DEVELOPMENTAL

BIOLOGY

Transforming

153,324-336

(1992)

Growth Factor ,B, Is an Epithelial-Derived That Influences Otic Capsule Formation

Signal Peptide

DOROTHYA.FRENZ,* VERAGALINOVIC-SCHWARTZ,* WEILIU,* KATHLEENC.FLANDERS,$ANDTHOMAS R. VANDE~ATER*~~ Departments of *Otolaryngolo~y and tNeuroscience, Albert Einstein College of Medicine, Bronx, New York 10461; and $Laboratory of Chemoprevention, National Cancer Institute, Bethesda, Maryland 208.92 Accepted May 22, 1992 Interactions between epithelial and mesenchymal tissues in the developing inner ear direct the formation of its cartilaginous capsule. Recent work indicates that many growth factors are distributed in the early embryo in viva in a temporal-spatial pattern that correlates with sites of ongoing morphogenetic events. We report here the localization of transforming growth factor & (TGF-&) in both epithelial and mesenchymal tissues of the mouse inner ear between 10 and 16 days of embryonic development (ElO-E16). In addition, utilizing a high-density culture system as an in vitro model of otic capsule chondrogenesis, we show that modulation of chondrogenesis by TGF-/3, in cultured mouse periotic mesenchyme mimics the in vitro effects of otic epithelium on the expression of chondrogenic potential. We provide evidence of a causal relationship of this growth factor to otic capsule formation in situ by demonstrating that the actual sequence of chondrogenic events that occur in the developing embryo is reproduced in culture by the addition of exogenous TGF-0, peptide. Furthermore, in cultures of mesenchyme containing otic epithelium, we demonstrate the localization of endogenous TGF-&, first within the epithelial tissue and later within both the epithelium and its surrounding periotic mesenchyme, contrasted to an absence of endogenous TGF-@, in cultures of mesenchpme alone. Our results suggest that TGF-0, is one of the signal molecules that mediate the effects of otic epithelium in influencing the formaI%,1992 Academic Press, Inc. tion of the cartilaginous otic capsule INTRODUCTION

Morphogenesis of the capsule of the mammalian inner ear involves a sequence of interactions between the epithelial cells of the otic anlage and the mesenchymal cells of the surrounding periotic tissue (Van de Water, 1981). Integral to these interactions are the inductive and inhibitory effects of otic epithelium on chondrogenie expression in vitro (Frenz and Van de Water, 1991), events that may play a significant role in chondrogenic patterning during capsular development in ~ivo. While the accumulation of hyaluronate or other matrix molecules has previously been implicated in signaling epithelial effects (Solursh et al., 1981; McPhee and Van de Water, 1986; McPhee et al., 1987), the specific factors involved in the control of otic morphogenesis have not been resolved. One factor that has been suggested to play an important role in the signaling of epithelial-mesenchymal tissue interactions is transforming growth factor & (TGF&), a member of the TGF-/3 superfamily of growth factors (Massague, 198’7; Sporn et ah, 1987). Not only are TGF-P, and TGF-o-like mRNAs widely distributed in the early embryo in &o (Weeks and Melton, 1987; Heine ef al., 1987; Rappolee et al., 1988), the temporalspatial distribution of TGF-& correlates with sites of ongoing morphogenetic events (Heine et ab, 1987). More001%1606/9S Copyright All rights

$5.00

cl 1992 by Academic Press, Inc. of reproduction in any form reserved.

324

over, the expression pattern of TGF-P, mRNA in differentiating epithelia (Lehnert and Akhurst, 1988; Akhurst et al., 1990; Fitzpatrick et al., 1990) corresponds with the distribution pattern of TGF-0, polypeptide in underlying mesenchymal structures (Heine et al., 198’7; Thompson et al., 1989). Recent studies in vitro have shown that transient exposure of limb mesenchymal cultures to TGF-0, causesa marked enhancement of chondrogenesis (Kulyk et al., 1989; Leonard et al., 1991). Because this is consistent with a role for TGF-0, as a regulator of chondrogenesis, and epithelial-mesenchymal interactions control chondrogenic responses in targeted inner ear mesenchyme (Frenz and Van de Water, 1991), we were interested in determining whether the tissue interactions directing otic capsule formation are mediated by TGF-8,. We have investigated in situ the relationship of the localization of TGF-& to sites of ongoing epithelial-mesenchymal interactions during morphogenesis of the embryonic mouse inner ear. We focused our attention particularly around the time of otic development when the presumptive capsule of the inner ear is forming (i.e., between embryonic days El0 and E14). In addition, later developmental stages (i.e., E15-E16) corresponding to the time of completion of otic morphogenesis were also examined in this study. We provide evidence of the localization of TGF-/3, in both epithelial and mesenchymal

tissues of the developing inner ear at sites of active morphogenesis and differentiation. Correspondingly, we demonstrate the in vitro localization of TGF-& in cultures of periotic mesenchyme containing otic epithelium, but not in cultures of mesenchyme alone. We show that exposure of cultured periotic mesenchyme to TGF@, parallels the effects of otic epithelium on the expression of mesenchymal cell phenotype. In addition, we demonstrate that the sequential stimulation and inhibition of cartilage formation during otic morphogenesis in viva are mimicked by the sequential addition of TGF-/3, in vitro. These results are consistent with the hypothesis that TGF-& is one of the signal peptides used by otic epithelium to influence formation of its otic capsule from the surrounding cephalic mesenchyme. MATERIALS

