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Keywords:

  • Tgfβ2;
  • chick embryo;
  • expression;
  • in situ hybridization

Abstract

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED

We performed a comprehensive analysis of the expression of transforming growth factor (TGF) β2 during chick embryogenesis from stage 6 to 30 (Hamburger and Hamilton, J Morphol 1951;88:49–92) using in situ hybridization. During cardiogenesis, Tgfβ2 was expressed in the endothelial/mesenchymal cells of the valvulo-septal endocardial cushion tissue and in the epicardium until the end of embryogenesis. During the formation of major arteries, Tgfβ2 was localized in smooth muscle progenitors but not in the vascular endothelium. During limb development, Tgfβ2 was expressed in the mesenchymal cells in the presumptive limb regions at stage 16, and thereafter it was localized in the skeletal muscle progenitors. In addition, strong Tgfβ2 expression was seen in the mesenchymal cells in the pharyngeal arches. Tgfβ2 mRNA was also detected in other mesoderm-derived tissues, such as the developing bone and pleura. During ectoderm development, Tgfβ2 was expressed in the floor plate of the neural tube, lens, optic nerve, and otic vesicle. In addition, Tgfβ2 was expressed in the developing gut epithelium. Our results suggest that TGFβ2 plays an important role not only in epithelial-mesenchymal interactions but also in cell differentiation and migration and cell death during chick embryogenesis. We also found that chick and mouse Tgfβ2 RNA show very similar patterns of expression during embryogenesis. Chick embryos can serve as a useful model to increase our understanding in the roles of TGFβ2 in cell–cell interactions, cell differentiation, and proliferation during organogenesis. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.

Transforming growth factor β (TGFβ) belongs to the TGFβ superfamily, and TGFβ1 (chick TGFβ4), 2, and 3 have been identified in mammals and avians (Kingsley,1994). TGFβ has been demonstrated to regulate many developmental processes. During lung development, TGFβ1 is localized in stromal cells and inhibits branching morphogenesis in organ culture (Heine et al.,1990; Serra et al.,1994), and TGFβs has been implicated in cartilage and bone formation (Alliston et al.,2008). TGFβ deficient mice show various phenotypes. For example, Tgfβ1 null mice show hematopoiesis and vasculogenesis defects (Dickson et al.,1995), whereas Tgfβ2 null mice have various developmental abnormalities including cardiac, lung, craniofacial, limb, spinal column defects, and so forth (Sanford et al.,1997). Mice lacking Tgfβ3 display delayed pulmonary development and defective palatogenesis (Kaartinen et al.,1995; Proetzel et al.,1995).

TGFβ activation is controlled by a variety of mechanisms (Dabovic and Rifkin,2008). In fact, in coculture systems, only 2–5% of total latent TGFβ is activated (Flaumenhaft et al.,1993). Understanding the spatiotemporal expression patterns of Tgfβ2 mRNA is also important for identifying the cells that produce its protein. In addition, chickens are a useful model for studying developmental processes, such as organogenesis, because chicken embryos can be manipulated more easily than mouse embryos. Studies of knockout mice have often been combined with microsurgical experiments on chick embryos to investigate limb development (Verheyden and Sun,2008), neural development (Nawabi et al.,2010), and other subjects. In such studies, gene expression profiling during embryogenesis is useful. Although there have been many reports about the expression patterns of Tgfβ genes during mouse embryogenesis (Heine et al.,1987; Lehnert and Akhurst1988; Pelton et al.,1989; Akhurst et al.,1990; Pelton et al.,1990; Flanders et al.,1991; Millan et al.,1991; Pelton et al.,1991; Dickson et al.,1993; Roelen et al.,1994), there have only been a few reports about the expression of Tgfβ during avian embryogenesis (Barnett et al.,1994; Jakowlew et al.,1994; Yamagishi et al.,1999; Aramaki et al.,2005). In this study, we comprehensively investigated the expression pattern of Tgfβ2 during chick embryogenesis. Our results showed that chick Tgfβ2 is expressed in various tissues, such as the epithelium, muscle, mesenchyme, and neural tissue during embryogenesis.

MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED

Chick Embryos

Fertilized chicken eggs (Gallus domesticus) were incubated at 37.8°C until the embryos reached the appropriate stages (from 24 hr to 8 days). The embryos were then collected in ice-cooled phosphate-buffered saline (PBS) and staged according to Hamburger and Hamilton (1951). Then, the staged embryos were subjected to cloning of their Tgfβ2 genes and in situ hybridization in order to examine the tissue distribution of their mRNA, as described below.

