Partial characterization of a morphogenetic factor in the developing chick limb

The development of the distinct pattern of asymmetry along the anteroposterior axis of the avian limb has been investigated intensely during the past 10–12 years, stimulated largely by Saunders’ discovery that posterior limb mesoderm is capable of inducing supernumerary limb development (Saunders & Gasseling, 1968). When transplanted to more anterior limb sites, this posterior tissue not only induces supernumerary structures, but also controls the polarity of the anteroposterior (a–p) axis. Supernumerary structures are always polarized with their posterior borders adjacent to the transplant. Polarized supernumerary structures can develop from donor tissue when the reciprocal experiment is done, i.e. anterior tissue transplanted to more posterior limb sites (Saunders & Gasseling, 1968; MacCabe, Lyle & Lence, 1979; Fallon & Thoms, 1979; Iten & Murphy, 1980). Thus in a variety of experimental situations anterior limb tissue responds to the posterior polarizing influence. We have exploited this ability of the anterior tissue to respond to the posterior polarizing influence in the development of an in vitro bioassay for polarizing activity (MacCabe & Parker, 1975). When the anterior responding tissue is excised and cultured with an adjacent piece of polarizing tissue, the ectodermal ridge remains thickened for at least 48 h. When cultured alone, or with tissue lacking polarizing activity, the ectodermal ridge flattens and macrophages appear in the mesoderm as they engulf debris from the death and autolysis of some of the mesodermal cells. The cause of this limited cell death is unknown, but it occurs both in vivo (Hinchliffe & Gumpel-Pinot, 1981) and in vitro (MacCabe & Parker, 1975) in the absence of polarizing activity. The appearance of macrophages provides an easy method for quantitating the in vitro assay, the total number of macrophages after a given period of culture being inversely related to the morphogenetic activity. The exposure to posterior tissue in vitro polarizes the a–p axis of the responding tissue as demonstrated by separating the two tissues after 20 h of culture and transplanting the responding tissue to a host embryo. Small wing tips develop that are clearly polarized along their a-p axes (MacCabe, Knouse & Richardson, 1981). This in vitro assay has an advantage over in vivo transplantation methods in that cell-free fractions from polarizing tissue can be tested for activity. We have not yet provided a direct demonstration that cell-free preparations are capable of polarizing the responding tissue. It does, how ever, clearly maintain a thickened apical ectodermal ridge, a requisite for limb outgrowth. The morphogen described here thus may correspond either to the polarizing factor, or to the apical ectoderm maintenance factor hypothesized by Zwilling (Zwilling & Hansborough, 1956). Previously we reported that morphogenetic activity could be found in particulate, high molecular weight (HMW) and low molecular weight (LMW) forms, depending on .the method of isolation (MacCabe, Calandra & Paiker, 1977; Calandra & MacCabe, 1978). When cells from the posterior half of the limb were homogenized in phosphate-buffered saline a particulate form was found. Homogenizing in high-salt-phosphate-buffered saline yielded a soluble HMW ( > 300000 daltons) form. When culture medium was conditioned with posterior limb tissue, a LMW (3500–12000 daltons) form was found in the medium. This report deals with the properties of the HMW form of the morphogenetic activity.

The chick embryos for these experiments were obtained from White Leghorn hens maintained by the University of Tennessee Department of Animal Science. The eggs were incubated at 37·5 °C for 3 days, 2 ml of albumin removed, windows cut in the shell and covered with Parafilm and the eggs returned to the incubator until the following day. Embryos of late stage 22 and early stage 23 (Hamburger & Hamilton, 1951) were used as the source of donor limbs for sonication. For each replicate experiment all four limb buds were excised from 24–36 embryos, cut into anterior and posterior halves and sonicated with an Artec Sonic 300 (Artec Systems Corporation, Farmingdale, New York) in 3 μl/limb-half of high-salt (0·25 M-KCI) phosphate-buffered saline (HS-PBS) using the method of Richardson & Spooner (1977). Cellular debris was removed by centrifugation for 2 min in an Eppendorf microfuge and the supernatant centrifuged at 100000g for 20 min in a Beckman Airfuge. Except as noted this 100000g supernatant was used in all experiments. The posterior limb halves were used as the source of morphogenetic activity and the anterior limb halves as inactive controls for comparison.

