ABSTRACT
To date, three closely-related TGFβ genes have been found in the mouse; TGFβ1, TGFβ2 and TGFβ3. Previous experiments have indicated that TGFβ1 and TGFβ2 may play important roles during mouse embryo-genesis. The present study now reports the distribution of transcripts of TGFβ3 in comparison to the other two genes and reveals overlapping but distinct patterns of RNA expression. TGFβ3 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, TGFβ3 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.
Introduction
Vertebrate embryonic development involves a sequence of specific interactions between different tissues and cell types. During these inductive processes, dissimilar cell types influence one another to follow alternative developmental pathways towards differentiation and morphogenesis. There is evidence that extracellular polypeptide factors, such as the transforming growth factor β (TGFβ) family of proteins, are involved in these types of cell-cell interactions (Whitman and Melton, 1989; Mercóla and Stiles, 1988). Although TGF/51 was first discovered and named because of its ability to elicit the transformed pheno-type in rodent fibroblasts (Moses et al. 1981; Roberts et al. 1981), it is now known that this molecule has a wide variety of other biological activities, many of which are important to embryonic development (reviewed in Pelton and Moses, 1990a). The isolation and characterization of purified polypeptides has identified at least 11 distinct TGFβ-related proteins in vertebrates, and recent PCR cloning studies indicate that the total number of genes may be much larger (Thompson and Melton, 1990; Ueno et al. 1990). In general, these factors can be grouped into three classes; a TGFβ1-hke group (TGFβs 1–5), a DPP-like group (DPP, Vg-1, Vgr-1, the BMPs), and another less well-defined group which includes the inhibins (activins) and Miillerian-inhibiting substance.
Within the TGFβ-like group, purified protein has so far been obtained for TGFβ1, TGFβ2 and TGFβ3. The overall structure of these three proteins is very similar in that each is synthesized as a pre-pro monomeric polypeptide which is cleaved at a multibasic peptide sequence to yield a 112-114 amino acid molecule (Miller et al. 1990). Active protein results from dimerization of the monomers. Although heterodimers between TGFβ1 and TGFβ2 are known (Cheifetz et al. 1987), to date no heterodimers involving TGFβ3 have been reported. Mature TGFβ3 protein has approximately 80% identity to both TGFβ1 and μ1, while TGFβ1 has approximately 72 % identity to TGFβ2 (ten Dijke et al. 1988; Derynck et al. 1988). The A-terminal regions of these three molecules show only 27 % sequence identity to each other. Although a few differences have been reported in the biological activities of TGFβ1 and TGFβ2 in vitro (Ohta et al. 1987; Jennings et al. 1988; Rosa et al. 1988), recent studies indicate that, in general, TGFβs 1, 2 and 3 have qualitatively similar activities when added to cells in culture (Graycar et al. 1989). Moreover, competition studies with TGFβ1, μ1 and β3 also indicate that all three proteins interact with the same cell-surface binding molecules (Graycar et al. 1989). Two important questions raised by these results are: (1) whether the effects obtained by adding exogenous TGFβ proteins to cells in culture reflects the normal activities of these proteins in vivo, and (2) whether TGFβ1, μl and μ1 have distinct biological functions in the intact animal. One possibility is that, in vivo, the different members of this TGFβ subfamily do in fact elicit qualitatively different responses in the same target tissue(s) and that receptors exist which preferentially bind one polypeptide rather than another. Alternatively, the three TGFβ isoforms may react with the same receptor(s) but bind to different subdomains in order to evoke their individual biological activities. Yet another hypothesis is that TGFβ1, β2 and β3 are functionally equivalent and elicit essentially the same responses in all target tissues, but their genes have acquired different sets of DNA regulatory elements controlling their temporal and spatial expression. In this way, the large number of regulatory elements needed to orchestrate the complex patterns of expression observed during development are divided among three (or more) separate genes. Such an arrangement could have evolved to cope with the more complex regulative tissue interactions characteristic of vertebrate development compared with embryos in which development is more deterministic.
In order to better understand the precise role of TGFβ-related genes in cellular interactions and embryonic inductions in vivo and to begin to answer some of the questions posed above, it is first necessary to know which tissues and cell types express these different genes. Previous studies have shown that TGFβ1 RNA (Sandberg et al. 1988,a; Sandberg et al. 1988,b; Lehnert and Akhurst, 1988; Wilcox and Derynck, 1988) and protein (Heine et al. 1987) as well as TGFβ2 RNA (Pelton et al. 1989) are expressed during embryogenesis in highly localized spatial and temporal patterns. Northern blot analysis has revealed that TGFβ3 is expressed in the embryo between 10.5 and 17.5 days post coitum (days p.c.) (Miller et al. 1989b). In this paper, we extend these studies by examining the temporal and spatial expression of TGFβ3 RNA in the mouse and contrasting its temporal and spatial expression patterns with TGFjSl and TGFβ2 RNAs.
Materials and methods
Mouse tissues
Staged embryos (Theiler, 1989) were obtained from matings of ICR outbred female (Harland Sprague Dawley) and Swiss-Webster male mice (Taconic Farms). Noon on the day of plug is 0.5 days p.c. The animals were killed via cervical dislocation and the embryos placed immediately in ice-cold 4% paraformaldehyde/phosphate-buffered saline (PBS).
