/ . Embryol. exp. Morph. Vol. 71, pp. 223-232, 1982
Printed in Great Britain © Company of Biologists Limited 1982
223
Effects of ultraviolet light on the
activity of an avian limb positional
signalling region
By LAWRENCE S. HONIG 1
From the Department of Biology as Applied to Medicine,
Middlesex Hospital Medical School, and Laboratory for
Developmental Biology, University of Southern California
SUMMARY
Cells from a region on the posterior margin of the developing avian limb bud (the polarizing region) can signal positional information to responding anterior cells. Polarizing activity
may be assayed by disaggregation of the tissue into single cells, followed by reaggregation
and grafting of cell pellet. This method enables treatments of the cells while in single-cell
suspension. The effects of ultraviolet radiation (254 nm) were studied to determine the role
of nucleic acids in polarizing activity. Ultraviolet radiation eliminated quail polarizing
activity over the same range of doses as it reduced cell spreading (20-60 J/m2). In contrast
to the published effects of y-radiation in which polarizing activity is affected only at doses
much higher than those that are cell lethal, ultraviolet apparently abolishes cell survival
and polarizing activity at comparable doses. Compared to other biological systems, polarizing activity is quite sensitive to ultraviolet light: the D37 is 18 J/m 2 ; the extrapolation
number, n, is 3*6. From pomparison of the effects of ultraviolet radiation to those published
for ionizing X- or y-radiation, one may conclude that ultraviolet and y-radiation abolish
limb polarizing activity at equivalent nucleic acid dosages, not at equivalent cell lethal doses.
INTRODUCTION
During chick limb embryonic development there is a region on the posterior
margin of the limb bud which, when transplanted to an anterior site on a host
limb, causes mirror-image duplication in the definitive limb pattern. This zone
of polarizing activity was originally identified by Saunders & Gasseling (1968)
and A. B. MacCabe, Gasseling & Saunders (1973). Evidence suggests that the
polarizing region is responsible for signalling positional information along the
limb anteroposterior axis (Tickle, Summerbell & Wolpert, 1975). It is active in
normal development (Summerbell, 1979), signalling over distances of tens of
cell diameters (Honig, 1981a). The signal seems universal in the sense that
homologous regions' from other amniote embryos including reptiles and
mammals also induce chick limb reduplications when grafted into chick limb
1
Author's present address: University of Southern California, Laboratory for Developmental Biology, GER 323, University Park, Los Angeles, California 90089-0191, U.S.A.
8-2
224
L. S. HONIG
buds (Fallon & Crosby, 1977; J. A. MacCabe & Parker, 1976; Tickle, Shellswell, Crawley & Wolpert, 1976). In a study of the nature of polarizing activity,
Smith, Tickle & Wolpert (1978) examined reduplications following grafts of
polarizing regions subjected to various doses of 60Co y-radiation. Very large
amounts of radiation, ~ 960 Gy (lGy = 100 rad) were required to abolish
positional signalling by the polarizing region, doses much larger than these were
sufficient to abolish cell multiplication (Smith, 1982) or normal DNA synthesis
(Honig, Smith, Hornbruch & Wolpert, 1981). These experiments showed that
polarizing activity does not require cellular contribution by the graft; similar
signalling behaviour has been shown during regeneration in Drosophila imaginal
discs (Adler & Bryant, 1977), and the amphibian limb (Holder, Bryant & Tank,
1979; Maden, 1979). However, since y-radiation adversely affects a multitude
of cell functions (e.g. Honig et al. 1981), the cellular nature of the polarizing
region signal could not be determined. Interest in assessment of the role of the
cell nucleus in positional signalling instigated use of ultraviolet irradiation
which would have at least 10-fold greater selectivity for nucleic acids than other
cell components. The penetrating power of actinic u.v. radiation (~ 254 nm)
is quite limited: the half-attenuation thickness for tissue is ~ 10 /tm. Therefore,
irradiation of whole eggs, embryos, or even isolated polarizing regions
(~ 200/mi in dimension) is not an effective procedure. To determine the u.v.
sensitivity of polarizing region cells, a different assay had to be used. This assay,
recently discussed by Tickle (1981) and Honig & Hornbruch (1982) utilizes the
ability of polarizing region cells to undergo complete disaggregation into single
cell suspension, followed by centrifugal reaggregation yielding a cell pellet which
then shows polarizing activity (Saunders,. 1972). Here I show the results of
treatment of polarizing region cells with u.v. light using this assay. Quail polarizing regions are used to facilitate comparison of these results with those of
Smith et al. (1978) and Smith (1982) who examined the effects of y-radiation on
quail tissue. Quail tissue is also advantageous since the cells form more cohesive
pellets following the disaggregation-centrifugation procedure (Honig & Hornbruch, 1982).
