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First published online 16 April 2008
doi: 10.1242/dev.020958

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Developmental origin of smooth muscle cells in the descending aorta in mice

Per Wasteson^1,2, Bengt R. Johansson³, Tomi Jukkola⁴, Silke Breuer^1,*, Levent M. Akyürek^1,2, Juha Partanen⁴ and Per Lindahl^1,2,

¹ Wallenberg Laboratory, Sahlgrenska University Hospital, University of Gothenburg, Gothenburg, Sweden.
² Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden.
³ Institute of Biomedicine, Electron Microscopy Unit, University of Gothenburg, Gothenburg, Sweden.
⁴ Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

View larger version (94K): [in this window] [in a new window]   Fig. 1. Hoxb6-cre activates the R26 reporter gene in lateral plate mesoderm cells before the first expression of the SMC marker SM22. (A-C) lacZ staining for the Hoxb6-cre-activated R26 reporter gene at three levels between the prospective forelimb and hindlimb regions at E8.5-E9.0 showed reporter expression in splanchnic (arrowheads) and somatic (arrows) lateral plate mesoderm. The reporter was also expressed in hindgut endoderm. (D,E) In posterior regions of the embryo, Hoxb6-cre had a broader expression domain and the reporter was also expressed in paraxial mesoderm cells (arrowheads in E; E shows boxed area in D) and cells in the neural plate (arrows in E). (F-H) The first expression of the SM22-lacZ gene in the forelimb bud region was detected at E9.0-E9.5 in splanchnic mesoderm adjacent to the aorta (arrows in F and G), and in cells situated between the dorsal aortas (arrows in H). d-v, dorsal-ventral axis; nt, neural tube; nf, neural folds; np, neural plate; pm, paraxial mesoderm; da, dorsal aorta; hg, hindgut; va, vitelline artery; c, coelomic cavity. Scale bars: 25 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 2. SMC differentiation is induced in lateral plate mesoderm-derived cells on the ventral side of the dorsal aorta at E9.5. (A-D) lacZ staining of stage- and location-matched sections from E9.5 Hoxb6-cre/R26 reporter and SM22-lacZ transgenic mice at three levels between the forelimb and hindlimb regions. SM22-lacZ was expressed around the full circumference of the aorta in the anterior sections (A), but was confined to the ventral side in more posterior sections (B,C). The Hoxb6-cre-activated R26 reporter gene was expressed in splanchnic and somatic mesoderm, but not in intermediate mesoderm (D). The reporter gene was expressed in all cells on the ventral side of the aorta, including the endothelial cells (arrowheads in A-C). The reporter was also expressed in a single layer of cells on the lateral and dorsal side of the aorta (arrows in A-C). (E-G) TEM of ultrathin sections from lacZ-stained E9.5 Hoxb6-cre/R26 reporter mice. On the dorsal side of the aorta (E,G), the lacZ staining, seen as an electron-dense cytoplasmic precipitate (arrows), was confined to endothelial cells. By contrast, the staining was found in endothelium and SMCs on the ventral side (F). The dorsal-ventral axis (indicated as d-v) runs from top to bottom if not otherwise indicated. nt, neural tube; nc, notochord; da, dorsal aorta; im, intermediate mesoderm; spl, splanchnic mesoderm; som, somatic mesoderm; g, gut; e, endothelium; s, SMC. Scale bars: 25 µm in A-D; 5 µm in E-G.

View larger version (68K): [in this window] [in a new window]   Fig. 3. Migration of dorsal mesenchymal cells towards the aortic wall. (A) Ultrastructure analysis of the space between the notochord and the aorta. Mesenchymal cells were widely spaced in a voluminous matrix. Cells that were located at greater distance from the vessel were polygonal (arrowheads), whereas cells closer to the aorta were more elongated (asterisks). Arrows indicate profiles of cytoplasmic extensions. (B,C) The asterisk-labeled cells in A displayed elongation in the direction of the arrows with cytoplasmic organelles distributed to the aorta-facing side of the nucleus. Scale bars: 20 µm in A; 5 µm in B,C.