Cloning of the Chick Tgfβ2 Gene

The chick Tgfβ2 gene (accession No. X50071) was isolated as described below. The following PCR primers (forward: 5′-GGAATTCCTCTCAGCCTGTCTACCTGC-3′ and reverse: 5′-GAGGATCCGCAGCATGGACAATGTA AGC-3′) were used. The PCR product amplified from stage 23 chick embryonic heart cDNA was subcloned into pBluescriptII KS (-) and characterized.

InSitu Hybridization

Digoxigenin-labeled single-strand RNA probes were prepared using a DIG RNA labeling kit (Roche Diagnostics, Tokyo, Japan) according to the manufacturer's instructions. To produce an antisense probe, Tgfβ2 (about 700 bp) was linearized using EcoRI and transcribed using T7 RNA polymerase. Embryos at appropriate stages were fixed in 4% paraformaldehyde in PBS and then embedded in paraffin. Then, the sections were deparaffinized, hydrated, digested with proteinase K (3 μg/mL), refixed with 4% paraformaldehyde, and acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0), before being dehydrated, air dried, and hybridized at 65°C. After the hybridization step, the sections were rinsed with 5× SSC before being treated with 50% formamide at 65°C, 2× SSC at 60°C, and 0.2× SSC at 65°C (twice). Hybridization was detected using alkaline-phosphatase-conjugated anti-digoxigenin antibody and BCIP/NBT. Whole mount in situ hybridization was performed essentially as described by Nieto et al., (1996).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED

Expression of cTgfβ2 mRNA During Cardiovascular Development

During early heart development, the heart is formed from the right and left lateral plate mesoderm of the precardiac region. This mesoderm fuses on the ventral side of the foregut, and a primitive single heart tube is formed. Then, the heart tube starts to become asymmetrical by looping to the right (forming a S-shape structure), and cells from the anterior heart field (AHF) or secondary heart field (SHF) are recruited into the anterior end of the heart. As development proceeds, various cardiac segments are formed: the outflow tract (OFT), primitive ventricle, atrioventricular canal, atrium, and sinus venosus (Dyer and Kirby,2009; Nakajima,2010). Although no Tgfβ2 gene expression was seen during the formation of the heart tube, its mRNA was detected in the endothelial cells and mesenchymal cells of the AV region at stage 16, but not in the myocardium (Fig. 1A,B). At stage 16–17 of the chick embryo, the endothelial cells of the OFT and AV regions are initiated transformtion to mesenchymal cells and invade the adjacent cardiac jelly, resulting in the formation of endocardial cushion tissue that constitutes the primordial valves and septa (Markwald et al.,1975,1977). During this process (at stage 18), intense Tgfβ2 mRNA signals were detected in the endothelial and mesenchymal cells in the OFT and AV regions, the epicardium, and the splanchnic mesoderm behind the heart (Fig. 1C–F). Thereafter, Tgfβ2 mRNA was detected in the endothelial and mesenchymal cells in the OFT and AV regions, and intense Tgfβ2 mRNA signals were observed in the epicardium at stage 23 (Fig. 1G,H). At stage 27, the expression of the Tgfβ2 gene was seen in the endothelial and mesenchymal cells in the OFT and AV regions and the epicardium (Fig. 1I,K). In the OFT region, the expression of the Tgfβ2 gene was detected in the myocardium (Fig. 1J). In the early fetus stage (stage 30), the expression of Tgfβ2 transcripts was detected in the endothelial and mesenchymal cells in the valve and cushion tissues but not in the myocardium (Fig. 1L,M).

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Figure 1. Localization of Tgfβ2 mRNA during heart development. (A) Sagittal section of the stage (st) 16 heart. Tgfβ2 mRNA was detected in the heart and the pharyngeal arches (ph). (B) High magnification of the boxed area shown in panel A. Tgfβ2 transcripts were detected in the endocardium (ec) and mesenchyme (mes) in the AV region. (C) Sagittal section of the stage (st) 18 heart. Tgfβ2 transcripts were localized in the outflow tract (oft), atrioventricular canal (av), sinus venosus (sv), and splanchnic mesoderm behind the heart (spm). (D) High magnification of the boxed area (D) shown in C. In the outflow tract (OFT) region, Tgfβ2 signals were seen in the endocardium (ec) and mesenchymal cells (mes). (E) High magnification of the boxed area (E) shown in C. In the AV region, Tgfβ2 signals were detected in the endocardium (ec) and mesenchymal cells (mes). (F) High magnification of the boxed area (F) shown in C. Strong expression of the Tgfβ2 gene was seen in the epicardium (epc).