Probe construction
In order to minimize the possibility of cross-hybridization between the three TGFβ RNAs, transcript specific probes were constructed by deleting the highly conserved regions of the three murine cDNAs. The resulting fragments were then subcloned into pGEM7Z(f+) (TGFβ1 and β3) or pSP73 (TGFβ2) so that antisense RNA is synthesized from the T7 promoter. The TGF/Î1 construct consists of nucleotides 421-1395 in the murine cDNA (Derynck et al. 1986). This region contains 764 base pair (bp) of the N-terminal glycopeptide region and 210bp of the mature region. The TGFβ2 construct consists of nucleotides 1511-1953 in the murine cDNA (Miller et al. 1989a) and contains 442 bp of the N-terminal glycopeptide region. The TGF/B construct contains nucleotides 831-1440 of the murine cDNA (Miller et al. 1989b) and covers 609 bp of the N-terminal glycopeptide region. Single-stranded antisense riboprobes were radiolabelled with [α-35S] UTP (1400 Ci mM-1, New England Nuclear) to a specific activity of ∼2x106 disint min-1 ng-1. The radiolabelled probes were reduced by limited alkaline hydrolysis to an average size of 100-150 bp and used at a final concentration of –4X104 counts min-1 gl-1. Previous studies with these probes demonstrate that when used for in situ hybridization and Northern blot analysis, different hybridization patterns are observed, indicating that each probe recognizes a specific TGFβ RNA and does not cross-hybridize with the other TGFβ transcripts (Lyons et al. 1990; Miller et al. 1989a,b).
In situ hybridization
Embryos were fixed overnight in 4% paraformaldehyde/ PBS, dehydrated by exposure to increasing concentrations of ethanol and embedded in paraffin wax. Sections of 5-7 μm were cut and floated onto slides coated with 3-triethoxysilylpropylamine (Sigma). The slides were then baked at 42°C overnight and the sections de-waxed through xylene, rehydrated through decreasing concentrations of ethanol and re-fixed in 4% paraformaldehyde/PBS. They were then treated with proteinase K (20/xgml-1 in 50 mM Tris, 5 HIM EDTA; Sigma) at room temperature for 5 min, re-fixed in 4% paraformaldehyde/PBS, acetylated (100 mM triethanolamine, 25 mM acetic anhydride) and dehydrated through ethanol. The slides were then hybridized at 55 or 57°C for ∼18h under siliconized coverslips in a solution containing 50% formamide, 10% dextran sulfate, 8mM DTT, 300 mM NaCl, 10mM Tris-HCl (pH7.4), SHIM EDTA, 10mM Na2PO4, lxDenhardt’s, and radiolabelled probe. After incubation in a humidified chamber for 18 h, the coverslips were removed in 5xSSC (lxSSC=150mM NaCl, 15mM sodium citrate), 10mM DTT at 50°C. The slides were then washed at 65°C in 2xSSC, 100mM DTT, 50% formamide, and then treated with RNase A (20ftgml-1; Sigma) at 37°C for 20min followed by washes in 2xSSC and 0.lxSSC at 65°C. Slides were then dehydrated through ethanol containing 0.3 M ammonium acetate, dipped in photographic emulsion (Ilford K.5) diluted 1:1 with 2% glycerol/water and exposed at 4°C for 2-3 weeks in the presence of desiccant. The slides were then developed (Kodak D19), counterstained with toluidine blue and analyzed on a Zeiss Axioplan microscope. Photographs were taken with an Olympus OMT on Panatomic X (Kodak) film using brightfield and dark-ground optics.
Results
In situ analysis of TGF03 RNA expression
Northern analysis of whole embryo RNA has demonstrated that TGFβ3 transcripts are expressed in the mouse during embryogenesis and are regulated in a temporal fashion (Miller 1989b). In order to localize TGFβ3 transcripts to specific cell types, and to compare the expression of this gene with that of TGFβ1 and β2, we hybridized 35S-labelled single-stranded antisense riboprobes derived mainly from the precursor regions of these genes (see Materials and methods) to sections of embryos from 10.5 days p.c. to birth. To control for non-specific binding, an 35S-labelled sense strand of TGFβ2 was used. As a positive control for expression in bone and as a negative control elsewhere, an 35S-labelled antisense strand of Spp I was used (Nomura et al. 1988). In these studies, TGFβ3 transcripts were found in a variety of cell types representing all three germ layers.
The skeletal system
The intervertebral discs, which separate the vertebrae of the spinal column, are derived from condensations of the metameric sclerotomes surrounding the notochord. These condensations form temporally in a gradient along the rostral-caudal axis in the embryo. At 11.5 days p.c., the segmented nature of the more rostral condensations is apparent as they begin to form between the anlagen of the vertebral bodies. A parasagittal section through an 11.5 days p.c. embryo (Figs 1 and 2) illustrates TGFβ3 RNA expression in the densely-packed regions of the perichordal mesenchyme which are destined to become the intervertebral discs. Transverse sections through the embryo at the same stage demonstrate that in the caudal region (Fig. 2A,E), these same cells express TGFβ3 transcripts (although at a lower level than those of the midthoracic region (Fig. 2B,F)) even before easily distinguishable perichordal condensations are visible in this area. After 11.5 days p.c., the perichordal mesenchyme continues to form compact segments of mesenchyme and by 13.5 days p.c., the intervertebral discs are well established and readily visible between the forming vertebrae. TGFβ3 transcripts in the intervertebral discs are clearly demarcated from the vertebral bodies at this stage (compare Figs 2C,G and 2D,H).
Expression of TGFβ3 RNA in the 11.5 days p.c. embryo as revealed by in situ hybridization. (A) Midsagittal section through the embryo hybridized with TGFβ3 antisense probe and photographed under brightfield illumination. Bar=lmm. (B) Same section as in A photographed under darkground illumination. Hybridization is seen in a number of tissues including meninges, choroid plexus, intervertebral discs, body wall, heart, trachea, liver capsule, lung, and extraembryonic membranes. (C) An adjacent section to A hybridized with TGFβ2 sense strand as a control.
Expression of TGFβ3 RNA in the 11.5 days p.c. embryo as revealed by in situ hybridization. (A) Midsagittal section through the embryo hybridized with TGFβ3 antisense probe and photographed under brightfield illumination. Bar=lmm. (B) Same section as in A photographed under darkground illumination. Hybridization is seen in a number of tissues including meninges, choroid plexus, intervertebral discs, body wall, heart, trachea, liver capsule, lung, and extraembryonic membranes. (C) An adjacent section to A hybridized with TGFβ2 sense strand as a control.
