/ . Embryol. exp. Morph. Vol. 67, pp. 1-12, 1982
Printed in Great Britain © Company of Biologists Limited 1982
Partial characterization of a morphogenetic
factor in the developing chick limb
By JEFFREY A. MACCABE1
AND KATHRYN E.Y.RICHARDSON1
From the Department of Zoology, University of Tennessee
SUMMARY
A morphogenetic substance from the posterior region of the developing chick limb appears
to be a glycoprotein with a molecular weight in the range of 370000-415000. This morphogen
is capable of maintaining the apical ectodermal ridge of the limb in culture and is probably
related to the previously hypothesized polarizing or maintenance factors in limb mesoderm.
INTRODUCTION
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 & Thorns,
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,
1
Authors' address: Department of Zoology, University of Tennessee, Knoxville, TN
37916, U.S.A.
2
J. A. MACCABE AND K. E. Y. RICHARDSON
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 cellfree 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-phosphatebuffered 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.
MATERIALS AND METHODS
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 /il/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 100000 g 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 morphogenetic factor in the limb
Table 1. Morphogenetic activity in fractions from
Sepharose 6B gel filtration
No. of macrophages .
A
Fraction no.
5
6
7
8
9
10
11
12
13
14
Anterior
l/2s
Posterior
l/2s
%max.
activity
53-8
55-8
54-8
530
1-5
0-5
561
45-8
56-4
56-4
520
511
520
55-5
56-2
50-6
8-3
42-7
51-8
46-2
530
58-2
-2-6
9-8
81-9
24-3
8-2
11-2
-3-7
-11-9
A molecular weight estimation was made by gel filtration on a 0-9 x 12-5 cm
Sepharose 6B (Pharmacia Fine Chemicals, Piscataway, New Jersey) column.
Two hundred microlitres of the 100 000 g 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 ju] 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 fi\ 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
J. A. MACCABE AND K. E. Y. RICHARDSON
0-1 mm
0-1 mm
Fig. 1 (a). Responding tissue cultured with fraction no. 9 (Sepharose 6B) of the
supernatant of posterior limb halves. No macrophages are apparent after 24 h of
incubation, indicating a high level of morphogenetic activity, (b) The responding
tissue detached from the culture dish and turned 90° to view the ectodermal ridge
(arrow) profile.
A morphogenetic factor in the limb
0-1 mm
0-1 mm
Fig. 2 (a). Responding tissue cultured 24 h with fraction no. 9 (Sepharose 6B) of the
supernatant of anterior limb halves. Many macrophages are visible at one end of the
tissue. This serves as a baseline (no morphogenetic activity) for the culture in the
previous figure, (b) The ectodermal ridge (arrow) of the responding tissue after
culture in anterior fraction no. 9 is shorter and thinner than that in Fig. 1 b.
J. A. MACCABE AND K. E. Y. RICHARDSON
30
20
Fraction number
Fig. 3. The absorbance curve from Sepharose 6B gel filtration of a 100 000 g
supernatant of sonicated posterior limb halves. The first peak represents the void
volume (Fo). Morphogenetic activity is found only in the stippled region, corresponding to a molecular weight of 370000-415000 daltons.
Table 2. Stability of morphogenetic activity to enzymatic degradation
Treatment
None
Carboxymethyl-cellulose
(CMC)
CMC-trypsin
CMC-protease
CMC-ribonuclease
Agarose-neuraminidase
Anterior l/2s
Posterior l/2s
Anterior l/2s
Posterior l/2s
Anterior l/2s
Posterior l/2s
Anterior l/2s
Posterior l/2s
Anterior l/2s
Posterior l/2s
Anterior l/2s
Posterior l/2s
No. of
cultures
Average no.
macrophages
% max.
activity
26
32
22
21
16
22
20
21
26
20
18
18
45-6
7-7
48-4
9-4
471
42-2
390
36-4
470
9-9
440
2-4
00
831
00
80-6
00
10-4
00
6-7
00
78-9
00
94-5
York). To the retained volume was added 1 mg/100/4 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
A morphogenetic factor in the limb
Table 3. Morphogenetic activity after affinity chromatography
with agarose-ConA
Fractions 1-5
Fractions 11-15
Anterior l/2s
Posterior 1/2s
Anterior 1/2s
Posterior l/2s
No. of
cultures
Ave. no.
macrophages
% max.
activity
12
11
12
12
44-8
40-9
482
24-2
00
8-7
00
49-8
12
Fig. 4. The absorbance curve from agarose - Con A affinity chromatography. Little
morphogenetic activity was found in the first peak. After the addition of a methylD-mannose (arrow) to dissociate Con A complexes, a small peak appeared that
contained most of the morphogenetic activity.
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 x 5-5 cm ConA-
8
J. A. MACCABE AND K. E. Y. RICHARDSON
Table 4. Morphogenetic activity in Triton X-100 treated supernatants
5000g supernatant
100000g supernatant
No. of
cultures
Ave. no.
macrophages
% max.
activity
12
26
11
12
54-9
7-8
60-3
62-9
00
85-8
00
-4-3
Anterior 1/2s
Posterior l/2s
Anterior 1/2s
Posterior l/2s
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 % a-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 ji\ 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 100 000 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.
RESULTS
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
A morphogenetic factor in the limb
9
Table 5. Heat stability of the morphogenetic activity
56 °C
100°C
Anterior 1/2s
Posterior l/2s
Anterior l/2s
Posterior l/2s
No. of
cultures
Ave. no.
macrophages
% max.
activity
6
6
12
12
69-8
80
71-6
600
00
88-5
00
16-2
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, fenitin 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
100 000 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 form 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).
10
J. A. MACCABE AND K. E. Y. RICHARDSON
DISCUSSION
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
A morphogenetic factor in the limb
11
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 gieater 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.
12
J. A. MACCABE AND K. E. Y. RICHARDSON
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{Received 15 April 1981, revised 20 July 1981)