up). bi, bilateral; abs, absent; inv, right-sided; wt, left-sided. Numbers in brackets represent number of embryos. Statistical significances were calculated using Pearson's chi-square test [11]. |