View larger version (118K): [in this window] [in a new window]   Fig. 4. Lateral plate mesoderm contributes to endothelial cells but not to SMCs in the descending aorta at E11.5. (A,B) A single layer of lacZ-expressing cells was detected in the aortic wall of Hoxb6-cre/R26 reporter mice. The aorta was surrounded by large areas of lacZ-negative cells in the anterior part of the descending aorta, shown here in the lung bud region (A), and by four or five layers of lacZ-negative cells in posterior parts, shown here in the gonadal ridge region (B). (C) Triple staining against Acta2 (green), CD31 (red) and lacZ (blue) showed that endothelial cells, but not SMCs, expressed the R26 reporter gene. (D,E) Similarly, double-staining against lacZ and Acta2 showed lacZ staining in endothelial cells but not in SMCs (high-power magnification of boxed area). (E) Cells that were double-labeled for lacZ and Acta2 were detected in a discrete region in which lateral plate mesoderm-derived cells were still found in proximity to the aorta (high-power magnification of boxed area). Arrows indicate lacZ-negative SMCs; arrowheads, lacZ-positive endothelial cells; asterisks, lacZ-positive SMCs; ao, aorta; lb, lung bud; cv, cardinal vein; gr, gonadal ridge; pc, peritoneal cavity. Scale bars: 25 µm in A-E; 10 µm in higher magnification views.

View larger version (48K): [in this window] [in a new window]   Fig. 5. Lateral plate mesoderm does not contribute to SMCs in the anterior and middle part of the descending aorta in adult mice. (A-F) Adult descending aorta from Hoxb6-cre/R26 reporter mice was stained for lacZ activity at different anterior-posterior levels (levels are indicated in A). lacZ staining was restricted to endothelial cells in the anterior and middle part of the vessel (B-D). In posterior parts of the vessel, expression was frequently found in SMCs and endothelial cells (E,F). Boxed areas in B,C show lacZ-expressing endothelial cells at higher magnification to confirm successful staining. Arrows, SMC; arrowheads, endothelial cells; al, aortic lumen; ra, renal artery; ia, iliac artery; m, media. Scale bars: 25 µm.

View larger version (85K): [in this window] [in a new window]   Fig. 6. Somite-derived cells replace lateral-plate mesoderm-derived SMCs at E10.5. (A-D) lacZ staining of E9.5 Meox1-cre/R26 reporter mice at two anterior-posterior levels between the forelimb and hindlimb regions. The reporter gene was expressed in myodermatome and in sclerotome in cells surrounding the dorsal part of the aorta. lacZ-expressing cells were found in close proximity to the aortic lumen on the dorsal and lateral sides of the vessel but not on the ventral or ventrolateral sides. (E-J) lacZ staining of E10.5 Meox1-cre/R26 reporter mice at three anterior-posterior levels between the forelimb and hindlimb regions. The reporter gene was homogenously expressed in dorsal mesoderm, but lacZ-expressing cells had also migrated along the ventrolateral walls of the aorta and replaced the lacZ-negative cells that were found in the ventral wall of the aorta at E9.5 (E-H). Meox1-cre/R26-expressing cells were still confined to the dorsal and lateral walls of the aorta in the posterior part of the embryo (I,J), which indicates that the migration of somite-derived cells along the ventrolateral walls of the aorta is initiated in the anterior part of the animal and progresses towards the posterior end. Arrows, Meox1-cre/R26-expressing peri-endothelial cells; arrowheads, lacZ-negative peri-endothelial cells; nt, neural tube; md, myodermatome; sc, sclerotome; da, dorsal aorta; hg, hindgut. Scale bars: 50 µm.

