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Development of the frontonasal mass (FNM), branchial arches, heart, and limbs depends on neural crest-mediated epithelial–mesenchymal (E–M) interactions. Teratogenesis by retinoic acid (RA) or blockade of serotonergic (5-HT) signaling by the pan-5-HT2 receptor antagonist, ritanserin, perturbs development of these embryonic structures. In both cases, resulting phenotypes include forebrain and olfactory placode anomalies, malformations of the face, eye and lens, as well as posterior neural tube and cardiac defects. Similar sites of malformations, together with the presence of RA response elements in the 5-HT2B receptor promoter, have led to the suggestion that a negative regulatory relationship may exist between RA and 5-HT2-mediated 5-HT signaling at sites of E–M interaction (Choi et al. 1997); however, another possibility is that RA and 5-HT act independently as opposing signals to regulate development of common embryonic targets. Together with recent evidence for opposite effects on chondrogenic differentiation in hindlimb micromass cultures (Bhasin et al. 2003a), results of the present study raise the possibility that these pathways may act as opposing signals for common targets in the mouse embryo. The RA receptors, co-factors and metabolic enzymes, and 5-HT2B receptors were found to be are coordinately expressed at sites of E–M interaction, including the FNM, in the embryonic day (E)10.5 mouse. Cell proliferation experiments using [3H]thymidine incorporation demonstrated that RA or activation of 5-HT2B receptors caused opposite effects in FNM explants, namely stimulation or inhibition of cell proliferation, respectively, 5-HT2B receptor activation did not appreciably alter patterning in FNM explants. While RA has been shown to regulate lateral patterning in the FNM (LaMantia et al. 2000), 5-HT2B receptor activation did not alter patterning in FNM explants. Quantification of 5-HT2B receptor transcripts by real-time PCR provided no evidence of negative regulation of 5-HT2B receptor expression by RA in FNM explants, although preliminary studies using in situ hybridization had suggested that this was a possibility in both explants and RA teratogenized embryos. Future studies using quantitative PCR may still show this to be the case in teratogenized embryos. Together with the finding of coordinate expression of 5-HT2B receptors and RA signaling molecules, results of the present study suggest that RA, and 5-HT mediated by 5-HT2B receptors, may act as opposing signals to regulate cell proliferation during craniofacial development in the mouse embryo. [ABSTRACT FROM AUTHOR]

Embryos and larvae of sea urchins (Lytechinus variegatus, Strongylocentrotus droebachiensis, Strongylocentrotus purpuratus, Dendraster excentricus), and starfish (Pisaster ochraceus) were investigated for the presence of a functional endocannabinoid system. Anandamide (arachidonoyl ethanolamide, AEA), was measured in early L. variegatus embryos by liquid chromatography/mass spectrometry. AEA showed a strong developmental dynamic, increasing more than 5-fold between the 8-16 cell and mid-blastula 2 stage. 'Perturb-and-rescue' experiments in different sea urchin species and starfish showed that AEA blocked transition of embryos from the blastula to the gastrula stage, but had no effect on cleavage divisions, even at high doses. The non-selective cannabinoid receptor agonist, CP55940, had similar effects, but unlike AEA, also blocked cleavage divisions. CB1 antagonists, AEA transport inhibitors, and the cation channel transient membrane potential receptor V1 (TrpV1) agonist, arachidonoyl vanillic acid (arvanil), as well as arachidonoyl serotonin and dopamine (AA-5-HT, AA-DA) acted as rescue substances, partially or totally preventing abnormal embryonic phenotypes elicited by AEA or CP55940. Radioligand binding of [(3)H]CP55940 to membrane preparations from embryos/larvae failed to show significant binding, consistent with the lack of CB receptor orthologs in the sea urchin genome. However, when binding was conducted on whole cell lysates, a small amount of [(3)H]CP55940 binding was observed at the pluteus stage that was displaced by the CB2 antagonist, SR144528. Since AEA is known to bind with high affinity to TrpV1 and to certain G-protein-coupled receptors (GPCRs), the ability of arvanil, AA-5-HT and AA-DA to rescue embryos from AEA teratogenesis suggests that in sea urchins AEA and other endocannabinoids may utilize both Trp and GPCR orthologs. This possibility was explored using bioinformatic and phylogenetic tools to identify candidate orthologs in the S. purpuratus sea urchin genome. Candidate TrpA1 and TrpV1 orthologs were identified. The TrpA1 ortholog fell within a monophyletic clade, including both vertebrate and invertebrate orthologs, whereas the TrpV1 orthologs fell within two distinct TrpV-like invertebrate clades. One of the sea urchin TrpV orthologs was more closely related to the vertebrate epithelial calcium channels (TrpV5-6 family) than to the vertebrate TrpV1-4 family, as determined using profile-hidden Markov model (HMM) searches. Candidate dopamine and adrenergic GPCR orthologs were identified in the sea urchin genome, but no cannabinoid GPCRs were found, consistent with earlier studies. Candidate dopamine D(1), D(2) or alpha(1)-adrenergic receptor orthologs were identified as potential progenitors to the vertebrate cannabinoid receptors using HMM searches, depending on whether the multiple sequence alignment of CB receptor sequences consisted only of urochordate and cephalochordate sequences or also included vertebrate sequences., (Copyright 2009 S. Karger AG, Basel.)