(G) Sagittal section of the stage (st) 23 heart. Intense signals for Tgfβ2 mRNA were detected in the OFT (oft) and AV (av) canal. (H) High magnification of the boxed area shown in G. Tgfβ2 signals were seen in the endocardium (ec), mesenchymal cells (mes), and epicardium (epc). (I) Frontal section of the stage (st) 27 heart. Tgfβ2 transcripts were detected in the OFT (oft) and cushion tissue (ct). (J) High magnification of the boxed area (J) shown in panel I. In the OFT (oft) region, Tgfβ2 transcripts were detected in the myocardium (my) and mesenchymal cells (mes). (K) High magnification of the boxed area (K) shown in panel I. In the AV canal, Tgfβ2 mRNA was localized in the endocardium (ec), mesenchymal cells (mes), and epicardium (epc).(L, M) Frontal section of the stage (st) 30 heart. M is a high magnification view of the boxed area shown in panel L. Tgfβ2 signals were detected in the endocardium (ec) and mesenchyme (mes). a, atrium; AV (av), atrioventricular canal; ct, cushion tissue; ec, endocardium; epc, epicardium; la, left atrium; lv, left ventricle; mes, mesenchymal cell; my, myocardium; OFT (oft), outflow tract; ph, pharyngeal arch; ra, right atrium; rv, right ventricle; spm, splanchnic mesoderm; st, stage; sv, sinus venosus; v, ventricle.

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The major vessels are formed by vasculogenesis. The dorsal aorta is formed from angioblasts originating in the splanchnic mesoderm. Tgfβ2 mRNA was detected in the mesenchymal cells underneath the endothelial cells of the dorsal aorta but not in endothelial cells or external mesenchymal cells at stage 18 (Fig 2A,B). These mesenchymal cells are thought to be progenitors of the smooth muscle cells surrounding the endothelial cells of the dorsal aorta (Hiruma and Hirakow,1992). As development proceeds (stage 23–24), these cells proliferate and form a smooth muscle cell layer. At this time these cells express Tgfβ2 mRNA, which is not detected at later stages (Fig. 2C,D).

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Figure 2. Expression of Tgfβ2 during arterial development. (A) At stage (st) 18, Tgfβ2 transcripts were expressed in the dorsal aorta (da), the neural tube (nt), the notochord (nc), and the sclerotome (sc). (B) High magnification of the boxed area shown in panel A. Tgfβ2 transcripts were detected in the mesenchymal cells subjacent to the endothelial cells (en) of the dorsal aorta. (C) At stage (st) 23, Tgfβ2 expression was seen in the dorsal aorta (da) and the sclerotome (sc). (D) High magnification of the boxed area shown in panel C. Tgfβ2 mRNA was detected in the mesenchymal cells but not in the endothelial cells (en). da, dorsal aorta; en, endothelial cell; nc, notochord; nt, neural tube; sc, sclerotome; st, stage.

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Expression of cTgfβ2 mRNA in the Pharyngeal Arches

The pharyngeal arches, which give rise to the skeletons and the muscles of the face and neck region, are formed on both sides of the pharyngeal foregut, starting from stage 14 during chick development. The outer layer of the pharyngeal arches is covered with ectoderm; the inner layer is covered with endoderm; and the core region is filled with mesenchymal cells, which are derived from the lateral plate mesoderm, somitomeres, and neural crest cells. During these developmental processes, the Tgfβ2 signal was first detected at stage 16 (Fig. 3A). In the first pharyngeal arch, Tgfβ2 transcripts were detected in the mesenchymal cells but not detected in the outer or inner epithelial cells (Fig. 3B). At stage 18, Tgfβ2 mRNA was seen in the first and second pharyngeal arches (Fig. 3C), and its mRNA was localized in the core region (arrow) of the mesenchymal cells in the pharyngeal arches (Fig. 3D). As development proceeded, intense Tgfβ2 mRNA signal were detected in the mesodermal core (arrows) and the mesenchymal cells of the first-sixth pharyngeal arches, but not in the ectoderm (Fig. 3E,F).