Expression of TGFβ3 in the developing vertebral column. Sections were hybridized with TGFβ antisense probe and photographed under brightfield (A-D) or darkground (E-H) illumination. (A,E) Transverse section through the caudal region of an 11.5 days p.c. embryo. Strong hybridization is seen in the mesenchymal cells surrounding the notochord (arrow) and, to a lesser extent, in a small region of condensing mesenchyme ventral to the notochord. No hybridization is observed in the notochord itself. Bar=325μm. (B,F) Transverse section through a more rostral region of the embryo than is shown in A. Again, strong hybridization is observed in the perichordal mesenchyme and in the condensing mesenchymal cells ventral to the notochord. Bar=1.5mm. (C,G) Sagittal section through the vertebral column of an 11.5 days p.c.. embryo. Hybridization can be seen in the condensations of perichordal mesenchyme (arrows) between each vertebral body. These condensations are the anlagen of the intervertebral discs. Bar=200μm. (D,H) Sagittal section through the vertebral column of a 13.5 days p.c. embryo. Strong signal is present in the intervertebral discs (arrows) which are well-defined by this stage. Bar=235 μm. Abbreviations: nt, neural tube; nc, notochord; v, vertebrae; ivd, intervertebral discs.
Expression of TGFβ3 in the developing vertebral column. Sections were hybridized with TGFβ antisense probe and photographed under brightfield (A-D) or darkground (E-H) illumination. (A,E) Transverse section through the caudal region of an 11.5 days p.c. embryo. Strong hybridization is seen in the mesenchymal cells surrounding the notochord (arrow) and, to a lesser extent, in a small region of condensing mesenchyme ventral to the notochord. No hybridization is observed in the notochord itself. Bar=325μm. (B,F) Transverse section through a more rostral region of the embryo than is shown in A. Again, strong hybridization is observed in the perichordal mesenchyme and in the condensing mesenchymal cells ventral to the notochord. Bar=1.5mm. (C,G) Sagittal section through the vertebral column of an 11.5 days p.c.. embryo. Hybridization can be seen in the condensations of perichordal mesenchyme (arrows) between each vertebral body. These condensations are the anlagen of the intervertebral discs. Bar=200μm. (D,H) Sagittal section through the vertebral column of a 13.5 days p.c. embryo. Strong signal is present in the intervertebral discs (arrows) which are well-defined by this stage. Bar=235 μm. Abbreviations: nt, neural tube; nc, notochord; v, vertebrae; ivd, intervertebral discs.
The bones of the appendicular skeleton are formed from lateral plate mesoderm through a process known as endochondral bone formation. A cartilage model of the bone is formed first and this is slowly replaced by osteogenic tissue. Fig. 3 shows a direct comparison of TGFβl, β2 and (53 in the embryonic cartilage of the hindlimb and demonstrates that hybridization to cells in the perichondrium is seen with all three TGFβ probes. Although a detailed quantitation of hybridization grains was not carried out, it appears that in the perichondrium the level of hybridization to each TGFjS RNA is different (Fig. 3). Using a heterologous (human) riboprobe, we have previously demonstrated that TGFβ2 RNA is localized to the perichondrial region of embryonic cartilage but not to the mature or hypertrophic cartilage (Pelton et al. 1989). This observation has now been confirmed using a homologous (murine) probe for TGFβ2. In addition, a comparison of TGFβ1, β2 and β3 RNA expression in the long bones showed that transcripts for all three molecules are found in the osteogenic cells of these tissues (data not shown).
Comparison of the expression of TGFβ1, β2 and β1 RNAs in the cartilage of a 16.5 days p.c. hind limb Sections were hybridized with TGFβ1, β2 or β1 antisense probe and then photographed under brightfield (A-C) or darkground (D-F) illumination. (A,D) Section hybridized with TGF/51 antisense probe. Hybridization signal is present in the perichondrium surrounding the mature cartilage. Bar=35μm. (B,E) Section hybridized with TGFβ antisense probe. Intense hybridization signal is detected in the perichondrium of the cartilage. No expression is seen in the mature cartilage. (C,F) Section hybridized with TGFβ3 antisense probe. A low level of hybridization signal is observed in the perichondral region surrounding the mature cartilage. Abbreviations: p, perichondrium; mctl, mature cartilage.
Comparison of the expression of TGFβ1, β2 and β1 RNAs in the cartilage of a 16.5 days p.c. hind limb Sections were hybridized with TGFβ1, β2 or β1 antisense probe and then photographed under brightfield (A-C) or darkground (D-F) illumination. (A,D) Section hybridized with TGF/51 antisense probe. Hybridization signal is present in the perichondrium surrounding the mature cartilage. Bar=35μm. (B,E) Section hybridized with TGFβ antisense probe. Intense hybridization signal is detected in the perichondrium of the cartilage. No expression is seen in the mature cartilage. (C,F) Section hybridized with TGFβ3 antisense probe. A low level of hybridization signal is observed in the perichondral region surrounding the mature cartilage. Abbreviations: p, perichondrium; mctl, mature cartilage.
In contrast to the bones of the axial and appendicular skeleton, in the skull osteogenic tissue (membrane bone) is formed directly by the differentiation of osteoblasts and osteocytes from neural crest mesenchyme. In situ hybridization suggests that all three TGF/5 molecules are involved in this process. Fig. 4 shows the distribution of TGFjSl, β2 and β3 RNA in the developing maxilla of the embryo. As in the perichondrium, all three probes show similar patterns, but different intensities, of hybridization.
