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Endocrinology Vol. 139, No. 9 3946-3957
Copyright © 1998 by The Endocrine Society

ARTICLES

Coordinate Expression of Transforming Growth Factor-ß1 and Adrenomedullin in Rodent Embryogenesis

Luis M. Montuenga1, Jennifer M. Mariano1, Margaret A. Prentice, Frank Cuttitta and Sonia B. Jakowlew

Department of Cell and Cancer Biology, National Cancer Institute, Medicine Branch, Rockville, Maryland 20850

Address all correspondence and requests for reprints to: Dr. Sonia B. Jakowlew, Department of Cell and Cancer Biology, National Cancer Institute, Medicine Branch, 9610 Medical Center Drive, Suite 300, Rockville, Maryland 20850. E-mail: jakowlews{at}bprb.nci.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transforming growth factor-ß (TGFß) and adrenomedullin (AM) are multifunctional regulatory peptides that are secreted by a variety of normal and malignant cells. The TGFßs are expressed in developing organs and adults, and their tissue distribution pattern has possible significance for signaling roles in many epithelial-mesenchymal interactions. AM is also expressed in a variety of embryonic and adult tissues. The present study reports a comparison of the patterns of expression of the proteins and messenger RNAs (mRNAs) for TGFß1 and AM in the developing mouse embryo. Immunohistochemical and in situ hybridization analyses were performed on formalin-fixed paraffin-embedded sections of developing embryonic mouse tissues using specific antibodies and complementary RNA probes for TGFß1 and AM. The early placenta, including the giant trophoblastic cells, showed high levels of staining and hybridization for TGFß1 and AM proteins and mRNAs. The heart was the first organ that showed expression of TGFß1 and AM during embryogenesis. The spatio-temporal patterns of expression of TGFß1 and AM in cardiovascular, neural, and skeletal-forming tissues as well as in the main embryonic internal organs showed striking similarities. The lung, kidney, and intestine, in which epithelial-mesenchymal interactions occur, showed similar patterns of TGFß1 and AM expression. These data show colocalization of TGFß1 and AM in specific cell types associated with several tissues in the developing mouse embryo. Additionally, RT-PCR amplification and Northern blot hybridization showed expression of TGFß1 and AM mRNAs in all embryonic and adult mouse and rat tissues examined. Our data show that the expression of TGFß1 and AM is regulated in a spatial and temporal manner such that overlapping patterns of expression of TGFß1 and AM occur in several tissues at the same stage of development and in the same cellular location in rodent embryogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AN INCREASING number of polypeptides have been shown to be involved in the long term modulation of growth and differentiation, not only in normal tissues but also in cancer, wound repair, and embryogenesis. The participation of polypeptide growth factors in mammalian embryonic development is well documented. For example, a variety of growth factors, including epidermal growth factor, insulin-like growth factor, transforming growth factor-{alpha} (TGF{alpha}), and TGFß, as well as their receptors have been detected in embryonic tissues and embryonic cell lines (for a review, see Ref. 1). An abundance of evidence suggests that these growth factors may be important in the regulation of cell differentiation and tissue morphogenesis as well as in the control of cell division. This is best illustrated by TGFß1 and its related family of proteins, which have a wide range of cellular effects. Sequence and structural similarities have been found between TGFß and other proteins, including Mullerian inhibiting substance, int, Notch, lin-12, decapentaplegic, bone morphogenetic proteins (BMPs), and vg-1, which have been independently identified by virtue of their effects during embryonic development (2, 3, 4, 5, 6, 7).

The TGFßs are a family of 25,000 mol wt homodimeric polypeptides that are linked by disulfide bonds. Five highly homologous, yet distinct, isoforms of TGFß have been identified: TGFß1, TGFß2, and TGFß3 from several species, including mammals (for reviews, see Refs. 8, 9) and birds (10, 11); TGFß4 from birds (12); and TGFß5 from amphibians (13). The highest level of amino acid sequence identity for the five TGFß isoforms is found at the carboxyl-terminal end of the molecules. In addition, the TGFß isoforms are synthesized as inactive precursor forms and are proteolytically cleaved to release the mature carboxyl-terminal peptide, which remains noncovalently associated with the amino-terminal peptide in an inactive complex. The latent peptide must be activated to give the mature, biologically active protein. The active form of TGFß has a vast range of regulatory activities. Initially discovered in an assay based on its ability to transform fibroblasts phenotypically in cell culture, TGFß has been shown to have profound effects on nearly all cell types, influencing their proliferation, their differentiation, or other aspects of their function. Many different types of cells have been demonstrated to synthesize TGFß, and essentially all cells have been shown to have a specific set of receptors for this growth factor. It is well established that TGFß plays a major role in adult physiology in processes including inflammation and tissue repair, control of hematopoiesis, and control of steroidogenesis (14, 15, 16). In addition, it has been demonstrated that TGFß plays a role in the control of differentiation and morphogenesis in embryonic development (17, 18, 19, 20, 21, 22, 23). The TGFßs are thought to regulate the signals by which primary and secondary inductions are initiated at different stages of embryogenesis (23). The TGFßs are expressed in developing organs, and their tissue distribution pattern has possible significance for signaling roles in epithelial-mesenchyme interactions during embryogenesis (22, 23). Although it is clear that the TGFßs are important molecules in the regulation of cellular differentiation and proliferation, it is still unknown how the activity of this growth factor is controlled under physiological conditions. The biological activity of TGFß is thought to be controlled in a number of ways, including messenger RNA (mRNA) expression and protein synthesis, cellular distribution of receptors, presence of the latent form of TGFß, activation of the latent form of TGFß, and inactivation of the active form of TGFß (for reviews, see Refs. 8, 9). Many of the diverse cellular effects of TGFß are the result of alterations in the expression of many different proteins, including extracellular matrix components, cell adhesion receptors, cell cycle components, transcription factors, and growth factors. TGFß1 regulates the expression of nuclear factors, including c-fos, c-jun, junB, and c-myc; growth factors, including TGFß1, platelet-derived growth factors A and B, and epidermal growth factor; and many other genes and cellular activities (for a review, see Ref. 8).