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Figure 3. Expression of Tgfβ2 mRNA during pharyngeal development. (A, B) A: Sagittal section of a stage (st) 16 embryo. B: High magnification of the boxed area shown in panel A. Tgfβ2 mRNA was localized in the mesenchymal cells of the first pharyngeal arch (ph1). (C, D) C: Sagittal section of a stage (st) 18 embryo. Tgfβ2 mRNA was seen in the first (ph1) and second pharyngeal arches (ph2). D: High magnification of the boxed area shown in panel C. Tgfβ2 transcripts were detected in the mesodermal core (arrow) in the first pharyngeal arch (ph1). (E, F) E: Frontal section of a stage (st) 23 embryo. Intense Tgfβ2 mRNA signals were detected in the mesenchymal cells in the pharyngeal arches. F: High magnification of the boxed area shown in panel E. Tgfβ2 transcripts were seen in the mesodermal core (arrows) and mesenchymal cells in the pharyngeal arches (ph1-6). aa, aortic arch; opv, optic vesicle; ph, pharyngeal arch; ph1′, maxillary arch; ph1″, mandibular arch; phx, pharynx.

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Expression of cTgfβ2 mRNA During Limb Development

The limb buds appear as small bulges on the lateral body wall and consist of a mesenchymal core of mesoderm, which is derived from the somatopleuric lateral plate mesoderm in the flank regions. This mesoderm differentiates into the bones, tendons, ligaments, and vasculature of the limbs, whereas the limb musculature is derived from a somatic mesoderm that migrates into the developing limb bud. At stage 18, Tgfβ2 mRNA was seen in the mesenchymal cells of the ventral regions of the limb buds (Fig. 4A1,A2). At stage 23, Tgfβ2 gene expression was seen in the dorsal and ventral muscle mass (Fig. 4B1,B2). At stage 27, Tgfβ2 mRNA signals were detected in the muscle mass and the axial mesenchymal condensation (Fig. 4C1,C2), and at stage 30, Tgfβ2 mRNA was seen in the chondrifying regions of the bone primordia (Fig. 4D1,D2).

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Figure 4. Expression of the Tgfβ2 gene during limb development. (A1) Whole mount in situ hybridization of a stage (st) 18 embryo. Tgfβ2 transcripts were observed in the primitive forelimb and hindlimb buds (arrowheads). (A2) In the frontal section of the forelimb shown in panel A1, Tgfβ2 mRNA signals were detected in the mesenchymal cells in the ventral region. (B1) Whole mount in situ hybridization of a stage (st) 23 embryo. Tgfβ2 transcripts were detected in the center of the forelimb and hindlimb buds (arrowheads). (B2) In the frontal section of the forelimb shown in panel B1, Tgfβ2 mRNA signals were seen in the mesenchymal cells of the muscle sections of the ventral and dorsal regions. (C1) Whole mount in situ hybridization of a stage (st) 27 embryo. Tgfβ2 mRNA signals were detected in the central regions of the forelimb and hindlimb buds (data not shown) and the anterior region of the forelimb (arrow). (C2) Cross-section of the forelimb shown in panel C1. Tgfβ2 transcripts were detected in the mesenchymal cells of the dorsal and ventral regions of the limb and the presumptive radius (r) and ulna (u) regions. (D1) Frontal section of the forelimb of a stage (st) 30 embryo. Tgfβ2 gene expression was detected in the ventral and the dorsal muscle regions (arrows) of the limb and osteogenic region. (D2) High magnification of the boxed area shown in panel D1. Tgfβ2 mRNA signals were detected in the epiphysis (epp) and perichondrium (arrowhead). epp, epiphysis; r, radius; st, stage; u, ulna.

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Expression of cTgfβ2 mRNA During Neural Tissue Development

The neural plate is formed from ectoderm folds along its midline to produce the neural tube. Then, the neural tube begins to differentiate into the brain and the spinal cord. In the spinal cord, the mantle layer of the spinal cord forms a pair of alar plates and a pair of basal plates. In addition, neural crest cells arise along the lateral margins of the neural folds and produce the peripheral nervous system and non-neural structures. During these processes, Tgfβ2 mRNA was detected in the floor plate and the basal plates at stage 23 (Fig. 5A). At stage 27, Tgfβ2 mRNA was seen in the floor plate and the spinal ganglion, which is derived from neural crest cells (Fig. 5B), and at stage 30, Tgfβ2 signals were seen in the visceral motor center of the spinal cord (Fig. 5C).

