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Fig. 1. Endothelial cells closely surround the thyroid mass, then epithelial follicles, which start producing thyroid hormones. Immunolabeling of control thyroid sections from e12.5 to e16.5 ((A), (B)) or at birth (C). (A) Topological relation between the thyroid epithelium and blood capillaries. At the thyroid bud stage (e12.5), endothelial cells (PECAM, red) closely surround the epithelial cell mass (Nkx2.1, green). During subsequent development, endothelial cells progressively invade the expanding cell mass, and generate therein a dense capillary network that eventually surrounds all individualized follicles at e16.5. Panels at e12.5 and e16.5 show frames selected from Supplementary Movies 1 and 2, respectively. (B) Polarization of thyrocytes. The e12.5 thyroid bud is composed of a mass of non-polarized epithelial cells (E-cad+; Ezrin−). Small intracellular dots labeled for ezrin appear at e14.5, sometimes seen as doublets just underneath both sides of an epithelial contact (arrowhead), but no lumen is detectable. At e16.5, essentially all epithelial cells are organized in follicular-like structures or “rosettes”, made of polarized monolayers, with apical ezrin delineating microlumina, which expand thereafter (see (C)). (C) Differentiation of thyrocytes. At birth, follicular cells (E-cad+) are apically labeled for iodothyroglobulin (T4 antibodies). See also Fig. S1 in the supplementary material. 6 W- x: |- L0 p; I; Q* Z) ^' l0 H w, `5 _; x( M
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Fig. 2. The developing thyroid strongly expresses Vegfa and is closely associated with Vegfr2-expressing endothelial cells. (A) Q-PCR profiling of VEGF family ligands and receptors. Vegfa and Vegfr2 are most expressed during thyroid development where expression increases from e14.5 to e17.5. (B) Vegfa and Vegfr2 localization by in situ hybridization and immunolabeling. At left, hybridization of the antisense probe for Vegfa on an e14.5 embryo shows the highest intensity in the thyroid region (boxed; enlarged at right). On the same section shown at upper right, the Vegfa pattern coincides with epithelial cells (E-cad; red), and is fully dissociated from endothelial cells (PECAM; green). At lower right, pattern for Vegfr2 mRNA is complementary to that of Vegfa and perfectly overlaps with endothelial cells (PECAM, green). All asterisks mark the cluster of epithelial cells originating from ultimobranchial bodies (E-cad+; Vegfa-). See also Fig. S2 in the supplementary material.6 v+ a3 x! {8 D/ N* W6 v% R
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Fig. 3. Thyroid-specific Vegfa KO demonstrates that recruitment and expansion of endothelial cells depends on production of VEGFA by epithelial cells. (A) Targeting and efficiency of Cre recombinase activity. β-galactosidase (lacZ) staining of Pax8Cre/+ thyroid gland demonstrates efficient Cre recombinase activity in the thyroid lobes and isthmus as early as e13.5. (B) Efficiency of Vegfa inactivation by RT-qPCR. Loss of one (heterozygote; het) or two (homozygote or Vegfa conditional knockout; cKO) Vegfa alleles causes a dose-dependent reduction in the expression of Vegfa, and of the endothelial markers Vegfr2 and Ve-cadherin in newborns (values are means±s.e.m. **p<0.001). (C) Efficiency of Vegfa inactivation as shown by in situ hybridization. Serial sections of control (upper row) or cKO e14.5 thyroids (lower row). cKO abrogates Vegfa antisense probe hybridization and decreases that for Vegfr2. (D) Decreased endothelial cell density in Vegfa cKO. Whole-mount immunolabeling for PECAM (green) of e17.5 control (upper row) and cKO thyroids (lower row). Left, individual confocal sections; right, 3D reconstruction from 43 confocal images (projection from 43-μm total thickness). In Vegfa cKO, thyroid microvascular density is strongly reduced. , T6 S- C. u4 q; |1 ? & r* q: }. m9 _! SFig. 4. Reduction in vascular density does not prevent thyrocyte differentiation but impairs calcitonin expression. (A) Thyroid-specific transcription factor analysis by RT-qPCR. Except for a slight increase of Pax8 expression level in Vegfa cKO and heterozygote thyroids (*p<0.05), the expression of Nkx2.1, Foxe1 and Hhex does not change in cKO newborns. (B) Differentiation marker analysis by RT-qPCR. In cKO P0 thyroids, the expression levels of thyroperoxidase (Tpo), thyroglobulin (Tg) and thyrotropin receptor (Tshr) are only marginally affected, but that of calcitonin is reduced by 50% (⁎p<0.01, ⁎⁎p<0.001). (C) Localization of transcription factors by in situ hybridization. The expression pattern of the four thyroid-specific transcription factors is comparable in e14.5 control and Vegfa cKO thyroids. (D) Imaging and quantification of endocrine differentiation. Immunolabeling (e17.5) and in situ hybridization (e18.5) on control and cKO thyroid sections. Although follicular differentiation is impaired by Vegfa cKO (see below), apical iodothyroglobulin production is qualitatively preserved, in line with RT-qPCR data (panel B). The number of calcitonin-expressing C-cells and their distribution within the lobes is not affected in cKO, but calcitonin mRNA level in each cell is significantly reduced, confirming RT-qPCR data (panel B).: V, Y% n7 c" ~5 ^ B ?