Adrenomedullin (AM) is also a multifunctional polypeptide that was originally isolated from extracts of human pheochromocytoma (24). Human AM is a 52-amino acid peptide with a 6-residue ring structure formed by an intramolecular disulfide bond that is essential for biological activity (24). AM shows slight structural homology with calcitonin gene-related peptide. AM and its gene-related peptide, proadrenomedullin N-terminal 20 peptide, are the two known bioactive amidated peptides generated by posttranslational enzymatic processing of the 185-amino acid prepro-AM molecule (25). AM has been shown to be secreted by a variety of normal and malignant cells (26, 27). Expression of AM has been shown in a variety of adult human, rat, and porcine tissues (26, 28) and more recently in embryonic tissues (29). The involvement of AM in the regulation of growth has also been suggested by its ability to stimulate DNA synthesis and cell proliferation of murine Swiss 3T3 fibroblasts acting through the elevation of intracellular cAMP levels (30) and by its ability to induce cell cycle progression from the G0 to the G1 phase and the expression of c-fos mRNA in cultured rat aorta muscle cells (31). Recently, TGFß1 has been shown to suppress the production of AM in cultured rat endothelial cells (32).

To facilitate more insight into the biology of AM, expression of AM was examined and compared with that of TGFß1 in embryonic rodent development. Our study is the first to compare localization of the mRNAs and proteins for TGFß1 and AM using in situ hybridization and immunohistochemical staining analyses in the developing mouse embryo. We show that expression of TGFß1 and AM mRNAs and proteins is colocalized in specific cell types associated with a number of tissues in the developing mouse embryo. In addition, Northern blot hybridization and RT-PCR analyses show expression of the mRNAs for TGFß1 and AM in a variety of embryonic and adult mouse and rat tissues. Our data show that the expression of TGFß1 and AM is regulated in a spatial and temporal manner, such that overlapping patterns of expression of TGFß1 and AM occur in several tissues at the same stage of development and in the same cellular location in embryogenesis. The colocalization of TGFß1 and AM may enable interaction between these two growth factors to influence development and differentiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissue samples
Sections of NIH Swiss mouse staged embryos (Novagen, Madison, WI) were used. Adult female animals were caged with adult males overnight. The presence of a vaginal plug the following morning was designated day 0 of pregnancy. For RNA extraction, embryonic (18 days after the vaginal plug was found) and adult (6-month-old) Sprague-Dawley rats (Science Applications International Corp., Frederick, MD) and adult (6-month-old) A/J mice (The Jackson Laboratory, Bar Harbor, ME) were used. Tissues were dissected and immediately used or were kept frozen at -80 C until extracted.

RNA extraction and Northern blot analysis
Total RNA was extracted from tissues by the method of Chirgwin et al. (33) using guanidine isothiocyanate and cesium chloride. For Northern blot analysis, equal amounts of total RNA were separated by electrophoresis on 1% agarose gels containing 0.66 M formaldehyde, transferred to Nytran filters (Scleicher and Schuell, Keene, NH), UV cross-linked, and baked for 3 h. Ethidium bromide (33 µg/ml) was included in both gels and running buffers to visualize the positions of ribosomal RNAs by UV illumination after electrophoresis. Blots were hybridized with 32P-labeled (3000 Ci/mmol; DuPont, Boston, MA) random primed probes at 65 C according to the method of Church and Gilbert (34), and then exposed for various times at -70 C using a DuPont Lightning Plus intensifying screen (DuPont). Densitometry of autoradiograms was performed using a scanning laser densitometer (Molecular Dynamics, Sunnyvale, CA).

RT-PCR amplification
The oligonucleotide primers were synthesized using a MilliGen/Biosearch 8700 DNA synthesizer (Millipore, Marlborough, MA). Primer sets were as follows: TGFß1 (mature), 620-bp product (nucleotides 782-1401): sense, 5'-GTGCCCGAACCCCCATTGCTGTCC-3'; antisense, 5'-GCGCCCGGGTTGTGTTGGTTGTAG-3'; and AM (mature), 539-bp product (nucleotides 603-1141): sense, 5'-ATTCGTGTCAAACGCTACCGCC-3'; antisense, 5'-GGTTTCTCACGGGGCATAAGCCT-3'.

RT-PCR was performed using 2 µg total RNA and the GeneAmp RNA PCR kit according to the manufacturer’s directions (Perkin-Elmer/Cetus, Norwalk, CT). Mouse complementary DNAs (cDNAs) from E7, E11, E15, and E17 embryos were purchased from Clontech (Palo Alto, CA). The RT procedure was performed using the antisense primers with the following conditions: RT at 42 C for 50 min, inactivation of reverse transcriptase at 70 C for 15 min, and ribonuclease H digestion at 37 C for 20 min. After RT, PCR was performed using the sense primers in a Perkin-Elmer 9600 thermal cycler as follows: 94 C for 15 sec, 60 C for 15 sec, and 72 C for 1 min for 30 cycles, followed by a 10-min incubation at 72 C. To visualize the PCR products, the samples were subjected to electrophoresis on 2% agarose gels containing ethidium bromide. The authenticity of the products was confirmed by Southern blot hybridization with nested internal primers and DNA sequencing.