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Figure 5. Expression of the Tgfβ2 gene during neural tissue development. (A) Cross-section of the trunk region at stage (st) 23. Tgfβ2 transcripts were detected in the floor plate (fp) and basal plate (bp) of the neural tube (nt). (B) Cross-section of a stage (st) 27 embryo. Tgfβ2 mRNA was detected in the floor plate (fp), spinal ganglion (spg), and sclerotome (sc), but not in the basal plate region at this stage. (C) Cross-section of the neural tube at stage (st) 30. Tgfβ2 gene expression was observed in the visceral motor center (vmc) and vertebra (ver). (D) Frontal section of the optic vesicle (opv) region of a stage (st) 18 embryo. Tgfβ2 mRNA signals were seen in the lens (l). (E) Frontal section of the optic vesicle region at stage (st) 23. The Tgfβ2 gene was expressed in the lens (l), the presumptive iris region of the retina (re; arrowhead), the optic stalk (os), and the progenitor cells of the extraocular muscle (em). (F) Frontal section of the optic vesicle region at stage (st) 27. Tgfβ2 transcripts were detected in the lens (l) and the optic nerve (on). (G) High magnification of the boxed area shown in panel F. In the lens, Tgfβ2 mRNA was detected in the lens epithelium (le), but not in the primary lens fiber (lf) of the lens body and the ectoderm (ecto). (I) High magnification of the boxed area shown in panel F. Tgfβ2 gene expression was detected in the optic nerve (on), but not in the retina (re). (I) Frontal section of a stage (st) 18 embryo. Tgfβ2 mRNA was seen in the epithelia of the otic vesicle (ov) and floor plate (fp). bp, basal plate; ecto, ectoderm; em, extraocular muscle; fb, fore brain; fp, floor plate; l, lens; le, lens epithelium; lf, lens fiber; nt, neural tube; on, optic nerve; opv, optic vesicle; os, optic stalk; ov, otic vesicle; re, retina; sc, sclerotome; spg, spinal ganglion; st, stage; ver, vertebra; vmc, visceral motor center.

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Tgfβ2 signals were also seen in the placodes of sensory organs. During eye development, Tgfβ2 signals were seen in the lens placode at stage 18 but not in the optic vesicle (Fig. 5D). At stage 23, an intense Tgfβ2 signal was observed in the lens placode, retina, and optic stalk (Fig. 5E), and then, at stage 27, Tgfβ2 mRNA was localized in the lens epithelium and optic nerve (Fig. 5F–H). During inner ear development, Tgfβ2 mRNA was expressed in the epithelium of the otic vesicle at stage 18 (Fig. 5I).

Expression of cTgfβ2 mRNA in Other Tissues

The notochord

The notochord arises in the embryo as a median structure from cells at the cephalic end of the primitive streak and is localized ventral to the neural tube. At this time (at stage 8), the Tgfβ2 gene was expressed in the notochord (Fig. 6A). Tgfβ2 expression was also seen at stage 14, but not at stage 18 (Fig. 6B).

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Figure 6. Expression of Tgfβ2 in other tissues. (A) Cross-section of the trunk region at stage (st) 8. Tgfβ2 transcripts were detected in the notochord (nc), but not in the neural plate (np) or somite (sm). (B) Cross-section of the trunk region at stage (st) 14. Tgfβ2 transcripts were seen in the notochord (nc) and the pronephric tubule (pt). (C) High magnification of the stage (st) 27 notochord region. Tgfβ2 transcripts were seen in the mesenchymal cells of the primitive vertebral body, but not in the notochord (nc). (D) Cross-section of the trunk region at stage (st) 23. Intense Tgfβ2 mRNA signals were detected in the mesentery (arrowhead), peritoneum (arrow), and the epithelium of the hindgut (hg). (E, F) High magnification of the boxed area shown in panel D. Tgfβ2 transcripts were localized in the visceral peritoneum (vp) and parietal peritoneum (pp). In panel F, Tgfβ2 signals were also seen in the mesenchymal cells subjacent to the parietal peritoneum (pp). (G) Sagittal section of the stage (st) 23 liver. Intense Tgfβ2 mRNA signals were seen in the visceral peritoneum (vp). (H) High magnification of the boxed area shown in panel G. Tgfβ2 transcripts were localized in the endothelial cells (en) of veins in the liver, but not in hepatocytes. (I) High magnification of the stage (st) 27 mesonephros. Tgfβ2 mRNA was detected in the endothelial cells (en) of the sinusoid (si). (J) Cross-section of the stage (st) 27 thoracic region. Tgfβ2 mRNA was detected in the lung as well as the parietal (arrow) and visceral pleura (arrowhead). (K) High magnification of the boxed area shown in panel J. Tgfβ2 transcript signals were observed in the mesenchymal cells of the lung, but not in the epithelium of the bronchial bud (br). (L) Cross-section of the posterior region at stage (st) 23. Tgfβ2 mRNA was observed in the epithelium (arrow) of the hindgut (hg). (M) Cross-section of the proventriculus region at stage (st) 30. Tgfβ2 signals were detected in the epithelial cells of the gland primordia (g) in the proventriculus but not in the epithelium of the lumen. br, bronchial bud; da, dorsal aorta; en, endothelial cell; g, gland primordia; hg, hind gut; nc, notochord; ng, neural groove; np, neural plate; nt, neural tube; pp, parietal peritoneum; pt, pronephric tubule; si, sinusoid; sm, somite; st, stage; vp, visceral peritoneum.

