Expression of the Tgfβ2 Gene During Chick Embryogenesis
Article first published online: 21 DEC 2011
Copyright © 2011 Wiley Periodicals, Inc.
The Anatomical Record
Volume 295, Issue 2, pages 257–267, February 2012
How to Cite
Yamagishi, T., Ando, K., Nakamura, H. and Nakajima, Y. (2012), Expression of the Tgfβ2 Gene During Chick Embryogenesis. Anat Rec, 295: 257–267. doi: 10.1002/ar.22400
- Issue published online: 11 JAN 2012
- Article first published online: 21 DEC 2011
- Manuscript Accepted: 5 AUG 2011
- Manuscript Received: 25 SEP 2010
- Grant-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Grant Number: 21590201
- Kawano Memorial Foundation for the Promotion of Pediatrics. Grant Number: 18-5.
- chick embryo;
- in situ hybridization
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
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.
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).
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).
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).
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).
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).
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).
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 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).
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).
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 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 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).
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.
|(smooth muscle progenitor cells)|
|Cartilage and bone|
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).
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.
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.
- 2007. Heart field: from mesoderm to heart tube. Annu Rev Cell Dev Biol 23: 45–68. , .
- 1990. TGF beta in murine morphogenetic processes: the early embryo and cardiogenesis. Development 108: 645–656. , , , .
- 2008. TGF-β family signaling in skeletal development, maintenance, and diesease. In: Derink R, Miyazono K, editors. The TGF-β family. Cold Springer Harbor: Cold Springer Harbor Laboratory Press. p 667–723. , , .
- 2005. Temporal and spatial expression of TGF-β2 in chicken somites during early embryonic development. J Exp Zool A Comp Exp Biol 303: 323–330. , , , , , .
- 1994. Cloning and developmental expression of the chick type II and type III TGF β receptors. Dev Dyn 199: 12–27. , , , , , , .
- 2005. The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development 132: 3151–3161. , .
- 2001. Double-outlet right ventricle and overriding tricuspid valve reflect disturbances of looping, myocardialization, endocardial cushion differentiation, and apoptosis in TGF-β2-knockout mice. Circulation 103: 2745–2752. , , , , , , , , .
- 1997. Chemotaxis of skeletal muscle satellite cells. Dev Dyn 208: 505–515. .
- 1999. TGFβ2 and TGFβ3 have separate and sequential activities during epithelial-mesenchymal cell transformation in the embryonic heart. Dev Biol 208: 530–545. , , , , , .
- 2005. Building the mammalian heart from two sources of myocardial cells. Nat Rev Genet 6: 826–835. , , .
- 1991. Comparative analysis of human and chicken transforming growth factor-β2 and -β3 promoters. J Mol Endocrinol 7: 175–183. , , .
- 2000. Immunolocalization of the transcription factor Slug in the developing avian heart. Anat Embryol 201: 103–109. , , , , , .
- 2008. TGF-β Bioavailability: latency, targeting, and activation. In: Derink R, Miyazono K, editors. The TGF-β Family. Cold Springer Harbor: Cold Springer Harbor Laboratory Press. p 179–202. , .
- 2005. Unraveling signalling cascades for the Snail family of transcription factors. Cell Signal 17: 535–547. , , .
- 2000. Delta 1-activated notch inhibits muscle differentiation without affecting Myf5 and Pax3 expression in chick limb myogenesis. Development 127: 5213–5224. , , , .
- 1995. Defective haematopoiesis and vasculogenesis in transforming growth factor-β1 knock out mice. Development 121: 1845–1854. , , , , , .
- 1993. RNA and protein localisations of TGFβ2 in the early mouse embryo suggest an involvement in cardiac development. Development 117: 625–639. , , , , .
- 2005. Balance of pro-apoptotic transforming growth factor-β and anti-apoptotic insulin effects in the control of cell death in the postnatal mouse retina. Eur J Neurosci 22: 28–38. , , , , , , , , .
- 2009. The role of secondary heart field in cardiac development. Dev Biol 336: 137–144. , .
- 1991. Localization and actions of transforming growth factor-βs in the embryonic nervous system. Development 113: 183–191. , , , , , , , , .
- 1993. Role of the latent TGF-beta binding protein in the activation of latent TGF-β by co-cultures of endothelial and smooth muscle cells. J Cell Biol 120: 995–1002. , , , , , , .
- 1986. Transforming growth factor-β. A very potent inhibitor of myoblast differentiation, identical to the differentiation inhibitor secreted by Buffalo rat liver cells. J Biol Chem 1986 261: 16509–16513. , , , , , .
- 2007. Heartening news for head muscle development. Trends Genet 23: 365–369. , .
- 1951. A series of normal stages in the development of the chick embryo. J Morphol 88: 49–92. , .
- 1987. Role of transforming growth factor-β in the development of the mouse embryo. J Cell Biol 105: 2861–2876. , , , , , , , .
- 1990. Colocalization of TGF-beta 1 and collagen I and III, fibronectin and glycosaminoglycans during lung branching morphogenesis. Development 109: 29–36. , , , , .
- 1992. Histogenesis of tunica media of the chick aorta. Kaibogaku Zasshi 67: 749–761. , .
