Teratogenic Effects of Serotonin Receptor 2B Disruption on the Migration and Cardiac Derivatives of the Cardiac Neural Crest

FIGURE 1. 1‐MP disrupts the migration patterns of cardiac neural crest in HH14 embryos. (A–D) Methodology used to measure the lengths of the R6 stream and the circumpharyngeal crest. (A) A reference embryo, stained for neural crest cells using the HNK1 antibody. The otic vesicle (OV) is indicated, as are the R6 stream (R6), the circumpharyngeal crest (cc) and the anterior tract (At). (B) The cardiac crest R6 stream and circumpharyngeal crest are schematized for added clarity in (C). (D) Landmarks used to carry out the measurements. The dorsal otic vesicle (dOV), which stains brightly with HNK1, serves as a point of reference for measuring the cardiac crest streams. (E–H) Embryos treated at HH8 with Ringers (control) or 20 μM 1‐MP and collected at HH14. HNK1‐positive cardiac neural crest cells (white box) are indicated on lower magnification images in (E) and (G). (E) An HH14 control embryo stained with HNK1, a marker of migrating neural crest cells, shows cardiac neural crest cells (white box) migrating caudal to the otic vesicle. (F) A close‐up image of the control embryo in (E), showing the length of the cardiac neural crest stream at rhombomere 6 (R6NCC; magenta arrow) as well as the…

FIGURE 2. Cell proliferation is altered in embryos treated with 1‐MP. (A–D) Control embryos. (A) DAPI staining shows cell nuclei. (B) HNK1 (red), Phospho‐histone H3 (PHH3; green) are overlaid atop DAPI (blue) in a merged image. C and D show close ups of PHH3 and HNK1, from the white box in (B). The dashed line in (C) delineates the HNK1‐positive region, to help discern which PHH3‐positive cells also express HNK1, from PHH3 cells that are located outside the HNK1 region and are therefore not neural crest cells. (E) Merge image of (C) and (D). The dashed line once again traces the HNK1‐positive cell population. (F) An embryo treated with 1‐MP, sectioned and stained with DAPI to show cell nuclei. (G) The merge image displays HNK1 (red), Phospho‐histone H3 (PHH3; green) and DAPI (blue). (H) and (I) are close ups of the area in the white box in (H). The dashed outline shows the HNK1‐positive region. (J) HNK1 and PHH3 staining are overlapped, with the dashes delimiting the HNK1‐stained population. (K) The percentage of neural crest cells within the cardiac neural crest region undergoing proliferation decreases following 1‐MP treatment at HH8, and this difference is statistically significant.…

FIGURE 3. Uncoupling the effects of 5‐HT2B from those of 5HT2C: Disruption of 5HT2B alone shows the migration defect induced by 1‐MP. (A–C) Migration patterns of cardiac neural crest cells (white box) in representative HH14 embryos—treatment with Ringer's alone at HH8 (A), 10 μM SB24 in Ringer's at HH8 (B), and 20 μM 1‐MP followed by 10 μM SB24 after an interval of 10 min (C). (D–F) Close‐up images of the cardiac neural crest in HH14 control embryos with the rhombomere 6 stream (R6NCC) length (magenta arrow) and circumpharyngeal neural crest stream (CirNCC) length (gold arrow), treatment with Ringer's only (D), treatment with 10 μM SB24 only (E), and treatment with 10 μM SB24 and 20 μM 1‐MP (F). (G) Ratios in R6 NC stream versus the circumpharyngeal stream in embryos treated with Ringer's alone, Ringer's followed by 1‐MP (see Figure 1G,H for an image of migrating cardiac crest streams in 1‐MP treated embryos), Ringer's followed by SB24, and 1‐MP followed by SB24. The difference in the ratios between embryos treated with Ringer's alone and those treated with 1‐MP, or those treated with Ringer's and SB24 or with SB24 followed by 1‐MP were statistically significant.

FIGURE 4. 1‐MP exposure induces tissue gaps in the aorticopulmonary septum at HH32. (A) Coronal section of the outflow tract in control HH32 chick embryo stained with hematoxylin and eosin (H&E) shows normal aorticopulmonary septum morphology between the aorta (ao) and the pulmonary artery (p) (n = 4). (B) The outflow tract of an HH32 embryo stained with H&E shows gaps in the aorticopulmonary septum (white arrowheads) (n = 4). (C) Antibody stain for smooth muscle Actin (SMA) shows normal expression patterns in the control embryo seen in (A). (D) Antibody staining for SMA in 1‐MP treated embryos from (B) show normal expression patterns. (E) DAPI stain of outflow tract showing normal morphology in the control embryo in (A). (F) DAPI stain of outflow tract showing the gap in the aorticopulmonary septum in the 1‐MP exposed embryo from (B). (G) Merge image of (C) and (E). (H) Merge panel of (D) and (F). Scale bars: A‐500 μm for A + B; C‐500 μm for C‐H. 1‐MP indicates 1‐methylpsilocin; ao, aorta; DAPI, 4,6‐diamidino‐2‐phenylindole; H&E, hematoxylin and eosin; p, pulmonary artery; SMA, smooth muscle actin.

FIGURE 5. Application of 1‐MP during neurulation prevents proper formation of the membranous interventricular septum at E10. (A) Control whole mount E10 embryo. (B) 1‐MP treated whole mount E10 embryo. (C) 14 μm cryogenic section of the heart of the control embryo in (A) along the sagittal axis to show the interventricular septum completely septating the heart (n = 4). (D) 14 μm cryogenic section of the heart of the 1‐MP treated embryo in (B) along the sagittal axis to show the interventricular septum with a septation defect (n = 3). Arrows in (E), (G), and (I) point to the fully formed, intact membranous interventricular septum in the control embryo in (A) and (C), whereas arrowheads in (F), (H), and (J) point to the defect in the interventricular septum in the 1‐MP treated embryo from panel (B) and (D). (E) High magnification of membranous septum indicated by the square (C), stained for smooth muscle Actin (SMA). The arrow points to the intact, fully‐formed membranous interventricular septum. (F) High magnification of membranous septum indicated by the square (D), stained for SMA. The arrowhead shows the defect in the membranous interventricular septum. (G) High magnification of…