Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional growth factor that has profound regulatory effects on many developmental and physiological processes. Disruption of the TGF-beta 1 gene by homologous recombination in murine embryonic stem cells enables mice to be generated that carry the disrupted allele. Animals homozygous for the mutated TGF-beta 1 allele show no gross developmental abnormalities, but about 20 days after birth they succumb to a wasting syndrome accompanied by a multifocal, mixed inflammatory cell response and tissue necrosis, leading to organ failure and death. TGF-beta 1-deficient mice may be valuable models for human immune and inflammatory disorders, including autoimmune diseases, transplant rejection and graft versus host reactions.

Targeted disruption of the mouse transforming growth factor-β1 gene results in multifocal inflammatory disease

In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells

During development and in the context of different morphogenetic events, epithelial cells undergo a process called epithelial to mesenchymal transition or transdifferentiation (EMT). In this process, the cells lose their epithelial characteristics, including their polarity and specialized cell-cell contacts, and acquire a migratory behavior, allowing them to move away from their epithelial cell community and to integrate into surrounding tissue, even at remote locations. EMT illustrates the differentiation plasticity during development and is complemented by another process, called mesenchymal to epithelial transition (MET). While being an integral process during development, EMT is also recapitulated under pathological conditions, prominently in fibrosis and in invasion and metastasis of carcinomas. Accordingly, EMT is considered as an important step in tumor progression. TGF-β signaling has been shown to play an important role in EMT. In fact, adding TGF-β to epithelial cells in culture is a convenient way to induce EMT in various epithelial cells. Although much less characterized, epithelial plasticity can also be regulated by TGF-β-related bone morphogenetic proteins (BMPs), and BMPs have been shown to induce EMT or MET depending on the developmental context. In this review, we will discuss the induction of EMT in response to TGF-β, and focus on the underlying signaling and transcription mechanisms.

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TGF-beta-induced epithelial to mesenchymal transition.

In the last 10 years, a large family of secreted signaling molecules has been discovered that appear to mediate many key events in normal growth and development. The family is known as the TGF-p superfamily (Massague 1990), a name taken from the first member of the family to be isolated (transforming growth factor-^l). This name is somewhat misleading, because TGF-p 1 has a large number of effects in different systems (Spom and Roberts 1992). It actually inhibits the proliferation of many different cell lines, and its original "transforming" activity may be due to secondary effects on matrix pro­ duction and synthesis of other growth factors (Moses et al. 1990). The two dozen other members of the TGF-p superfamily have a remarkable range of activities. In Diosophila, a TGF-p-related gene is required for dorsoventral axis formation in early embryos, communication between tissue layers in gut development, and correct proximal distal patterning of adult appendages. In Xenopus, a TGF-p-related gene is expressed specifically at one end of fertilized eggs and may function in early signaling events that lay out the basic body plan. In mammals, TGF-p-related molecules have been found that control sexual development, pituitary hormone production, and the creation of bones and cartilage. The recognition of TGF-p superfamily members in many different organ­ isms and contexts provides one of the major unifying themes in recent molecular studies of animal growth and development. The rough outlines of the TGF-p family were first rec­ ognized in the 1980s. Since that time, a number of ex­ cellent reviews have appeared that summarize the prop­ erties of different family members (Ying 1989; Massague 1990; Lyons et al. 1991; Spom and Roberts 1992). Here, I will focus on four areas that have seen major progress in the last 3 years: structural characterization of the signal­ ing molecule, isolation of new family members, cloning of receptor molecules, and new genetic tests of the func­ tions of these factors in different organisms.

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The TGF-beta superfamily: new members, new receptors, and new genetic tests of function in different organisms.

The growth and differentiation factor transforming growth factor-beta2 (TGFbeta2) is thought to play important roles in multiple developmental processes. Targeted disruption of the TGFbeta2 gene was undertaken to determine its essential role in vivo. TGFbeta2-null mice exhibit perinatal mortality and a wide range of developmental defects for a single gene disruption. These include cardiac, lung, craniofacial, limb, spinal column, eye, inner ear and urogenital defects. The developmental processes most commonly involved in the affected tissues include epithelial-mesenchymal interactions, cell growth, extracellular matrix production and tissue remodeling. In addition, many affected tissues have neural crest-derived components and simulate neural crest deficiencies. There is no phenotypic overlap with TGFbeta1- and TGFbeta3-null mice indicating numerous non-compensated functions between the TGFbeta isoforms.

TGFbeta2 knockout mice have multiple developmental defects that are non-overlapping with other TGFbeta knockout phenotypes

Isoform-specific antibodies to TGF beta 1, TGF beta 2, and TGF beta 3 proteins were generated and have been used to examine the expression of these factors in the developing mouse embryo from 12.5-18.5 d post coitum (d.p.c.). These studies demonstrate the initial characterization of both TGF beta 2 and beta 3 in mammalian embryogenesis and are compared with TGF beta 1. Expression of one or all three TGF beta proteins was observed in many tissues, e.g., cartilage, bone, teeth, muscle, heart, blood vessels, lung, kidney, gut, liver, eye, ear, skin, and nervous tissue. Furthermore, all three TGF beta proteins demonstrated discrete cell-specific patterns of expression at various stages of development and the wide variety of tissues expressing TGF beta proteins represent all three primary embryonic germ layers. For example, specific localization of TGF beta 1 was observed in the lens fibers of the eye (ectoderm), TGF beta 2 in the cortex of the adrenal gland (mesoderm), and TGF beta 3 in the cochlear epithelium of the inner ear (endoderm). Compared to the expression of TGF beta mRNA transcripts in a given embryonic tissue, TGF beta proteins were frequently colocalized within the same cell type as the mRNA, but in some cases were observed to localize to different cells than the mRNA, thereby indicating that a complex pattern of transcription, translation, and secretion for TGF beta s 1-3 exists in the mouse embryo. This also indicates that TGF beta 1, beta 2, and beta 3 act through both paracrine and autocrine mechanisms during mammalian embryogenesis.

