<i>bmp2b</i> is necessary for pharyngeal pouch progenitor specification.

(A) mCherry fluorescence at the 17-somite stage in Tg(nkx2.3:mCherry) embryos injected with 4 ng cMO, 0.25 ng bmp2b MO, 2 ng bmp4 MO, and 4 ng bmp7a MO, respectively. Scale bar, 50 μm. (B-C) Quantification of the number of the mCherry+ pouch progenitors (B) and the pericardial progenitors (C) from embryos of each group shown in (A). Student’s t-test, ****PD-E) Alteration of the nkx2.3 expression pattern in swrta72 mutants at the 14-somite stage (D) and embryos injected with 0.25 ng bmp2b MO at the 17-somite stage (E). Black dotted lines indicate the region where the pouch progenitors are located. (F) mCherry fluorescence at the 17-somite stage in Tg(nkx2.3:mCherry) embryos injected with 1 ng cMO or bmp2b MO. Scale bar, 50 μm. (G) The expression of nkx2.3 in wild-type embryos injected with indicated MOs. (H-I) mCherry fluorescence in heated Tg(nkx2.3:mCherry;hsp70l:caBmpr1b-GFP) embryos. Embryos injected with 0.25 ng cMO or bmp2b MOs were heat shocked at 9 hpf for 20 min, and then harvested at the 17-somite stage for confocal imaging (H). Scale bar, 50 μm. The numbers of mCherry+ pericardial and pouch progenitors were quantified from heat shocked-embryos with or without GFP expression (I). The group values are expressed as mean±s.d. Student’s t-test, ****PJ) p-Smad1/5/8 levels were dramatically reduced in in the pharyngeal endoderm of bmp2b morphants. cMO or bmp2b MO-injected Tg(sox17:GFP) embryos at the 6-somite stage were stained for anti-p-smad1/5/8 and anti-GFP antibodies. Representative dorsal confocal images were shown in the upper panels. Boxed areas are enlarged and presented in the middle panels. The orthogonal sections taken from the boxed areas were shown in the lower panels. Scale bar, 50 μm. (K-L) Knocking down bmp2b resulted in the failure of pharyngeal pouch progenitor specification. Tg(nkx2.3:mCherry;sox17:GFP) embryos were injected with ng bmp2b MO at one-cell stage and harvested at the 10-somite stage for confocal imaging analysis (K). Arrowheads indicate the pouch progenitors expression both mCherry and GFP. Scale bar, 20 μm. The number of mCherry+/GFP+ cells was statistical analyzed in (L). Student’s t-test, ****P < 0.0001.</p

BMP signal inhibition results in a severe loss of <i>nkx2</i>.<i>3</i>⁺ pouch epithelium.

(A-B) In situ hybridization analysis of the expression of foxa1 (A), nkx2.7, nkx2.3, and pdgfαa (B) at 26 hpf in embryos treated with different dose of BMP inhibitors from bud stage to the 17-somite stage. Black arrowheads in (A) indicate the first pouch. Black dotted lines in (B) highlight the pharyngeal region. (C) The expression of pdgfαa in DMH1-treated embryos injected with or without sox32 MO. Note that when DMH1-treated embryos were injected with 8 ng sox32 MO, the expression of pdgfαa was abolished. (D) The expression of nkx2.7 and pdgfαa in nkx2.3+ pouch progenitor-depleted embryos. Tg(nkx2.3:KalTA4-p2a-mCherry;UAS:NTR-mCherry) embryos were treated with 50 mM MTZ from the 32-cell stage to the 17-somite stage, and then harvested for in situ hybridization at 36 hpf. (E-F) Double-fluorescence in situ hybridizations for nkx2.3 and pdgfαa at 26 hpf, In situ hybridization analysis of pdgfαa at 26 hpf. Note that nkx2.3 (green) and pdgfαa (red) display nested expression patterns along the dorsal-ventral axis of the pouch epithelium. Schematic expression of nkx2.3 and pdgfαa was shown in (F). Scale bar, 50 μm.</p

BMP signal inactivation leads to a decrease of pouch progenitors.