- 1994. Expression of transforming growth factor-β2 and β3 mRNAs and proteins in the developing chicken embryo. Differentiation 55: 105–118. , , , , .
- 1995. Abnormal lung development and cleft palate in mice lacking TGF-β3 indicates defects of epithelial-mesenchymal interaction. Nat Genet 11: 415–421. , , , , , , .
- 1994. The TGF-β superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev 8: 133–46. .
- 2000. Reduction of endogenous transforming growth factors β prevents ontogenetic neuron death. Nat Neurosci 3: 1085–1090. , , , , , , .
- 1988. Embryonic expression pattern of TGF β type-1 RNA suggests both paracrine and autocrine mechanisms of action. Development 104: 263–273. , .
- 2001. TGF-β inhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3. Genes Dev 15: 2950–2966. , , .
- 1977. Structural development of endocardial cushions. Am J Anat 148: 85–119. , , .
- 1975. Sturctural analysis of endocardial cytodifferentiation. Dev Biol 42: 160–80. , , .
- 1991. Embryonic gene expression patterns of TGF β1, β2 and β3 suggest different developmental functions in vivo. Development 111: 131–143. , , , .
- 2003. Expression patterns of Tgfβ1-3 associate with myocardialisation of the outflow tract and the development of the epicardium and the fibrous heart skeleton. Dev Dyn 227: 431–444. , , , , , , , .
- 2002. Altered apoptosis pattern during pharyngeal arch artery remodelling is associated with aortic arch malformations in Tgfβ2 knock-out mice. Cardiovasc Res 56: 312–322. , , , , , , .
- 2010. Second lineage of heart forming region provides new understanding of conotruncal heart defects. Congenit Anom 50: 8–14. .
- 2000. Mechanisms involved in valvuloseptal endocardial cushion formation in early cardiogenesis: roles of transforming growth factor (TGF)-β and bone morphogenetic protein (BMP). Anat Rec 258: 119–127. , , , .
- 1998. An autocrine function for transforming growth factor (TGF)-β3 in the transformation of atrioventricular canal endocardium into mesenchyme during chick heart development. Dev Biol 194: 99–113. , , , , .
- 2008. The contribution of Islet1-expressing splanchnic mesoderm cells to distinct branchiomeric muscles reveals significant heterogeneity in head muscle development. Development 135: 647–657. , , , , , , , , .
- 2010. A midline switch of receptor processing regulates commissural axon guidance in vertebrates. Genes Dev 24: 396–410. , , , , , , , , , , , , .
- 1996. In situ hybridization analysis of chick embryos in whole mount and tissue sections: In: Bronner-Fraser M, editors. Methods in cell biology. San Diego: Academic Press, p 219–235. , , .
- 1999. Differentiation of avian craniofacial muscles: I. Patterns of early regulatory gene expression and myosin heavy chain synthesis. Dev Dyn 216: 96–112. , , , .
- 2006. Transforming growth factor-β stimulates epithelial-mesenchymal transformation in the proepicardium. Dev Dyn 235: 50–59. , , , .
- 1991. Cell death during development of the nervous system. Annu Rev Neurosci 14: 453–501. .
- 1990. In situ hybridization analysis of TGFβ3 RNA expression during mouse development: comparative studies with TGFβ1 and β2. Development 110: 609–620. , , , .
- 1989. Expression of transforming growth factor β2 RNA during murine embryogenesis. Development 106: 759–767. , , , .
- 1991. Immunohistochemical localization of TGFβ1, TGFβ2, and TGFβ3 in the mouse embryo: expression patterns suggest multiple roles during embryonic development. J Cell Biol 115: 1091–1105. , , , , .
- 1995. Transforming growth factor-β3 is required for secondary palate fusion. Nat Genet 11: 409–414. , , , , , , , , .
- 2008. Embryonic development of the proepicardium and coronary vessels. Int J Dev Biol 52: 229–36. , , .
- 1994. Expression of TGF-βs and their receptors during implantation and organogenesis of the mouse embryo. Dev Biol 166: 716–728. , , , , .
- 1999. Slug is a mediator of epithelial-mesenchymal cell transformation in the developing chicken heart. Dev Biol 212: 243–254. , .
- 1997. TGFβ2 knockout mice have multiple developmental defects that are non-overlapping with other TGFβ knockout phenotypes. Development 124: 2659–2670. , , , , , , , .
- 1994. TGFβ1 inhibits branching morphogenesis and N-myc expression in lung bud organ cultures. Development 120: 2153–2161. , , .
- 2008. An Fgf/Gremlin inhibitory feedback loop triggers termination of limb bud outgrowth. Nature 2008. 454: 638–641. , .
- 2005. Ablation of the secondary heart field leads to tetralogy of Fallot and pulmonary atresia. Dev Biol 284: 72–83. , , , .
- 2009. Roles of TGFβ and BMP during valvulo-septal endocardial cushion formation. Anat Sci Int 84: 77–87. , , .
- 1999. Expression of TGFβ3 RNA during chick embryogenesis: a possible important role in cardiovascular development. Cell Tissue Res 298: 85–93. , , .