AND

METHODS

Experimental unirnuls. Hybrid CBA/C57 BL6 mouse embryos of gestation age ElO-El6 were obtained by crossmating CBA-J and C57BL6-J mice (Jackson Laboratories). Gestational age was estimated by the vaginal plug method, with the day of plug occurrence designated as Day 1 (El). After death of the gravid females by cervical dislocation, their embryos were excised and immediately placed into Dulbecco’s phosphate-buffered saline (PBS, Gibco Laboratories). Embryonic age was determined by a combination of somite count and external features (Theiler, 1972). Antibodies. Rabbit polyclonal antibodies (i.e., antibody LC and antibody CC) to unconjugated peptides corresponding to the amino-terminal 30 amino acids of TGF-0, were prepared as previously described (Flanders ef ul., 1988). These antibodies give distinct staining patterns for the presence of intracellular TGF/3, (antibody LC) and extracellular TGF-0, (antibody CC). In enzyme-linked immunosorbent assays, Western blots, and immunoprecipitation assays, antisera showed no reactivity against TGF-& (Flanders ef ab, 1988). I~,~?nu~1oh%‘st0~hemica.l stainix,cl in S&L. The distribution of TGF-/3, was determined by immunohistochemical staining utilizing the peroxidase-antiperoxidase method (Dako Kit). Cross sections were prepared from ElO-El6 mouse inner ears as described below. Specimens were fixed in methacarn fixative (Puchtler et al., 1970), dehydrated in methanol, cleared in methyl benzoate (Fisher) and Histoclear (National Diagnostics), and embedded in paraffin (Surgipath) at 59°C. Specimens 5-6 pm thick were collected on alcohol-cleaned, uncoated slides and deparaffinized. Nonspecific binding was blocked by preincubation with normal swine serum (30 min, 20°C). Some specimens were pretreated with testicular hyaluronidase (1 m&ml, Sigma H-3884) in 0.1 M sodium acetate (pH 5.5) with 0.85Yo NaCl for 30 min

at 37°C. Sections were incubated with antibody CC or antibody LC (0.05 mg/ml) at 4°C overnight in a humidified chamber. Controls were done by replacing the antiTGF-fl rabbit IgG by bovine serum albumin or preimmune serum from the anti-LC rabbit serum. Following a final incubation with 3-amino-9-ethylcarbazole substrate solution, specimens were counterstained with Mayer’s hematoxylin (Sigma). Specimens were mounted in crystal mount (Biomedia) with final mounting in Permount (Fisher). Tissue distribution of TGF-/3, was subjectively evaluated by the density of the peroxidase reaction product, with relative stain intensity rated on a scale of l-4+. Six different specimens were rated for each developmental stage. Interspecimen stainingvariation was not greater than 0.5 unit on the stain intensity rating scale for each stage evaluated by immunostaining. Micromass culture. Otoeysts were excised with their associated periotic mesenchyme from E10.5 to El4 mouse embryos. Periotic mesenchyme was dissected free of epithelial tissue and cultured utilizing modifications of the micromass technique (Ahrens et al., 1977; Frenz and Van de Water, 1991). Briefly, periotic mesenthyme was dissociated with 0.05% trypsin-0.53 mM EDTA in Hanks’ balanced salt solution without calcium and magnesium for 1.5 min at room temperature. Dissociated mesenchymal cells were resuspended at a density of 2.5 x lo7 cells/ml in Ham’s F-12 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco). Tenmicroliter spots of cell suspension (i.e., 2.5 X lo5 cells/ spot) were plated dropwise into the centers of wells of a 24-well tissue culture plate (Costar 3424) or a 4-well tissue culture plate (Nunc 134673). After a 1-hr incubation period at 37”C, 1 ml of culture medium was added to each culture. Nutrient solution containing added human platelet-derived TGF-P, (Assoian et al., 1983) was introduced into culture for a 24-hr period either at this time (i.e., 1 hr after seeding) or after 18 hr of incubation, following exchange of the initial milliliter of culture medium. Subsequent changes of medium (tTGF-&) occurred every 48 hr. Control cultures were not exposed to added growth factor. Cultures were maintained for a period of 7 days in vita. Quantitution of cell condensations. Twenty-four hours following exposure to added TGF-&, cultures were monitored by phase contrast or Hoffman modulation contrast microscopy for identification of condensed mesenchymal cells. In phase-contrast images, mesenehymal condensations can be recognized by their characteristic features of cell packing and orientation; with Hoffman modulation optics, condensations appear as dark images against a lighter background (Frenz ef al., 1989). Between the second and third days of culture, cell condensations were counted and these counts recorded.