View larger version (111K): [in this window] [in a new window]   Fig. 7. Ectopic expression of the Meox1-cre/R26 reporter gene in cardiac outflow tract and renal cortex. (A) Real-time RT-PCR of Meox1 mRNA expression at E9.5-E18.5 showed that Meox1 expression peaked during somitogenesis at E9.5-E10.5 and thereafter declined rapidly. (B-E) X-gal staining of cryosections (B-D) and whole-mount preparations of organs (E) from P2 mice confirmed reporter gene expression in somite-derived tissues, including dermis, vertebrae, skeletal muscle and microvessels. Arrows in B indicate dermal papilla and arrowheads indicate epidermal follicular epithelium. Thin arrow in C indicates chondrocytes, thick arrow indicates ossification centre in ventral part of vertebrae, arrowhead indicates an intervertebral disc that derive from the notochord. Arrows in D indicate the external oblique muscle surrounded by white adipose tissue. Arrowheads in E indicate blood vessels that spread across the surface of the unlabelled uterus. (F-I) Ectopic expression was detected in the arterial pole of the heart: whole-mount staining showed expression in ascending aorta (white arrow in F) and aortic arch, but also in patches of atrial myocardium (black arrow in F). Paraffin sections of whole-mount stained hearts confirmed expression in SMCs in proximal aorta (ao) and pulmonary trunk (pt) (G). Expression was also detected in a fine caliber branched network (arrowheads in H) on the heart surface that seemed to be distinct from blood vessels (arrows in H). Double staining against lacZ and Acta2 on paraffin sections confirmed that the network on the heart surface (arrowheads in I) were distinct from the coronary arteries (arrows in I). (J-M) Ectopic expression in kidney: en bloc X-gal staining of kidney slices revealed reporter expression in the cortex (white asterisk in J) but not in the pelvic region (black asterisk). Detailed inspection of the cortex in paraffin sections revealed that collection ducts (arrows in K) lacked expression, whereas other components were homogenously stained (asterisk indicates s-shaped nephron anlagen). Other derivates of intermediate mesoderm such as adrenal cortex (asterisk in L) and gonads (white arrow in M) lacked staining. Arrows in L indicate reporter-expressing cells in the adrenal capsule, arrowheads in M indicate blood vessels; ed, epidermis; de, dermis.

View larger version (148K): [in this window] [in a new window]   Fig. 8. SMCs in the adult descending aorta originate from the somites. (A-F) Descending aorta from adult Meox1-cre/R26 reporter mice was stained for lacZ activity at different anterior-posterior levels (levels are indicated in A). SMCs stained positive for the reporter gene at all investigated levels, also in the posterior part of the aorta. (G) Double staining against lacZ and Acta2 confirmed reporter gene expression in SMCs. (H-M) SMCs in the major branches of the descending aorta were investigated in Meox1-cre/R26 reporter mice at P2 to determine the distal borders of somite contribution. Whole-mount staining of internal organs with attached aorta showed lack of reporter expression in the coeliac artery (arrowheads in H) and superior mesenteric artery (arrows in H and I). The renal arteries were homogenously stained (arrows in J). The aorta was displaced to expose the branch points. X-gal staining of cryosections confirmed lack of somite contribution to the coeliac and superior mesenteric arteries (K,L). The transition from lacZ-positive to negative SMCs occurred at the branch points (thin arrows in K and L), but not in a strict manner. Asterisk in L indicates somite-derived SMCs that extended into the superior mesenteric artery (sm). Arrowheads in K and L indicate the walls of the coeliac artery and superior mesenteric artery, respectively. The renal artery was entirely occupied by somite-derived SMCs (M). Boxed area in M shows renal arterial wall at higher magnification. Arrows in M indicate lacZ-positive SMCs, arrowheads indicate lacZ-negative endothelial cells. al, aortic lumen; ao, aorta; ca, coeliac artery; dm, dorsal mesentery; ia, iliac artery; in, intestines; m, tunica media; ra, renal artery; sm, superior mesenteric artery; asterisks, endogenous X-gal activity. Scale bars: 25 µm in B-G; 50 µm in K-M.

View larger version (14K): [in this window] [in a new window]   Fig. 9. A model for the dynamics of SMC-lineages during aortic development. Blue stripe indicates lateral plate mesoderm; red stripe, sclerotome; solid blue, lateral plate mesoderm-derived SMCs; solid red, somite-derived SMCs; solid green, serosal mesothelium-derived SMC; nt, neural tube; g, gut; ra, renal artery; sm, superior mesenteric artery.

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