Journal: Frontiers in Cell and Developmental Biology

Article Title: Hoxa5 Activity Across the Lateral Somitic Frontier Regulates Development of the Mouse Sternum

doi: 10.3389/fcell.2022.806545

Figure Lengend Snippet: LPM-derived HOXA5 expression can be seen laterally as early as E11.5 and E12. (A – J ″ ) E12.5 Prx1-Cre-RFP embryo in which alternate sections were labelled for SOX9 (A – E ″ ) or HOXA5 (F – J ″ ) . Images are shown with RFP and either SOX9 (A – E) or HOXA5 (F – J) ; RFP alone (A ′– E ′; F ′– J ′ ) , or SOX9 (A ″– E ″ ) or HOXA5 (F ″– J ″ ) alone. Pink arrows in (A,B) indicate SOX9 expression associated with developing clavicles and IC elements. (K) Whole mount in situ hybridization for Hoxa5 in an E11.5 embryo. Black arrow indicates LPM expression anterior to the forelimb; red arrow points to LPM expression adjacent to the forelimb; green arrow marks additional HOXA5 expression that remains untranslated. (L,P) RFP and HOXA5 expression in sections through an E11.5 Prx1-Cre RFP embryo at the level indicated in (K) by black arrow (L) , or red arrow (P) . (M,N) expression of Hoxa5 (M) or EBF3 and HOXA5 (N) in the area indicated by blue square in (L) . (O) SOX9 expression in an alternate section of same embryo shown in (L) . (Q,R) expression of HOXA5 (Q) or EBF3 and HOXA5 (R) in area indicated by blue square in (P) . (S) SOX9 expression in an alternate section of same embryo shown in (P) . Yellow arrow in (N,R) indicates region of HOXA5 and EBF3 co-expression. Yellow arrow in (O,S) shows corresponding region of SOX9 expression. Inset in (O) shows SOX9 expression in the context of Prx1-Cre-RFP. Scale bar: (A – J) , 200 μM; (K) , 25 mm; (L – S) , 100 μM. SB, sternal bar; T1, thoracic rib 1.

Article Snippet: Primary antibodies: HoxA5 ( ; 1:5000); Sox 9 (Millipore-Sigma AB5535, 1:500 or RnD Systems AF3075-SP 1: 500); RFP (Chromotek 5F8, 1:1000); Ebf3 (RnD Systems AF5166, 1:1000); Tenascin (Sigma-Aldrich T3413, 1: 100); PCNA (Santa Cruz sc-56 1:200), Cleaved Caspase 3 (CST 9661 1:200).

Techniques: Derivative Assay, Expressing, In Situ Hybridization

Journal: Development (Cambridge, England)

Article Title: Transient and lineage-restricted requirement of Ebf3 for sternum ossification

doi: 10.1242/dev.186239

Figure Lengend Snippet: Essential roles of Ebf3 in sternum ossification. (A) Whole-mount immunostaining of E10.5 mouse embryo with antibodies against Ebf3 (green), Pax7 (cyan) and Sox9 (red). Ebf3 was expressed in the lateral plate mesoderm, as well as in syndetomes located between Pax7 + dermomyotomes and Sox9 + sclerotomes. Insets 1 and 2 show higher magnifications of the indicated regions. (B,B′) Ebf3 (green) was expressed in Prx1:Tdtomato + cells (red) at the thoracic lateral plate mesoderm and in syndetomes adjacent to Pax7 + dermomyotomes (cyan) at E10.5. B′ shows an enlarged view of the boxed region. (C) WISH for Ebf1 , Ebf2 and Ebf3 in E10.5 and E11.5 mouse embryos. Red arrowheads indicate that Ebf3 , but not other Ebfs, was expressed in the thoracic lateral plate mesoderm tissue at E10.5 and show highest expression of Ebf3 in the sternal primordial bars at E11.5. (D) Schema of sternum development during murine embryogenesis. Red regions represent sternal bars at E12.5 and ossified zones of the sternum at E18.5. (E) Ossification and chondrogenesis analyses of Ebf3 flox/flox CAG-Cre and Ebf3 flox/+ CAG-Cre embryos at E18.5 and E17.5 based on skeletal preparations. Ebf3-KO mice exhibited defective sternum ossification without any defects in chondrogenesis. Scale bars: 100 μm (B); 1 mm (C); 2 mm (E).