326

DEVELOPMENTAL BIOLOGY

Quantitative Al&an blue staining of cultures. After a period of ‘7 days, cultures were fixed for 5 min with a solution of 10% formalin containing 0.5% cetylpyridinium chloride (CPC). Cell spots were washed with a 3% acetic acid solution (adjusted to pH 1.0 with HCl) and stained overnight with a 0.5% solution of Alcian blue 8GX (Sigma) in 0.1 NHCl (pH 1.0). To remove unbound stain, cell layers were washed twice with 3% acetic acid solution (pH 1.0). Matrix-bound Alcian blue stain, an index of accumulated sulfated glycosaminoglycans (SGAG) (Lev and Spicer, 1964), was extracted from each cell layer with 300 ~1 of a 8 M guanidine hydrochloride solution and measured by spectrophotometric quantitation (Hassell and Horigan, 1982) using a Cambridge Technology Inc. microplate reader equipped with a 600nm-wavelength filter. S-GAG accumulation in TGF-&treated cultures was compared to that in mesenchymal cultures that were grown in the presence of otic epithelium, as described (Frenz and Van de Water, 1991). Incorporation of radiolabeled sulfate into S-GAG. Mesenchymal cells were grown as described, but with the addition of 2-4 PCi of Na35S0,/ml (carrier free, 1 Ci = 3’7 GBq, Amersham) to the culture medium. Medium was collected at every change and pooled for each cell spot. After a period of 7 days in vitro, each cell spot was homogenized in 1 ml of medium. Homogenates and pooled media were digested overnight (37°C) with 100 /*g of proteinase K/ml. Fifty-microliter aliquots of each digest were spotted onto strips of Whatman 3MM filter paper as described (Leonard et al., 1989). After completely drying, these strips were washed five times in 1% CPC in 0.3 M NaCl (Wasteson et al., 1973). Strips were dried, cut, and suspended in Aquasol for liquid scintillation counting. DNA determination in chondrogenic cultures. A modification of the method of LaBarca and Paigen (1980) was utilized. Briefly, cell spots were harvested on Culture Day 7, and samples homogenized in phosphate-saline buffer (0.05 M NaPO,, 2 M NaCl, 2 X lop3 M EDTA, pH 7.4). Aliquots of the homogenate (15 ~1) were mixed with 1.5 ml phosphate-saline buffer containing 10 ~1Hoechst compound 33258. Fluorescence measurements were made on a Hoefer Scientific Instruments DNA fluorometer (TKO 100). The assay was checked for linearity by making dilutions of calf thymus DNA (i.e., a reference standard, ICH Laboratories Inc.) and plotting a standard curve. Optical density readings correspond to DNA concentrations in the pg/ml range. Indirect immunojluorescent staining of micromass cultures. Mesenchymal cultures containing otic epithehum, prepared as previously described (Frenz and Van de Water, 1991), or cultures of mesenchyme alone were grown on coverslips that were inverted into the wells of four-well tissue culture plates (Nunc). Cultures were

VOLUME 153,1992 TABLE 1 IMMUNOLOCALIZATION OF TGF-P, PEPTIDE IN THE INNER EAR OF THE DEVELOPING MOUSE Epithelium (antibody LC)

Embryo age

Vestibule

Cochlea

El0 E10.5

2+” 3+

2+ 3+

E11.5

ND 1+

ND

El2 El3 El4 El5

El6

3+ 2+

2+ 3+ 2f

+/0

+/0

Mesenchymal matrix (antibody CC) Vestibule

Cochlea

1+

1+ 1+ 1+

2+ 3f 3+ 4+/3+b o/4+ o/4+ o/3+

2+ 4+/3+b o/4+ o/2+

o/o

a Relative stain intensity en a scale of 1 to 4+ judged subjectively. * Chondrogenic mesenchyme/perilymphatic space mesenchyme.

fixed in cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min and then washed three times for 5 min each with 0.1 M phosphate buffer. Antibody directed against TGF-& peptides (anti-CC) (Flanders et ab, 1988) was applied for 30 min at room temperature. Cells were washed free of primary antibody with 0.1 M phosphate buffer. Controls were prepared by omitting application of primary antibody to the samples or replacing the antibody with either preimmune serum or bovine serum albumin. A secondary antibody conjugated to fluorescein isothiocyanate (FITC) was then applied for 30 min at room temperature. Cells were washed free of secondary antibody, and the coverslips mounted in glycerol/phosphate buffer (9/l). Specimens were examined for the presence of bound antibody on a Zeiss Axiophot using a FITC epifluorescence blue-wavelength (450-490 nm) excitation filter set.

RESULTS

In Situ Localization of Intracellular TGF-(3,

and Extracellular

Two antibodies raised against a TGF-0, synthetic peptide (Flanders et al., 1988) define the intracellular (anti-LC) and extracellular (anti-CC) temporal-spatial distribution of this growth factor in the epithelial and mesenchymal tissues of the developing inner ear from El0 to El6 (Table 1). TGF-P, was present in the epithelial cells (anti-LC antibody) at the earliest stage (i.e., El0 otocysts) of otic development studied, and persisted until El4 (Fig. 4A) when otic morphogenesis is essentially complete. The developmental stages that had the densest immunostain deposition over their otic epithelium (i.e., El0 and E13; see Table 1, Figs. 1A and 3A)

FRENZ

ET AL.

TGF-p, Injuences

correlate respectively with the events of initiation (E10.5) and inhibition (E13) of chondrogenesis, both of which are known to be mediated by epithelial-mesenchymal interactions (Frenz and Van De Water, 1991). Reaction product indicating the intracellular presence of TGF-/3, was sparse over the epithelial tissue of the El2 otocyst except for the most ventral aspect of the forming cochlear duct, which stained moderately (i.e., 2f) for the presence of this signal peptide (see Fig. 2A). The epithelial tissue of El6 mouse inner ears did not stain for the presence of intracellular TGF-P,. Localization of extracellular TGF-fi, (anti-CC antibody) in the otic mesenchyme showed great variation of the immunostaining pattern. Stain deposition over the periotic mesenchyme surrounding the El0 otocyst was uniform and light (Fig. 1B) in contrast to the very dense deposits of reaction product over the extracellular matrix of the periotic mesenchymal cells of the El2 otocyst that are dorsal and lateral to the developing horizontal semicircular duct of the vestibular apparatus (Fig. 2B). This dense immunostain associated with the dorsal portion of the vestibule was in contrast to the moderate stain deposition over the lateral mesenchyme that was adjacent to the mid- and ventral vestibular structures