In situ hybridization
Detection of the mRNAs for TGFß1 and AM was performed using in situ hybridization. The cDNAs generated using RT-PCR, as outlined previously for TGFß1 (35) and AM (29), were ligated into the pcDNA1 vector (Invitrogen, San Diego, CA) following the manufacturer’s procedures and used to generate riboprobes. The plasmids were linearized with EcoRV and BamHI and used as templates to synthesize digoxigenin-labeled sense and antisense RNA transcripts. Hybridization was performed in a moist chamber at 65 C for 20 h in a 50-µl volume containing the antisense probes (35). After stringency washes, visualization of digoxigenin was performed using a digoxigenin detection kit (Boehringer Mannheim, Indianapolis, IN). Sense probes were used as controls.

cDNA probes
Hybridization was performed using the following cDNA probes: 0.9-kb XbaI-HindIII fragment of rat TGFß1, plasmid pRTGFß1 (36), and a 0.55-kb EcoRI fragment of mouse AM, plasmid pcRIIrAM.

Immunohistochemistry
For the immunocytochemical localization of TGFß1 and AM in paraffin sections, the avidin-biotin peroxidase complex technique was employed (Vector Laboratories, Burlingame, CA). An affinity-purified polyclonal antibody to TGFß1 [TGFß1(V)] was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A previously reported, well characterized rabbit antiserum (no. 2343) was used to localize AM immunoreactivity (29). After deparaffinization and blocking of endogenous peroxidase in hydrogen peroxide-methanol, the sections were blocked with 1.5% normal goat serum-0.5% BSA, incubated overnight at 4 C with the affinity-purified antisera for TGFß1 at 0.5 µg/ml and at a 1:1000 dilution for AM, washed extensively, and then incubated with biotinylated goat antirabbit IgG and avidin-biotin-enzyme complex. Sections were stained with 3,3-diaminobenzidine (Sigma Chemical Co., St. Louis, MO) and hydrogen peroxide, and counterstained with Gill’s hematoxylin. Controls include 1) using primary antisera preincubated with a 20-fold excess of the appropriate peptide for 2 h at room temperature, and 2) replacing primary antiserum with normal rabbit IgG. Antibody specificity has been previously demonstrated using Western blotting (26, 35).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of TGFß1 and AM in mouse development
To investigate the localization of TGFß1 and AM proteins in mouse embryo development, we examined the immunohistochemical staining patterns of TGFß1 and AM in several embryonic tissues ranging in age from 8–16 days (E8–E16) using specific antibodies. Table 1Go summarizes a comparison of the immunocytochemical detection of TGFß1 and AM during the organogenetic period of the mouse. Widespread expression of both TGFß1 and AM proteins was detected throughout the embryonic development period. The difference in the pattern and intensity of the brown immunohistochemical staining suggests that expression of both TGFß1 and AM is regulated in a spatio-temporal manner during embryogenesis. Whereas relatively low levels of TGFß1 and AM immunoreactivity were detected in the embryo at the beginning of organogenesis (E8), except for extraembryonic tissues, the levels of immunostaining increased for both proteins during late organogenesis (E14–E16). Although the intensity of staining for TGFß1 and AM in late organogenesis was similar in the cardiovascular, nervous, and skeletal systems as well as in chondrocytes and lung, the staining intensity for TGFß1 was generally higher than that for AM in intestine, liver, thymus, kidney, and perichondrium. The distribution of the mRNAs for TGFß1 and AM also colocalized with the respective proteins in a spatio-temporal manner.


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  		Table 1. Tissue distribution of immunohistochemically detectable TGFß1 and adrenomedullin in mouse organogenesis

 
Expression of TGFß1 and AM in developing placenta
Figure 1Go, A and C, shows the immunohistochemical staining pattern of TGFß1 and AM in the early placenta of E8 embryos. Immunohistochemical staining for TGFß1 and AM was detected in the early placenta, particularly in the giant trophoblastic cells and the maternal decidual cells. Immunohistochemical staining for TGFß1 and AM was completely blocked when the antibodies were preincubated with solutions of peptides against which they were raised (Fig. 1Go, B and D).



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  		Figure 1. Expression of TGFß1 and AM in the mouse early placenta. A, Immunohistochemical detection of TGFß1. B, Absorption control. C, Immunohistochemical detection of AM. D, Absorption control. E, In situ hybridization for TGFß1 mRNA. F, Control using a sense TGFß1 cRNA probe. G, In situ hybridization for AM mRNA. H, Control using a sense AM cRNA probe. Note the intense staining in the giant trophoblastic cells in A, C, E, and G and blocking in B, D, F, and H. Indicated are yolk sac (ys), giant trophoblastic cells (gt), and decidual cells (d). Immunostaining and hybridization in this and subsequent figures are representative of three separate experiments. Magnification: E–H, x100; A–D, x200.

 
To examine the tissue-specific distribution of TGFß1 and AM mRNA expression, in situ hybridization was performed on these embryo sections using nonradioactive digoxygenin-labeled complementary RNA (cRNA) probes. Hybridization with antisense and sense (negative control) cRNA probes and washing were performed at high stringency to ensure specific hybridization. Negative control sections were hybridized with sense TGFß1 and AM cRNA probes. Figure 1Go, E and G, shows prominent hybridization as a purple precipitate to TGFß1 and AM mRNAs in several cell types of the developing placenta, especially the giant trophoblastic cells. Hybridization was also performed using digoxigenin-labeled sense TGFß1 and AM cRNA probes as controls for specificity; Fig. 1Go, F and H, shows the absence of hybridization of these probes to the early placenta.