(A-B) Wild-type embryos were exposed to different inhibitors from bud stage to the 17-somite stage and harvested at 36 hpf for in situ hybridizations with nkx2.3 probe. Dorsal views, anterior to the top. Note that embryos treated with 20 μM Dorsomorphin (A) or 10 μM DMH1 (B) showed a clear reduction of nkx2.3 expression in pharyngeal pouches. (C) Live confocal images of malformed pharyngeal pouches in 10 μM DMH1 treated Tg(sox17:GFP) embryos at 36 hpf. Scale bar, 50 μm. (D) In situ hybridization of thymus marker ccl25a in DMH1 treated embryos at 48 hpf. Lateral views, anterior to the left. (E-F) Representative confocal sections showing mCherry+ progenitors in embryos treated with or without 10 μM DMH1 from bud stage to the 17-somite stage (E). The lateral pharyngeal endoderm is indicated by white dotted lines. Scale bar, 50 μm. Quantification of the numbers of pericardial and pouch progenitors positive for mCherry in DMSO and DMH1 conditions was shown in (F). The group values are expressed as mean±s.d. Student’s t-test, ****PG-H) mCherry fluorescence in 9 hpf-heat shocked Tg(nkx2.3:mCherry;hsp70l:dnBmpr1a-GFP) embryos at the 17-somite stage (G). Scale bar, 50 μm. The numbers of mCherry+ progenitors were quantified from heat shocked-embryos with or without GFP expression (H). Student’s t-test, ****PI) The expression of nkx2.3 in embryos treated with 10 μM DMH1 from bud stage to the 17-somite stage. Black dotted lines indicate the region where the pouch progenitors are located. (J-M) Overactivation of BMP signaling has no effect on the formation of nkx2.3+ pouch progenitors and pouch epithelium. Tg(nkx2.3:mCherry;hsp70l:caBmpr1b-GFP) embryos were heat shocked at 9 hpf for 20 min, and then harvested at the indicated developmental stages for in vivo confocal imaging (J-L) and in situ hybridization (M). Scale bars, 50 μm. The number of mCherry+ progenitors was calculated from heat shocked-embryos in (J) and presented in (K). ns, non-significant.</p

BMP signal is essential for pouch progenitor specification.

(A) Time-lapse confocal images showing the specification of nkx2.3+ pouch progenitors from the pharyngeal endoderm. White arrowhead indicate the mCherry+/GFP+ cells in Tg(nkx2.3:mCherry;sox17:GFP) embryos. Scale bar, 20 μm. (B) Detection of p-Smad1/5/8 in Tg(sox17:GFP) embryos at the 6-somite stage. Representative dorsal confocal images were shown in the left three panels. Boxed areas are enlarged in the right upper panels. The orthogonal sections taken from the boxed areas were shown in the right lower panels. Arrowheads indicate the endodermal cells with p-Smad1/5/8 expression. Scale bars, 50 μm. (C-D) BMP signal inhibition impairs pouch progenitor specification. Tg(nkx2.3:mCherry;sox17:GFP) embryos were treated with 10 μM DMH1 from bud stage to the 10-somite stage. Representative confocal images were shown in (C). Scale bar, 20 μm. The number of mCherry+/GFP+ cells was statistical analyzed in (D). Student’s t-test, ****P < 0.0001.</p

<i>bmp2b</i> is an important regulator in pouch formation.