/ Q* } l, k7 J9 c( Z8 P + M6 |$ p. V9 a$ M) G: @% ]Fig. 5. In vivo reduction in vascular density impairs epithelial reorganization and follicle formation. Representative immunofluorescence views of control ((A)–(E)) and cKO ((A′)—(E′)) thyroid sections or full projections over 18.5 μm ((D), (D′)). ((A)–(B)) At e15.5, control epithelial masses (E-cad+; dotted lines) have started to form pre-follicular structures or “rosettes”. Concerted polarization of epithelial cells is also initiated, as demonstrated by pluricellular apical ezrin+ structures and ZO-1 belts (arrowheads in (A), (B)). In contrast, the epithelium of Vegfa cKO thyroids remains a mass of non-polarized cells with random small intracellular ezrin dots (arrows in A′) and rare, exclusively bicellular ZO-1 staining (B′). ((C)–(E)) Around birth, control thyrocytes (E-cad+) are organized into follicles, where ezrin delineates well-defined apical lumina shared by 7 to 9 cells per profile (numbered in (C)), showing multiple contacts with blood capillaries (PECAM+). The enlarged size of closed lumina and the density of surrounding microvasculature are best appreciated by the full projection at (D). In cKO, thyrocytes are still clustered as a multilayered mass, wherein only a minority of cells have engaged into the formation of small ezrin+ structures, shared by 2 to at most 4 cells per profile (numbered in (C′)). Other cells show intracellular ezrin dots (arrow in (C′)), which mimick those seen at e15.5 in controls (arrow in (A)). ((D), (D′)) 3D reconstruction of 50 confocal images evidences a higher number of smaller ezrin+ structures between fewer vessels in Vegfa cKO. ((E), (E′)) Opposite localization of apical cellular (ezrin) and basal extracellular (laminin) markers in WT confirms full thyrocyte polarity in independent follicles. In cKO, epithelial cells (E-cad+) exhibit defective apico-basal polarization: cells displaying ezrin at one pole do not assemble laminin at the opposite pole, and vice versa (arrows in (E′)). (F) Quantification of the number and size of ezrin+ structures at birth. As compared to controls, the number of ezrin+ profiles per section in cKO glands is twice higher but their mean size is twice smaller, exceptionally reaching 50 μm2, as opposed to ～150 μm2 in controls. ((G)–(I)) Transmission electron microscopy at e18.5. (G) A control follicle, composed of at least 5 polarized thyrocytes in this part of the section, surrounds a closed lumen (tight junctions at the upper part of the lateral membrane marked by arrows) into which numerous microvilli project. (H) In cKO thyroids, follicular structures are smaller due to fewer cells, displaying apical differentiation (tight junctions) and shared lumen with microvilli. (I) Some cKO cells exhibit apparently similar structures (red asterisk), yet entirely intracellular (no visible junction). + T6 S8 O) G3 r7 k0 f. |# `0 g V: O3 p' P j2 G/ q
: ~1 d0 e# _( W( X! YFig. 6. Key steps of thyroid development are reproduced ex vivo: bilobation, vascularization, polarization and folliculogenesis, and dual endocrine differentiation. (A) Expansion and bilobation. Whole-mount immunolabeling of e12.5 thyroid explants before and after 4 days of culture. At e12.5, the thyroid midline anlage (th; E-cad+, Nkx2.1+) is positioned ventrally to the trachea (tr; E-cad+ only), while the ultimobranchial bodies (ub; E-cad+, Nkx2.1+) are lateral and caudal. After 4 days ex vivo, the thyroid midline anlage has migrated laterally and fused with the ultimobranchial bodies to form two lobes on each side of the trachea. (B) Vascularization, polarization and folliculogenesis. After 4 days ex vivo, almost all epithelial cells are polarized (ezrin+) and organized in follicular structures closely surrounded by a capillary network (PECAM+), despite lack of blood flow. Panel at left shows a frame selected from Supplementary Movie 3. (C) Dual endocrine differentiation. PCR profiling of differentiation markers in the cultured thyroid explants. After 4 days ex vivo (e12.5+4), expression of thyroperoxidase (Tpo), thyroglobulin (Tg), TSH receptor (Tshr), Na+I− symporter (Nis) and calcitonin a (calca), undetectable one day after culture onset (e13.5), was comparable to that observed in vivo (e17.5). & O# D* N8 r! h* ^! A+ C8 z# U, K: c. d$ F: {