Expression of TGFß1 and AM in developing heart
On day 8 of development (E8), although most of the cells of the mouse embryo were not stained, moderate staining for TGFß1 and AM was detected in the walls of the primitive cardiac tube (Table 1Go and Fig. 2Go, A and E). By day 9 (E9), the myocytes of the walls of the ventricle and atrium were more intensely stained for both TGFß1 and AM (Fig. 2Go, B, C, F, and G). Weak staining for TGFß1 and AM was also detected in the primitive mesenchyme at this time. By day 11 (E11; Fig. 2Go, D and H), the intensity of staining for both TGFß1 and AM increased in the heart as the cardiac myocytes proliferated with progressive development. Immunohistochemical staining analysis of the cardiac musculature showed similar localization and intensity of staining for TGFß1 and AM beginning on E10 (Table 1Go). In situ hybridization analysis showed localization of both TGFß1 and AM mRNAs in the developing mouse heart in a manner analogous to the corresponding protein immunostaining (Fig. 2Go, I–L).



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  		Figure 2. Expression of TGFß1 and AM in the developing mouse heart. A–D, Immunohistochemical detection of TGFß1. E–H, Immunohistochemical detection of AM. I–J, In situ hybridization for TGFß1 mRNA. K–L, In situ hybridization for AM mRNA. A and E, E8 embryos. Note that cardiac tube and some mesenchymal cells are positive. B, C, F, G, I, and K, E9 embryos. Atrium and ventricle are positive. D, H, and L, E11 embryos. Cardiomyocytes in the ventricle are positive. J, E10 embryos. Indicated are cardiac tube (ct), ventricular chamber (vc), ventricle wall (vw), and atrial chamber (ac). Magnification: B and F, x40; A, C–E, and G–L, x100.

 
Expression of TGFß1 and AM in the developing skeletal and nervous system
The detection of immunohistochemically stained TGFß1 and AM was observed at low levels in a diffuse pattern of groups of mesenchymal cells beginning on E9. The patterns of expression of TGFß1 and AM by these groups of cells seemed to follow organized patterns of distribution, as the intensity of staining of the tissue varied from region to region as development proceeded. Detection of immunohistochemically stained TGFß1 and AM in the developing skeletal system was not observed before E12 (Table 1Go). At the E12 stage, expression of both TGFß1 and AM was detected as the differentiation of cartilage proceeded from condensed mesenchyme and precartilage to mature chondrocytes. Figure 3Go, A and E, shows the detection of TGFß1 and AM in the epithelial cells and sclerotomal cells of the somites of the caudal area of E12 embryos; although both proteins were detected in these two types of cells, staining for TGFß1 was more prominent in the epithelial cells, whereas AM was more prominently displayed in cells of the sclerotomal area. Maturing cartilage cells, including perichondrium and chondrocytes, were positively stained for TGFß1 and AM at the E13 stage (Fig. 3Go, B and F); however, although the staining intensity of TGFß1 in the perichondrium increased continuously to the E16 stage, the intensity of AM staining remained constant to the E14 stage in this tissue (Table 1Go). The intensity of staining for TGFß1 and AM in chondrocytes was similar and remained detectable for both proteins to the E16 stage. Figure 3Go, I and J, shows localization of TGFß1 and AM mRNAs in cartilage cells of the developing vertebrae as well as in the surrounding muscle of E14 embryos; here, expression of TGFß1 mRNA was detected in both perichondrium and chondrocytes (Fig. 3IGo), whereas AM mRNA was detected mainly in mature chondrocytes (Fig. 3JGo).



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  		Figure 3. Expression of TGFß1 and AM in the developing mouse skeletal and nervous systems. A–D, Immunohistochemical detection of TGFß1. E–H, Immunohistochemical detection of AM. I and K, In situ hybridization of TGFß1 mRNA. J and L, In situ hybridization of AM mRNA. A, E12, TGFß1; E, E12, AM. Cells of the epithelium and sclerotomal area of the somites are positive. B, E13, TGFß1; F, E13, AM. Chondrocytes and muscle of the developing vertebrae are positive. C, E13, TGFß1; G, E13, AM. Note the staining of the developing nerve trunks and cartilage of the vertebrae. D, E13, TGFß1; H, E13, AM. Note the positive staining in the epithelium of the choroid plexus. I, E14, TGFß1 mRNA; J, E14, AM mRNA. Chondrocytes of the developing vertebrae and surrounding muscle express TGFß1 and AM mRNAs. K, E14, TGFß1 mRNA, L, E14, AM mRNAs. The epithelium of the choroid plexus and underlying neural tissue express TGFß1 and AM mRNAs. Indicated are striated muscle (stm), cartilage (cg), dorsal root ganglion (drg), and epithelium (e), Magnification: C, D, and G–L, x100; A, B, E, and F, x200.