Article Snippet: After washing with PBS-T (PBS with 0.05% Tween 20), the embryo tissues were blocked for 60 min at RT in Blocking One (Nacalai Tesque) and stained with primary antibodies for Ebf3 (1:1000; R&D Systems, AF5166), Sox9 (1:1000; Millipore, AB5535), tenascin (1:1000; Millipore, AB19013), GFP (1:500; Nacalai Tesque, 04404-84), osterix (1:1000; ab22552, Abcam), Runx2 (1:500; Abcam, ab23981), Islet1 (1:1000; Abcam, ab109517), MF20-c (1:200; DSHB, AB_214781) and laminin (1:1000; Sigma-Aldrich, L9393).

Techniques: Immunostaining, Expressing

Journal: Development (Cambridge, England)

Article Title: Transient and lineage-restricted requirement of Ebf3 for sternum ossification

doi: 10.1242/dev.186239

Figure Lengend Snippet: Ebf3 is required in lateral plate mesoderm-derived cells for sternum osteoblast differentiation. (A) Left: The generation of lateral plate mesoderm-specific Ebf3-KO mice embryos by crossing the Ebf3 flox/+ Prx1-Cre line with Ebf3 flox/flox Rosa26 Tdtomato/Tdtomato mice. Right: The expression pattern of Prx1-Cre-activated Tdtomato in the sternum tissue and muscle connective tissues based on longitudinal sections of E15.5 embryos together with immunostaining for tenascin (green). (B) Ossification analysis based on Alizarin Red skeletal preparations of thoracic bones in Ebf3 flox/flox Prx1-Cre embryos at E18.5. (C) Longitudinal sections of the sternum of Ebf3 flox/flox Prx1-Cre and Ebf3 flox/+ Prx1-Cre embryos at E18.5, immunostained for osteoblast (green, osterix) and pre-osteoblast (green, Runx2) markers. Upper panels show the distribution of lateral plate mesoderm-derived cells labeled with Prx1-Cre-activated TdTomato (red) in interstitial cells surrounding the sternum, which was decreased in Ebf3-KO embryos ( n =7) compared with heterozygous mice ( n =7). Lower-enlarged panels show much fewer osterix/Runx2-positive lateral plate mesoderm-derived cells in the periosteum of Ebf3-KO mice compared with heterozygous mice (white arrowheads). Scale bars: 100 μm.

Article Snippet: After washing with PBS-T (PBS with 0.05% Tween 20), the embryo tissues were blocked for 60 min at RT in Blocking One (Nacalai Tesque) and stained with primary antibodies for Ebf3 (1:1000; R&D Systems, AF5166), Sox9 (1:1000; Millipore, AB5535), tenascin (1:1000; Millipore, AB19013), GFP (1:500; Nacalai Tesque, 04404-84), osterix (1:1000; ab22552, Abcam), Runx2 (1:500; Abcam, ab23981), Islet1 (1:1000; Abcam, ab109517), MF20-c (1:200; DSHB, AB_214781) and laminin (1:1000; Sigma-Aldrich, L9393).

Techniques: Derivative Assay, Expressing, Immunostaining, Labeling

Journal: Development (Cambridge, England)

Article Title: Transient and lineage-restricted requirement of Ebf3 for sternum ossification

doi: 10.1242/dev.186239

Figure Lengend Snippet: Decreased number of Runx2 + pre-osteoblasts in Ebf3-deficient thoracic lateral plate mesoderm at E12.5. (A) Transverse section of thoracic lateral plate mesoderm (as indicated in the schematic) at E12.5 immunostained with anti-Ebf3 and anti-Runx2 antibodies. Most but not all Ebf3-expressing cells were Runx2 positive. White and yellow arrowheads show Ebf3/Runx2 double-positive cells. (B) Decrease in the number of Prx1: Tdtomato + Runx2 + cells in Ebf3 flox/flox Prx1-Cre compared with that in heterozygous embryos (white arrowheads). Immunostained transverse sections of the thoracic lateral plate mesoderm at E12.5 are shown. A and upper panels of B show images of Ebf3/Runx2-double-positive cells and those of Prx1: Tdtomato + Runx2 + LPMs, respectively, prepared from a single section of a heterozygous embryo stained with antibodies against Ebf3, Runx2 and DAPI. Yellow arrowheads indicate Prx1: Tdtomato + Ebf3 + Runx2 + LPMs. (C) The ratio of Runx2 + cells in Prx1: Tdtomato + thoracic LPMs was lower in Ebf3 flox/flox Prx1-Cre KO mice than in Ebf3 heterozygous mice at E12.5. Error bars represent s.e.m. ** P <0.01 ( n =4). (D) Ratio of Sox9 + cells in Prx1: Tdtomato + thoracic LPMs at E12.5. Error bars represent s.e.m.; ns, non-significant ( n =4). Scale bars: 100 μm.

