Journal: bioRxiv

Article Title: BMP signaling underlies the craniofacial heterochrony in phyllostomid bats, a hyperdiverse mammal group

doi: 10.1101/2021.05.17.444516

Figure Lengend Snippet: Immunofluorescence targeting the CNC (Pax7), mitotic cells (PH3), cartilage (Sox9), and BMP signaling (pSMAD) in bat embryos are represented by three sagittal sections capturing the medial (middle) and lateral aspects of the midface. Pax7 and PH3 immunostaining are performed on the same section, whereas pSMAD and Sox9 immunostaining are performed on adjacent sections approximately 10 and 20um apart, respectively, from the Pax7;PH3 double-immuno. Histological staining with trichrome (top of panel) match the three different levels. Expression of Pax7 is detected in multiple cranial regions, but is primarily enriched in the skin (epithelium), the upper lip mesenchyme (L), nose mesenchyme (nm), leaf nose (If), and the developing olfactory turbinates (om). In the cartilage (ca), a distinct spatial pattern is observed, with Pax7 expressed in lateral, but not in medial cartilages. PH3-positive mitotic cells are observed throughout the midface, but the medial cartilage has fewer mitotic cells. Sox9 expression is observed in the early developing cartilages of the midface with distinct patterns in the lateral and medial regions among species. pSMAD expression is observed in both Pax7 (undifferentiated mesenchyme, m) and in Sox9 (early cartilage commitment) spatial domains, as well as the developing secondary palate. CP ( Carollia perspicillata ); MN ( Miniopeturus natalensis ); AJ ( Artibeus jamaicensis ); GS ( Glossophaga soricina ). C = condensed mesenchyme; ca = cartilage; e = epithelium; L= lip; If= leaf nose; m = mesenchyme; n= naris; nc= nasal cavity; nm= nasal mesenchyme; ns= nasal septal cartilage; oe = olfactory epithelium; om = olfactory mesenchyme; pal = palate; t= tooth; to = tongue. Scale: 500um

Article Snippet: Immunostaining for BMP signaling (pSMAD1/5/9,1:500, Cell Signaling) was done, followed with a biotinylated secondary antibody (1:300, Jaksonlmmuno), amplified by a streptavidin-horseradish peroxidase conjugate (1:300) and detected with TSA tyramid-Cy3 (1:50, PerkinElmer).

Techniques: Immunofluorescence, Immunostaining, Staining, Expressing

Journal: bioRxiv

Article Title: BMP signaling underlies the craniofacial heterochrony in phyllostomid bats, a hyperdiverse mammal group

doi: 10.1101/2021.05.17.444516

Figure Lengend Snippet: Immunofluorescence targeting the CNC (Pax7), mitotic cells (PH3), and BMP signaling (pSMAD) in bat embryos at CS18. Top panel shows histological staining with trichrome highlighting cartilage and connective tissue development (blue) and each fluorescence image below match the three sagittal sections capturing the medial (middle) and lateral aspects of the midface. Expression of Pax7 is detected anteriorly in the developing upper lip (L) and posteriorly in the condensing (c) mesenchyme of the olfactory turbinates. It is also observed in the developing leaf-nose (If), tongue (to), and around the naris (n). It is not present in the mature, differentiated cartilage. Based on histology and Pax7 expression, the olfactory turbinates are more mature in Artibeus jamaicensis (AJ), which express less Pax7, than other bats at CS18. Laterally (far left), Carollia perspicillata (CP) is highly expressed along the cheek tissue and condensed mesenchyme (c) of the developing turbinates. PH3-positive cells are observed in the midface, but are more highly present in AJ and CP than in GS and MN. Arrowheads highlight some PH3-positive cells in GS, which have an excess in non-specific background staining. In all species, pSMAD expression is similarly distributed throughout the midface in the olfactory epithelium (oe), cartilage (ca), condensing mesenchyme (c), palate (pal), tongue (to), nasal septal cartilage (ns), and leaf-nose (If). pSMAD is more highly expressed in the developing palate and the olfactory turbinates of AJ. pSMAD is more highly expressed throughout the nasal septal (ns) cartilage of GS. We note an excess in background staining in our pSMAD immunos for GS. Pax7 and PH3 immunostaining are performed on the same section, whereas pSMAD immunostaining is performed on an adjacent serial section approximately 10um from the Pax7;PH3 double-immuno. CP ( Carolliaperspicillata ); MN ( Miniopeturus natalensis ); AJ ( Artibeus jamaicensis ); GS ( Glossophaga soricina). C = condensed mesenchyme; ca = cartilage; e = epithelium; L= lip; If= leaf nose; m = mesenchyme; n= naris; nc= nasal cavity; nm= nasal mesenchyme; ns= nasal septal cartilage; oe = olfactory epithelium; om = olfactory mesenchyme; pal = palate; t= tooth; to = tongue. Scale: 500um