 
Immunohistochemically detectable TGFß1 and AM was observed in the nervous system by day 10 (E10); at this time, both the brain and spinal cord were positively stained for TGFß1 and AM (Table 1Go). On E11, TGFß1 and AM could be detected in the choroid plexus, pituitary, and dorsal root ganglia. Although the intensity of staining for TGFß1 in the dorsal root ganglia increased rapidly between E11 and E12, staining for AM in this tissue increased only gradually, so that maximal levels of staining for AM occurred on E15. Figure 3Go, C and G, shows intense immunohistochemical staining for TGFß1 and AM in the developing nerve trunks of E13 embryos; in E14 embryos, both nerve trunks and neural cell bodies were intensely stained for TGFß1 and AM (data not shown). Figure 3Go, D and H, shows expression of TGFß1 and AM in the epithelium of the choroid plexus; weak staining for AM was also observed in the surrounding neural tissue. Expression of TGFß1 and AM mRNAs was detected in the choroid plexus and surrounding neural tissue by in situ hybridization (Fig. 3Go, K and L).

Expression of TGFß1 and AM in embryonic internal organs
In addition to the heart, several other internal organs expressed TGFß1 and AM during development in the mouse embryo, including the intestine, liver, thymus, lung, kidney, and adrenal gland (Table 1Go). Expression of TGFß1 and AM was detected at the same developmental stage in the intestine (E11), liver (E11), lung (E11), and adrenal gland (E13). In contrast, expression of TGFß1 was detected earlier than that of AM in the developing thymus (E13 vs. E15) and kidney (E12 vs. E13). Figure 4Go, A–H, shows positive immunostaining for TGFß1 and AM in the lung, liver, intestine, and adrenal gland of E14 embryos. In the lung, immunostaining for TGFß1 and AM was detected in the bronchial epithelium and developing respiratory epithelium as well as in the developing blood vessels (Fig. 4Go, A and E); immunostaining for AM was also detected in the surrounding mesenchyme of this tissue (Fig. 4EGo). In addition, the cells surrounding the developing bronchi that will differentiate into smooth muscle cells stained intensely for TGFß1 and AM (data not shown). Positive staining for TGFß1 and AM was also detected in the hepatocytes and megakaryocytes of the liver (Fig. 4Go, B and F), the intestinal epithelium and surrounding muscle fibers (Fig. 4Go, C and G), and the adrenal gland (Fig. 4Go, D and H). Expression of TGFß1 and AM continued to be detected in these tissues up to E16. Figure 4Go, I–L, shows expression of TGFß1 and AM in the tracheal epithelium as well as in the epithelium of the esophagus and the intestinal epithelium and surrounding muscle at this later stage. Expression of the mRNAs for TGFß1 and AM was also detected at similar locations as the corresponding proteins in these organs using in situ hybridization (data not shown).



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  		Figure 4. Expression of TGFß1 and AM in several organs of the developing mouse. Figures show immunohistochemical staining. A–H show E14 embryos. I–L show E16 embryos. A, Lung, TGFß1. Note positive staining in the bronchiolar epithelium, respiratory epithelium, and blood vessels. B, Liver, TGFß1. C, Intestine, TGFß1. Note positive staining in the intestinal epithelium and surrounding muscle. D, Adrenal gland primordia, TGFß1. E, Lung, AM. Note positive staining in the bronchiolar epithelium, respiratory epithelium, and blood vessels. F, Liver, AM. G, Intestine, AM. Note positive staining in the intestinal epithelium and surrounding muscle. H, Adrenal gland primordia, AM. I, Esophagus and trachea, TGFß1. Note the intense staining in tracheal and esophageal epithelium, cartilage, and muscle. J, Intestine, TGFß1. Positive staining is detected in intestinal epithelium and muscle. K, Esophagus and trachea, AM. L, Intestine, AM. Indicated are bronchus (b), respiratory airway (r), mesenchyme (m), blood vessel (bv), smooth muscle (sm), adrenal gland (ag), esophagus (e), cartilage (cg), trachea (tr), and intestinal lumen (il). Magnification: C, D, and G–L, x100; A and E, x200; B and F, x400.

 
RT-PCR amplification of TGFß1 and AM mRNAs in embryonic mouse
In addition to in situ hybridization, RT-PCR amplification was used to evaluate the expression of TGFß1 and AM mRNAs in embryonic mouse. Figure 5Go shows the 620- and 539-bp PCR products for TGFß1 and AM, respectively, that were generated from cDNAs synthesized by RT of the mRNAs and that were detected by ethidium bromide staining with the appropriate DNA mol wt markers and were confirmed by Southern blotting with antisense nested probes (data not shown). The expected RT-PCR products for TGFß1 and AM mRNAs were produced in all of the mouse embryos examined (E7–E17). Although expression of TGFß1 mRNA was less in E7 embryos than in older embryos, that of AM mRNA was approximately equal in all of the mouse embryos examined.



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  		Figure 5. RT-PCR amplification analysis of TGFß1 and AM mRNAs in embryonic mouse. Two micrograms of cDNA from E7, E11, E15, and E17 mouse embryos were used for each PCR. The sizes of the amplified TGFß1 (lanes 2–5) and AM (lanes 7–10) cDNA fragments are indicated based on ethidium bromide staining with mol wt DNA markers. The gel shown is representative of two separate experiments.