Article Snippet: After washing with PBS-T (PBS with 0.05% Tween 20), the embryo tissues were blocked for 60 min at RT in Blocking One (Nacalai Tesque) and stained with primary antibodies for Ebf3 (1:1000; R&D Systems, AF5166), Sox9 (1:1000; Millipore, AB5535), tenascin (1:1000; Millipore, AB19013), GFP (1:500; Nacalai Tesque, 04404-84), osterix (1:1000; ab22552, Abcam), Runx2 (1:500; Abcam, ab23981), Islet1 (1:1000; Abcam, ab109517), MF20-c (1:200; DSHB, AB_214781) and laminin (1:1000; Sigma-Aldrich, L9393).

Techniques: Expressing, Staining

Journal: Development (Cambridge, England)

Article Title: Transient and lineage-restricted requirement of Ebf3 for sternum ossification

doi: 10.1242/dev.186239

Figure Lengend Snippet: Ebf3 plays a crucial role in sternum ossification at approximately E9.5-E10.5. (A) Generation of tamoxifen-induced temporal Ebf3-KO mouse embryos by crossing the Ebf3 flox/+ Ubc-CreER T2 line with Ebf3 flox/flox mice. (B) Strategy to determine Ebf3 functional timing in sternal ossification in Ubc-CreER T2 Ebf3-KO embryos. (C) Evaluation of sternum phenotypes in Ebf3 flox/flox Ubc-CreER T2 embryos upon tamoxifen injection at different developmental stages (E8.5-E12.5) based on Alizarin Red skeletal preparations. Embryonic days when tamoxifen was injected into pregnant dams are indicated.

Article Snippet: After washing with PBS-T (PBS with 0.05% Tween 20), the embryo tissues were blocked for 60 min at RT in Blocking One (Nacalai Tesque) and stained with primary antibodies for Ebf3 (1:1000; R&D Systems, AF5166), Sox9 (1:1000; Millipore, AB5535), tenascin (1:1000; Millipore, AB19013), GFP (1:500; Nacalai Tesque, 04404-84), osterix (1:1000; ab22552, Abcam), Runx2 (1:500; Abcam, ab23981), Islet1 (1:1000; Abcam, ab109517), MF20-c (1:200; DSHB, AB_214781) and laminin (1:1000; Sigma-Aldrich, L9393).

Techniques: Functional Assay, Injection

Journal: Development (Cambridge, England)

Article Title: Transient and lineage-restricted requirement of Ebf3 for sternum ossification

