Journal: The Journal of Neuroscience

Article Title: BMP/SMAD Pathway Promotes Neurogenesis of Midbrain Dopaminergic Neurons In Vivo and in Human Induced Pluripotent and Neural Stem Cells

doi: 10.1523/JNEUROSCI.1540-17.2018

Figure Lengend Snippet: BMP5/7 are necessary for the generation of postmitotic mDA neurons. mRNA in situ hybridization (A–C, E, H–M′) and immunohistochemistry (D, F, G, N–P′) on parasagittal (A–C, H–M′) and coronal (D-G, N–P′) sections of the mesencephalic flexure at different developmental ages are represented. Bmp5 (A), Bmp6 (B), and Bmp7 (C) mRNAs are expressed at the mesencephalic flexure at E12.5. At the same developmental time point, BMPR1B is expressed specifically in the mDA progenitor domain (D). At E10.5, Bmp7 mRNA is expressed lateral to the midline in the mDA progenitor domain (E). Red arrows indicate BMP5 immunoreactivity in the pial surface and mantle layer of mDA progenitor domain (F). White arrowheads point to cluster of cells with phosphorylated SMAD-1/5/8 (p-S1/5/8) immunoreactivity within the mDA progenitor domain (G). Single Bmp5, Bmp6, and Bmp7 gene deletion does not affect the formation of mDA neurons, as shown by the comparison of Dat mRNA expression on parasagittal sections of WT and Bmp5−/− (H, H′), Bmp6−/− (I, I′), and Bmp7−/− (J, J′) mutants at P0. Dat mRNA expression is unchanged in Bmp5−/−;Bmp6−/− mutants at P0 (K, K′). Bmp6−/−;Bmp7−/− mutants did not show any obvious alterations in mDA development, as indicated by normal Th mRNA expression at E11.5 (L, L′). In contrast, in Bmp5−/−;Bmp7−/− mutants at E10.5, Nurr1 mRNA (M, M′) and NURR1 (N–O") immunoreactivity were absent. In addition, the postmitotic neuronal marker TUJ1 was coexpressed in NURR1+ cells at this time point in WT (N–N"′). Adjacent ocular motor neurons visualized by ISLET1/2 immunoreactivity were normally induced in mutants at E9.5 (P′) compared to WTs (P). Scale bars: A–D, G–L′ 100 μm. G, P, P′; 25 μm. E, F, N–O" 25 μm.

Article Snippet: Primary antibodies used for the in vivo study were as follows: mouse anti-SHH (1:10), mouse anti-NKX6.1 (1:10), mouse anti-ISLET1/2 (1:10), mouse anti-MSX1/2 (1:10), mouse anti-FOXA2 (1:10), mouse anti-NESTIN (1:10), mouse anti-EN1 (1:50) - all from DSHB, rabbit anti-LMX1A (1:400, catalog #AB10533; Millipore), rabbit anti-LMX1B (1:1000, gift from Dr. C. Birchmeier, MDC, Berlin), rabbit anti-NURR1 (1:100, catalog #SC991; Santa Cruz Biotechnology), mouse anti-NURR1 (1:100, catalog #376984; Santa Cruz Biotechnology) mouse anti-neurogenin-2 (1:800, catalog #MAB3314; R&D Systems), rabbit anti-TH (1:200, catalog #AB152; Millipore), mouse anti-TH (1:200, catalog #MAB318; Millipore), rabbit anti-phospho-SMAD1/5/8 (1:200, #9511S Cell Signaling), sheep anti-BMP5 (1:10, catalog #AF6176; R&D Systems), rabbit anti-phospho-histone-H3 (1:1000, catalog #06-570; Millipore), rabbit anti-phospho-P38 (1:1000, catalog #4511 Cell Signaling), rabbit anti-GIRK2 (1:200, catalog #APC-006; Alomone Laboratories), rabbit anti-SOX6 (1:500, catalog #ab30455; Abcam), mouse anti-POU4F1 (1:300, catalog #sc8429; Santa Cruz Biotechnology), mouse anti-N-cadherin (1:200, catalog #610920; BD Biosciences), rabbit anti-ZO1 (1:100, catalog #40-2200; Invitrogen), rabbit anti-KI67 (1:100, catalog # ab16667; Abcam), rabbit anti-cleaved-caspase 3 (1:100, catalog #3661; Cell Signaling Technology), rabbit anti-MAP-2 (1:200, catalog #sc20172; Santa Cruz Biotechnology), rabbit anti-β-catenin (1:400, catalog #9587s; Cell Signaling Technology), rabbit anti-SHH (1:50, catalog #sc9024; Santa Cruz Biotechnology), rabbit anti-BMPR1B (1:100, catalog #10537; Orbigen), rabbit anti-PITX3 (catalog #38-2850; Thermo Fisher Scientific), mouse anti-BrdU (catalog #B2531; Sigma-Aldrich), rabbit anti-CCND1(1:150, catalog #sc-450; Santa Cruz Biotechnology), rabbit anti-phospho-β-catenin (1:200, catalog #9561s; Cell Signaling Technology), rabbit anti-calbindin (1:500, catalog # d -28k; Swant), and rabbit anti-β III tubulin/TUJ1 (1:500, catalog #302 302; Synaptic Systems).