 
Northern blot analysis of TGFß1 and AM mRNAs in mouse and rat development
Steady state expression of the mRNAs for TGFß1 and AM was examined in several tissues in embryonic rat and adult mouse and rat using Northern blot analysis. Expression of the 2.5-kb TGFß1 mRNA was readily detectable at varying levels in most tissues of the 18-day-old (E18) embryonic rat with the exception of liver (Fig. 6AGo); TGFß1 mRNA was also detected in liver with longer exposure (data not shown). Table 2Go shows the relative amounts of TGFß1 mRNA in these tissues after normalizing with 28S ribosomal RNA. Expression of TGFß1 mRNA was especially prominent in the umbilical cord as well as in the limb, lung, and intestine. After dehybridization of the nylon membrane and overnight exposure to film to ensure complete dehybridization, the membrane was reprobed with an AM cDNA probe. Figure 6BGo shows expression of two AM mRNA transcripts of 1.6 and 2.1 kb in several embryonic rat tissues; the level of expression of both AM transcripts was 3- to 6-fold higher in placenta, umbilical cord, and yolk sac than in embryonic brain, heart, lung, intestine, and kidney (Table 2Go). As a control, the gel was stained with ethidium bromide and photographed to show the amounts of RNA that had been applied to the gel (Fig. 6CGo).



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  		Figure 6. Northern blot analysis of TGFß1 and AM mRNAs in rat embryo tissues. Total RNA (2 µg) was isolated from rat embryo tissues, separated by electrophoresis on a 1% agarose-formaldehyde gel, and transferred to a Nytran filter as described in Materials and Methods. Hybridization was performed with 32P-labeled random primed cDNA probes for TGFß1 (A) and AM (B) as described in Materials and Methods. Blots for A and B were exposed for 2 and 6 days, respectively. C, The ethidium bromide-staining pattern of the gel showing 18S and 28S ribosomal RNA. The blots shown in this figure are representative of two separate experiments.

 

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  		Table 2. Relative amounts of TGFß1 and AM mRNAs in rat embryo tissues

 
Expression of TGFß1 mRNA was also readily detectable in several tissues of the 6-month-old adult mouse and rat, including heart, lung, and kidney, with lower levels in mouse and rat brain and mouse intestine (Fig. 7AGo). As in the embryo, expression of TGFß1 mRNA could be detected in adult mouse and rat liver only after prolonged exposure (data not shown). After dehybridization of the nylon membrane as before, the membrane was reprobed with an AM cDNA probe. Figure 7BGo shows expression of a single 1.6-kb AM transcript in the adult mouse and rat tissues examined, with prominent AM mRNA transcripts in mouse heart, lung, and kidney and in rat heart and lung and lower levels in brain and intestine. As in the embryo, AM mRNA expression could be detected in adult mouse and rat liver only after prolonged exposure (data not shown). As a control, the gel was stained with ethidium bromide and photographed to show the amounts of RNA that had been applied to the gel (Fig. 7CGo).



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  		Figure 7. Northern blot analysis of TGFß1 and AM mRNAs in adult mouse and rat tissues. Total RNA (10 µg) was isolated from 6-month-old adult mouse (lanes 1–6) and rat (lanes 7–12) tissues, separated by electrophoresis on a 1% agarose-formaldehyde gel, and transferred to a Nytran filter as described in Materials and Methods. Hybridization was performed with 32P-labeled random primed cDNA probes for TGFß1 (A) and AM (B) as described in Materials and Methods. Blots for A and B were exposed for 1 and 2 days, respectively. C, The ethidium bromide-staining pattern of the gels showing 18S and 28S ribosomal RNA. The blots shown in this figure are representative of two separate experiments.

 
RT-PCR amplification of TGFß1 and AM mRNAs in embryonic and adult rat tissues
RT-PCR amplification was also used to evaluate TGFß1 and AM mRNAs in a variety of embryonic and adult rat tissues. Figure 8AGo shows the 620-bp RT-PCR products for TGFß1 mRNA that were detected by ethidium bromide staining with appropriate DNA mol wt markers and confirmed by Southern blotting with antisense nested probes (data not shown). The cloned PCR products for embryonic and adult lung were further verified as authentic fragments of TGFß1 mRNA by nucleotide sequencing in either direction using primers at the T7 and SP6 promoter regions. The expected 620-bp TGFß1 RT-PCR product was produced in all of the embryonic and adult tissues tested. Figure 8BGo shows the 539-bp RT-PCR products for AM mRNA in embryonic and adult rat tissues that were also detected by ethidium bromide staining and confirmed by Southern blotting and DNA sequencing as for TGFß1 (data not shown). As for TGFß1 mRNA, all of the embryonic and adult tissues examined were shown to produce the expected 539-bp AM RT-PCR product.



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  		Figure 8. RT-PCR amplification analysis of TGFß1 and AM mRNAs in rat embryo and adult tissues. Two micrograms of total RNA extracted from rat embryonic (lanes 2–11) and adult (lanes 13–20) tissues was used for each RT-PCR. The sizes of the amplified TGFß1 (A) and AM (B) cDNA fragments are indicated at the right based on ethidium bromide staining with mol wt DNA markers. The gels shown are representative of two separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In a recent report, we suggested that the pattern of distribution of AM immunoreactivity and AM mRNA in mouse development might be reminiscent of that of TGFß (29). In the present study, we have examined and compared the expression and localization of TGFß1 and AM proteins and their corresponding mRNAs during rodent embryogenesis using the techniques of Northern blot hybridization, RT-PCR, in situ hybridization, and immunohistochemical staining analyses. Our results indicate that TGFß1 and AM proteins and their corresponding mRNAs are expressed in a variety of tissues in mouse and rat embryos. Furthermore, this expression is regulated in a spatial and temporal manner, such that overlapping patterns of detection of TGFß1 and AM proteins and mRNAs occur in several tissues at the same stage of development and in the same cellular location in mouse embryogenesis. This indicates an intricate pattern of gene transcription, translation, secretion, storage, and degradation of TGFß1 and AM during mouse embryogenesis.