Article Snippet: Immunostaining for BMP signaling (pSMAD1/5/9,1:500, Cell Signaling) was done, followed with a biotinylated secondary antibody (1:300, Jaksonlmmuno), amplified by a streptavidin-horseradish peroxidase conjugate (1:300) and detected with TSA tyramid-Cy3 (1:50, PerkinElmer).

Techniques: Immunofluorescence, Staining, Fluorescence, Expressing, Immunostaining

Journal: bioRxiv

Article Title: BMP signaling underlies the craniofacial heterochrony in phyllostomid bats, a hyperdiverse mammal group

doi: 10.1101/2021.05.17.444516

Figure Lengend Snippet: Relative expression changes in overall BMP-signaling pathway (pSMAD 1/5/9 immunohistochemistry). These representative parasagittal sections highlight species-specific differences in pSMAD molecular expression (magenta) and related cranial morphology (DAPI, blue) in the fruit-eating (A, B) and nectareating bats (C). The pSMAD expression along the basicranium relates to facial length morphology: pSMAD is less expressed throughout the facial cartilages of A. jamaicensis compared to C. perspicillata and pSMAD is more highly expressed throughout the nasal septal cartilage and basicranium (bc) of G. soricina (C, C’) compared to A. jamaicensis (A’) and C. perspicillata (B’). The elevated pSMAD in the developing secondary palate (sdp) of A. jamaicensis (A, A’) might relate to the width of the palate. Cranial features relating to elevated pSMAD expression are the leaf-nose, nasal epithelium, choroid plexus (cp), tongue (to), and larynx (lx), bc= basicranium; cb= cerebellum; cp = choroid plexus; ctx = cortex, ge = ganglionic eminence; L = lip; lx = larynx; mo = medulla oblongata; mv = mesencephalic ventricle; nc = nasal cavity; nph = nasopharynx; o = oropharynx; ol = olfactory lobe; on= optic recess; p= pons; pg = pituitary gland; sc = spinal cord; sdp = secondary palate; sp = soft palate; th = thalamus; to = tongue; vc = vertebrae cartilage.

Article Snippet: Immunostaining for BMP signaling (pSMAD1/5/9,1:500, Cell Signaling) was done, followed with a biotinylated secondary antibody (1:300, Jaksonlmmuno), amplified by a streptavidin-horseradish peroxidase conjugate (1:300) and detected with TSA tyramid-Cy3 (1:50, PerkinElmer).

Techniques: Expressing, Immunohistochemistry

Journal: bioRxiv

Article Title: BMP signaling underlies the craniofacial heterochrony in phyllostomid bats, a hyperdiverse mammal group

doi: 10.1101/2021.05.17.444516

Figure Lengend Snippet: Variation in BMP signaling at E12.5 affects facial morphology in E16.5 mouse embryos. Relative to control littermates (left), the Wnt1 mutant mice (right) have enlarged facial features. The facial length is longer and extends beyond the lower jaw (arrowheads). The rhinarium, or nose, is larger in mutants (arrow).

Article Snippet: Immunostaining for BMP signaling (pSMAD1/5/9,1:500, Cell Signaling) was done, followed with a biotinylated secondary antibody (1:300, Jaksonlmmuno), amplified by a streptavidin-horseradish peroxidase conjugate (1:300) and detected with TSA tyramid-Cy3 (1:50, PerkinElmer).