Otic Capsule Fomtutiou

327

and forming cochlear duct, observed in El2 otocysts (Fig. 2B). Extracellular matrix staining for TGF-/3, of periotic mesenchyme that forms the cartilaginous otic capsule was at its greatest intensity at El3 (Fig. 3B). Intense immunostaining of the forming perilymphatic spaces was also detected in El3 specimens (Fig. 3D). However, on El4 almost no reaction product was present over the condensed chondrifying mesenchymal cells. This was in sharp contrast to the loosely organized mesenchymal cells that compose the developing perilymphatic spaces that stained intensely for the presence of extracellular TGF-& (Table 1, Fig. 4B). By El6 the epithelial tissues of the inner ear no longer stained for the intracellular presence of the TGF-& signal peptide, but there was still a highly localized intense extracellular staining for this growth factor in areas of perilymphatic space formation surrounding the semicircular ducts (Table 1, Figs. 5A and 5B). Enzymatic treatment of tissue sections with hyaluronidase to remove matrix in general decreased, and in some instances (e.g., E12) completely abolished immunostaining for TGF-/3,. The only exception to this was noted at El6 when enzymatic treatment enhanced the staining of the matrix of the cartilaginous capsule of the inner ear.

FIG. 1. El0 mouse embryo inner ear, TGF-P, antibodies, PAP method. (A) LC antibody: Reaction product (orange-brown color) is concentrated over the otocyst (0), epithelium (2+), and neuroepithelium (2+), of the adjacent rhombencephalon (b), while only a diffuse (k) immunoreaction product is present over the cells of the periotic mesenchyme (m). (B) CC antibody: Neuroepithelium is unlabeled in contrast to the light immunolabel (l+) present over the surrounding periotic mesenchyme (m). FIG. 2. El2 mouse embryo inner ear, TGF-0, antibodies, PAP method. (A) LC antibody: Densest immunoprecipitate (2+) is over the epithelium of the forming cochlear duct (cd), with lighter immunolabel (I+) accumulated over the epithelium of the vestibule (v) and forming lateral semicircular duct (d). (B) CC antibody: Densest reaction product (3+), indicating extracellular TGF-/3, peptide is present over the condensed mesenchyme (m) that is opposite the anlage of the lateral semicircular duct, while the aggregated mesenchyme lateral to the forming cochlear duct shows moderate (2+) accumulation of immunolabel. The loosely organized mesenchyme at the tip of the cochlea and medial to the cochlear duct is only lightly (l+) labeled for the presence of this growth factor. FIG. 3. El3 mouse embryo inner ear, TGF-0, antibodies, PAP method. (A) LC antihody: Dense immunolocalization of TGF-p, (3+) is now uniformly present over both vestihular (i.e., utricle, u; saccule, s; superior semicircular duct, d) and auditory (i.e., cochlea, cd) sensory structures while rhomhencephalic tissue and the vestibular (vg) and acoustic (ag) ganglia display moderate levels (2+) of immunolabel. (B) CC antibody: The densest (4+) accumulation of immunoprecipitate is localized over the condensed mesenchyme that is fated to form the otic capsule (c) and the footplate of the stapes (f), with the mesenchyme of forming perilymphatic spaces (p) also showing a dense accumulation (3+) of immunolabe1 for TGF-P,. (C) The lateral semicircular duct of Fig. 3A at higher magnification shows dense (3+) intracellular (LC antibody) label for TGF-lji to he predominantly concentrated over the epithelial cells that compose this duct, (D) The extracellular (CC antibody) immunolocalization of this growth factor is in sharp contrast with Fig. SC, with immunoprecipitate exclusively localized over the matrix of the periotic mesenchyme that surrounds the lateral semicircular duct that was shown at lower magnification in B. Figures 1,2, 3A, and 3B: bar = 100 pm; Figs. 3C and 3D: har = 25 pm. FIG. 4. El4 mouse inner ear, TGF-0, antibodies, PAP method. (A) LC antibody: Moderate concentration (2+) of immunoprecipitate is present over both the vestibular (i.e., utricle, u; saccule, s) and auditory (i.e., cochlear duct, cd) receptor epithelium and only light (l+) immunostaining is present over the ganglia (i.e., vg, ag) that innervate these receptors. The neuroepithelium of the rhombencephalon is almost devoid (t-) of labcll for intracellular TGF-&. (B) CC antibody: Immunolabel of TGF-0, is no longer evident over the forming cartilaginous capsule (c) of the inner ear, contrasted by the presence of dense (4t) immunoprecipitate over the cellular matrix of the forming perilymphatic spaces (p), There is no labeling of the epithelial structures (i.e., u, s, cd) of the inner ear. FIG. .5. El6 mouse inner ear, TGF-0, antibodies, PAP method, CC antibody. (A) Immunolabeling of the perilymphatic spaces (p) is more restricted to sites of active morphogenesis. The perilymphatic spaces that surround the developing semicircular ducts (i.e., superior semicircular duct, d) are completely labeled (3-t) while the perilymphatic spaces surrounding the developing utricle (u) and saccule (s) are labeled only at thr interface between the forming perilymphatic spaces and the cartilaginous capsule (arrowheads). (8) A high-power view of the superior semicircular duct of Fig. 5A showing the perilymphatic spaces (p) and cartilaginous capsule (c) surrounding this superior semicircular duct, revealing the exclusive concentration of immunolabel for extracellular TGF-0, over the cellular matrix of the forming perilymphatic space. Figures 4 and 5A: bar = 100 pm; Fig. 5B: bar = 25 pm.

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DEVELOPMENTALBIOLOGY

VOLUME 153,1992

FRENZETAL.