Using Northern blot hybridization and RT-PCR analyses, we examined the expression of TGFß1 and AM mRNAs in embryonic and adult mice and rats. We showed that the pattern of expression of TGFß1 and AM mRNAs was similar in adult mouse and rat tissues. Rats were used to examine individual embryonic tissues instead of mice because of the need for sufficient amounts of RNA. Although most of the broad studies examining the localization of TGFß1 in rodent tissues have been conducted in the mouse, more recent studies examining the roles of TGFß in renal function, cardiac ischemia, liver regeneration, and lung branching morphogenesis have employed the rat because of its size and ease of use. In addition, many of the original studies examining the function of TGFß have been conducted using normal rat kidney cells (for reviews, see Refs. 8, 9). Similarly, with respect to AM, we have shown in an earlier report that localization of expression of AM during rat embryogenesis parallels that of mouse embryogenesis (29). Our examination of expression of TGFß1 mRNA in the embryonic and adult rat parallels that of an earlier report of TGFß1 mRNA expression in the mouse (37), and we have shown expression of TGFß1 mRNA in all of the rat embryonic tissues examined at levels similar to those found in the mouse. Like the mouse, TGFß1 mRNA was detected at approximately equal levels in many of the embryonic tissues examined, including the placenta, yolk sac, brain, heart, and kidney; a higher level of TGFß1 mRNA was detected in umbilical cord, lung, and intestine. A low level of TGFß1 mRNA was detected in embryonic rat liver; this is similar to the low level of TGFß1 mRNA in the embryonic mouse liver (37). Our ability to detect expression of TGFß1 mRNA in embryonic rat tissues was also shown using RT-PCR analysis with specific oligonucleotide primers for TGFß1. We have shown expression of AM mRNA in embryonic rat and adult mouse and rat tissues using Northern blot hybridization; with this method of analysis, we have demonstrated expression of two transcripts of 1.6 and 2.1 kb for AM in approximately equal amounts in the embryonic mouse and a single AM transcript of 1.6 kb in the adult mouse and rat. Recent studies have shown expression of the 1.6-kb AM transcript in a variety of adult rat tissues (38, 39). Our study shows that there is at least one additional transcript for AM in the embryonic rat. The 2.1-kb AM transcript may be the product of an alternative splicing event or a differential polyadenylation addition as has been shown for porcine TGFß1 (40). A recent report has shown that three additional transcripts (2.7, 3.6, and 6.0 kb) exist for AM mRNA in human pheochromocytoma along with the major 1.6-kb transcript (41). Additional studies are needed to determine the derivation of these AM transcripts. Interestingly, adult mouse and rat tissues showed only the 1.6-kb AM transcript. Use of RT-PCR with specific oligonucleotides to synthesize a 539-bp AM product, although not quantitative, showed amplification of the AM cDNA fragment in all of the embryonic and adult rat tissues examined. Using Northern blot hybridization, AM mRNA was detected in every embryonic rat tissue examined, with higher levels of AM mRNA in placenta, umbilical cord, yolk sac, limb, and liver than in brain, heart, lung, intestine, and kidney. This relative distribution is in accordance with other recent reports (38, 39)

Relatively low levels of TGFß1 and AM expression were detected in the mouse embryo at the beginning of organogenesis (E8), except in extraembryonic tissues (29). Immunohistochemical staining and in situ hybridization detected both TGFß1 and AM proteins and mRNAs in the early placenta, particularly in the giant trophoblastic cells and maternal decidual cells. Our localization of TGFß1 in the developing mouse placenta is consistent with that of earlier reports (42, 43). TGFß1 and AM proteins and mRNAs show similar intensities of staining in the primitive placenta, but comparison of relative protein and transcript levels of different genes by immunohistochemical and in situ hybridization analyses is difficult. In addition to TGFß1, other members of the TGFß superfamily, including BMP4, BMP8a, and BMP8b, have been shown recently to play a role in development of the mouse placenta (44, 45). Furthermore, in the past few years, a number of genes encoding transcription factors have been detected in the trophoblast cells of the developing placenta (reviewed in Refs. 46, 47). The colocalization of expression of TGFß1, AM, BMPs, and transcription factor proteins may reflect complex interactions among these proteins in regulating the growth and differentiation of the placenta.

The role of TGFß1 in mouse development has been examined by developing mice that lack both TGFß1 alleles (48). TGFß1 null mice born from a homozygous null female die within 1 day of birth due to severe cardiac abnormalities, suggesting that TGFß1 may play an important role in cardiac development (49). TGFß1 has been shown to be expressed in very early endocardial cells, then becomes limited to the endothelial cells that contribute to mesenchymal cushion tissue (17). Although most the cells of the developing E8–E9 embryo in our study were not stained, the walls of the atrial and ventricular chambers of the primitive heart were moderately stained for both TGFß1 and AM. The intensity of immunostaining for TGFß1 and AM continues to be comparable to at least E16 in the cardiovascular system. These data support the suggestion that although TGFß1 does not play a role in very early embryogenesis, it is only later, at the beginning of organogenesis, that TGFß1 starts to play an important role in mouse development. The similar immunostaining and in situ hybridization patterns of AM protein and mRNA in early embryogenesis also suggest that AM probably plays a role in early organogenesis in the mouse.

