DEVELOPMENTAL DYNAMICS 195276289 (1992)

Pattern of Expression Of Transforming Growth Factor-f54 mRNA and Protein in the Developing Chicken Embryo SONIA B. JAKOWLEW, GARY CIMENT, ROCKY S. TUAN, MICHAEL B. SPORN, AND ANITA B. ROBERTS Laboratory of Chemoprevention, National Cancer Institute, Bethesda, Maryland 20892 (S B J , M B S , A B R ), Department of Cell Biology and Anatomy, Oregon Health Sciences University, Portland, Oregon 97201 (G C 1, Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 (R S T )

ABSTRACT Expression of TGF-P4 mRNA and protein was studied in the developing chicken embryo using specific cDNA probes and antibodies for chicken TGF-P4. Expression of TGF-P4 mRNA was detected b y day 4 of incubation (Hamburger and Hamilton stage 22, E4) by RNA Northern blot analysis and increased with developmental age until day 12 of incubation (stage 38, E12) where it was detected in every embryonic tissue examined, with expression being highest in smooth muscle and lowest in the kidney. The steady-state level of expression of TGF-P4 mRNA remained relatively constant in most embryonic tissues through day 19 (stage 45, E19). In situ hybridization analysis detected TGF-P4 mRNA as early a s the “definitive primitive streak” stage (stage 4); during neurulation (stage 101, TGF-P4 mRNA was detected in all three germ layers, including neuroectoderm. Following neurulation, TGF-P4 mRNA was detected in the neural tube, notochord, ectoderm, endoderm, sclerotome, and myotome, but not dermotome a t stage 16. By day 6 of incubation (stage 29, E6), TGF-P4 mRNA was localized in several tissues including heart, lung, and gizzard. Immunohistochemical staining analysis also showed expression of TGF-P4 protein in all three germ layers as early as stage 4 in various cell types in qualitatively similar locations as TGF-P4 mRNA. These results suggest that TGF-P4 may play a n important role in the development of many tissues in the chicken. c 1993 Wiley-Liss, Inc. Key words: TGF-P-4, Expression, Development, Chicken INTRODUCTION The transforming growth factor-betas (TGF-ps) are polypeptide growth factors that regulate many functions, such as cell growth and differentiation, in nearly all cells examined (for reviews, see Sporn and Roberts, 1990; Roberts and Sporn, 1990; Massague, 1990). Thus far, five highly conserved, yet functionally similar TGF-ps have been described. All of the five TGF-ps show 64-82% identity a t the amino acid level and share essential structural features including synthesis from a large precursor and conservation of all nine cysteine residues in the processed peptide. In addition, the C,

1993 WILEY-LISS, INC

TGF-P isoforms are 390 to 414 amino acids long, and contain a hydrophobic signal peptide sequence of about 22 amino acids preceding the first methionine, three or four potential N-linked glycosylation sites in the precursor region, a multibasic cleavage site that is presumed to be necessary for processing of the precursor peptide to the biologically active, mature protein, and the presence of 112 N-terminal amino acids after the multibasic cleavage site. In addition to similarities based on amino acid sequence, TGF-ps 1, 2, 3, and 5 have analogous biological activities (Cheifetz et al., 1987; Graycar et al., 1989; Roberts and Sporn, 1990). TGF-ps 1, 2, and 3 have been shown t o be localized in both embryonic mammalian tissues (Heine et al., 1987, 1990; Lehnert and Akhurst, 1988; Miller et al., 1989a,b; Pelton et al., 1989, 1990, 1991; Millan et al., 19911, and in adult mammalian tissues (Roberts et al., 1986; Thompson et al., 1988, 1989). In previous work, we have cloned and characterized avian homologs of the various TGF-ps (Jakowlew et al., 1988a,b, 1990).TGF-p4, which has only been identified in the chicken so far, is unique with respect to the other TGF-Ps in that there is a n insertion of two amino acids (glycine and aspartic acid) near the N-terminus of the processed peptide, which would result in a 114 amino acid mature protein after cleavage from the precursor. To further characterize the biology of TGF-P4, we chose to examine the expression of TGF-p4 during embryonic chicken development. Using RNA Northern blot, in situ hybridization, and immunohistochemical staining analyses of several embryonic tissues of different developmental ages, expression of TGF-P4 mRNA and protein is found in many tissues of the developing chicken embryo. RESULTS Northern RNA Blot Analysis of TGF-P4 mRNA During Embryonic Chicken Development Steady-state expression of TGF-P4 mRNA was first examined in whole chicken embryos of developmental ages ranging between 4 days and 19 days of incubation (Hamburger and Hamilton 1951 stages 22 and 45, E4 and E19) using RNA Northern blot analysis. As shown _~ Received November 3, 1992. Sonia B. Jakowlew is now a t National Cancer Institute, Biomarkers Prevention and Research Branch, Rockville, MD 20850. Address reprint requests there.