Techniques: In Situ Hybridization, Immunohistochemistry, Expressing, Marker

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heterogeneous Origin of Gonadotropin Releasing Hormone-1 Neurons in Mouse Embryos Detected by Islet-1/2 Expression

doi: 10.3389/fcell.2020.00035

Figure Lengend Snippet: Islet-1/2 expression delineates two populations of GnRH neurons. E11.5 (A,B) and E12.5 (C,D) stained parasagittal sections of nasal region. Low magnification (A,C) and higher magnification of boxed area (B,D) is shown. At E11.5 (A,B) GnRH neurons (blue-black cells, black arrows) within or adjacent to the VNO are detected. At E12.5 (C,D) GnRH neurons (blue-black) are detected migrating out of VNO along olfactory axons (brown) to the nasal-forebrain junction (NFJ). (E–H) Fluorescent expression of Islet-1/2 (red) in GnRH neurons (green). (E,F) At E11.5, GnRH neurons (green)/Islet-1/2(+) (red, white asterisk) and GnRH neurons Islet-1/2(–) (white arrow) were detected within or adjacent to the VNO (boxed area in E shown at higher magnification in F ). (G,H) At E12.5, both Islet-1/2(+) (red, white asterisk) and Islet-1/2(–) (white arrow) GnRH cells (green) were detected along the migratory tracts (boxed area in G shown at higher magnification in H ). (I) Percentage of Islet-1/2 negative GnRH neurons at E11.5 and E12.5, N = numbers of animals. FB, forebrain; OE, olfactory epithelium; VNO, vomeronasal organ; NFJ, nasal forebrain junction; LV, lateral ventricle; 3V, third ventricle; MGE, medial ganglionic eminence; T, tongue. Scale (A,C) (250 μm), (B,D) (100 μm), (E–H) (10 μm).

Article Snippet: Primary antibodies used were: Rabbit polyclonal anti-GnRH-1 (SW-1, 1:15000) , chicken anti-GnRH (1:100, Aves), mouse monoclonal anti-Islet-1/2 [1:400, Developmental Studies Hybridoma Bank (DSHB)], rabbit polyclonal anti-Islet-1/2 (1:1000, Abcam), chicken anti-peripherin (1:1000, Aves) and chicken anti-GFP (1:2000, Abcam).

Techniques: Expressing, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heterogeneous Origin of Gonadotropin Releasing Hormone-1 Neurons in Mouse Embryos Detected by Islet-1/2 Expression

doi: 10.3389/fcell.2020.00035

Figure Lengend Snippet: Islet-1/2 staining identifies placodal ectodermally derived GnRH cells at E11.5. Representative sections of the VNO from 3 different E11.5 embryos stained for GnRH, Wnt1-GFP and Islet-1/2. Left panels , schematics indicating VNO area from which the immunofluorescent images were taken (asterisk, boxed area). Middle panels , triple labeling of GnRH (gray), Wnt-1GFP (green) and Islet-1/2 (red). The green and red boxes in middle panels are shown at a higher magnification ( right panels ) at all three individual wavelengthes. Green box: GnRH neurons negative for Islet-1/2 but positive for Wnt1-GFP. Red box: GnRH neurons positive for Islet-1/2 but negative for Wnt1-GFP (white asterisk). FB: forebrain, OE, olfactory epithelium; RE, respiratory epithelium; Scale bar, 10 μm. Clusters of GnRH cells, as seen in upper middle panel on right edge of photomicrograph were omitted from analysis due to inability to discern single cells.

Article Snippet: Primary antibodies used were: Rabbit polyclonal anti-GnRH-1 (SW-1, 1:15000) , chicken anti-GnRH (1:100, Aves), mouse monoclonal anti-Islet-1/2 [1:400, Developmental Studies Hybridoma Bank (DSHB)], rabbit polyclonal anti-Islet-1/2 (1:1000, Abcam), chicken anti-peripherin (1:1000, Aves) and chicken anti-GFP (1:2000, Abcam).

Techniques: Staining, Derivative Assay, Labeling

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heterogeneous Origin of Gonadotropin Releasing Hormone-1 Neurons in Mouse Embryos Detected by Islet-1/2 Expression

doi: 10.3389/fcell.2020.00035

Figure Lengend Snippet: Islet-1/2 staining identifies placodal ectodermally derived GnRH cells at E12.5. Left panel schematic of GnRH cells on migratory tracts (boxed area, green, sensory axons, blue, GnRH). Middle panel shows triple labeling of GnRH (gray), Wnt1-GFP (green) and Islet-1/2 (red). The green and red boxes indicate the two population of GnRH neurons negative and positive for Islet-1/2, respectively. Right panel shows magnified boxed areas in the middle panel at all three individual wavelengths. Green box: GnRH neurons negative for Islet-1/2, positive for Wnt1-GFP. Red box: GnRH neurons positive for Islet-1/2, negative for Wnt1-GFP. Asterisks, Islet-1/2 (+) GnRH cells. FB, forebrain; OE, olfactory epithelium; VNO, vomeronasal organ; III, third ventricle; T, tongue; Scale bar, 10 μm.

Article Snippet: Primary antibodies used were: Rabbit polyclonal anti-GnRH-1 (SW-1, 1:15000) , chicken anti-GnRH (1:100, Aves), mouse monoclonal anti-Islet-1/2 [1:400, Developmental Studies Hybridoma Bank (DSHB)], rabbit polyclonal anti-Islet-1/2 (1:1000, Abcam), chicken anti-peripherin (1:1000, Aves) and chicken anti-GFP (1:2000, Abcam).

Techniques: Staining, Derivative Assay, Labeling