TGF-0, IWJ~UI

329

TABLE2 EFFECTSO~FTGF-&ONMESENCHYMALCONDENSATION Condensation Mesenchyme

age at culture E10.5 El20 E13.0 E14.0

Control 0 15 rt 1 115 + 7 55 -t 9

Note. Values represent the mean number of condensations to five cultures per experimental group. All cultures were exogenous TGF-6, (1 nglml) 18 hr after initial cell seeding period.

Eflects

qf TGF-/I,

on Mesenchymal

number t TGF-8, 0 34 rt 4 34 + 9 14 rt 2 for three exposed to for a Z4-hr

Condensation

Previous studies have shown that the enhancement of limb chondrogenesis by TGF-P is preceded by a stimulation of mesenchymal condensation (Leonard et a,l., 1991). On the basis of the hypothesis that TGF-& mediates the effects of otie epithelium on chondrogenesis, it was of interest to determine whether this factor influences otic chondrogenesis by an early stimulation or suppression of the condensation process. Mesenehymal condensations similar in appearance to those associated with otic chondrogenesis in sitzc develop in high-density cultures of periotic mesenchyme (Frenz and Van de Water, 1991). Just prior to the formation of these condensations (i.e., 18 hr after cell seeding), we introduced exogenous TGF0, (1 rig/ml, 24-hr exposure) to cultures of E12-El4 mesenchyme. Cultures were monitored by microscopic examination and mesenchymal condensations counted between the second and third days of culture. Mean values for condensation counts are given in Table 2. Comparison of values for experimental (.t TGF-&) and control cultures of El2 mesenehyme indicates that condensation number was increased by 55% in response to added TGF-P,. In contrast, suppression of mesenchymal condensation by exogenous TGF-&, indicated in Table 2 by a 70% decrease in condensation number, began on E13. In cultured El4 mesenchyme, comparison of experimental values with controls indicates a decrease in condensation number by 75% in response to exogenous TGF-& (Table 2). Eflects

of TGF-/I,

on S-GAG Accum.ulation

If TGF-0, is a factor that regulates capsular development, we reasoned that the effects on mesenehymal condensation (described above) should be reflected in the later chondrogenic event of S-GAG accumulation (Frenz and Van de Water, 1991). We therefore examined the extent of chondrogenesis in response to exogenous

TGF-0, by measuring the accumulation of Alcian bluepositive matrix at pH 1.0. In previous studies in periotic mesenchyme cultures, the presence of type II collagen was used as a marker of cartilage differentiation, and was shown to parallel the results for binding of Alcian blue stain, pH 1.0 {Frenz and Van de Water, 1991). Comparison of Alcian blue staining results for experimental (+TGF-fl,, 1 n&ml) and control cultures of El2 mesenthyme, given in Table 3, indicates that in response to exogenous growth factor, a striking increase of 51% in S-GAG accumulation had occurred. A similar increase in the quantity of Alcian blue-positive extracellular matrix (ECM) in cultures treated with TGF-& was observed in limb mesenchyme (Kulyk et al., 1989; Leonard et ah, 1991). In contrast, addition of TGF-P, to cultured El3 or El4 mesenchyme resulted in a marked decrease in bound Alcian blue stain and, therefore, S-GAG accumulation, by 47% in each case (Table 3). Concentrations of TGF-& as low as 0.5 rig/ml resulted in similar modulation of S-GAG accumulation, but stimulation and suppression were both less dramatic (Table 3). However, addition of TGF-0, at a concentration of 2 nglml had no effect on accumulation of S-GAG (not shown). Moreover, introduction of TGF-0, at a time subsequent to the onset of the condensation process (i.e., between 24 and 72 hr of incubation) did not enhance or suppress S-GAG accumulation. The observed modulation of S-GAG accumulation by TGF-0, may represent an effect of this growth factor on matrix retention. To address this possibility, we biosynthetically labeled S-GAG using 35SO;2 in cultures grown in the presence or absence of added TGF-/3,. All radioactivity incorporated into CPC-precipitable GAG of the cell spots at 7 days in culture (Wasteson et al., 1973; Leonard et ah, 1991) and of the pooled media collected over the ?-day culture period was separately as-

TABLE

3

EFFECTSOFTGF-PI ONS-GAG ACCUMULATION INCULTUREDMESENCHYME -tTGF-& Control E10.5 E12.0 E13.0 E14.0

0.027 0.473 1.421 0.458

2 + i f

0.5 n&ml 0.004 0.060 0.213 0.064

0.035 0.568 1.094 0.369

i: 0.002 i 0.076 ztz 0.096 c 0.023

1 rig/ml 0.125 0.954 0.750 0.241

_t i+t

0.040 0.246 0.229 0.087

Nota Values represent mean optical densities of matrix-bound Alcian blue stain (pfi 1.0) for three to six cultures per experimental group follotving extraction with 8 M guanidine hydrochloride. E12El4 mesenchgme cultures were exposed to TGF-8, 18 hr after initial cell seeding for a 24-hr period; E10.5 cultures were exposed to TGF-,S, for the entire culture period (i.e., 7 days).

FRENZETAL.