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The vertebra

Somites divide into three kinds of mesodermal primordia: myotomes, dermatomes, and sclerotomes. The sclerotome cells migrate toward the midline of the embryo to surround the neural tube and notochord, where they subsequently form the vertebral arches and vertebral bodies, respectively. Tgfβ2 expression was seen in the sclerotome at stage 18 and 23 (Fig. 2A,C), and then, Tgfβ2 mRNA was seen in the mesenchymal cells around the notochord and neural tube at stage 27 and 30 (Figs. 5B,C and 6B).

The kidney

In the urogenital system during avian and mammal development, pronephros, mesonephros, and metanephros are formed in succession. These organs arise from the intermediate mesoderm lateral to the somite. The pronephros is formed in the anterior region and is represented by tubules. Tgfβ2 mRNA was seen in the pronephric tubule at stage 14 (Fig. 6B), and at stage 27, Tgfβ2 mRNA was detected in the endothelial cells of the mesonephros (Fig. 6I). Endothelial expression of the Tgfβ2 gene was also seen in the developing liver at stage 23 (Fig. 6H).

The serous membranes

The lateral plate mesoderm gives rise to the serous membranes. The lateral plate mesoderm consists of the two layers, the somatic and splanchnic layers. The somatic layer coats the inner surface of the body wall (somatopleure), and the splanchnic layer ensheathes the lung, the liver, and the gut (splanchnopleure). Tgfβ2 signals were seen in the somatopleure and splanchnopleure of the thoracic and abdominal regions (Fig. 6D–G,J), and Tgfβ2 mRNA was also expressed in the mesenchymal cells in the dorsal mesentery region (Fig. 6D).

The lung

The lung is a composite of endodermal and mesodermal tissues. The ventral foregut endoderm gives rise to the lung bud and differentiates into various epithelial cell types lining the inner surface of the developing lung and trachea. The lung mesenchyme originates from lateral plate mesoderm and gives rise to the lung vasculature, cartilage, and muscle tissue. During lung development Tgfβ2 mRNA was seen in the mesenchymal cells around the bronchial buds, but not in the epithelium of the bronchial bud (Fig. 6J).

The gut

The primitive gut is divided into three compartments, the foregut, midgut, and hindgut. The epithelia of these guts are derived from the endoderm. Tgfβ2 mRNA was seen in the hindgut epithelium at stage 27 (Fig. 6I). In birds, the stomach consists of two portions, the proventriculus and gizzard. Tgfβ2 mRNA was localized in the glandular epithelium of the proventriculus, but not in the luminal epithelium at stage 30 (Fig. 6M).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED

In this study, we have described the mRNA localization of chick Tgfβ2 in chick embryos from stage 6 through to stage 33 in detail. Our results are summarized in Table 1. While the gene expression patterns of Tgfβ2 and Tgfβ3 overlap in some tissues, the pattern for Tgfβ2 was distinctly different from that observed for Tgfβ3 (Yamagishi et al.,1999). Our results clearly indicate that Tgfβ2 is widely expressed in a variety of organs derived from the endoderm, mesoderm, and ectoderm. Indeed, Tgfβ2 null mice exhibit a wide range of developmental defects, including heart, lung, craniofacial, limb, spinal column, eye, inner ear, and urogenital defects (Sanford et al.,1997). These results suggest that TGFβ2 plays an essential role in the development of a wide range of tissues and organs.

Table 1. Embryonic cell types expressing chick Tgfβ2 and Tgfβ3 and mouse Tgfβ2
 cTgfβ2cTgfβ3amTgfβ2b
  • + indicates that a positive signal was seen (stage 6–30).

  • − indicates not detectable by in situ hybridization.

  • uk indicates unknown expression of Tgfβ2.

  • a

    Data taken from Yamagishi et al. (1999).

  • b

    Data taken from Millan et al. (1991).

  • c

    Data taken from Molin et al. (2003).