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eral tissues a t different stages of development using RNA Northern blot analysis. Expression of the 1.7 Kb TGF-P4 mRNA in embryonic heart was found to increase between 4 days and 8 days of incubation (stages 22 and 34, E4 and ES), reach a plateau, and then decrease between 16 days and 19 days of incubation (stages 42 and 45, E l 6 and E19) (Fig. 2). Expression of the 1.7 Kb TGF-(34 mRNA was also detected in brain, eye, striated muscle, smooth muscle, and liver RNAs extracted from day 4 to day 19 (stages 22-45, E4-El9) chicken embryos. However, in contrast to the heart, expression of TGF-P4 mRNA was relatively constant throughout embryonic development in most other tissues (Fig. 2). Expression of the 1.7 Kb TGF-p4 mRNA was highest in smooth muscle and lowest in the kidney. Expression of the 2.5 Kb TGF-P4 mRNA was also detectable in most tissues, but a t a level significantly lower than that of the 1.7 Kb mRNA, just a s in the whole embryo. Also as in whole embryos, expression of both of the TGF-P4 mRNAs was significantly lower in each embryonic tissue examined compared to their level of expression in cultured chondrocytes.

Fig. 1, RNA Northern blot analysis of TGF-P4 mRNA in whole chicken embryos. Total RNA (15 pg) isolated from 4- to 19-day-old (stages 22-45, E4-EI9) chicken embryos was electrophoresed on a 1% agarose-formaldehyde gel, and transferred to a Nytran filter as described in Materials and Methods. The developmental ages of the embryonic chicken RNAs are labeled above each lane. A: Hybridization was performed with [3'P]-labeled random-primed plasmid pTGFB-Ch63. The positions of TGF-p4 mRNAs are shown as 1.7 and 2.5 Kb. B: The ethidium bromide profile of the gel showing 18 S and 28 S rRNAs.

in Figure lA, expression of the 1.7 Kb TGF-64 mRNA was detectable by 4 days (stage 22, E4), and increased between 8 days and 12 days of incubation (stages 34 and 38, E8 and E12) before reaching a plateau. In contrast, expression of the 2.5 Kb TGF-P4 mRNA was detectable by 8 days (stage 34, E8) and was significantly lower than the 1.7 Kb mRNA. Moreover, the level of expression of TGF-P4 mRNA was significantly lower a t all stages of embryonic growth compared to the level in primary cultures of chondrocytes isolated from 15-dayold (stage 41, E15) embryonic chicken sterna, the source from which TGF-64 cDNA was originally cloned (Jakowlew et al., 198813). As a control, the gel was stained with ethidium bromide to assure that approximately equal amounts of RNA had been applied to the gel (Fig. 1B). To identify sites of TGF-P4 mRNA synthesis, we then examined the expression of TGF-P4 mRNA in sev-

