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BACKGROUND: Consistent left-right (LR) asymmetry is a fascinating problem in developmental and evolutionary biology. Conservation of early LR patterning steps among vertebrates as well as involvement of nonprotein small-molecule messengers are very poorly understood. Serotonin (5-HT) is a key neurotransmitter with crucial roles in physiology and cognition. We tested the hypothesis that LR patterning required prenervous serotonin signaling and characterized the 5-HT pathway in chick and frog embryos. RESULTS: A pharmacological screen implicated endogenous signaling through receptors R3 and R4 and the activity of monoamine oxidase (MAO) in the establishment of correct sidedness of asymmetric gene expression and of the viscera in Xenopus embryos. HPLC and immunohistochemistry analysis indicates that Xenopus eggs contain a maternal supply of serotonin that is progressively degraded during cleavage stages. Serotonin's dynamic localization in frog embryos requires gap junctional communication and H,K-ATPase function. Microinjection of loss- and gain-of-function constructs into the right ventral blastomere randomizes asymmetry. In chick embryos, R3 and R4 activity is upstream of the asymmetry of Sonic hedgehog expression. MAO is asymmetrically expressed in the node. CONCLUSIONS: Serotonin is present in very early chick and frog embryos. 5-HT pathway function is required for normal asymmetry and is upstream of asymmetric gene expression. The microinjection data reveal asymmetry existing in frog embryos by the 4-cell stage and suggest novel intracellular 5-HT mechanisms. These functional and localization data identify a novel role for the neurotransmitter serotonin and implicate prenervous serotonergic signaling as an obligate aspect of very early left-right patterning conserved to two vertebrate species.
Figure 1.
Pharmacological Screen Implicates Serotonergic Signaling in LR Asymmetry
(AâC) Control embryos exposed to drug vehicle alone exhibited the normal invariant asymmetry of the heart, gut, and gall bladder. Heterotaxic embryos exhibited independent randomization of laterality of the three organs; the embryo in (B) exhibits reversal of all three, whereas the embryo in (C) has reversal of the heart alone. Red arrowheads indicate apex of heart; yellow arrowheads indicate apex of gut; green arrowheads indicate gall bladder.
(D) Pharmacological blockers targeting 5-HT receptor types R1âR7 were applied to batches of embryos from fertilization to stage 12. Only blockers of R3 and R4 induced heterotaxia (statistical analysis is presented in Tables S2âS6).
(E) Exposure to several different blockers of R3 and R4 also randomized laterality, confirming their role in LR asymmetry. Each error bar is 50% of the standard deviation above and below the datapoint.
(F) Monoamine oxidase inhibitors targeting either the A and B isoform induced heterotaxia in embryos.
(G) Serotonin content was determined by HPLC in samples of 90â150 embryos taken from three pooled batches at a range of stages.
Figure 2.
Asymmetric Gene Expression Is Randomized in Xenopus by Inhibition of 5-HT Signaling through R3 or R4
Embryos were exposed to blockers of R3 or R4 during early stages and processed for in situ hybridization with the laterality markers XNR-1 and Xlefty. Embryos exposed to vehicle only (controls) exhibited the normal left-sided pattern of XNR-1 (A) expression. In contrast, embryos in which the activity of MAO, R3, or R4 was inhibited exhibited bilateral (C), right-sided (D), or absent (E) expression of XNR-1 in addition to the left-sided expression (B). This effect was statistically significant (see numerical data and analysis in Table S9). Red arrows indicate regions of expression; white arrows indicate lack of expression.
Figure 3.
Dynamic, Asymmetric Localization of Serotonin in Early Xenopus Embryos Depends on Functional Gap Junctions and H+/K+-ATPase Activity
Frog embryos at various stages were fixed and sectioned for immunohistochemistry with an antibody specific for serotonin as previously described [33]. Red arrows indicate signal; white arrows indicate lack of signal. Panels show sections taken perpendicular to the animal-vegetal axis, except those marked with “av†(B, D, and J), which were cut parallel to the animal-vegetal axis. In unfertilized eggs, maternal serotonin is localized circumferentially at the vegetal cortex (A). In fertilized eggs before cleavage, the serotonin is spread more than halfway toward the animal pole (B). The distribution of serotonin is symmetrical in the cytoplasm of blastomeres at the 2-cell stage (C). By the 4-cell stage, dynamic finger-like projections can be observed reaching from the pool of 5-HT in the vegetal half of the embryo toward the animal pole (D). During subsequent cleavages, serotonin is localized in a ring-like pattern (E). As cleavage proceeds, serotonin signal becomes weaker and is progressively confined to a specific subset of blastomeres at the 32- and 64-cell stages (F and G). Analysis of embryos lineage labeled with rhodamine-dextran (green arrow) in the right-ventralblastomere at the 4-cell stage reveals that the serotonin signal becomes restricted to the rightventral cells (H; white line indicates midline; LV, leftventral; RV, rightventral). By stage 6, 5-HT is no longer detected by immunohistochemistry (I and J). In embryos exposed to blockers of gap junctions, serotonin did not localize to specific blastomeres and exhibited a diffuse pattern in several blastomeres (K). In embryos in which H+/K+-ATPase activity was abrogated, serotonin likewise did not localize but remained symmetrically distributed (L). Exposure of embryos to nocodazole did not affect the ability of serotonin to localize to specific blastomeres (M).