TGF-p,

Injuences

Otic Capsule Fomtrtim

331

TABLE4 INCORPORATIONOF%O;~INTOGLYCOSAMINOGLYCANS INCHONDROGENICCULTURES Age at culture

Treatment

El2

Control +TGF-0, Control +TGF-[I, Control +TGF-fi, Control tTGF-P,

El3

Fraction Cell Cell Media Media Cell Cell Media Media

cpm 78* 9 131 k 26 182? 7 240 + 7 2135 + 67 673 f 153 820 + 105 277 f 161

layer layer

layer layer

Note. Mesenchymal cells were grown in the presence or absence of TGF-$, (1 &ml, 24 hr) with the addition of 2-4 &i of Na%O,/ml to the culture medium. Cultures were maintained for a period of 7 days i)~ vifro. Each value represents the mean for two to four cultures per experimental group.

sayed. Representative experiments in El2 and El3 mesenchymal cultures are given in Table 4. The relative amounts of S-GAG in TGF-&-treated and control cultures (i.e., cell layers) corresponded to the Alcian blue staining results in Table 3. The amounts of S-GAG exported into the media of TGF-&-treated cultures were essentially proportional to the amounts exported into the media of control cultures, indicating that TGF-& did not act by increasing or inhibiting matrix retention.

Exposure of Mesenchyme

Sequentid

to TGF-/3,

To mimic the sequential stimulation and inhibition of chondrogenesis that occur during capsule formation in uiuo, we introduced exogenous TGF-0, (1 rig/ml) into cultures of El2 mesenchyme at two distinct times during the culture period. The first exposure of the cell cultures to TGF-8, (1 rig/ml) was prior to the condensation of mesenchyme (i.e., 18 hr after seeding). After a 24-hr period, the growth factor was removed, and precartilaginous cell condensations were now evident in all cultures. The second exposure to growth factor (TGF-&, 1 rig/ml, 24 hr) was on Day 3 (i.e., 66 hr after seeding). Control

TABLE

5

EFFECTOFSEQUENTIALEXPOSURETO TGF-&ON&GAG ACCUMULATIONINCULTURED El2 MESENCHYME Single

exposure

0.815 k 0.197

Double

FIG.6. High-density cultures of El2 periotic mesenchyme that were exposed to exogenous TGF-PI (A) once during the culture period, just prior to cell condensation, and (B) twice during the culture period, on Days 1 and 3. Comparison of A with B demonstrates a marked suppression of chondrogenesis following sequential exposure to growth factor. Note that the cells express a chondrogenic phenotype (arrow) in A but not in B. Phase-contrast micrographs, bar = 35 pm.

exposure

0.029 ? 0.032

,Vofe. Values represent mean optical densities of matrix-bound Alcian blue stain (pH 1.0) for two or three cultures per experimental group. Cultures were exposed to TGF-@, (1 n&ml) on either Day 1 (24 hr) or Days 1 and 3 (24 hr, each exposure).

cultures did not receive a second dose of exogenous growth factor. By Day ‘7in control cultures (i.e., a single 24-hr exposure to TGF-& on Day l), sites of precartilage condensation had become chondrogenic (measured by S-GAG accumulation; Table 5, Fig. 6A). In contrast, in cultures that were sequentially exposed two times to

332

DEVELOPMENTAL

c] Mesenchyme

BIOLOGY

+ Otic Epithelium

El 0.5

El3

El2 Age At Culture

FIG. 7. Comparison of the effects of TGF-@, and otic epithelium on S-GAG accumulation by cultured mesenchyme. Note that TGF-PI mimieks the effects of otic epithelium in E12, E13, and El4 mesenthyme and partially mimicks these effects in E10.5 mesenchyme. Depicted values represent the mean optical densities for S---IO cultures per experimental group following extraction of matrix-bound Alcian blue stain (pH 1.0).

TGF-PI (i.e., two separate exposures to growth factor, Days 1 and 3), there was no further development of the condensed periotic mesenchyme into a chondrogenic phenotype and, correspondingly, accumulation of SGAG did not occur (Table 5, Fig. 6B). Moreover, when an additional set of cultures of El2 mesenchyme were exposed to TGF-,&, on Day 3 (i.e., 66 hr after seeding) but not on Day 1, chondrogenesis was neither enhanced nor suppressed. Modulation TGF-0,

but Not Initiation

of Otic Chondrogen,esis by

We have previously shown that isolated E10.5 inner ear mesenchyme does not differentiate into cartilage in culture (Frenz and Van de Water, 1991). We were therefore interested in determining whether TGF-/3, can act to initiate chondrogenesis in this early mesenchyme. Cultures of El05 periotic mesenchyme were prepared as described. TGF-& was introduced into cultures at 0.5, 1.0,5.0, or 10.0 rig/ml either on the initial day of culture {i.e., 1 hr after seeding) or 18 hr after seeding of the mesenchymal cells. Exposure to added growth factor was varied between 24 hr and 7 days (i.e., continuous). Under none of these conditions did cellular condensations form and develop into chondrogenic foci, thus demonstrating the absence of chondrogenic initiation by TGF-6,. However, TGF-8, (l-10 rig/ml, 7 days) did stimulate mesenchymal cells to synthesize to a limited extent an Alcian blue-positive ECM (Table 3).

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153,1%X2

Modulation of S-GAG accumulation in cultured mesenchyme in response to exogenous TGF-& was compared to the effects of otic epithelium on S-GAG accumulation in cultured mesenchyme (Frenz and Van de Water, 1991) and to S-GAG accumulation in control cultures (i.e., no TGF-PI or epithelium; Fig. 7). While the addition of TGF-& (I rig/ml, 24 hr) closely paralleled the stimulation and suppression of S-GAG aecumulation by otic epithelium in cultured El2 and E13-El4 mesenchyme, respectively, it partially mimicked the enhanced S-GAG accumulation by epithelium in cultured E10.5 mesenchyme (TGF-& at 1 rig/ml, ‘7 days). Furthermore, the increased or decreased cell condensation number that respectively occurred when El2 or E13-El4 mesenchyme was grown in the presence of otic epithelium (Frenz and Van de Water, 1991) was also reproduced by the presence of TGF-& in the culture medium (Table 2).