Northern RNA Blot Analysis of TGF-fi4 mRNA in Hatchling and Adult Chickens To compare the embryonic levels of expression of TGF-P4 mRNA with those of hatchlings and adults, additional RNA Northern blot analyses were performed. Figure 3 shows that expression of both the levels of the 1.7 and 2.5 Kb TGF-P4 mRNAs was found to be lower, in general, in most hatchling tissues compared to 12-day-old (stage 38, E12) embryonic tissues except in striated muscle, smooth muscle, cardiac muscle, and lung (panels A and B). Examination of the expression of the TGF-P4 mRNAs in adult chicken tissues showed higher expression of the TGF-P4 mRNAs in adult striated muscle, smooth muscle, and cardiac muscle than in other adult tissues including lung (Fig. 3C). Expression of the TGF-P4 mRNAs in both the liver and kidney was just barely detectable in the hatchling and below the level of detection in the adult. In Situ Hybridization of TGF-fi4 mRNA During Embryonic Chicken Development To examine the tissue-specific distribution of TGFP4 mRNA expression in more detail, in situ hybridization was performed on developing embryos ranging in age from 18 h r old (stage 4) to 6 days old (stage 29, E6) using a biotinylated TGF-P4 cDNA. This cDNA includes the entire coding region of the TGF-P4 gene plus 218 nucleotides of the 5'-untranslated region and 135 nucleotides of the 3'-untranslated region (Jakowlew et al., 198813). To minimize possible cross-hybridization with closely related gene products such a s TGF-Ps 2 and 3, hybridization and washing were performed at high stringency. Use of this probe in hybridization studies using RNA Northern blot analysis showed no cross-hybridization to the other TGF-P isoforms. Neg-

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Fig 2 RNA Northern blot analysis of TGF-P4 mRNA in chicken embryo tissues Total RNA (15 pg) isolated from tissues extracted from 4- to 19-day-old (stages 22-45, E4-El9) embryonic chicken tissues was electrophoresed on a 1% agarose-formaldehyde gel, transferred to a Nytran filter, and hybridized as described in Materials and Methods The striated

muscle and smooth muscle was obtained from pectoral muscle and gizzard, respectively The developmental ages of the tissue RNAs are labeled above each lane The ethidium bromide profile of the gel showing 18 S and 28 S rRNAs is shown to the right of each blot

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Fig. 3. RNA Northern blot analysis of TGF-p4 mRNA in embryonic, hatchling and adult chicken tissues. Total RNA isolated from tissues extracted from (A) 12-day-old embryonic, (B) I-day-old hatchling, and (C) 3-month-old adult chickens was electrophoresed on a 1% agarose-form-

aldehyde gel, transferred to a Nytran filter, and hybridized as described in Materials and Methods. The tissues used are labeled above each lane. The ethidium bromide profile of the gel showing 18 S and 28 S rRNAs is shown to the right of each blot.

ative control sections were hybridized with vector plasmid DNA containing no insert. Figure 4 shows adjacent transverse tissue sections through the anterior primitive groove (panels A and B) and the posterior primitive groove (panels C and D) of a “definitive primitive streak” stage chicken embryo (stage 4) hybridized with either TGF-P4 cDNA (panels A and C ) or control plasmid DNA (panels B and D). In these photomicrographs, hybridization to TGF-P4 mRNA can be detected in the ectoderm, mesoderm, and endoderm. In slightly older embryos, the pattern of hybridization of TGF-P4 mRNA to cells in all three germ layers continued. Figure 5 shows transverse sections through pharyngeal regions of stage 8 embryos. Panels A and B are adjacent sections just caudal to the anterior intestinal portal of a stage 8 embryo and hybridization to TGF-P4 mRNA can be detected in the ectoderm, neuroectoderm of the neural tube, notochord, and endo-

derm. Hybridization can also be detected in the paraxial mesoderm, which gives rise to the somites, and the splanchnic mesoderm, which gives rise to the mesenchyme of the gut (see higher magnification in panel C). In stage 10 embryos, expression of TGF-P4 mRNA continues in the endoderm following closure of the anterior intestinal portal, as well as in the ectoderm, neural tube, and in the various mesodermal derivatives, such as the notochord and head mesenchyme (data not shown). By 2.5 days of incubation (stage 16, E2.51, the overall intensity of the TGF-P4 mRNA hybridization signal was found to increase. Panels D-F of Figure 5 show a stage 16 chicken embryo hybridized with biotinylated TGF-P4 cDNA or vector DNA; specific hybridization was detected in the neural tube, sclerotomal and myotomal mesoderm, and in the endoderm and splanchnic mesoderm of the developing gut. In the 6-day-old (stage 29, E6) chicken embryo, ex-