Fig. 7. Ex vivo ablation of endothelial cells impairs follicle formation. Whole-mount immunolabeling of control or treated explants (VEGFR2 inhibitor SU5416). (A) 3D reconstruction from a Z-stack of 79 confocal images (every 0.37 μm). The dense endothelial cells network (PECAM+, red) is absent when explants are cultured in the presence of the VEGFR2 inhibitor (VEGFR2i). Structures labeled by ezrin are much smaller. (B) Corresponding orthogonal views better show that VEGFR2i causes maintenance of multilayering and the lack of large lumina, abundant in untreated controls, with ezrin mostly limited to intracellular dots (arrows). Upper rectangles boxed in green correspond to XZ reconstructions from positions indicated by horizontal green lines in the central XY squares. Right rectangles boxed in red correspond to to YZ reconstructions at the red vertical red lines. See also Fig. S7 in the supplementary material.8 _1 ]' L6 B' w4 R: [
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Fig. 8. Induction of follicle formation and calcitonin expression, in thyroid explants with endothelial cell ablation, by exogenous factor(s) derived from embryonic endothelial progenitor cell (EPC). E12.5 thyroid explants were first treated with the VEGFR2 inhibitor (VEGFR2i) for 40 h to ablate endogenous endothelial cells, washed, then incubated for 2 days with either exogenous endothelial progenitor cells (EPCs), or their concentrated conditioned media (c.m.). ((A), (B)) RT-qPCR analysis. (A) Treatment of e12.5 thyroid explants with VEGFR2i causes a strong reduction in Tie2 and Vegfr2 expression, two endothelial markers. Subsequent addition of EPCs restores Tie2 but not Vegfr2 expression. (B) Calcitonin expression is enhanced by EPCs or their conditioned medium. (C) Immunolabeling. In untreated control explants, epithelial cells (E-cad+) are polarized (apical ezrin, upper row; basal laminin, lower row) and organized in follicles surrounded by a microvascular network (PECAM+). VEGFR2i ablates endothelial cells, and prevents thyrocyte polarization (asterisk on multilayered mass) and lumen coalescence (arrowheads). Subsequent incubation of VEGFR2i-treated explants with EPCs or their c.m. accelerates enlargement of polarized follicles as compared to untreated controls. 6 v: K0 g$ f3 n7 n: e2 o" S$ \5 u9 @. [7 L3 ]6 \8 s+ U! M
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Fig. 9. A working model: role of endothelial cells in thyroid follicle formation and C-cells differentiation. VEGFA released by thyroid epithelial cells (blue) triggers VEGFR2+ endothelial cells (red) to invade the epithelial mass. Reorganization of the mass goes along with the appearance of ezrin+ structures (green) that coalesce to form early lumina. Follicles, i.e. monolayers of polarized epithelial cells, with ezrin (green) at the apical pole and laminin (purple) at the basal pole, are thus formed and remain surrounded by a dense microvascular network. C-cells (orange) are scattered between follicles. In the absence of Vegfa expression (in vivo cKO) or VEGFR2 activity (ex vivo inhibition), endothelial cells density is reduced. Epithelial cells remain as a multilayered mass and cannot efficiently polarize, thus harboring more numerous smaller ezrin+ structures that fail to coalesce. In cKO, C-cells are still scattered within the epithelial mass but express less calcitonin (light orange). Conversely, when explants with ablated endogenous endothelial cells are rescued by eEPCs or their conditioned medium (c.m.), folliculogenesis is accelerated and calcitonin expression is enhanced (dark orange).作者: bioon 时间: 2013-9-11 07:37
本帖最后由 bioon 于 2013-9-11 07:52 编辑 / d" q7 n% ]9 u. F
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