Defective development of calvarial mesenchymal cells by loss of β-catenin in neural crest cells.

<p><i>In situ</i> hybridization of <i>Col2a1</i> was conducted to show the condensing calvarial mesenchymal cells at E14.5 (a). Bottom panels show mediodorsal <i>Col2a1</i>⁺ mesenchymal cells (b). An arrow indicates infiltrating <i>Col2a1</i>⁺ cells in the Sox10-Cre;Foxc1flx/flx mutants. Scale bars = 100 µm.</p

Wnt-responding mesenchymal cells expand in the dorsal interhemispheric region.

<p><b>A</b>) Staining of Pdgfrß⁺ neural crest-derived mesenchymal cells from E10 to E14. Dashed lines highlight the dorsomedial mesenchymal cells, which expand at E10 and spread laterally at later ages. At E14, perivascular cells strongly express Pdgfrß. <b>B</b>) A diagram showing mesenchymal cells at the level of dorsoventral axis in the forebrain; the dorsal (Md), lateral (Ml), and ventral (Mv) mesenchymal cells. The derivatives of the neural crest cells are listed with markers used in this study. Ep = epidermis, Ch = cortical hem, Cp = choroid plexus, P = pericytes, m = meninges. <b>C</b>) X-gal staining of E10.5 Bat-gal transgenic embryos. A high power image of the boxed area of <b>C-a</b> is presented in <b>C-b</b>. X-gal⁺ mesenchymal cells are localized in the interhemispheric region (red dashed lines). <b>D</b>) X-gal staining of E10 <i>ROSA-lac</i>Z Cre reporter mice crossed with Sox10-Cre, a neural crest driver. A high magnification image of the boxed area in <b>D-a</b> is shown in <b>D-b</b>. Red dashed lines mark the area with mesenchymal cells. Scale bars = 100 µm.</p

Ectopic generation of smooth muscle cells by loss of β-catenin in neural crest cells.

<p><b>A</b>) A schematic drawing shows the three regions (a, b, and c) used to stain markers for smooth muscle cells. <b>B</b>) Isolectin IB4 staining shows distribution of blood vessels in the epidermis and meninges. Fewer mesenchymal cells were seen in the space between the blood vessels of Sox10-Cre;Ctnnb1(lof)flx/flx mutant embryos. <b>C</b>) SM22a, a marker for the smooth muscle cells, and CD44, a marker for MSCs, were used to characterize the mesenchymal cells in the regions a–c. <b>C′</b>) Sox10-Cre;Ctnnb1(gof) mutant embryos double-stained for SM22a and CD44. Yellow bars indicate the thickness of the mesenchyme. <b>D</b>) aSMA, a marker for smooth muscle cells, and Desmin, a marker for pericytes, were used to reveal the ectopic generation of smooth muscle cells from neural crest cells of Sox10-Cre;Ctnnb1(lof)flx/flx mutant embryos at E14.5. Arrows indicate the incompetent spreading of mesenchymal cells into the ‘b’ region of Sox10-Cre;Ctnnb1(lof)flx/flx mutants. Scale bars = 100 µm.</p

Failure of mesenchymal coverage of the neocortex after loss of ß-catenin signaling in neural crest.

<p><b>A</b>) Sections of E12.5 embryos were stained for Pdgfrß. Dorsomedial Pdgfrß+ mesenchymal cells are shown in the top panel. Higher magnification images of neocortical mesenchymes are presented in the lower panel (corresponding to the boxed region). White bars indicate the distribution of Pdgfrß⁺ mesenchymal cells. <b>A′</b>) A schematic drawing shows two sources of migrating neural crest cells to the neocortex. Md = dorsal mesenchyme, Ml = lateral mesenchyme, Mv = ventral mesenchyme. <b>B</b>) Sections from E14.5 embryo heads were stained for CD44 and Cav1 to show MSCs and meningeal blood vessels, respectively. Thickness of the CD44 domain is reduced in both Sox10-Cre;Ctnnb1(lof)flx/flx and Sox10-Cre;Foxc1flx/flx mutants than the control (as marked by white bars). <b>B′</b>) A schematic drawing shows the region where images were taken (Ml). Scale bars = 100 µm.</p

Expansion of mesenchymal cells by activation of β-catenin in neural crest cells.

<p><b>A</b>) Mesenchymal cells of Sox10-Cre;Ctnnb1(gof) mutant at E16.5 marked by <i>Col2a1</i> expression (top) and alkaline phosphatase activities (bottom, osteoblasts) obtained from adjacent sections. Higher magnification images of the boxed areas are shown in <b>A′</b>. <b>B</b>) Mesenchymal cells were labeled for Pdgfrα and Ki67 to show the proliferating mesenchymal cells. <b>B′</b>) A graph shows thickness of dermal mesenchymal cells in the midline at E16.5 (white lines of <b>B</b>, n = 3). Error bar indicates SEM. Scale bars = 100 µm.</p

Failure of telencephalic midline invagination after loss of β-catenin in neural crest cells.