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Fig 4 Localization of TGF-f34 mRNA in stage 4 chicken embryos Hamburger and Hamtlton stage 4 embryo sections at the anterior [A,B) and posterior (C,D) regions of the primitive streak were hybridized with either biotinylated TGF-f34 cDNA (A,C) or biotinylated control vector plasmid DNA (B,D) The ectoderm (ec), mesoderm (me), and endoderm (en)

are shown in B and D and the primitive groove (pg) in C The midline (dotted line) is shown in each panel Note intense hybridization in ail cell layers in A and C and the lack of hybridization in analogous regions in B and D Hybridizations were performed three times Bar :100 pm

pression of TGF-P4 mRNA continued to increase in most tissues. Figure 6A shows high levels of TGF-P4 mRNA expression in the cardiac muscle fibers. Similar high levels of expression of TGF-P4 mRNA were also detected in the muscle layers of the arteries of the aortic arch (Fig. 6C). Figure 6E shows a high level of expression of TGF-P4 mRNA in the granular smooth muscle layer of the gizzard while no expression of TGF-P4 mRNA is detected in either the epithelial muscle layer or the sub-granular muscle layer. Figure 6G shows significant hybridization for TGF-P4 mRNA in the mesenchyme of the lung and in the muscle surrounding the bronchioles and alveoli. No hybridization was detected in the epithelium of the bronchioles or alveoli. Hybridization of adjacent tissue sections with control plasmid DNA showed no hybridization signal (Fig. 6B,D,F,H). High levels of expression of TGF-64 mRNA were also detected in the sub-dermal layer of the developing feather germ, while little hybridization was detected in the epidermis of the feather (data not shown).

Immunohistochemical Staining Analysis of TGF-p4 Protein During Embryonic Chicken Development To examine expression of TGF-P4 protein, and to compare its location with that of its mRNA, indirect immunoperoxidase staining studies were performed. Figure 7 illustrates the immunohistochemical staining pattern of TGF-P4 in a transverse section through the rostral region of a stage 4 chicken embryo; immunoreactive material was detected in the endoderm, mesoderm, and hypoderm of the embryo proper, as well as in the area opaca of the extraembryonic membrane using anti-P 8-15(4) antibody (Fig. 7A,B). Expression of TGF-P4 in all three germ layers was also observed in more caudal sections, including the primitive knot (Fig. 7 0 , and in rostral and caudal regions of the primitive groove (Fig. 7D,E, respectively). In these regions of the primitive groove, the hypoblast typically stained more intensely than the ectoderm. Similar patterns of immunostaining were obtained using anti-Pre 135-

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Fig. 5. Localization of TGF-64 mRNA in tissue sections through the pharyngeal region of stage 8 (A-C) and lower cervical region of stage 16 chicken embryos (D-F).Sections were hybridized with either biotinylated TGF-P4 cDNA (A,C,D,F) or biotinylated control vector plasmid DNA (B,E). The ectoderm (ec), endoderm (en), notochord (no), neural tube (nt), paraxial mesoderm (pm), splanchnic mesoderm (spm), dorsal aorta

(da), dermomyotome (dm), foregut (fg), and sclerotome (sc) are shown. Note TGF-64 mRNA expression in the endoderm, neural tube, both the paraxial and splanchnic mesoderm, and in particular, in the ectoderm. C and F are higher magnifications of A and D, respectively. The dark spot at the neural folds denoted by the heavy arrow in A is an artifact. Bar = 100 wm.