Besides the cardiovascular system, the nervous system is one of the first embryonic systems to arise, yet one of the last to complete development. Expression of TGFß1 and AM could be localized in the nervous system of the mouse embryo by E10; the nervous system also illustrates the complexities of the patterns of expression of these proteins. For example, although approximately equal levels of expression of TGFß1 and AM can be detected in the brain on E10 that persist to at least E16, the level of TGFß1 immunostaining is higher than that of AM in the pituitary and spinal cord at all stages of development examined. In the choroid plexus, although expression of TGFß1 increases continuously from E10 before peaking on E14, expression of AM is detected at approximately equal levels throughout development to E16. Although expression of both TGFß1 and AM is detected at approximately equally low levels in the dorsal root ganglia at stage E11, expression of TGFß1 increases at a faster rate than that of AM; however, by E15, the intensities of both TGFß1 and AM are approximately equal. Our localization of TGFß1 immunohistochemical staining to the developing embryonic nervous system of the mouse is consistent with the earlier observations of Heine et al. (17). An additional report by Flanders et al. (50) investigated the localization of TGFß in mouse embryo development and showed limited immunohistochemical staining by TGFß1 in the nervous system and more intense staining by antibodies to TGFß2 and TGFß3. We have also detected immunohistochemical staining for TGFß2 and TGFß3 in the developing embryonic mouse nervous system (data not shown), but the staining for TGFß1 is as prominent as that for the other two TGFß isoforms, especially in the central nervous system. An explanation for these differences in TGFß staining patterns includes the use of a different TGFß1 antibody that may recognize different epitopes of the TGFß1 protein. The earlier studies employed an antibody that was raised to the amino-terminal portion of the mature TGFß1 protein (50). In our study, the antibody that was used to detect TGFß1 was generated against a peptide that was localized in the middle portion of the mature TGFß1 molecule. Additional antibodies for TGFß1 will need to be examined in future studies to address these differences.

A third system in which similarities between TGFß1 and AM were noted was the skeletal system. Here, expression of the proteins and mRNAs for TGFß1 and AM was detected in such skeletal structures as muscle, chondrocytes, perichondrium, cartilage, and osteoblasts, with staining for TGFß1 generally being more intense than that for AM except in hypertrophic cartilage and osteoblasts. Several reports have shown the localization of TGFß1 in the developing mouse skeletal system (17, 18, 19, 20, 21, 22, 23). These studies have shown that TGFß1 contributes to segmentation of the axial skeleton; specifically, TGFß1 has an organizational role in the morphogenesis of vertebral bodies that are derived from sclerotomal mesenchyme of somites, and in subsequent chondrification and ossification. Using Northern blot hybridization and RT-PCR analyses, we have shown expression of transcripts for TGFß1 and AM in embryonic rat limbs. The presence of TGFß1- and AM-immunoreactive material is observed in a diffuse pattern in groups of mesenchymal cells starting in E9 embryos. In the E12 embryo, when condensed mesenchyme and precartilage differentiate toward more mature chondrocytes, both TGFß1- and AM-immunoreactive proteins are detected. Maturing cartilage cells are increasingly positive for both proteins from stage E12 on, as are osteoblasts from stage E15 on. The ultimate formation of these skeletal structures involves a series of chemotactic, proliferative, and differentiation events that result in a sequence of modeling and remodeling events to generate a mature skeletal structure. It has been established that extracellular matrix proteins have a direct role in promoting and stabilizing specific protein synthesis during this process, including the formation of such structures as somites, the differentiation of somitic mesenchyme to form sclerotome, and the subsequent chondrification and ossification to form vertebrae. TGFß1 has been shown to be of major importance in controlling the formation and destruction of many components of the extracellular matrix, including collagen, fibronectin, and proteoglycans. Because of the similarities in the localizations of TGFß1 and AM, it should be possible to design additional experiments to learn more about the role of AM in these differentiation processes.

It has been pointed out in several previous reports dealing with mouse and human embryos that TGFß1 is expressed in embryonic epithelia undergoing intense morphogenetic interactions with the underlying mesenchyme (17, 18, 19, 20, 21, 22, 23). It has also been shown that AM is expressed at high levels in tissues in which strong mesenchymal-epithelial interactions take place, including kidney, lung, tooth primordia, and hair follicles (29). The expression of AM in these structures is tightly regulated and restricted to particular cell types in any given stage of embryogenesis. In all cases in which TGFß1 mRNA was detected in epithelia, the protein was found to be deposited in the underlying mesenchyme. This has led to the suggestion that TGFß1 is involved in paracrine interactions with the underlying mesenchyme and that TGFß1 facilitates epithelial-mesenchymal interactions in regions of morphogenesis. Our study has shown that the same may be true of AM. Our more extensive knowledge of the biology of TGFß could provide clues to the functions of AM and guide further experimentation concerning the role of AM. Because of their colocalization, TGFß1 and AM may be able to coordinately act to influence development and differentiation. Future experiments will be needed to be performed in vivo and in vitro to examine interactions between TGFß1 and AM; this includes examination of TGFß1 and AM in TGFß1 null mice and AM null mice in which both TGFß1 or AM alleles have been deleted and analysis of the effects on cell targets of TGFß1 and AM. Also, future studies will be needed to examine and compare the localization of additional TGFß isoforms, including TGFß2 and TGFß3, with AM in embryogenesis. In addition, more extensive studies must be conducted in the cardiovascular, nervous, and skeletal systems to more clearly define the interaction between the TGFßs and AM in these different systems.


   Acknowledgments
 
The authors thank Dr. S. W. Qian (NCI) for rat TGFß1 cDNA. They are grateful to Drs. K. Flanders (NCI) and J. Hill (NICHHD) for helpful suggestions.


   Footnotes
 
1 The first two authors contributed equally to the research. Back

Received February 3, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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