(A) bmp2b expression during early somite stages. In situ hybridization of bmp2b in whole mount embryos at the 6-somite stage (left panel). Sagittal section of the same embryo was shown in the right panel. The black dotted line indicates the plane of the section. (B) Whole-mount RNAscope assays showing expression of egfp in lateral pharyngeal endoderm (green) and bmp2b in pharyngeal ectoderm (red) of Tg(nkx2.3:EGFP-CAAX) transgenic embryos at the 10-somite stage. Left panel shows the merged sections in X-Y view (Scale bar, 50 μm); the right panel shows higher magnification orthogonal section of the boxed area in the left panel (Y-Z view; Scale bar, 10 μm). (C) In situ hybridization of nkx2.3 at 36 hpf in wild-type embryos injected with 4 ng cMO, 0.25 ng bmp2b MO, 2 ng bmp4 MO, and 4 ng bmp7a MO, respectively. Note that the expression of the pouch marker nkx2.3 was not notably disrupted in bmp4 MO or bmp7a MO-injected embryos, but was depleted in bmp2b morphants. (D) Malformed lateral pharyngeal endoderm (green) in 0.25 ng bmp2b MO-injected Tg(sox17:GFP) embryos at 36 hpf. Scale bar, 50 μm. (E) Overactivation of BMP signaling rescues the pouch formation in bmp2b-deficient embryos. Tg(hsp70l:caBmpr1b-GFP) embryos injected with 0.25 ng cMO or bmp2b MOs were heat shocked at 9 hpf for 20 min, and then harvested at 36 hpf for in situ hybridizations with nkx2.3 probe. (F-I) The expression of foxa1 (F), nkx2.3 (G), nkx2.7 (H), and pdgfαa (I) at 26 hpf in bmp2b MO-injected embryos. The black dotted lines in (G, H and G) indicate the pharyngeal regions. The black arrowheads in (F) indicate the first pouch.</p

<i>nkx2</i>.<i>3</i> is expressed in the lateral pharyngeal endoderm during early somite stages.

(A) Tg(nkx2.3:mCherry) embryos at 24, 28 and 36 hpf exhibiting fluorescence in the pericardium (yellow arrowhead) and pharyngeal pouches (white arrowhead). PC, pericardium; PP, pharyngeal pouch. Scale bars, 50 μm. (B) mCherry-positive cells (red) were located between EGFP-labeled cranial neural crest cells (green) in Tg(nkx2.3:mCherry;fli1:EGFP) embryos (left panel), and co-localized with Zn8 labeled pharyngeal pouch cells (green) in Tg(nkx2.3:mCherry) embryos (right panel). Scale bars, 50 μm. (C) mCherry fluorescence in Tg(nkx2.3:mCherry) embryos from the 10- to 18-somite stages. Embryos were dorsal views with anterior to the top. n, notochord; ss, somite stage. Scale bar, 50 μm. (D) Tg(nkx2.3:mCherry;sox17:GFP) embryos with mCherry-positive cells (red) and GFP-labeled endodermal cells (green) at the 16- and 17-somite stages. The lower panels are optical transverse sections (XZ) taken at the level of white lines in their respective upper panels. Scale bar, 50 μm. (E-F) mCherry (red) and GFP (green) fluorescence in Tg(nkx2.3:mCherry;sox17:GFP) embryos injected with 8 ng control MO (cMO) or 8 ng sox32 MO at the 17-somite stage (E) and 36 hpf (F). In panel F, the GFP fluorescence was shown in the inset. The ratios of affected embryos are indicated. White dotted lines highlight the lateral pharyngeal endoderm. Scale bars, 50 μm. (G-H) In situ hybridization of sox17 (G) and nkx2.3 (H) expression in wild-type embryos at indicated developmental stages. Black arrowheads in (G) indicate the KVs. (I) Alteration of nkx2.3 expression pattern in 8 ng sox32 MO injected embryos at the 17-somite stage. Black dotted lines show the lateral pharyngeal endoderm.</p

<i>nkx2</i>.<i>3</i>⁺ progenitors give rise to pharyngeal pouches.