Comparative expression of TGFβ1, β1 and β3 RNAs during formation of the bones of the skull. (A) A low power brightfield photomicrograph of the head of a 15.5 days p.c. embryo to illustrate the location of the developing maxilla (arrow). Bar=375 μm. (B) A higher power darkfield photomicrograph of a section through the maxilla hybridized with TGF/31 antisense probe. Hybridization grains are present over the osteoblasts giving rise to this bone. Bar=100μm. (C) A section similar to that in B hybridized with TGFβ2 antisense probe. Hybridization is again seen in the osteoblasts. (D) A section similar to B and C hybridized with TGFβ3 antisense probe. The hybridization signal observed with the TGFβ3 probe is similar to TGFβ1 and βl and is found in the osteoblasts. A low level of TGFβ3 signal is present in the mesenchyme surrounding the bone. Abbreviations: mx, maxilla; t, tongue.
Comparative expression of TGFβ1, β1 and β3 RNAs during formation of the bones of the skull. (A) A low power brightfield photomicrograph of the head of a 15.5 days p.c. embryo to illustrate the location of the developing maxilla (arrow). Bar=375 μm. (B) A higher power darkfield photomicrograph of a section through the maxilla hybridized with TGF/31 antisense probe. Hybridization grains are present over the osteoblasts giving rise to this bone. Bar=100μm. (C) A section similar to that in B hybridized with TGFβ2 antisense probe. Hybridization is again seen in the osteoblasts. (D) A section similar to B and C hybridized with TGFβ3 antisense probe. The hybridization signal observed with the TGFβ3 probe is similar to TGFβ1 and βl and is found in the osteoblasts. A low level of TGFβ3 signal is present in the mesenchyme surrounding the bone. Abbreviations: mx, maxilla; t, tongue.
The mesenchymal components of the teeth are also derived from neural crest mesoderm. Fig. 5 demonstrates embryonic teeth at two different stages of development hybridized with TGFβl, β2 and βi probes. At 13.5 days p.c., the invaginated buds of the oral epithelium are surrounded by condensing neural crest mesenchyme. While TGFβl RNA appears to be expressed in both the epithelium and the condensed mesenchyme of the bud stage tooth (Fig. 5A,D), TGFβ3 transcripts are found only in the loose mesenchyme adjacent to and surrounding the condensed mesenchyme (Fig. 5C,F). Although TGFβ3 RNA is detectable in neither the epithelial nor the condensed mesenchymal layers early in tooth development, hybridization is seen in the stellate reticulum of the enamel organ during the cap and bell stages (data not shown). TGFβ2 RNA is found only at very low levels in the non-condensed mesenchyme surrounding the tooth bud (Fig. 5B,E). By 16.5 daysp.c., the tooth has developed distinct ameloblast and odontoblast layers. At this stage, the highest levels of TGFβ2 and RNA hybridization signal are seen in the odontoblast layer, although a lower level of signal is seen in the mesenchymal primordium of the pulp lying subjacent to the odontoblasts (Fig. 5H,K,I,L). In contrast, TGFβ1 transcripts are found at the highest level in the mesenchymal primordium of the pulp and only at background levels in the odontoblast layer (Fig. 5 G,H). No hybridization for any of the TGFβs is seen in the ameloblast layer.
Comparison of expression of TGFβ1, β2 and β1 RNAs in developing teeth of mouse embryos. Sections are of 13.5 days p.c. (A-F) and 16.5 days p.c. (G-L) teeth analyzed by in situ hybridization and photographed under brightfield (A-C, G-I) and darkground (D-F, J-L) illumination. (A,D) Section through a 13.5 days p.c. bud stage tooth hybridized with TGF/U antisense probe. Hybridization signal is present in both the condensed neural crest mesenchyme as well as in the oral epithelium giving rise to the ameloblast primordium. Bar=65μm. (B,E) Section similar to that in A hybridized to TGFβ2 antisense probe. Only very low levels of hybridization are detectable in the non-condensed mesenchyme. Signal is not observed in the condensed mesenchyme or epithelium. (C,F) Section similar to that in A hybridized with TGFβ3 antisense probe. Hybridization grains are present in the non-condensed mesenchyme adjacent to and surrounding the condensed mesenchyme. (G,J) Section through a 16.5 days p.c. tooth hybridized with TGFβ1 antisense probe. Intense hybridization signal is present in the mesenchymal primordium of the pulp but is absent from the ameloblast and odontoblast layers. Bar=75μm. (H,K) Section similar to that in G hybridized with TGFβ2 antisense probe. Strong TGFβ2 signal is observed in the odontoblast layer. The ameloblast cells do not show hybridization but a low level is present in the pulp. (I,L) Section similar to G hybridized with TGFβ3 antisense probe showing hybridization grains over the odontoblast layer and at lower levels in the pulp. The ameloblast layer is negative for TGFβ3 signal. Abbreviations: rn, mesenchyme; ap, ameloblast primordium; a, ameloblast; o, odontoblasts, mpp, mesenchymal primordium of the pulp.
Comparison of expression of TGFβ1, β2 and β1 RNAs in developing teeth of mouse embryos. Sections are of 13.5 days p.c. (A-F) and 16.5 days p.c. (G-L) teeth analyzed by in situ hybridization and photographed under brightfield (A-C, G-I) and darkground (D-F, J-L) illumination. (A,D) Section through a 13.5 days p.c. bud stage tooth hybridized with TGF/U antisense probe. Hybridization signal is present in both the condensed neural crest mesenchyme as well as in the oral epithelium giving rise to the ameloblast primordium. Bar=65μm. (B,E) Section similar to that in A hybridized to TGFβ2 antisense probe. Only very low levels of hybridization are detectable in the non-condensed mesenchyme. Signal is not observed in the condensed mesenchyme or epithelium. (C,F) Section similar to that in A hybridized with TGFβ3 antisense probe. Hybridization grains are present in the non-condensed mesenchyme adjacent to and surrounding the condensed mesenchyme. (G,J) Section through a 16.5 days p.c. tooth hybridized with TGFβ1 antisense probe. Intense hybridization signal is present in the mesenchymal primordium of the pulp but is absent from the ameloblast and odontoblast layers. Bar=75μm. (H,K) Section similar to that in G hybridized with TGFβ2 antisense probe. Strong TGFβ2 signal is observed in the odontoblast layer. The ameloblast cells do not show hybridization but a low level is present in the pulp. (I,L) Section similar to G hybridized with TGFβ3 antisense probe showing hybridization grains over the odontoblast layer and at lower levels in the pulp. The ameloblast layer is negative for TGFβ3 signal. Abbreviations: rn, mesenchyme; ap, ameloblast primordium; a, ameloblast; o, odontoblasts, mpp, mesenchymal primordium of the pulp.