Techniques: Control, Mutagenesis

Journal: bioRxiv

Article Title: BMP signaling underlies the craniofacial heterochrony in phyllostomid bats, a hyperdiverse mammal group

doi: 10.1101/2021.05.17.444516

Figure Lengend Snippet: Model of Bmp signaling implicated in skeletal cell differentiation and facial length phenotype. Bmp signaling and its modulation are involved in craniofacial skeletal development. Neural crest derived mesenchymal progenitors are specified to chondro-progenitor cells (Sox9+) prior to cartilage maturation. When Bmp signaling is low, chondrocyte proliferation is promoted, resulting in short faces. When the BMP is elevated, cell proliferation is suppressed, resulting in an enlarged midface. Bmp signaling in the Sox9+ chondrocyte lineage affects the size of cartilage by suppressing or promoting cell proliferation. Bmp signaling and its modulation are involved in craniofacial skeletal development. Neural crest derived mesenchymal progenitors are specified to chondro-progenitor cells (Sox9+) prior to cartilage maturation. When Bmp signaling is low, chondrocyte proliferation is promoted, resulting in short faces. When the BMP is elevated, cell proliferation is suppressed, resulting in an enlarged midface. Bmp signaling in the Sox9+ chondrocyte lineage affects the size of cartilage by suppressing or promoting cell proliferation. Our proposed model implicates Bmp signaling in skeletal cell differentiation in the cartilage lineage (Sox9+) as one important aspect underlying facial length phenotype through direct regulation of cell behavior.

Article Snippet: Immunostaining for BMP signaling (pSMAD1/5/9,1:500, Cell Signaling) was done, followed with a biotinylated secondary antibody (1:300, Jaksonlmmuno), amplified by a streptavidin-horseradish peroxidase conjugate (1:300) and detected with TSA tyramid-Cy3 (1:50, PerkinElmer).

Techniques: Cell Differentiation, Derivative Assay

Journal: eLife

Article Title: Completion of neural crest cell production and emigration is regulated by retinoic-acid-dependent inhibition of BMP signaling

doi: 10.7554/eLife.72723

Figure Lengend Snippet:

Article Snippet: Antibody , anti-pSmad1/5/9 (Rabbit monoclonal) , Cell Signaling Technology , Cat#CST13820; RRID: AB_2493181 , IF(1:500).

Techniques: Recombinant, Plasmid Preparation, Sequencing, Software, Microscopy, Staining

Journal: Thorax

Article Title: Relationship between impaired BMP signalling and clinical risk factors at early-stage vascular injury in the preterm infant

doi: 10.1136/thoraxjnl-2021-218083

Figure Lengend Snippet: Significant reduction in BMP signalling at the earliest stage of lung vascular pathology is accompanied by microvessel loss in postnatal lung injury. (A) Preclinical mouse model of bronchopulmonary dysplasia (BPD) with induction of lung injury by exposure of neonatal mice to mechanical ventilation (MV) and/or hyperoxia (FiO 2 =0.4) for 8 hours or 24 hours. (B) Immunoblot analysis reveals a significant decrease in pSMAD 1-5-9 and Id-1 protein levels in WT mice in the early course of postnatal lung injury provoked by short-term MV-O 2 and O 2 exposure. (C) Representative images of immunofluorescence (IF) staining for cleaved caspase-3 (green) and VE-cadherin positive cells (red) in lung tissue sections (4 µM, 400X, upper panel; nuclei (DAPI, blue) from 5 to 8 day old pups show a significant increase in EC apoptosis, confirmed by quantitative analysis (lower-left panel). Histological analysis reveals a significant decrease in the number of micro vessels (20–100 µm diameter) in the lungs of mice undergoing only 8 hours of O 2 and/or MV when compared with room air (RA) controls (lower-right panel). (D) In BMPR2 deficient mice, immunoblot analysis demonstrates significantly lower VE-cadherin protein expression in the neonatal lung at baseline (RA) when compared with WT pups (left panel). Postnatal exposure to moderate O 2 further decreases VE-cadherin protein expression in BMPR2 deficient mice (right panel). (E) Representative images of immunofluorescence (IF) staining for BMPR2 (green) and VE-cadherin positive cells (red) in lung tissue sections (4 µM, 400X, upper panel; nuclei (DAPI, blue) following 24 hours MV show a prolonged and significant decrease in BMPR2 expression, confirmed by quantitative analysis (right panel). (F) ISH demonstrates a persistent decrease in BMPR2 transcription in neonatal mouse PCLS exposed to O 2 for 24 hours when compared with RA control samples. Data are mean±SD *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=3–4 mice/group compared with RA controls. Quantification of if in 10 fields of view (FOV) per section in two sections per animal, normalisation of positive cells to 100 nuclei; arrows point to positive cells. ISH quantification in 212.55 µm x 212.55 µm FOV. BMP, bone morphogenetic protein; BMPR2, BMP receptor 2; ISH, RNA in situ hybridization; PCLS, precision cut lung slices; VE, vascular endothelial; WT, wild type.