143(4) antibody suggesting that some of the immunoreactivity may be due to the presence of pre-pro TGFp4 (data not shown). Immunohistochemical staining for TGF-P4 was completely blocked when anti-P 8-15(4) antibody was preincubated with a solution of the peptide against which i t was raised (Fig. 7F). It has not yet been possible to demonstrate the specificity of these antibodies for authentic TGF-P4 using Western blotting analysis because no abundant natural source of TGF-P4 has been identified and recombinant TGFp4 has not been expressed in sufficient quantities. However, these antibodies have been demonstrated not to react with 100 ng of porcine TGF-ps 1 and 2 or recombinant chicken TGF-P3 (data not shown). Moreover, immunohistochemical staining for TGF-P4 was not blocked when anti-P 8-15(4) antibody was preincubated with the synthetic peptides used to raise antibodies for TGF-(3s 1, 2, and 3 (data not shown). With formation of the neural tube in stage 13 chicken embryos, the overall levels of TGF-P4 immunostaining appeared qualitatively to decrease. Immunostaining studies of sections through truncal and pharyngeal regions of stage 13 embryos showed detectable levels of TGF-P4 in the neural tube, notochord, somites, and lateral plate somatopleural and splanchnopleural mesoderm (Fig. 8A,C). Weaker immunostaining for TGF-P4 was also detected in the ectoderm and endoderm, although pharyngeal endoderm showed significantly more staining than more caudal endoderm. Again, negative control experiments using pre-

absorbed anti-P 8-15(4) antibody showed significantly lower levels of overall immunostaining (Fig. 8B,D). In older chicken embryos, the sites of TGF-64 immunostaining were found to be more localized. For example, in the central nervous system of stage 16 embryos, TGF-P4 immunostaining was more highly concentrated in presumptive white matter than in the proliferating cells of the germinal matrix, although there were sites of localization of TGF-P4 in the roof plate and occasional cells along the ventricular surface (Fig. 9C). In the peripheral nervous system, TGF-P4 immunostaining was more highly concentrated in peripheral nerves, such a s the ventral roots (Fig. 9C,G), and in various peripheral ganglia, such a s the dorsal root ganglia (Fig. 9C). Although there were low levels of TGFp4 immunostaining in most tissues examined, particularly high levels of expression of TGF-p4 were observed in the myotome (Fig. 9G) and in the notochord (Fig. 9E). Again, negative control experiments using preabsorbed anti-P 8-15(4) antibody showed significantly lower levels of staining (Fig. 9B,D,F,G).

DISCUSSION Highly conserved homologs of the TGF-p family have been identified in the chicken. Two of the TGF-(3 isoforms (TGF-ps 2 and 3) have been shown to have mammalian counterparts and their patterns of expression during mammalian development have been studied (Heine et al., 1987; Lehnert and Akhurst, 1988; Pelton et al., 1989, 1990, 1991; Millan et al., 1991; Schmid et

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al., 1991). The remarkable conservation of TGF-@sequences between species and the ability of TGF-P to modulate cellular differentiation and cellular function, together with its specific effects on matrix protein formation (Roberts et al., 1986; Ignotz and Massague, 1986, 1987; Ignotz et al., 1987), on cellular chemotaxis (Moses et al., 1985; Robey et al., 1987; Wahl e t al., 1987; Postlethwaite et al., 1987), and on angiogenesis (Roberts et al., 1986; Yang and Moses, 1990), all strongly suggest that TGF-@plays a fundamental role in morphogenesis. Although TGF-@4has been detected only in the chicken thus far, its amino acid sequence shows significant identity with that of mammalian [email protected] data presented in this study suggest that like TGF-@lin mammals, TGF-P4 is likely to play a major role in a variety of developmental processes in the chicken. Also like mammalian TGF-@I,the function of TGF-@4 apparently involves diverse mechanisms of action in tissues of different lineages. Using in situ hybridization and immunohistochemical staining analysis, we have shown that TGF-@4 mRNA and protein can be detected in chicken embryogenesis as early as the definitive primitive streak stage (stage 4). These transcripts and proteins are present in all three germ layers and in both the mesenchymal and epithelial components of different tissues. Although we have not studied earlier stages for technical reasons, i t is possible that TGF-@4plays a role in chicken embryogenesis prior to stage 4 since expression of TGF-@ mRNA and protein is already quite prominent by stage 4.This may be similar to mouse embryogenesis where expression of TGF-@lmRNA has been shown to occur as early as the 2-cell stage (Rappolee et al., 1988). Expression of TGF-@1remains high throughout the remainder of development of the mouse embryo (Heine et al., 1987; Lehnert and Akhurst, 1988; Pelton et al., 1989, 1990, 1991; Millan et al., 1991; Schmid et al., 1991) and on into neonatal and adult life (Thompson et al., 1989). The precise role of the TGF-@sin developing avian embryos is unclear. Cell proliferation, differentiation, and patterning are all key elements in the development of organized structures. Since the TGF-Ps have significant effects on proliferation and differentiation of avian cells in culture (Jakowlew et al., 1991, 19921, these isoforms may also be important in these processes during embryonic development. It has been postulated that many of the effects of TGF-@in embryogenesis in mammals are probably mediated by its effects on extracellular matrix (Heine et al., 1987; Thompson et al., 1988; Schmid et al., 1991). The composition and organization of the extracellular matrix is an important determinant of cellular behavior in that it regulates cellular adhesion, migration, proliferation, and differentiation (Ekblom et al., 1986; Ruoslahti and Pierschbacher, 1987). TGF-@lhas been shown to activate gene transcription and increase synthesis and secretion of several matrix proteins including collagen and fibronectin (Roberts et al., 1986; Ignotz and Mas-