The internal organs
Lateral plate mesoderm gives rise to the visceral and parietal mesoderm which collectively form the mesenchymal components of the body wall and internal organs as well as the pleura and mesenteries which cover these organs. A low power photomicrograph (Fig. 6A,B) provides examples of high levels of TGFβ3 RNA hybridization signal to three different tissues derived from lateral plate mesoderm: the atrioventricular cushions of the heart, the body wall and the capsule of the liver. At a higher magnification of the liver sections (Fig. 6A,B,E,F), it is clear that the TGFβ3 transcripts are localized to a layer of tissue 3-4 cells thick which surrounds this organ. Hybridization to TGFβ3 RNA is seen neither in the hepatocytes which constitute the liver parenchyma nor in the ventricle of the heart (Fig. 6E,F). Specificity of the probes is demonstrated by hybridization of an adjacent section with a probe for TGFβ1 RNA (Fig. 6G,H). Although, as previously reported (Wilcox and Derynck, 1988; Lehnert and Akhurst, 1988), high levels of TGFβ1 RNA are found in the embryonic megakaryocytes in the liver, no hybridization is detectable in the liver capsule. In addition, strong hybridization to TGFβ3 RNA is found in the mesenchymally derived tissue of the trachea (Fig. 6C,D; see also Fig. 1) and esophagus (data not shown) but is not observed in the epithelial lining of these organs. Transcripts are also seen in the mesenteries surrounding the digestive organs (Fig. 1) and pleura of the embryonic lung (Fig. 7C,D; see also Fig. 1). Expression of TGFβ3 RNA is observed in a variety of other embryonic tissues including the mesenchyme surrounding the umbilical arteries and vein, many of the large blood vessels throughout the embryo, the amnion and in the mesenchyme of the fetal portion of the placenta (Fig. 1 and data not shown). In contrast to TGFβ1 and TGFβ (Lehnert and Akhurst, 1988; Pelton et al. 1989), no transcripts for TGFβ3 are detectable in the mesoderm-derived cell types (e.g. fibroblasts and smooth muscle) supporting the epithelial layer of the stomach, intestines or lung.
Expression of TGFβ3 in the mesenchymal component of embryonic internal organs. Sections were hybridized with TGFβ3 (A-F) or TGFβ3 (G-H) and photographed under brightfield (A,C,E,F) or darkground (B,D,F,H) illumination. (A,B) Section through the heart of an 11.5 days p.c. embryo hybridized with TGFβ3 probe. Strong hybridization signal is present in the atrioventricular cushions of the heart but is absent from the ventricle. Note the intense TGFβ3 signal found in the body wall and liver capsule (arrows). Bar =165 am. (C,D) Section through the trachea of a 15.5 days p.c. embryo hybridized with TGFβ3 antisense probe. Hybridization grains are present in the submucosa of the trachea but are not detected in the tracheal epithelium. Hybridization is seen in the perichondrial region of the tracheal cartilage as well as in the cartilage lying adjacent to the trachea. (E,F) Section of 11.5 days p.c. liver hybridized with TGFβ3 antisense probe showing TGFβ3 signal in the liver capsule. No hybridization is found in the liver parenchyma. Bar=85μm. (G,H) Section adjacent to that in E hybridized with TGFβ3 probe. Although hybridization grains are found over the megakaryocytes in the liver (single arrow), the liver parenchyma and capsule (double arrow) are negative for TGF® signal. Abbreviations: av, atrioventricular valves; bw, body wall; v, ventricles; Ic, liver capsule; t, trachea; ctl, cartilage; Ip, liver parenchyma; m, megakaryocytes.
Expression of TGFβ3 in the mesenchymal component of embryonic internal organs. Sections were hybridized with TGFβ3 (A-F) or TGFβ3 (G-H) and photographed under brightfield (A,C,E,F) or darkground (B,D,F,H) illumination. (A,B) Section through the heart of an 11.5 days p.c. embryo hybridized with TGFβ3 probe. Strong hybridization signal is present in the atrioventricular cushions of the heart but is absent from the ventricle. Note the intense TGFβ3 signal found in the body wall and liver capsule (arrows). Bar =165 am. (C,D) Section through the trachea of a 15.5 days p.c. embryo hybridized with TGFβ3 antisense probe. Hybridization grains are present in the submucosa of the trachea but are not detected in the tracheal epithelium. Hybridization is seen in the perichondrial region of the tracheal cartilage as well as in the cartilage lying adjacent to the trachea. (E,F) Section of 11.5 days p.c. liver hybridized with TGFβ3 antisense probe showing TGFβ3 signal in the liver capsule. No hybridization is found in the liver parenchyma. Bar=85μm. (G,H) Section adjacent to that in E hybridized with TGFβ3 probe. Although hybridization grains are found over the megakaryocytes in the liver (single arrow), the liver parenchyma and capsule (double arrow) are negative for TGF® signal. Abbreviations: av, atrioventricular valves; bw, body wall; v, ventricles; Ic, liver capsule; t, trachea; ctl, cartilage; Ip, liver parenchyma; m, megakaryocytes.