Blood island
  Endothelial cells
  Blood cells
Blood vessel
  Endothelial cells++
  Myoblasts++uk
 (smooth muscle progenitor cells)   
Cartilage and bone
  Precartilagenous blastemas+++
  Perichondria+++
Cardiac tissue
  Myocardium+(OFT)++
  Endocardium++
  Cushion mesenchyme+++
  Epicardium++c
Neuronal tissue
  Spinal cord++
  Forebrain++
  Retina++
  Optic nerve+uk
Somite++uk
Mesothelia
  Pleura++
  Peritoneum++
Limb
  Mesenchyme+
  Epithelium
Notochord+
Others
  Otic vesicle++
  Bronchial mesenchyme+
  Olfactory epithelium+
  Palate++
  Gut epithelium+
  Lens epithelium++
  Pharyngeal arch+uk

Cardiovascular Development

During heart development, Tgfβ3 mRNA is localized to the premyocardium, endocardial cushion tissue, and ventricular myocardium (Yamagishi et al.,1999; Nakajima et al.,1998). In this study, our data show that Tgfβ2 is also expressed in the developing heart. However, no Tgfβ2 RNA was seen in the myocardium, except for in a section of the OFT region at stage 27. Previous studies by other investigators have also demonstrated that Tgfβ2 RNA is expressed in the chick embryonic heart (Barnett et al.,1994; Boyer et al.,1999). For example, Boyer et al. (1999) reported that Tgfβ2 mRNA was expressed throughout the entire myocardium and endocardium at stages 14 and 19. In addition, Barnett et al. (1994) reported that Tgfβ2 mRNA was observed in the AV cushion and the OFT from stage 18 to 26 and in the AV groove at stage 26. Our current findings coincide with Barnett's. The difference between Boyer's results, and ours and Barnett's can be attributed to the probe region used. Although Boyer et al. (1999) used a 2 kb chick Tgfβ2 cDNA probe that was hybridized at 52°C, the probes used by us and Barnett consisted of sequences from the precursor region of chick Tgfβ2 (see Material and methods), which is dissimilar to the Tgfβ3 sequence, and our hybridizing and washing conditions were stringent (65°C). Hence, the probes used by us and Barnett were unlikely to undergo cross-hybridization.

Many reports have shown that TGFβ signaling is involved in the epithelial-mesenchymal transition (EMT) during cushion tissue formation (Nakajima et al.,2000; Yamagishi et al.,2009). This study shows that Tgfβ2 RNA was expressed in endothelial and mesenchymal cells when the endothelial cells began their transformation to mesenchymal cells. As development proceeded; that is, during the later cushion tissue formation stage, the Tgfβ2 signals in the endothelial and mesenchymal cells of the cushion tissue became weak (Fig. 1H). These results suggest that Tgfβ2 mediates initial endothelial cell-cell separation during EMT and that the expression of Tgfβ2 mRNA is induced by other soluble EMT signals (Boyer et al.,1999). Intense Tgfβ2 RNA signals were also seen in the epicardium. The proepicardium migrates over the heart until it has completely covered it, and then a subpopulation of epicardial cells undergoes EMT to generate mesenchymal cells (Ratajska et al.,2008), before these cells invade the myocardium and give rise to cardiac fibroblasts and vascular smooth muscle cells. A previous in vitro experiment demonstrated that TGFβ1 and TGFβ2 induce epicardial EMT (Olivey et al.,2006). During heart development, the expression pattern of Tgfβ2 is consistent with that of the Slug gene, a member of the snail family that is known to play a critical role in EMT (Carmona et al.,2000; Barrallo-Gimeno and Nieto,2005; De Craene et al.,2005). In fact, during the development of endocardial cushions, slug is an essential target of Tgfβ2 (Romano and Runyan, 2000). Taken together, these data suggest that TGFβ2 plays an important role in the EMT processes that occur during heart development.

A previous study demonstrated that Tgfβ2 null mice exhibit morphological abnormalities of the cardiovascular system, including a double-outlet right ventricle, ventricular septal defects, and hypoplasia of the aortic arch, and so forth (Sanford et al.,1997; Bartram et al.,2001; Molin et al.,2002). It has been suggested that the cardiovascular system defects seen in Tgfβ2 null mice result from increased neural crest cell apoptosis or disrupted neural crest cell migration and homing (Sanford et al.,1997; Bartram et al.,2001). The expression of Tgfβ2 in the pharyngeal arches overlaps with the expression patterns of MyoD, Myf5, and Isl-1 (arrows in Fig. 3D and F; Noden et al.,1999; Nathan et al.,2008). This result reveals that Tgfβ2 is expressed in the cranial pharyngeal mesoderm, which extends into the pharyngeal arches (in the AHF). In addition, the Tgfβ2 expression of the splanchnic mesoderm behind the heart was found to coincide with that of the SHF (Fig.1C). The myocardium of the OFT is derived from SHF and AHF cells (Buckingham et al.,2005; Abu-Issa and Kirby,2007; Nakajima,2010). In fact, ablation of the SHF induces abnormal OFT development (Ward et al.,2005). Taken together, in addition to playing several roles in cardiac neural crest cells, TGFβ2 might also affect the AHF and SHF during OFT development.