Rather than directly affecting cell differentiation, TGF-/3, may have resulted in modulation of the condensation process and S-GAG accumulation by influencing the rate of cell proliferation. To address this possibility, we measured DNA content by microfluorometric quantitation in chondrogenic cultures grown in the presence or absence of added growth factor. Comparison of TGF&-treated El&E13 cultures with El2El3 controls on Day 7 indicated similar quantities of DNA &g/ml) under both culture conditions (Fig. 8).

30.

25

El2

Age at Culture

I

Control

B

+ TGF-P, (Ing/ml)

El3

FIG. 8. Microfluorometric quantitation of DNA in TGF-&-treated and control cultures of El2 and El3 periotic mesenchyme. Absorbance values are expressed as DNA concentration in fig/ml. DNA contents in TGF-&-treated and nontreated mesenehymal cultures are essentially the same.

IVI w unolocalixation

of TGF-p, in Culture

The correspondence between the effects of otic epithelium and the addition of TGF-& on otic chondrogenesis led us to investigate the localization of endogenous TGF-8, in cultured mesenchyme and epithelium of the developing mouse inner ear. High-density cultures of El4 mesenchyme alone or El4 mesenchyme containing otic epithelium were maintained for a period of l-3 days i?r vitro in the absence of added growth factor, and then fixed and immunofluorescently labeled for the presence of endogenous TGF-P, Prior to examination of the cultures for the presence of bound anti-TGF-P, antibody, the fixed cultures were observed by differential interference contrast microscopy to identify epithelium and/or mesenchyme. In cultures of mesenchyme alone, no immunostaining for TGF-0, was present on any of the culture days examined (i.e., Days l-3), even at sites of incipient cellular condensation. However, in Day 1 cultures of mesenchyme containing otic epithelium (Fig. 9A), this was contrasted by the presence of intense staining for TGF-& in the otic epithelium (Fig. 9B). When similar Day 2-3 cultures were immunofluorescently labeled, TGF-P, was detected not only in the epithelial tissue, but in the surrounding mesenchyme as well (Fig. 9C). In control specimens, no immunostaining occurred. Similar results were obtained in cultures of El%El3 mesenthyme and E10.5-El3 mesenchyme containing otic epithelium. DISCUSSION

The pattern of TGF-0, distribution in the epithelial and mesenchymal tissues of the anlagen of the inner ear between El0 and El6 and the in vitro effects of TGF-& on the chondrogenic potential of a staged series of periotic mesenchyme cultures imply a specific regulatory role for this growth factor in otic capsule morphogenesis. The presence of TGF-0, at sites of incipient cartilage formation in the forming otic capsule (ElO-E13, Figs. 1-3) concurs with earlier observations in which participation of this signal peptide in cartilage formation was suggested for both axial skeleton and craniofacial structures (Heine et al., 1987). We have further defined a relationship between sites of chondrogenic suppression (e.g., formation of perilymphatic spaces) and localization of TGF-0, polypeptide (E13-14, Figs. 3 and 4). Inhibition of chondrogenesis during capsule remodeling and perilymphatic space formation (E13-14, Figs. 3 and 4), in addition to initiation of otic chondrogenesis (E10.5, Fig. 1) and condensation of chondrifying mesenchymal cells (El& Fig. 2), are events regulated by otic epithelium (Frenz and Van de Water, 1991). Identification of TGF-0, first in the epithelium (El0 and E12, Figs. 1 and 2), then in the mesenchyme fated to form the presump

FIG. 9. Immunolocalization of endogenous TGF-p, in vitro. (A) Differential interference contrast photomicrograph of a Day 1 culture of El4 periotic mesenchyme containing El4 otic epithelium. (B) Immunofluorescent photomicrograph of the same field, demonstrating the localization of endogenous TGF-0, in the otic epithelium (E) only. (0 Immunofluoreseent photomicrograph of a similar Day 2 culture of El4 periotic mesenchyme containing El4 otic epithelium now shows the binding of anti-TGF-0, antibody to both the otic epithelium and its surrounding periotic mesenchyme.

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tive otic capsule and perilymphatic spaces (E13-E14; Figs. 3 and 4), clearly implicates TGF-P, as a factor involved in this epithelial control. Furthermore, expression of TGF-& mRNA in the sensory epithelia of the developing inner ear (Gatherer et al., 1990) supports otic epithelium as a source of TGF-&-like factors. In a complementary series of in vitro experiments, high-density cultures of embryonic mesenchyme were utilized to provide an effective model for investigating the function of TGF-& in regulation of chondrogenic differentiation. Consistent with studies of limb bud chondrogenesis by Leonard et al. (1991), a correspondence between mesenchymal condensation and otic capsule chondrogenesis occurred in response to treatment with TGF-/3, (Tables 2 and 3). Not only was stimulation of the condensation process in inner ear mesenchyme paralleled by enhancement of otic chondrogenesis (measured by S-GAG accumulation and 35S0,2 incorporation), suppression of both indices corresponded as well (Tables 2 and 3). In addition, the lack of a stimulatory or inhibitory response by the periotic mesenchymal cells to TGF-P, that was administered subsequent to the development of cellular condensations (i.e., after 18 hr of culture) supports TGF-/3 as a regulator of condensation formation (Leonard et al, 1991). In accord with the studies of Kulyk et al. (1989), TGF-P, did not act on in vitro chondrogenesis by affecting cell proliferation, since there was essentially no difference in DNA content of TGF-@,-treated and control cultures (Fig. 8). Moreover, 35SO;2 incorporation into GAG (Table 4) indicated that modulation of S-GAG accumulation by TGF-0, in cultured mesenchyme resulted from its effects on S-GAG synthesis, not matrix retention. A role for a TGF-P-like molecule in chondrogenic pattern formation has been suggested in the embryonic limb (Newman, 1988; Leonard et al., 1991). Complex patterns of chondrogenesis are also involved in establishing a distinct otic capsular morphology and in sculpting of the perilymphatic spaces. These morphogenetic processes are likely to be controlled through the action of local regulatory molecules that either stimulate chondrogenesis to permit capsular growth, or selectively inhibit this process to allow for capsular remodeling. We have shown that not only can TGF-0, elicit both of these cellular responses as a function of the developmental age of the treated mesenchyme (i.e., E12, stimulation; E14, inhibition; see Tables 2 and 3), but also the same cellular responses of stimulation and inhibition of chondrogenesis can be sequentially elicited in the same population of cultured mesenchyme cells. Thus, the addition of TGF-0, in vitro mimics the actual sequence of events that occurs in the embryo during inner ear development (Figs. l-5). Addition of TGF-/3, to cultures of mesenthyme of the earliest stage committed to chondrifica-