Expression of TGFβ3 and TGFβ2 in epithelium. Sections were hybridized with either TGF/33 or TGFβ2 antisense probe and photographed under brightfield (A,C,E,G) or darkground (B,D,F,H) illumination. (A,B) Sagittal section of a 15.5 days p.c. mouse head hybridized with TGFβ3 antisense probe. Intense TGFβ3 hybridization signal is observed in the epithelium (arrow) of the secondary palate. Bar=160μm. (C,D) Section of a 15.5 days p.c. embryonic lung hybridized with TGFβ3 antisense probe. Hybridization is present in the pleura surrounding the embryonic lung as well as in the proximal (columnar) bronchial epithelium. Note that the more distal (cuboidal) epithelium is negative for TGFβ3 RNA.
Expression of TGFβ3 and TGFβ2 in epithelium. Sections were hybridized with either TGF/33 or TGFβ2 antisense probe and photographed under brightfield (A,C,E,G) or darkground (B,D,F,H) illumination. (A,B) Sagittal section of a 15.5 days p.c. mouse head hybridized with TGFβ3 antisense probe. Intense TGFβ3 hybridization signal is observed in the epithelium (arrow) of the secondary palate. Bar=160μm. (C,D) Section of a 15.5 days p.c. embryonic lung hybridized with TGFβ3 antisense probe. Hybridization is present in the pleura surrounding the embryonic lung as well as in the proximal (columnar) bronchial epithelium. Note that the more distal (cuboidal) epithelium is negative for TGFβ3 RNA.
Epithelial-derived tissues
TGFβ3 RNA is found in the epithelial component of several tissues. The most intense epithelial TGFβ3 (E,F) Sagittal section through the choroid plexus of an 11.5 days p.c. embryo hybridized with TGFβ3 antisense probe, A high level of TGFβ3 signal is present in the ependymal epithelium at the base of the choroid plexus but a low level of signal is also seen in the choroid plexus. Bar=80μm. (G,H) Sagittal section through the head of a 16.5 days p.c. mouse embryo hybridized with TGFβ2 antisense probe demonstrating high levels of TGFβ2 RNA in the ciliated epithelium of the cochlea. Abbreviations: sp, secondary palate; t, tongue; pe, proximal epithelium; de, distal epithelium; pl, pleura; ee, eppendymal epithelium; cp, choroid plexus; ce, cochlear epithelium.
RNA hybridization signal is found in the medial edge epithelium of the secondary palate (Fig. 7A,B). A less intense but equally specific TGFβ3 RNA signal is seen in the epithelial lining of the major airways in the embryonic lung (Fig. 7C,D). This hybridization pattern of TGFβ3 RNA in the embryonic lung is quite distinct from patterns of TGFβ1 and TGFβ2 RNA in this organ. Lehnert and Akhurst (1988) reported TGFβ1 RNA expression in the alveoli of the embryonic lung, while TGFβ2 transcripts have been localized to the subepithelial regions of the major airways and subendothelial regions of the large pulmonary blood vessels (Pelton et al. 1989). Moreover, in the adult lung, TGFβ3 transcripts are found in the smooth muscle cells and connective tissue fibroblasts lying subjacent to the epithelial lining of the large airways (Pelton et al. 19906). No expression of TGFβ3 RNA is detected in the epithelium of the adult lung. Expression of TGFβ3 RNA is also observed in the ependymal epithelium of the choroid plexus. The choroid plexus, located in the four ventricles of the brain, is composed of a modified secretory ependymal epithelium and serves to produce cerebrospinal fluid. Although expression is present in the villous processes of the choroid plexus throughout the brain, the highest levels of expression are found at the base of this tissue. Yet another type of modified epithelial cell is found in the cochlea of the inner ear. Although high levels of TGFβ2 RNA are found in these epithelial cells (Fig. 7G,H) neither TGFβ1 nor TGFβ3 RNAs are detected in these cell types (data not shown).
The vomeronasal (or Jacobson’s) organs are paired tubular structures lying along either side of the nasal septum and are lined by a specialized accessory olfactory neuroepithelium. These organs have separate connections to the brain from the olfactory bulbs and are thought to play a role in the behavioral response of lower mammals to specific odors such as pheromones. Fig. 8 demonstrates a comparison of TGFβ1, β2 and (33 RNA expression in the vomeronasal organs. TGFβ1 RNA expression is found in a punctate pattern in individual mesenchymal cells lying directly adjacent to the epithelial cells of the vomeronasal organs (Fig. 8A,B). In direct contrast, transcripts for TGFβ2 are found at very high levels in the epithelium of the vomeronasal organs but are not seen on the adjacent mesenchymal cells except in the perichondrium surrounding the cartilage of the nasal septum (Fig. 8C,D). Finally, transcripts for TGFβ3 are found neither in the epithelium nor in a punctate pattern in the adjacent mesenchymal cells but are instead found diffusely throughout the nasal mesenchyme and in the perichondria! cells of the nasal cartilage (Fig. 8E,F).
Comparative expression of TGF/Î1, p2 and (33 RNA in the vomeronasal (Jacobson’s) organ. Frontal sections of a 13.5 days p.c. embryo were hybridized with either TGF/U, (32 or (33 antisense probes. Brightfield photomicrographs are located on the left and darkground are on the right. (A,B) Section hybridized with antisense TGFβ1 probe. Punctate hybridization to TGFβ1 RNA can be detected in the mesenchyme directly adjacent to the neuroepithelium of the vomeronasal organ, but not within the epithelium itself. Bar=65μm. (C,D) Section hybridized with TGFβ2 antisense probe. TGFβ2 transcripts are found within the neuroepithelium but not in the surrounding mesenchyme. (E,F) Section hybridized with TGFβ3 antisense probe. TGFβ3 transcripts are found in a diffuse pattern in the mesenchyme surrounding the vomeronasal neuroepithelium but are not detected in the epithelium itself. Abbreviations: v, vomeronasal epithelium.