Skeletal Muscle Development

Tgfβ2 RNA is expressed in the dorsal and ventral muscle masses in the developing limbs and the mesodermal core of the pharyngeal arches; that is, the myogenic cells that form the jaw and the extra-ocular muscles (Figs. 3F and 5E; Grifone and Kelly,2007). The expression of Tgfβ2 during limb and pharyngeal arch development follows the same patterns as MyoD and Myf5 expression (Noden et al.,1999; Delfini et al.,2000). Invitro experiments have revealed that the TGFβ inhibits myoblast differentiation and induces migration of skeletal muscle satellite cell that is thought to become myogenic progenitor cell (Florini et al.,1986, Bischoff,1997). Therefore, TGFβ2 might be one of the factors regulating the differentiation and migration of myoblast cells during muscle development of the limb and head regions.

Chondrogenesis and Osteogenesis During Craniofacial Development

The cranial neural crest cells, which arise from the embryonic midbrain and cranial portion of the hindbrain, migrate into the pharyngeal arches. During the development of the first pharyngeal arch, these cells contribute to cartilage and bone. In this study, Tgfβ2 RNA was not only seen in the mesodermal core but also in the neural crest cells in the pharyngeal arches. Tgfβ2 null mice show a number of morphogenetic mandibular defects (Sanford et al.,1997). Therefore, TGFβ2 could be indispensable for skeletogenesis during the craniofacial development of mammalian and avian embryos.

Comparisons of Chick and Mouse Tgfβ2 Expression in Other Developing Tissues

Chick Tgfβ2 expression was detected in the central nervous and sensory organ systems, as was found for mouse Tgfβ2. Additionally, intense chick Tgfβ2 signals were detected in the optic nerve. Neuronal cell death plays an important role in normal neural development and is seen in different types of neurons in both the central and peripheral nervous systems, such as in motoneurons in the spinal cord, dorsal root ganglion, and others (Oppenheim,1991). TGFβ regulates ontogenetic neuron cell death in the parasympathetic ciliary ganglion, sensory dorsal root ganglion, and the lumbar spinal motoneuron column (Krieglstein et al.,2000), and the eyes of Tgfβ2 null mice show an enlarged inner neuroblastic layer and cellular infusion (Sanford et al.,1997). In an invitro retina culture system, the addition of exogenous TGFβ induced programmed cell death (Duenker et al.,2005). Thus, TGFβ2 might regulate cell death during nervous system development.

During lung development, chick Tgfβ2 RNA was detected in the mesenchyme, which is derived from the mesoderm, whereas mouse Tgfβ2 RNA was seen in the epithelium of the growing terminal end bud and alveolar epithelium, which are derived from the endoderm (Millan et al.,1991). During lung development, the expression pattern of chick Tgfβ2 was similar to that of mouse Tgfβ1 (Pelton et al.,1991). During gut development, chick Tgfβ2 RNA was detected in the epithelium, which is derived from the endoderm, while mouse Tgfβ2 RNA was seen in the submucosal layer, which is derived from the mesoderm (Pelton et al.,1989).

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED

The Tgfβ2 expression that occurs during chick embryogenesis is very similar to that seen in the mouse embryo (Table 1). Indeed, a comparison of chicken and human TGFβ2 promoter regions surrounding the major transcription start site, including in the cyclic AMP-responsive element, TATA box, and AP-2 sequence motif, revealed sequence homology between the two genes (Burt et al.,1991). These results suggest that the mechanisms of TGFβ2 gene regulation are conserved between avian and mammalian development. Taken together, chick embryos are a useful model that will increase our understanding of the roles of TGFβ2 in the cell–cell interactions, cell differentiation, and proliferation that occur during organogenesis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED

The authors thank Ms. K. Yoneyama for her excellent technical assistance. The authors are also grateful to Drs. S. Nishimatu and T. Masuda for their helpful discussions.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSIONS
  7. Acknowledgements
  8. LITERATURE CITED
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