(A-B) Tg(nkx2.3:EosFP) embryos at the 17-somite stage before (green) and after (red) photoconversion in the right-side nkx2.3+ cluster (A). At 36 hpf, embryos were imaged in the green and red channels (B). Cells in the right-side nkx2.3+ cluster remained un-photoconverted as an internal control and their derivatives were imaged and shown in the inset. Scale bars, 50 μm. (C-D) The posterior part of the right-side nkx2.3+ cluster was photoconverted at the 17-somite stage (C). Images of the pharyngeal pouches in the same embryos at 36 hpf are shown in (D). Scale bars, 50 μm. (E-F) Tg(nkx2.3:EosFP) embryos were photoconverted in the right-side nkx2.3+ cluster at the 10-somite stage (E), and then these embryos were imaged in the red and green channels (inset) at 28 hpf (F). Scale bars, 50 μm. (G-J) Tg(nkx2.3:KalTA4-p2a-mCherry;UAS:NTR-mCherry) embryos were treated with 50 mM MTZ from the 32-cell stage to the 17-somite stage. Subsequently, these embryos were harvested at the indicated developmental stages for in vivo confocal imaging (G), in situ hybridization (H-I) and Alcian Blue staining (J). m, Meckel’s; pq, palatoquadrate; hs, hyosymplectic; bh, basihyal; ch, ceratohyal; cb, ceratobranchial. Scale bars, in panel G, 50 μm; in panel J, 100 μm.</p

BMP signaling is required for <i>nkx2</i>.<i>3</i>-positive pharyngeal pouch progenitor specification in zebrafish

Pharyngeal pouches, a series of outpocketings that bud from the foregut endoderm, are essential to the formation of craniofacial skeleton as well as several important structures like parathyroid and thymus. However, whether pharyngeal pouch progenitors exist in the developing gut tube remains unknown. Here, taking advantage of cell lineage tracing and transgenic ablation technologies, we identified a population of nkx2.3+ pouch progenitors in zebrafish embryos and demonstrated an essential requirement of ectodermal BMP2b for their specification. At early somite stages, nkx2.3+ cells located at lateral region of pharyngeal endoderm give rise to the pouch epithelium except a subpopulation expressing pdgfαa rather than nkx2.3. A small-scale screen of chemical inhibitors reveals that BMP signaling is necessary to specify these progenitors. Loss-of-function analyses show that BMP2b, expressed in the pharyngeal ectoderm, actives Smad effectors in endodermal cells to induce nkx2.3+ progenitors. Collectively, our study provides in vivo evidence for the existence of pouch progenitors and highlights the importance of BMP2b signaling in progenitor specification.</div

Table1_Immune-Related Molecular Profiling of Thymoma With Myasthenia Gravis.DOC

Background: Approximately 50% of thymoma patients also show myasthenia gravis (MG), which is an autoimmune disease; however, the pathogenesis of MG-associated thymoma remains elusive. Our aim was to investigate immune-related lncRNA profiles of a set of candidate genes for better understanding of the molecular mechanism underlying the pathogenesis of thymoma with or without MG.Methods: Molecular profiles of thymoma with or without MG were downloaded from The Cancer Genome Atlas, and Pearson’s correlation analysis was performed to identify immune-related lncRNAs. T test was used to examine the differential expression and differential methylation between thymoma patients with or without MG. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to predict the function of target genes of immune-related lncRNAs.Results: Analyses of the 87 thymoma samples with complete MG information revealed that 205 mRNAs and 56 lncRNAs showed up-regulated expression in thymoma with MG patients, while 458 mRNAs and 84 lncRNAs showed down-regulated expression. The methylation level of three immune-related lncRNAs (AP000787.1, AC004943.1, WT1-AS, FOXG1-AS1) was significantly decreased in thymoma tissues, and the methylation level of these immune-related lncRNAs (WT1-AS: Cor = 0.368, p Conclusion: Our results revealed the immune-related molecular profiling of thymoma with MG and without MG and identified key pathways involved in the underlying molecular mechanism of thymoma-related MG. These findings provide insights for further research of potential markers for thymoma-related MG.</p

MOESM6 of 18F-labeled magnetic nanoparticles for monitoring anti-angiogenic therapeutic effects in breast cancer xenografts

Additional file 6: Figure S6. Prussian blue staining of heart (a), liver (b), spleen (c), lung (d), kidney (e), intestine (f) and tumor (g) after injection of placebo solution. Magnification: ×200