A molecular weight estimation was made by gel filtration on a 0 · 9 × 12 · 5 cm Sepharose 6B (Pharmacia Fine Chemicals, Piscataway, New Jersey) column. Two hundred microlitres of the 100000g supernatant was applied to the column and eluted with HS-PBS. Twenty-drop (0 · 85 ml) fractions were stored frozen until thawed and concentrated to 150 – 180 μl by vacuum dialysis against MEM. Foetal bovine serum was added to 10 % and each fraction assayed for morphogenetic activity with 12 replicate cultures. Each culture consisted of the responding tissue, excised from the anterior region of the limb as previously reported (MacCabe & Parker, 1975), in 12 μl of the sample in a Falcon ‘microtest’ culture plate and incubated for 24 h at 37 °C in an atmosphere of 5 % CO₂ – 95 % air. The cultures were evaluated as described previously (MacCabe & Parker, 1975). While the ectodermal ridge thickness was evaluated subjectively to verify the relationship between the appearance of macrophages and ridge loss, it is not included in the tables since it does not contribute to the quantitation of the assay. In addition to the average number of macrophages per culture, the percentage of the maximum detectable activity (no macrophages) is calculated by dividing the average number of macrophages in the anterior samples minus that in the posterior samples by the number in the anterior samples and multiplying by one-hundred.

Experiments designed to test the sensitivity of morphogenetic activity to various degradative enzymes were performed with immobilized enzymes to allow for enzyme removal before the bioassay. Trypsin, protease and ribonuclease were obtained attached to carboxymethylcellulose (CMC) (Miles Laboratories, Inc., Elkhart, Indiana) and neuraminidase was attached to garose (Sigma Chemical Company, Saint Louis, Missouri). The 100000g supernatants were dialyzed for 24 h against 1000 volumes of Minimum Essential Medium (MEM) (Grand Island Biological Company, Grand Island, New York). To the retained volume was added 1 mg/100 μl of the washed immobilized enzyme (specific activities: trypsin 0 · 364 units/mg, protease 0 · 087 units/ mg, ribonuclease 0 · 211 units/mg and neuraminidase 0 · 04 units/mg). The reaction mixture was rocked for 18 h at 0 · 4 °C, the enzyme removed by centrifugation, the sample diluted 1:3 with MEM, and foetal bovine serum added to a 10 % level. Controls were treated the same way but without immobilized enzyme or with CMC alone. The samples were then tested for morphogenetic activity by culturing the responding tissues in them as before.

For ribonuclease and neuraminidase, control enzyme assays with known substrates were required to confirm the presence of activity under the conditions used. Ribonuclease was assayed by the method of Zimmerman & Sandeen (1965) in PBS using polycytidylic acid (Miles Laboratories, Inc., Elkhart, Indiana) as a substrate. Neuraminidase was assayed using the Worthington method (Decker, 1977) in a citrate buffer with a ten-fold reduction in scale using bovine mucin as a substrate.

The ability of the morphogenetically active component to bind Concanavalin A (ConA) was tested by affinity chromatography on a 0 · 5 × 5 · 5 cm ConA-Agarose column. Two-hundred microlitres of posterior 100000 g HS-PBS supernatant were added to the column and eluted with glucose-free HS-PBS at room temperature. Elution was monitored with an ISCO model UA-5 absorbance monitor (Instrumentation Specialties Company, Lincoln, Nebraska). Absorbance at 280 nm was no longer detected after 5 ml of eluent had come off the column, indicating the sample minus that binding to ConA, had come through. An additional 5 ml of eluent was collected and then the eluting buffer changed to 5 % α -methyl-D-mannoside (Sigma Chemical Company, Saint Louis, Missouri) in glucose-free HS-PBS. The next 5 ml was also retained. The first and last 5 ml fractions were concentiated to 150 – 180 μl by vacuum dialysis against MEM, foetal bovine serum added to 10 % and the sample assayed for morphogenetic activity as before.