Comparative expression of TGF/Î1, p2 and (33 RNA in the vomeronasal (Jacobson’s) organ. Frontal sections of a 13.5 days p.c. embryo were hybridized with either TGF/U, (32 or (33 antisense probes. Brightfield photomicrographs are located on the left and darkground are on the right. (A,B) Section hybridized with antisense TGFβ1 probe. Punctate hybridization to TGFβ1 RNA can be detected in the mesenchyme directly adjacent to the neuroepithelium of the vomeronasal organ, but not within the epithelium itself. Bar=65μm. (C,D) Section hybridized with TGFβ2 antisense probe. TGFβ2 transcripts are found within the neuroepithelium but not in the surrounding mesenchyme. (E,F) Section hybridized with TGFβ3 antisense probe. TGFβ3 transcripts are found in a diffuse pattern in the mesenchyme surrounding the vomeronasal neuroepithelium but are not detected in the epithelium itself. Abbreviations: v, vomeronasal epithelium.
TGFβ3 RNA expression in the CNS
The olfactory bulbs of the vertebrate embryo arise as outgrowths of the telencephalon and are readily apparent by 13.5 daysp.c. in the mouse. By 14.5 days p.c., the intermediate zone surrounding the ventricular epithelium contains a sparse population of differentiating mitral cells which over the next twenty-four hours will coalesce to form a distinct mitral cell layer (Brunjes and Frazier, 1986; Hinds, 1972). TGFβ3 expression is first observed in the developing olfactory bulb at 14.5 days p.c.. At this stage, transcripts appear to be dispersed throughout the intermediate zone but are not detected in the ventricular epithelium (Fig. 9A-F). By 15.5 days p.c., TGFβ3 RNA is restricted to the rostral periphery of the olfactory bulb and transcripts appear to be localized to the condensing mitral cell layer (Fig. 9G-I). The perichondrium of developing nasal cartilage also expresses low levels of TGF/53 RNA, but transcripts are not found in the olfactory epithelium. At 16.5 days p.c., TGFβ RNA expression remains confined to the mitral cell layer and RNA is detected along the entire periphery of the olfactory bulb (Fig. 9J-L and data not shown). TGFβ3 transcripts are also observed in the meningeal layers surrounding the brain (Fig. 9) and spinal cord (Fig. 1) throughout development. As previously discussed, transcripts for TGFβ are found in the ependymal epithelium of the choroid plexus in the CNS as well.
Expression of TGFβ3 in the embryonic olfactory system. Sections were hybridized with TGFβ3 antisense probe and photographed under brightfield (first two columns) or background (righthand column) illumination. (A) Frontal section of the anterior brain showing the olfactory bulbs of a 14.5 days p.c. embryo hybridized to TGFβ3 antisense probe. Bar=200μm. (B,C) Higher magnification of the section in A. Hybridization is detected in the intermediate zone of the developing olfactory bulb and in the meningeal layers around the inside of the brain cavity. The ventricular neuroepithelium is negative for TGFβ3 signal. Bar=100μm. (D) Sagittal section of a embryo head at the same stage as in A-C, hybridized with TGFβ3 antisense probe. Bar=535μm. (E,F) Higher magnification of the section in D. Strong hybridization is observed in the olfactory bulb as well as in the meningeal layers surrounding the brain. Bar=275μm. (G) Sagittal section of a 15.5 days p.c. head hybridized to TGFβ3 antisense probe. Bar=500μm. (H,I) Higher magnification of the section in G. TGFβ3 hybridization is seen in the mitral cell layer on the rostral periphery of the olfactory bulb. The meningeal layers are also positive for TGFβ3 signal (arrow). Bar=250μm. (J) Frontal section of a 16.5 days p.c. mouse embryo head hybridized with TGFβ3 antisense probe. Bar=500μm. (K,L) Higher magnification of the section in J. Strong hybridization signal can be observed in the peripheral mitral cell layer (double arrow) in the olfactory bulbs and in the meningeal layers (single arrow). Abbreviations: m, meninges; ve, ventricular epithelium; i’z, intermediate zone; ob, olfactory bulbs; nc, nasal cavity; mcl, mitral cell layer.
Expression of TGFβ3 in the embryonic olfactory system. Sections were hybridized with TGFβ3 antisense probe and photographed under brightfield (first two columns) or background (righthand column) illumination. (A) Frontal section of the anterior brain showing the olfactory bulbs of a 14.5 days p.c. embryo hybridized to TGFβ3 antisense probe. Bar=200μm. (B,C) Higher magnification of the section in A. Hybridization is detected in the intermediate zone of the developing olfactory bulb and in the meningeal layers around the inside of the brain cavity. The ventricular neuroepithelium is negative for TGFβ3 signal. Bar=100μm. (D) Sagittal section of a embryo head at the same stage as in A-C, hybridized with TGFβ3 antisense probe. Bar=535μm. (E,F) Higher magnification of the section in D. Strong hybridization is observed in the olfactory bulb as well as in the meningeal layers surrounding the brain. Bar=275μm. (G) Sagittal section of a 15.5 days p.c. head hybridized to TGFβ3 antisense probe. Bar=500μm. (H,I) Higher magnification of the section in G. TGFβ3 hybridization is seen in the mitral cell layer on the rostral periphery of the olfactory bulb. The meningeal layers are also positive for TGFβ3 signal (arrow). Bar=250μm. (J) Frontal section of a 16.5 days p.c. mouse embryo head hybridized with TGFβ3 antisense probe. Bar=500μm. (K,L) Higher magnification of the section in J. Strong hybridization signal can be observed in the peripheral mitral cell layer (double arrow) in the olfactory bulbs and in the meningeal layers (single arrow). Abbreviations: m, meninges; ve, ventricular epithelium; i’z, intermediate zone; ob, olfactory bulbs; nc, nasal cavity; mcl, mitral cell layer.