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Immunohistochemical localization of TGF beta 1, TGF beta 2, and TGF beta 3 in the mouse embryo: expression patterns suggest multiple roles during embryonic development

Bone morphogenetic protein-2A (BMP-2A) is a member of the transforming growth factor beta (TGF beta) gene family that has been implicated in cartilage and bone formation. Here we use in situ hybridization to show that BMP-2A RNA is expressed in a variety of embryonic epithelial and mesenchymal tissues outside of the developing skeletal system, including cell populations known to play important roles in morphogenesis. Thus, high levels of transcripts are found in developing limb buds (ventral ectoderm and apical ectodermal ridge), heart (myocardium of the atrioventricular canal), whisker follicles (ectodermal placodes, hair matrix and precortex cells), tooth buds (epithelial buds, dental papilla and odontoblasts), and craniofacial mesenchyme, as well as a number of other sites. The expression patterns of BMP-2A are different from those of TGF beta-1, -2 and -3, and this is illustrated in detail in the developing whisker follicles. These results suggest that BMP-2A plays multiple roles in morphogenesis and pattern formation in the vertebrate embryo.

Organogenesis and pattern formation in the mouse: RNA distribution patterns suggest a role for bone morphogenetic protein-2A (BMP-2A).

The murine Vgr-1 (Vg-related) and BMP-2a (bone morphogenetic protein 2a) genes are members of the decapentaplegic subgroup of the transforming growth factor-beta (TGF beta) superfamily. Although genetic and biochemical studies suggest that the members of this subgroup play important roles in development, little is known about their function in mammals. Therefore, we investigated the expression of Vgr-1 and BMP-2a RNAs in embryonic, newborn, and adult tissues by in situ hybridization. Vgr-1 RNA is maternally encoded in ovarian oocytes but declines in fertilized eggs and is undectable by the two- to four-cell stage. Only low levels of transcripts are seen in blastocysts and early postimplantation stages. From mid-gestation on, Vgr-1 RNA is expressed at high levels in developing skin, especially in the suprabasal cells of the proliferating epidermis but not in the dermis or hair follicles, both of which contain TGF beta 1 and/or TGF beta 2 RNAs. In contrast, BMP-2a transcripts are seen only in the hair follicles in the cells of the hair bulb cortex. Temporally and spatially distinct patterns of BMP-2a, Vgr-1, TGF beta 1, and TGF beta 2 expression are also seen in different populations of mesenchymal cells in the developing skeletal system (cartilage and bone). Our results suggest that the coordinated expression of several members of the TGF beta superfamily is required to control the progression of specific cell types through their differentiation pathways.

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Patterns of expression of murine Vgr-1 and BMP-2a RNA suggest that transforming growth factor-beta-like genes coordinately regulate aspects of embryonic development.

To date, three closely-related TGF beta genes have been found in the mouse; TGF beta 1, TGF beta 2 and TGF beta 3. Previous experiments have indicated that TGF beta 1 and TGF beta 2 may play important roles during mouse embryogenesis. The present study now reports the distribution of transcripts of TGF beta 3 in comparison to the other two genes and reveals overlapping but distinct patterns of RNA expression. TGF beta 3 RNA is expressed in a diverse array of tissues including perichondrium, bone, intervertebral discs, mesenteries, pleura, heart, lung, palate, and amnion, as well as in central nervous system (CNS) structures such as the meninges, choroid plexus and the olfactory bulbs. Furthermore, in several organ systems, TGF beta 3 transcripts are expressed during periods of active morphogenesis suggesting that the protein may be an important factor for the growth and differentiation of many embryonic tissues.

In situ hybridization analysis of TGF beta 3 RNA expression during mouse development: comparative studies with TGF beta 1 and beta 2.

Murine transforming growth factor-β2 (TGF-β2) cDNAs were isolated from cDNA libraries derived from a differentiated murine embryonic carcinoma cell line, PCC3. The composite cDNA sequence is 4267 nucleotides long, including a 1217 nucleotides 5′-untranslated sequence, and encodes a murine TGF-β2 precursor of 414 amino acids with 96% identity to its human counterpart. Several consensus polyadenylation sequences are present in the 1807 nucleotides 3′-untranslated sequence. Five TGF-β2 mRNA species are observed in the developing mouse fetus and they show different patterns of expression during development. TGF-β2 mRNA expression was also examined in adult mouse tissues, in which four of the five RNA species were observed. TGF-β2 mRNAs were present in all adult mouse tissues examined, except liver, and was most abundant in placenta, the male submaxillary gland and lung. The patterns of expression suggest a physiological role for TGF-β2 both in embryonic development and in the maintenance of adult tissues.

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Ron W. Pelton

Papers

Immunohistochemical localization of TGF beta 1, TGF beta 2, and TGF beta 3 in the mouse embryo: expression patterns suggest multiple roles during embryonic development

Organogenesis and pattern formation in the mouse: RNA distribution patterns suggest a role for bone morphogenetic protein-2A (BMP-2A).

Patterns of expression of murine Vgr-1 and BMP-2a RNA suggest that transforming growth factor-beta-like genes coordinately regulate aspects of embryonic development.

In situ hybridization analysis of TGF beta 3 RNA expression during mouse development: comparative studies with TGF beta 1 and beta 2.

Murine Transforming Growth Factor-β2 cDNA Sequence and Expression in Adult Tissues and Embryos