V0~~~~153,1992

tion (i.e., E12) (Frenz and Van de Water, 1991) results in a pronounced stimulation of chondrogenesis. However, when this same culture of El2 mesenchyme cells is exposed to TGF-P, a second time, at a period corresponding to El4 in viva (i.e., during perilymphatic space formation), suppression of chondrogenesis occurs. This inhibitory response is specific only to El2 mesenchyme that has previously been exposed to TGF-/3,, since addition of TGF-P, on Day 3 to nontreated mesenchyme cultures did not result in chondrogenic suppression. Thus, the sequential effects of TGF-P, on otic chondrogenesis in vitro parallel the natural occurrences of this growth factor in the otic epithelium (Table 1). Moreover, there is a striking resemblance between the influences of TGF-& and the effects of otic epithelium on in vitro chondrogenesis (Fig. 7). This correspondence is consistent with a mechanism of action involving the transfer of epithelial-derived growth factor to underlying mesenchymal cells (Lehnert and Akhurst, 1988). Furthermore, the localization of endogenous TGF-/3, first in cultured otic epithelium and then in otic epithelium and its surrounding periotic mesenchyme (Figs. 9b and c) provides strong evidence in support of TGF-P, as a paracrine mediator of otic capsule formation. In addition, the absence of this endogenous growth factor in cultures of mesenchyme alone does not indicate an autocrine regulatory effect on chondrogenic differentiation. In mesenchyme isolated from fetal rat muscle explants, TGF-& and TGF-P, can induce chondrogenesis and stimulate the synthesis of cartilage-like proteins (Seyedin ef al., 1985,1986,1987). Interestingly, TGF-6, is localized in the otic epithelium at 10.5 days of embryonic development (Fig. 1A). However, in cultured E10.5 periotic mesenchyme, TGF-/?, does not initiate otic chondrogenesis, whereas otic epithelium does (Frenz and Van de Water, 1991). Increased cell packing and S-GAG accumulation (Table 3) occur in response to added growth factor, suggesting that TGF-0, alone is not an adequate inductive signal to initiate cartilage differentiation in E10.5 periotic mesenchyme, but that it may play some role in this process. TGF-P, is only one member of a family of peptides that are expressed during active morphogenesis and differentiation in vivo and in vitro (Jakowlew et al., 1991; Pelton et al., 1990a,b) and that can interact with a family of cell receptors (Cheifetz et al., 1987; Ignotz and Massague, 198’7). Therefore, induction of chondrogenesis in this early periotic mesenchyme may require the action of some other growth factor(s) or synergistic interaction between TGF-/3, and another growth factor(s). Synergy between growth factors occurs during the induction of mesoderma1 differentiation in Xenopus embryos (Kimelman and Kirschner, 1987). The striking correlation between the distribution of

endogenous TGF-PI in the epithelium and mesenchyme of the developing inner ear in vivo and in vitro and the effects of exogenously administered TGF-P, in cultured periotic mesenchyme indicate a role for this signal peptide as a paracrine regulator of the epithelial-mesenchyma1 interactions that mediate the patterns of otic capsule chondrogenesis. It remains to be determined whether the activity of this epithelial-derived growth factor may also be autocrine, directly affecting the differentiation of the otic epithelium itself (Lehnert and Akhurst, 1988). Furthermore, it is of interest to determine whether the expression of different members of the TGF-fl superfamily, including BMP-2a and Vgr-1, coordinately regulates otic morphogenesis as has been suggested in other embryonic systems (Lyons et tsl., 1989; Lyons and Hogan, 1990). Finally, to further define its role in the initiation and modeling of the developing dtic capsule, it will be important to ascertain the relationship between TGF-0, and other growth-related factors produced by otic epithelium with respect to the initiation of chondrogenesis in competent but uncommitted periotic mesenchyme (i.e., El05 mesenchyme). This Grant D.A.F.,

work was supported NS-07098 to D.A.F., and NIDCD Research

by NINDS Neuropathology NIDCD Research Grant Grant DC-00088 to T.R.V.

Training DC-00081 to

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A maternal mRNA localized to eggs codes for a growth factor

Transforming growth factor beta 1 is an epithelial-derived signal peptide that influences otic capsule formation.

Interactions between epithelial and mesenchymal tissues in the developing inner ear direct the formation of its cartilaginous capsule. Recent work ind...
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