sague, 1986; Varga and Jimenez, 1986; Fine and Goldstein, 1987; Van Obberghen-Schilling et al., 19881,proteoglycans (Rasmussen and Rapraeger, 1988), and cell adhesion receptors (Heino et al., 1989; Ignotz and Massague, 1987), as well as decrease synthesis of proteolytic enzymes that degrade matrix proteins and increase synthesis of protease inhibitors that block the activity of these enzymes (Laiho et al., 1986; Edwards et al., 1987). Recent studies have shown that TGF-p induces the differentiation of periosteal mesenchymal cells of bone into chondrocytes and osteoblasts in mammalian and avian species (Joyce et al., 1990; Thorp et al., 1992). In previous studies, we have shown localization of TGF-@in avian cartilage and bone (Jakowlew et al., 1991). TGF-@has also been shown to stimulate proliferation and synthesis of the extracellular matrix proteins characteristic of bone and cartilage (Joyce et al., 1990). Changes in expression of TGF-@are also associated with alterations in osteogenic and chondrogenic cell fate during intramembranous bone development (Sato and Tuan, 1992). The participation of TGF-P in deposition of extracellular matrix is consistent with our observation of TGF-P4 immunoreactivity in the notochord, a site of significant extracellular matrix production. In addition, TGF-@has also been shown to have effects on neural crest cell development, causing precocious emigration from the neural tube, inhibiting their commitment to the melanogenic lineage, and promoting their emigration from the neural tube (Stocker et al., 1991; Rogers et al., 1992; Delannet and Duband, 1992). Although the data presented here suggest that expression of TGF-@4mRNA is important in development of the chicken, a n understanding of the molecular mechanisms governing this expression will require isolation of the 5’-upstream regulatory region of the TGF-@4gene and a n analysis of the TGF-@4promoter. It is hoped that characterization of the chicken TGF-@4 promoter will reveal possible controlling elements in regulation and help us understand more about the role of TGF-@4in the developing chicken embryo.

EXPERIMENTAL PROCEDURES RNA Extraction and RNA Northern Blot Analysis Total RNA was extracted from embryonic, neonatal, and adult white Leghorn chicken tissues that had been

Fig 6 Localization of TGF-p4 mRNA in sections through the heart great vessels, gizzard, and lung primordia of a 6-day-old (stage 29, E6) chicken embryo Sections were hybridized with either biotinylated TGF-p4 cDNA (left) or biotinylated control vector plasmid DNA (right) Note the high level of expression of TGF-P4 mRNA in cardiac muscle (A), smooth muscle of the great vessels (C) and gut (E) Also note the high level of TGF-64 mRNA expression in the mesenchyme of the lung and in the basal cells associated with the basement membrane, whereas the epithelium is negative (G) The smooth muscle (sm), lung epithelium (ep) and lung mesenchyme (me) are shown Bar = 100 pn