Exposure of a pregnant woman to physical and/or psychological stress might affect her offspring by promoting the development of various learning, behavioral and/or mood disorders in later life. The 5-HT1A and 5-HT2A receptors are prominently implicated in the modulation of anxiety and mood-related behaviors. Using a semi-quantitative radiolabel immunocytochemical analysis (immunobinding), we studied the effect of prenatal stress on binding of these two receptor subtypes in the hippocampus of 4-week-old male and female Fischer 344 rats. Levels of 5-HT1A immunobinding in the ventral hippocampus, which is primarily implicated in emotional processing, were significantly decreased in male offspring after prenatal stress. A trend towards a decrease was observed in the ventral hippocampus of females. In contrast, 5-HT1A immunobinding within the dorsal hippocampus, which is mainly related to learning and memory, was not affected by prenatal stress in offspring of either gender. Likewise, no significant differences between control and prenatally stressed rats were observed for levels of 5-HT2A immunobinding in either part of the hippocampus or gender. The observed reduction in hippocampal 5-HT1A receptor binding in male offspring after prenatal stress may have important consequences for adult anxiety- and depressive-like behavior.

To investigate the putative role of serotonin (5-HT) in auditory brainstem development, the expression of the 5-HT transporter (5-HTT) was evaluated in the normal mouse brainstem at 6 different postnatal ages. The brains of C3H/HeJ mice at birth (P0) and P1, P8-P9, P13, P21-P22, P35-P36 and P48-P50 were collected and processed immunohistochemically with an antibody raised against the 5-HTT. 5-HTT immunoreactivity (5-HTT-IR) was first observed in P8 mice and was localized to cell bodies in the ventral cochlear nucleus (VCN) and principal nuclei of the superior olivary complex, including the medial nucleus of the trapezoid body. Labeled neurons were found in similar regions in older mice except at P48-50, where labeled neurons were observed in the VCN only. 5-HTT-IR was especially prominent in VCN neurons at P21 and was observed in all of the brains examined at this age. These results indicate that auditory brainstem neurons of the normal inbred mouse express the 5-HTT postnatally. The presence of 5-HTT-IR in neurons located in the VCN indicates a regional expression of the 5-HTT that is related to the ascending auditory pathway. The timing of 5-HTT expression indicates that 5-HT may modulate developmental processes that rely on cochlear input., (Copyright 2005 S. Karger AG, Basel.)

Retinoic acid (RA) synthesizing and metabolizing enzymes are coordinately expressed with serotonin 2B (5-HT2B) receptors at sites of epithelial-mesenchymal (E-M) interaction in the mouse embryo (Bhasin et al., 1999). The promoter of the 5-HT2B receptor contains potential RA response element (RAREs) as well as an AP-2 site. Because both retinoid and serotonergic signaling have been implicated in the regulation of chondrogenic differentiation, the present study investigated whether these signals may work together to regulate this morphogenetic process in hindlimb bud micromass cultures. Results indicate that 5-HT promotes [35S]sulfate incorporation (chondrogenic differentiation) by activation of 5-HT2B receptors, which use the mitogen activated protein kinase (p42 MAPK) signal transduction pathway, whereas RA dose-dependently inhibits sulfate incorporation and promotes expression of RARbeta, which could lead to inhibition of p38 MAPK. No evidence was found to support the possibility that RA negatively regulates expression of 5-HT2B receptors. Taken together, these results suggest that 5-HT and RA may act as opposing signals to regulate chondrogenic differentiation in the developing hindlimb, possibly mediated by different MAPK signal transduction pathways., (Copyright 2004 Wiley-Liss, Inc.)

The Smith-Lemli-Opitz syndrome (SLOS) is a malformation/mental retardation syndrome resulting from an inborn error in 3beta-hydroxysteroid Delta7-reductase (DHCR7), the terminal enzyme required for cholesterol biosynthesis. Using a targeting strategy designed to virtually eliminate Dhcr7 activity, we have created a SLOS mouse model that exhibits commissural deficiencies, hippocampal abnormalities, and hypermorphic development of serotonin (5-HT) neurons. The latter is of particular interest with respect to current evidence that serotonin plays a significant role in autism spectrum disorders and the recent clinical observation that 50% of SLOS patients present with autistic behavior. Immunohistochemical analyses have revealed a 306% increase in the area of 5-HT immunoreactivity (5-HT IR) in the hindbrains of mutant (Dhcr7-/-) mice as compared to age-matched wild type animals. Amount of 5-HT IR was measured as total area of IR per histological section. Additionally, a regional increase as high as 15-fold was observed for the most lateral sagittal hindbrain sections. In Dhcr7-/- mice, an expansion of 5-HT IR into the ventricular zone and floor plate region was observed. In addition, the rostral and caudal raphe groups exhibited a radial expansion in Dhcr7-/- mice, with 5-HT IR cells present in locations not seen in wild type mice. This increase in 5-HT IR appears to represent an increase in total number of 5-HT neurons and fibers. These observations may help explain the behavioral phenotype seen in SLOS, and provide clues for future therapeutic interventions that utilize pharmacological modulation of the serotonergic system.