The ability of Triton X-100 (Rohm and Haas, Inc., Knoxville, Tennessee) to yield the HMW form of the morphogenetic activity from the particulate form was examined by sonicating in PBS and adding Triton X-100 to the 5000g supernatant to a level of 0 · 1 %. After 20 min at 0 – 4 °C the deteigent was removed by treatment with Bio-Rad SM-2 Biobeads (Bio-Rad Laboratories, Richmond, California) for two hours. The Biobeads were removed by centrifugation and half the sample centrifuged at 100000 g. The 5000 and 100000 g supernatants were then dialyzed overnight against 1000 volumes of MEM. The retained volumes were diluted to one-fourth their starting volumes, foetal bovine serum added to 10 % and assayed for morphogenetic activity as before.

The heat stability of the HMW form was tested by incubating the HS-PBS 100000 g supernatants at the designated temperatures for 10 min, then dialyzing, diluting, adding serum and assaying as above.

The size of the HMW factor was estimated by gel filtration on Sepharose 6B after preliminary experiments revealed that activity remained in the void volume using Sephadex G-200. Fifteen 20-drop (0 · 85 ml) fractions were collected, concentrated and tested for activity. Only fraction no. 9 from posterior limb halves inhibited the appearance of macrophages and resulted in a thickened ectodermal ridge (Table 1, Figs 1 and 2). The possible low level of activity in fraction no. 10, though not found consistently in repeat experiments, suggested the activity might be largely at the low molecular weight end of fraction 9. The fraction size was cut in half (10 drops) and the ones (no. 17 and no. 18) corresponding to fraction 9 in the previous experiment were concentrated and assayed. In this case activity was found only in fraction 18 (Fig. 3), corresponding to a molecular weight range of 370000 – 415000 daltons using globular protein standards (aldolase, catalase, ferritin and thyroglobulin). The u.v. absorbance profile of inactive anterior supernatants was identical to that of the posterior supernatants.

In an attempt to determine the nature of the HMW molecule it was subjected to enzymatic degradation, then tested for morphogenetic activity (Table 2). The proteolytic enzymes trypsin and protease eliminated morphogenetic activity, while ribonuclease,neuraminidase and carboxymethyl-cellulose without attached enzyme had no significant effect. Ribonuclease and neuraminidase were assayed under identical conditions using known substrates or substrate plus the 100000 g supernatant from posterior limb halves. These controls showed the enzymes were active under the conditions used and the supernatants did not inhibit enzyme activity.

We examined the possibility that polysaccharides were associated with the HMW morphogenetic activity by affinity chromatography with ConA-Agarose. After application of posterior limb half supernatants to the column and elution with HS-PBS, little activity was detected (Table 3). Subsequent elution with methyl-D-mannoside, which dissociates ConA complexes (So & Goldstein, 1967), resulted in a small peak containing much of the activity (Table 3, Fig. 4).

An attempt was made to recover the soluble HMW form of the morphogenetic activity from the particulate from using Triton X-100. Treating the particulate form (isolated by sonication of posterior limb halves in PBS) with 0 · 1 % Triton X-100 failed to yield a soluble activity as indicated by its absence in 100000 g supernatants (Table 4). Activity was found in the 5000 g supernatant however, indicating the detergent didn’t destroy morphogenetic activity.

Heating the HMW form of the activity to 100 °C for 10 min destroyed morphogenetic activity but it was stable upon heating to 56 °C for 10 min (Table 5).

The experiments reported here provide a partial characterization of a morphogenetic factor(s) from the chick limb bud that gives the same response in an in vitro bioassay as tissue with polarizing activity and that maintains the ectodermal ridge in vitro. Polarizing tissue induces limb outgrowth and controls a – p polarity when in contact with appropriate responding tissue in vivo (Saunders & Gasseling, 1968) or in vitro (MacCabe et al. 1981). However, we have been unable to demonstrate significant stimulation of limb outgrowth after exposure of the responding tissue to the HMW factor even though the in vitro response is identical to that using intact polarizing tissue. This is not totally unexpected however, showing some similarity to results obtained with intact polarizing cells. When limb mesoderm is dissociated to a suspension of single cells, then reaggregated and covered with limb ectoderm, a limb develops without a-p polarity, i.e. symmetrically. When polarizing tissue is placed at the anterior or posterior end of the ectoderm, a limb polarized according to the position of the polarizing tissue develops (MacCabe, Saunders & Pickett, 1973). On the other hand if polarizing cells are distributed randomly throughout the dissociated and reaggregated mesoderm, the limb fails to develop (Crosby & Fallon, 1975). Thus both with polarizing cells or the HMW factor, a uniform distribution of activity .fails to stimulate outgrowth. The significance of these observations is not clear, particularly since transplanting responding tissue to a host limb 90° to the a-p axis, results in symmetrical outgrowth (MacCabe et al. 1979). The properties the polarizing tissue and the cell-free preparations have in common and the in vitro maintenance of the ectodermal ridge, appear to warrant the tentative identification of this cell-free activity as that of the polarizing factor or an ectodermal ridge maintenance factor. The distinction between these two hypothesized factors is not clear, being defined by transplantation and tissue recombination experiments. Both result in the maintenance of a thickened ectodermal ridge by subjacent mesoderm. It may not be reasonable with the evidence now available to attempt a distinction between the two.

