a, Illustration of the in vivo injection procedure: PCPA or vehicle solution was injected into the labyrinth zone of the placenta. After 30 min, dams were euthanized and tissue collected and processed for HPLC. UtA: uterine artery; uc: umbilical cord; Ut: uterus. b, Compared to vehicle injection (untreated, n=6), placental PCPA injection (+PCPA, n=4), has no significant (n.s.) effect on 5-HT concentration in the hindbrain, whereas it significantly lowers 5-HT concentration in the forebrain and the placenta (statistical significance versus 5-HT levels in untreated tissue was analyzed by Student’s t test; *, p<0.05; data are presented as means ± s.e.m.). c, Model of the progressive switch of the source of 5-HT in the fetal forebrain, from an early exogenous (placental, blue line) to a later endogenous (5-HT axons, red line) source. The green boxes represent the amount of 5-HT measured in the Pet-1^−/− mice, which lack most of the endogenous neuronal source.

a, Placental extracts were incubated with the co-factor tetrahydrobiopterin (BH4; control) or BH4 and tryptophan (+Trp+BH4) and 5-HT neosynthesis measured after 30 min. b, Similar experiments conducted using human placenta tissue collected at 11 weeks of gestation (11 GW) show that human fetal villi synthesize 5-HTP and 5-HT (statistical significance versus control analyzed by Student’s t test; *, p<0.005; n=3; data are presented as means ± s.e.m.). c, Immunostaining for the monocarboxylate transporter MCT1, a marker of syncytiotrophoblastic cells (sc) in the labyrinth (la) region on the fetal side (fet) of an E14.5 mouse placenta. On the maternal side (mat) the decidua (dc) is devoid of staining. Asterisks indicate red blood cells of maternal origin. d–h, Higher magnifications of the region boxed in c; MCT1 is expressed on the apical side (arrows) of syncytiotrophoblasts facing the maternal blood space (mbs). The 5-HT synthetic enzymes TPH1 (f, g) and AADC (h) are expressed in overlapping patterns in the cytoplasm of syncytiotrophoblastic cells (arrows). i, Schematics of the ex-vivo dual perfusion system for the mouse placenta; UtA: uterine artery, UA umbilical artery, UV umbilical vein. g, 5-HT neosynthesis in ex vivo dually-perfused mouse placentas at E17.5. L-tryptophan (100 μM) was injected through the UtA. 5-HT, neo-synthesized from maternal tryptophan and released into the UV, is evident within 15 min of precursor injection (statistical significance of 5-HT levels variation across time was analyzed by one-way ANOVA; *, p<0.05; **, p<0.01; data from 3 independent experiments are presented as means ± s.e.m.).

a, 5-HT concentration in the Pet-1^−/− (KO) hindbrain is significantly lower than in wild type (WT) littermates at every age tested. b, In contrast 5-HT concentrations in the Pet-1^−/− forebrain are not significantly different from wild type littermates from E10.5 to E15.5 but become significantly lower at E16.5 and E17.5 (n>=6 embryos per genotype per age; *, p<0.005; one-way ANOVA; data are presented as means ± s.e.m.). c–h, Serotonergic axons (SERT+) and dorsal thalamic (DT) axons (NetG1a+) immunostained on sagittal sections at E14.5 in wild type (c) and Pet-1^−/− (f) embryos (regions shown correspond to the red box in the drawings). In wild type E14.5 embryos (d) SERT+ axons grow ventrally into the forebrain through the medial forebrain bundle (mfb, white arrowheads); SERT also labels DT axons at this age (open arrowhead). In comparison, very few SERT+ axons remain in the Pet-1^−/− (g). The pattern and density of SERT+ DT axons appear unaffected. Scale bars: 50 μm. The rostral-most extent of ingrowing serotonergic axons immunolabeled with 5-HT in the wild type (e) shows numerous 5-HT+ axons, some of which diverge toward the hypothalamus (Hyp). In contrast, only few 5-HT+ axons remain in the Pet-1^−/− forebrain (h). Scale bars: 20 μm. i, Densitometric analysis of 5-HT+ axons in the most rostral part of the mfb at E14.5 (region indicated in the right panel) confirms fewer axons in the Pet-1^−/−. j, AADC staining identifies DA neurons in the substantia nigra pars compacta (SNC) along with their and serotonergic axons coursing through the ventral forebrain at E14.5, and also AADC+ catecholaminergic neurons present in the hypothalamus (Hyp, black box). Scale bar: 100 μm. k-k″, AADC+ neurons in the hypothalamic region (red box in bottom right drawing; k′, white arrowheads) are 5-HTP-negative. Open arrowheads indicate fluorescence from blood vessels. Scale bar: 25 μm.