Discussion
Earlier reports have suggested that TGFβ1 and TGFβ2 play a role in differentiation and morphogenesis during mouse development (for a recent review see Pelton and Moses, 1990a). Here, we have extended these studies by directly comparing the expression of RNA transcripts for TGFβ3 with TGF/S1 and TGFβ2. In some tissues, e.g. the perichondrium and bone, all three genes appear to be expressed in the same cell types. In contrast, in other tissues the TGFβ genes show different distribution patterns. For example, in the vomeronasal organ, while TGFβ2 RNA is expressed in the neuroepithelium, TGFβ1 RNA is found in a subpopulation of mesenchymal cells lying adjacent to the epithelium and TGFβ3 RNA is located diffusely throughout the mesenchyme surrounding the organ. Differential expression of TGFβ RNAs is also observed in other embryonic tissues such as the lung, tooth bud, liver, whisker follicle (Lyons et al. 1990) and secondary palate (Pelton et al. 1990c). These distinct patterns of gene expression indicate that TGFβ1, β2 and β3 have different DNA regulatory elements that control the temporal and spatial expression of each TGFβ isoform. It should be noted that in several organ systems in which the TGFβs show differential gene expression during the early stages of tissue formation, at later stages when morphogenesis and differentiation are complete, the RNAs for all three TGFβs show very similar patterns of expression (for example in the whisker follicle (Lyons et al. 1990) and lung (Pelton et al. 1990b). Although the significance of this change in RNA expression is not known, it may reflect changing roles for the proteins at different stages of development. Lastly, it is important to note that our data demonstrating differential expression patterns of the TGFβ1, β2 and β3 genes neither support nor disprove the hypothesis that the individual TGFβ proteins may have isoform-specific activities.
While in many cases TGFβ3 expression patterns are the same as, or overlap with TGFβ1 and β2, in a few instances TGFβ3 is the only member of this group which is expressed. One example is in the embryonic olfactory bulbs. The development of the vertebrate olfactory system involves complex cell interactions between the olfactory bulb and innervating afferent axons. Other researchers have shown that the development of the olfactory bulb is arrested if receptor axons are prevented from innervating the forebrain. Moreover, hypertrophy of peripheral cell layers occurs when additional axons innervate this region (for a review see Graziadei and Graziadei, 1978), suggesting that neurotrophic factors supplied by incoming neurons stimulate the growth and development of the olfactory bulbs (Baker, 1988). Nonetheless, at present, little is known about how cell proliferation in the olfactory bulbs is stimulated by neuronal ingrowth or what factors regulate growth control in this system. TGFβ3 transcripts are first observed in the intermediate zone of the developing olfactory bulb at 14.5 daysp.c.. and over the next two days transcripts become progressively confined to the periphery of the embryonic olfactory bulb. This expression pattern of TGFβ3 RNA temporally and spatially coincides with the formation of the mitral cell lamina, suggesting that TGFβ3 may be involved specifically in the proliferation and/or differentiation of neuronal or glial cells within the mitral cell layer.
The localization of TGFβ3 transcripts in the developing vertebral column demonstrates yet another example of TGFβ gene-specific expression in the embryo. As early as 11.5 days p.c., TGFβ3 RNA is localized in a somewhat diffuse pattern in the mesenchyme surrounding the notochord. This perichordal mesenchyme gives rise to the vertebrae and intervertebral discs of the spinal column. Over a period of days, the perichordal mesenchyme condenses along the anterior-posterior axis of the embryo in a temporal gradient to form the intervertebral discs. During this time TGFβ3 transcripts become progressively localized to the compact intervertebral mesenchyme as it condenses. Because of their similar patterns of distribution, it is of interest to compare the pattern of TGFβ3 RNA expression in the developing vertebral column to the expression patterns observed for Pax-1 RNA (Deutsch et al. 1988) and XlHbox 1 protein (Oliver et al. 1988). Pax-1 and XlHbox 1 are vertebrate genes which contain strong sequence similarity to the Drosophila paired box and homeobox sequences, respectively. Like TGFβ3, Pax-1 RNA is first localized diffusely in a subset of sclerotomal cells lateral to the notochord and then becomes progressively localized, in a rostral-caudal gradient, to the condensing mesenchyme which gives rise to the intervertebral discs (Deutsch et al. 1988). Similarly, localization studies in the mouse vertebral column have shown that XlHbox 1 protein is first expressed in the sclerotome-derived mesenchyme adjacent to the notochord. The observed staining pattern becomes metameric as the mesenchyme condenses to form the intervertebral discs (Oliver et al. 1988). Although the similar expression patterns of Pax-1 RNA and XlHbox 1 protein with TGFβ3 RNA do not establish a causal relationship between these molecules, they do suggest that these molecules may interact in vivo in the formation of the vertebral column.
In summary, we demonstrate the localization of TGFβ3 RNA during mouse embryogenesis in an array of organs and tissues which are undergoing morphogenesis. Moreover, a direct comparison of RNA expression for TGFβ1, β2 and β3 has been presented. While the patterns of gene expression for the three TGFβ isoforms overlap in some tissues, the pattern observed for TGFβ3 is distinctly different from that observed for the TGFβ1 and TGFβ2 genes. These results indicate that the DNA elements regulating the temporal and spatial expression of RNA for each TGFβ gene are different. The interchange of these elements between the various TGFβ genes and subsequent incorporation into transgenic animals should provide clues as to whether TGFβ1, β2 and β3 proteins elicit the same biological effects in vivo as they do in some cell types in vitro.
ACKNOWLEDGEMENTS
The authors thank Dr Frank Margolis for many helpful discussions concerning the development of the vertebrate olfactory system. We also thank Dr Chris Wright for helpful criticisms of the manuscript. This work was supported by grants CA-42572 and CA-48799 from the National Cancer Institute, U.S. Public Health Service. R.W.P. was supported by NIH grant no. T32-GM07347 for the medical scientist training program (MSTP).