Earlier we reported three size categories for the cell-free morphogenetic activity, particulate, HMW ( > 300000) and LMW (3500 – 12000) (MacCabe et al. 1977). The experiments reported here deal primarily with the HMW form of morphogenetic activity. The results of enzymatic degradation experiments suggest the morphogen is proteinaceous or at the least is protein requiring. Its activity is resistant to ribonuclease, neuraminidase and a 10 min exposure to 56 °C but is lost after boiling for 10 min. The factor binds to ConA, a lectin that binds to certain sugars, polysaccharides and glycoproteins (Goldstein, Hollerman & Merrick, 1965; Leon, 1967). These data suggest the HMW factor is a glycoprotein. The HMW form is not obtained from the particulate form with Triton X-100, .but is with HS-PBS (Calandra & MacCabe, 1978 and MacCabe & Richardson, unpublished). The results of gel filtration with Sepharose 6B suggests the HMW factor corresponds in size to globular proteins with molecular weights within the range of 370000 – 415000 daltons. The u.v. absorbance profile of both anterior and posterior supernatants were identical and there was no discernible peak where activity was found in posterior samples, suggesting the morphogenetic factor is present in very low amounts. The relationships between this HMW factor and the particulate and LMW forms is not clear. The fact that the HMW form can be obtained from the particulate form by washing with HS-PBS indicates these two forms are not separate entities. In addition, the LMW form is obtained by conditioning culture medium with polarizing tissue, suggesting this form is exported by source cells. With these facts in mind, at least three possible relationships between the particulate/HMW and LMW forms can be readily envisioned. The particulate/HMW form may be a precursor to the LMW form. Crick (1970) suggested an aggregate storage form for morphogens that establish a diffusion gradient as a way to keep the gradient constant. The concentration of the morphogen would thus depend upon the solubility of the morphogen rather than its rate of synthesis. A second alternative is the particulate/HMW form results from the attachment of the LMW form to a larger molecule in or on receptor cells in the mesoderm. This might serve to stabilize a gradient formed by a LMW diffusible morphogen as in the model suggested by Tickle, Summerbell & Wolpert (1975). In this instance the LMW form has the size required for a diffusible polarizing factor and the particulate/HMW form the more stable properties of the ‘apical ectoderm maintenance factor’ proposed by Zwilling & Hansborough (1956). The inability of the attached form to diffuse out of the limb could also account for the continued outgrowth of the limb after removal of the zone of polarizing activity. The attachment of the factor to the outside of mesodermal cells could also serve as a mechanism for closer cell-to-cell communication of the type suggested by Iten (1980). A third possibility is the particulate/HMW form results from fortuitous binding upon homogenization, and has no real developmental significance. We are currently trying to distinguish between these three possibilities experimentally. Preliminary evidence indicates that the LMW morphogen can be converted to a HMW form by a 90-min incubation with a high molecular weight fraction from anterior limb halves. This result appears to favour the later two possibilities over the first. For the present it seems clear that we have identified molecules of morphogenetic significance, probably the polarizing factor or related morphogens, that are found in the posterior region of the developing limb. Of even greater interest will be the challenge of determining the involvement of these molecules in establishing the pattern of asymmetry along the anteroposterior axis of the limb.

This work was supported by NIH grant HDO7282 and NIH-RCDA HD-00228 to J. A. M.