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Fig. 7. Localization of TGF-04 protein in stage 4 and 6 chicken embryos. Stage 4 embryo sections at the anterior (A,B), middle (C) and posterior (D) regions of the primitive streak were reacted with anti-P 8-15(4) (A-D). Stage 6 embryos (E,F) were reacted with anti-P 8-15(4) (E) or anti-P 8-15(4) preincubated with its synthetic peptide (F). Immu-

nohistochemical staining was performed three times. The ectoderrn (ec), mesoderm (me), endoderm (en), hypoblast (hy), area opaca (ao), prirnitive knot (pk), and primitive groove (pg), neuroectoderm (ne) are shown. Note TGF-64 immunoreactivity in cells of all three germ layers. Bar : 100 pm.

excised and quick-frozen in liquid nitrogen according to the LiC1-urea procedure described by Auffray and Rougeon (1980). In some instances, poly (A + mRNA was isolated using oligo d(T)-cellulose affinity chromatog-

raphy (Collaborative Research, Waltham, MA) according to Aviv and Leder (1972). For RNA Northern blot analysis, equal amounts of RNA (15 pg of total RNA and 5 pg of poly (A + mRNA) were electrophoresed on

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Fig. 8. Localization of TGF-p4 protein in stage 13 chicken embryos. Transverse sections of stage 13 embryos at the truncal and pharyngeal regions were reacted with anti-P 8-15(4) (A,C) and anti-P 8-15(4) preincubated with its synthetic peptide (B,D). The endoderm (en), ectoderm (ec), notochord (no), neural tube (nt), somites (som), dorsal aorta (da),

pharynx (ph), dermomyotome (dm), and somatopleural (sop) and splanchnopleural (spp) mesoderm are shown. Note TGF-p4 immunoreactivity in the neural tube, notochord, somites, and lateral plate somatopleural and splanchnopleural mesoderm. Bar = 100 pm.

1%agarose gels containing 0.66 M formaldehyde and transferred to "Nytran" filters (Schleicher and Schuell, Keene, NH). Ethidium bromide (33 pg/ml) was included in both the gels and running buffers in order to visualize the positions of ribosomal RNAs by UV illumination following electrophoresis. Before blotting, the gels were pretreated with 50 mM NaOH in I X SSC (150 mM sodium chloride/l5 mM sodium citrate) for 20 min a t room temperature and then rinsed two times in 10 x SSC for 20 min each. Blots were hybridized using [32P]-labeled(3,000 Ciimmole, New England Nuclear, Boston, MA) random-primed probes a t 65"C, washed a t 65°C according to Church and Gilbert (1984), and exposed for various times a t - 70°C using a n intensifying screen.

and stored in 70% ethanol before embedding in paraffin and sectioning a t 5 pm onto slides treated with 3-aminopropyl-triaethoxysilane.

Preparation of Tissues for I n Situ Hybridization Tissues were excised, rinsed in phosphate-buffered saline (PBS), fixed for 48 h r in filtered 10% neutralbuffered formalin, treated for 30 min each in PBS and then saline, followed by 15 min each in 50% saline/50% ethanol, and then two times in 70% ethanol for 15 min,

In Situ Hybridization In situ hybridization was performed essentially as described previously (McDonald and Tuan, 1988; Sato and Tuan, 1992). Sections were deparaffinized with xylene followed by Americlear (Polysciences, Warrington, PA) for 20 min each, hydrated through a graded series of ethanol (loo%, 95%, 70%, and 50%) for 1 min each, equilibrated with 2 x SSC for 1 min, and demineralized in 0.25 M HC1 for 30 min. After being rinsed in water, the sections were incubated with 2 pgiml proteinase K (Boehringer-Mannheim, Indianapolis, IN), predigested at 37"C, rinsed in water and 2 x SSC, refixed in formaldehydei2 x SSC for 2 h r a t room temperature, dehydrated in a graded series of ethanol as before, and air dried. Sections were denatured in 95% deionized formamide/O.l x SSC a t 70°C for 15 min immediately before hybridization, immersed into icecold 0.1 x SSC for 2 min, washed in water, and dehy-