Article Snippet: Immunoblotting on Bis-Tris or Tris-Acetate gels (Life Technologies, Germany, #NP0321BOX, #EA0375BOX) included detection of CYP1A1 (A-9, Santa Cruz Biotechnology #SC-393979), ID1 (Abcam #ab168256), SMAD2/3 (Cell Signalling Technologies #3102), pSMAD2 (Cell Signalling Technologies #3101), SMAD 1 (Cell Signalling Technology, #6944), pSMAD 1-5-9 (Cell Signalling Technology, #9511), SMAD4 (Santa Cruz Biotechnology, #7966), SPARC (D10F10, Cell Signalling Technologies #8725), VE-Cadherin (H-72, Santa Cruz Biotechnology #28644), VEGF-A (147, Santa Cruz Biotechnology #507), VEGFR2 (R and D system, #AF644) and and β-actin (Santa Cruz Biotechnology, #sc-81178) (secondary antibodies goat anti-rabbit IgG (Santa Cruz Biotechnology, #2301) or goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, #sc-81178)).

Techniques: Western Blot, Immunofluorescence, Staining, Expressing, Control, RNA In Situ Hybridization

Journal: Thorax

Article Title: Relationship between impaired BMP signalling and clinical risk factors at early-stage vascular injury in the preterm infant

doi: 10.1136/thoraxjnl-2021-218083

Figure Lengend Snippet: Impaired BMPR2 signalling is ameliorated on treatment with FK506 in vitro and in vivo. (A) Exposure of human pulmonary microvascular endothelial cells (HPMEC) to cyclic stretch or O 2 (FiO 2 =0.4) for 24 hours in vitro mimics postnatal injury. (B) Immunoblot analysis shows a significant downregulation of pSMAD 1-5-9 protein expression in HPMECs after exposure to O 2 and/or stretch, (C) enhanced by TGF-β (D) and translated into decreased ID-1 and (E) Smad 4 protein expression. (F) Immunoblot analysis shows significant upregulation of pSMAD 1-5-9 protein levels in HPMECs on treatment with a single dose of 2 ng/mL FK506 in the presence of O 2 in combination with stretch and/or TGF-β incubation. (G) The significant reduction in BMPR2 mRNA levels after O 2 treatment is reversed by FK506 in HPMECs. (H) In vivo, a single intraperitoneal dose of 2 ng/mL FK506 in neonatal mice at the onset of O 2 or MV-O 2 exposure for 2 hours resulted in an upregulation of pulmonary CD31 mRNA expression levels in neonatal mice when compared with PBS treated control mice. Data are mean±SD *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=3–4 mice/group. BMPR2, bone morphogenetic protein receptor 2; ns, not significant; PBS, phosphate buffered saline.

Article Snippet: Immunoblotting on Bis-Tris or Tris-Acetate gels (Life Technologies, Germany, #NP0321BOX, #EA0375BOX) included detection of CYP1A1 (A-9, Santa Cruz Biotechnology #SC-393979), ID1 (Abcam #ab168256), SMAD2/3 (Cell Signalling Technologies #3102), pSMAD2 (Cell Signalling Technologies #3101), SMAD 1 (Cell Signalling Technology, #6944), pSMAD 1-5-9 (Cell Signalling Technology, #9511), SMAD4 (Santa Cruz Biotechnology, #7966), SPARC (D10F10, Cell Signalling Technologies #8725), VE-Cadherin (H-72, Santa Cruz Biotechnology #28644), VEGF-A (147, Santa Cruz Biotechnology #507), VEGFR2 (R and D system, #AF644) and and β-actin (Santa Cruz Biotechnology, #sc-81178) (secondary antibodies goat anti-rabbit IgG (Santa Cruz Biotechnology, #2301) or goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, #sc-81178)).

Techniques: In Vitro, In Vivo, Western Blot, Expressing, Incubation, Control, Saline