<p><b>A</b>) <i>In situ</i> hybridization of midline markers. E10.5 embryos from Sox10-Cre;Ctnnb1(lof)flx/+ and Sox10-Cre;Ctnnb1(lof)flx/flx were used to show expression of <i>Lmx1a</i>, <i>Ttr</i>, and <i>Lhx2</i>. The dashed red lines highlight gene expression domains and the inverted dorsomedial telencephalon in Sox10-Cre;Ctnnb1(lof)flx/flx mutants. <b>B</b>) E14.5 embryos from Sox10-Cre;Ctnnb1(lof)flx/+ and Sox10-Cre;Ctnnb1(lof)flx/flx were used to examine the expression of midline markers, <i>Lmx1a</i>, <i>Ttr</i>, <i>Lhx2</i>. Arrows indicate the area of gene expression and highlight the failure of dorsal midline invagination in Sox10-Cre;Ctnnb1(lof)flx/flx mutants. Scale bars = 100 µm.</p

Conservation of the meninges in neural crest cells lacking β-catenin.

<p>Expression of meningeal markers, <i>Raldh2</i> and <i>Cxcl12</i>, in the embryonic midline at E15.5. Raldh2 was also expressed in the choroid plexus. Ctx = cortex, Cp = choroid plexus, m = meninges. Scale bars = 100 µm.</p

Normal expansion of neural crest-derived mesenchymal cells is affected by the loss of ß-catenin signaling.

<p><b>A</b>) Immunofluorescence for mesenchymal cell markers Pdgfrß and Vimentin in Sox10-Cre;Ctnnb1(lof)flx/+ and Sox10-Cre;Ctnnb1(lof)flx/flx embryos at E10.5. Higher magnification images of <b>A-a</b> are shown in <b>A-b</b>. Arrows in <b>A-b</b> indicate mesenchymal cells in the dorsal midline bordered by the dashed lines. <b>A′</b>) Quantification of Pdgfrß⁺ mesenchymal cells in the interhemispheric region (n = 3). <b>B</b>) Ki67+ proliferating cells were counted from a region adjacent to the cortical hem (CH) at E12.5 (top) and E14.6 (bottom). <b>B′</b>) The drawing shows the area used to count Ki67+ cells in the dashed line and a graph represents quantification of Ki67+ cells (n = 3). <b>C</b>) Ki67+ cells were counted from mesenchymal tissues adjacent to the neocortex at E14.5. <b>C′</b>) The drawing shows the area used to count Ki67+ cells in the dashed line and a graph represents quantification of Ki67+ cells from the area (n = 3). Error bars indicate SEM. Md = dorsal mesenchyme, Ml = lateral mesenchyme. Scale bars = 100 µm.</p

Multiple Precursor Ion Scanning of Gangliosides and Sulfatides with a Reversed-Phase Microfluidic Chip and Quadrupole Time-of-Flight Mass Spectrometry

Precise profiling of polar lipids including gangliosides and sulfatides is a necessary step in understanding the diverse physiological role of these lipids. We have established an efficient method for the profiling of polar lipids using reversed-phase nano high-performance liquid chromatography microfluidic chip quadrupole time-of-flight mass spectrometry (nano-HPLC-chip Q-TOF/MS). A microfluidic chip design provides improved chromatographic performance, efficient separation, and stable nanospray while the advanced high-resolution mass spectrometer allowed for the identification of complex isobaric polar lipids such as NeuAc- and NeuGc-containing gangliosides. Lipid classes were identified based on the characteristic fragmentation product ions generated during data-dependent tandem mass spectrometry (MS/MS) experiments. Each class was monitored by a postprocessing precursor ion scan. Relatively simple quantitation and identification of intact ions was possible due to the reproducible retention times provided by the nano-HPLC chip. The method described in this paper was used to profile polar lipids from mouse brain, which was found to contain 17 gangliosides and 13 sulfatides. Types and linkages of the monosaccharides and their acetyl modifications were identified by low-energy collision-induced dissociation (CID) (40 V), and the type of sphingosine base was identified by higher energy CID (80 V). Accurate mass measurements and chromatography unveiled the degree of unsaturation and hydroxylation in the ceramide lipid tails

DR ATAC-seq

ATAC-seq raw datasets for C57BL6/J dorsal raphe nuclei</p