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drated. Hybridizations were performed in 1x SSCP (0.12 M NaC1, 15 mM sodium citrate, 20 mM sodium phosphate, pH 6.0) with 0.025% Denhardt’s solution and 10% dextran sulfate in 50% deionized formamide. The probes used for hybridization consisted of total plasmid cDNAs labeled by random-priming in the presence of biotinylated dUTP (16-mer, Enzo Diagnostics, New York, NY) using standard procedures (Maniatis et al., 1982). Hybridization was allowed to proceed at 37°C for 48 hr. Slides were washed four times in 30% formamidel2 x SSC a t 40°C for 5 min each, followed by two washes each in 2 x SSC, 1x SSC, and 0.5 x SSC for 3 min a t 40°C. Slides were processed for detection as recommended by Enzo Diagnostics using streptavidinbiotinylated alkaline phosphatase. All specimens were examined and photographed using Nomarski differential interference microscopy.

Preparation of TGF-P4 Specific Antibodies Synthetic peptides corresponding to unique segments of the pre-pro and mature regions of chicken TGF-P4 were purchased from Bachem Biochemicals (Torrance, CAI. Polyclonal antibodies, anti-Pre 134153(4) and anti-P 8-15(4), were generated to peptides corresponding to amino acids 134-153 of the pre-pro region of chicken TGF-f34,and amino acids 8-15 of the mature region of chicken TGF-P4, with tyrosine residues added to both the amino- and carboxyl-terminals. Antibodies were purified by passage over 1.5 ml columns of synthetic peptide conjugated to Affi-Gel 10 or 15 as described by Flanders et al. (1988). Preparation of Tissues for Immunohistochemical Staining Excised tissues were washed in PBS, fixed for 48-96 hr in 10% neutral-buffered formalin, treated with Bouin’s solution for 6 h r a t room temperature, and stored in 70% ethanol before embedding in paraffin and sectioning a t 5 pm. Immunohistochemical Staining TGF-P was localized in sections as described by Heine et al. (1987), using avidin-biotin-peroxidase kits (Vector Laboratories, Burlingame, CAI. After deparaffinization, blocking of endogenous peroxidase in hydrogen peroxidelmethanol, and permeabilization with hyaluronidase, the sections were blocked with 1.5%

Fig. 9. Localization of TGF-f34 protein in stage 16 chicken embryos. Transverse sections of stage 16 embryos at the region of the dorsal aorta were reacted with anti-P 8-15(4) (A,C,E,G) and anti-P 8-15(4) preincubated with its synthetic peptide (B,D,F,H). The amnion (am), ectoderm (ec), notochord (no), neural tube (nt), dermomyotome (dm), dermotome (de), myotome (myo), sclerotome (scl), dorsal aorta (da), cardinal vein (cv), dorsal root ganglia (drg), roof plate (rp), floor plate (fp), ventral root (vr), and ventricular surface (ve) are shown. Note TGF-p4 immunoreactivity in the presumptive white matter, peripheral nerves, myotome, and notochord. Bar = 100 pm.

normal goat serum/0.5% BSA, incubated overnight a t 4°C with affinity-purified antisera at 3-5 p,g/ml, washed extensively, and then incubated with biotinylated goat anti-rabbit IgG and avidin-enzyme complex. Sections were stained with 3,3’-diaminobenzidine (Sigma, St. Louis, MO) and hydrogen peroxide, and counterstained with Mayer’s hematoxylin. Controls include 1)replacing primary antisera with normal rabbit IgG; 2) using primary antisera that had been preincubated with a 20-fold molar excess of the appropriate peptide for 2 h r a t room temperature before this mixture was applied to the section. The distribution pattern of TGF-P4 observed in the developing chicken embryo was found to be independent of the fixative used. Fixation with formaldehyde a t neutral pH alone, or fixation with formaldehyde followed by Bouin’s fixative at pH 2, resulted in identical staining patterns. Because the majority of immunohistochemical staining studies for TGF-f3 in mammals have been conducted using Bouin’s fixative, this fixation process was used exclusively in the study of the developmental patterns of TGF-P4 reported here.

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