doi: 10.1242/dev.186239

Figure Lengend Snippet: Ebf3 downstream analysis in mesenchymes of the thoracic lateral plate mesoderm by RNA-seq. (A) Thoracic LPMs were excised from Ebf3 flox/flox Prx1-Cre and Ebf3 flox/+ Prx1-Cre embryos at E10.5, and Prx1: Tdtomato + cells were isolated by FACS ( n =3). (B) Differentially expressed genes based on RNA-seq data between Ebf3 flox/flox Prx1-Cre and Ebf3 flox/+ Prx1-Cre embryos (cut off: 10 read, P ≤0.1) were categorized by their biological functions using IPA software into three groups: (1) genes required for the differentiation of connective-tissue (CT) cells, (2) genes required for the differentiation of bone cells and osteoblasts, and (3) genes required for the differentiation of mesenchymal cells. (C) Venn diagram of these three groups. Genes in red were significantly upregulated and genes in blue were significantly downregulated in Ebf3 flox/flox Prx1-Cre compared with expression in Ebf3 flox/+ Prx1-Cre embryos ( P <0.05) (from RNA-seq data). (D) Relative expression levels of MCT cell/tenocyte-associated genes in Ebf3 flox/flox Prx1-Cre embryos compared with those in Ebf3 flox/+ Prx1-Cre embryos (from RNA-seq data). ** P <0.01, * P <0.05; ns, not significant ( n =3). (E) Relative expression levels of osteoblast-associated genes in Ebf3 flox/flox Prx1-Cre embryos compared with those in Ebf3 flox/+ Prx1-Cre embryos. ** P <0.01, * P <0.05 (from RNA-seq data) ( n =3). (F) Relative expression level of Islet1 in Ebf3 flox/flox Prx1-Cre embryos was increased significantly compared with that in Ebf3 flox/+ Prx1-Cre embryos. ** P <0.01 (from RNA-seq data) ( n =3). (G) Islet1 was expressed in Prx1:Tdtomato + Ebf3 + cells at the lateral somitic frontier of the thoracic lateral plate mesoderm (as indicated in the schematic) at E10.5 (white arrowheads). (H) Increase in the number of Prx1:Tdtomato + Islet1 + cells in Ebf3 flox/flox Prx1-Cre ( n =3) compared with that in heterozygous embryos ( n =3) (white arrowheads). Immunostained transverse sections of the lateral somitic frontier at E10.5 are shown. (I) The ratio of Islet1 + cells in Prx1:Tdtomato + LPMs was higher in Ebf3 flox/flox Prx1-Cre compared with that in heterozygous mice at E10.5. ** P <0.01 ( n =3). Error bars represent s.e.m. Scale bars: 100 μm.

Article Snippet: After washing with PBS-T (PBS with 0.05% Tween 20), the embryo tissues were blocked for 60 min at RT in Blocking One (Nacalai Tesque) and stained with primary antibodies for Ebf3 (1:1000; R&D Systems, AF5166), Sox9 (1:1000; Millipore, AB5535), tenascin (1:1000; Millipore, AB19013), GFP (1:500; Nacalai Tesque, 04404-84), osterix (1:1000; ab22552, Abcam), Runx2 (1:500; Abcam, ab23981), Islet1 (1:1000; Abcam, ab109517), MF20-c (1:200; DSHB, AB_214781) and laminin (1:1000; Sigma-Aldrich, L9393).

Techniques: RNA Sequencing, Isolation, Software, Expressing

Journal: Development (Cambridge, England)

Article Title: Transient and lineage-restricted requirement of Ebf3 for sternum ossification

doi: 10.1242/dev.186239

Figure Lengend Snippet: Ebf3 regulates mesenchymal progenitors in Scx-Cre lineage cells of the thoracic lateral plate mesoderm. (A) Ebf3 was expressed in Prx1:Tdtomato + Scx:GFP + cells at the lateral somitic frontier of the thoracic lateral plate mesoderm at E10.5 (white arrowheads). Scale bar: 100 μm. Schematic shows Ebf3, Scx:GFP and Islet1 expression in the lateral somitic frontier. (B) Ossification analysis based on Alizarin Red skeletal preparations of thoracic bones in Ebf3 flox/flox Scx-Cre embryos at E18.5. Red rectangle highlights ossification defects of the sternum. (C) Schematic summarizing the working hypothesis on the roles of Ebf3 in sternum ossification. Osteo-chondrogenic progenitors in the lateral plate mesoderm give rise to osteo-fibro progenitors marked with Islet1 + cells in the lateral somitic frontier (green area). Ebf3 regulates differentiation of these cells towards Runx2 + pre-osteoblasts (light blue), which differentiate into osteoblasts and osteocytes to form the sternum bone. In the absence of Ebf3, osteo-fibro progenitors differentiate towards fibroblastic cells (yellow) or self-renew.

Article Snippet: After washing with PBS-T (PBS with 0.05% Tween 20), the embryo tissues were blocked for 60 min at RT in Blocking One (Nacalai Tesque) and stained with primary antibodies for Ebf3 (1:1000; R&D Systems, AF5166), Sox9 (1:1000; Millipore, AB5535), tenascin (1:1000; Millipore, AB19013), GFP (1:500; Nacalai Tesque, 04404-84), osterix (1:1000; ab22552, Abcam), Runx2 (1:500; Abcam, ab23981), Islet1 (1:1000; Abcam, ab109517), MF20-c (1:200; DSHB, AB_214781) and laminin (1:1000; Sigma-Aldrich, L9393).

Techniques: Expressing