MATERIALS AND METHODS
Fertile chick eggs were obtained from a local flock (Needle Farm, Enfield,
U.K.) and fertile eggs of Japanese quail (Coturnix coturnix japonica) were
obtained from a colony kept at the Middlesex Hospital Medical School.
To assay polarizing region activity, about thirty quail embryos at stages
21-23 (Hamburger & Hamilton, 1951) were placed in cold calcium- and
magnesium-free Hank's Balanced Salts Solution (HBSScmf, Gibco). The wing
and leg buds were excised, rinsed, and incubated in 2 % trypsin (Gibco, 1:250)
in HBSScmf at 0° for 2 h. After four rinses with cold cmf Dulbecco's phosphatebuffered saline (PBScmf; Oxoid) the limb mesenchymes were freed of their
ectodermal jackets by dissection at 5-10 °C. Polarizing regions were cut out
Effects of ultraviolet on avian limb polarizing activity
225
Table 1. Effects ofu.v. irradiation on polarizing activity
Dose (J/m2)
0
No. operations
4-reduplication
3-reduplication
2-reduplication
no reduplication
Polarizing activity
32
24
4
2
2
85
Spreading
+ +
14
28
35
42
59
71
130 230 2700
7
5
2
0
0
10
6
2
1
1
7
0
5
2
0
6
0
5
1
0
8
0
2
4
2
13
0
0
3
10
13
0
1
2
10
11
0
0
0
11
6
0
0
0
6
6
0
0
0
6
90
77
57
61
33
8
10
0
0
0
++
+
+
0
0
0
0
0
4
+
+ +
+
+ +
+
+
from the mesenchymes, placed in 250 [A. of 0-25 % trypsin in PBScmf, and
incubated for ~ 12 min at 38°, interrupted three times by gentle vortexing.
After dissociation into single cells, 1 ml was added of cold Minimal Essential
Medium (Gibco) containing Hank's Salts, 25 mM HEPES, 2 mM L-glutamine,
10% foetal calf serum, and 1% antibiotic-antimycotic (HMEM). The cell
suspension was mixed, counted, and centrifuged down at 50g for 10 min. The
cells were resuspended in 300 /d of HMEM containing 3 mg/ml soybean trypsin
inhibitor, recentrifuged and resuspended in PBS. The cell suspension was kept
at 0 °C and divided equally into 1 ml aliquots (ca. 2 x 106 cells each) placed in
35 mm diameter open tissue-culture Petri dishes which were then irradiated at
4 °C at a rate of 35 /tW/cm 2 at a distance of 28 cm from a 12 in., 15 W bactericidal low-pressure mercury vapour lamp with ozone-suppressing liquid filter
(Hanovia Model 16, Slough, U.K.). The u.v. radiation was 9 5 % composed of
the 253-7 nm mercury line. The cells were then removed into tubes, HMEM
was added, and they were centrifuged, resuspended in HMEM, re-centrifuged
and incubated at 38° for 1-3 h. In some cases 50 /*g/ml concanavalin A (Sigma,
London) was present in the reaggregation medium to promote cell-cell adhesion
Reaggregated cell pellets were cut into pieces ~ 300 /on square by 100 /on thick,
consisting of about 104 cells, and transplanted into stage-19 to -22 host chick
wings (average stage 20 for each dose point), using the method of Saunders,
Gasseling & Cairns (1959). The test tissue was positioned between uplifted
apical ectodermal ridge and subjacent limb mesenchyme in an anterior position
opposite somites 16/17.
Operated chick embryos were sacrificed after an additional 6-8 days incubation and the limbs were fixed and stained with Alcian Green 2GX (Summerbell,
1979). Polarizing activity was quantified (Honig et al. 1981) by assigning 3 points
to limbs with duplicated digits 4 (Fig. 1 a), 2 points to those containing duplicated digits 3 (but not 4; e.g. Fig. \b), 1 point to limbs showing only duplicated digits 2 (Fig. 1 c) and 0 points to limbs having no extra digits (Fig. 1 d).
Even if a digit was not fully or well-formed it was counted providing positive
226
L. S. H O N I G
Fig. 1. Wholemounts of operated limbs which have had grafts of treated polarizing
region cell pellets. Doses of (A) 0, (B) 28, (C) 42 and (D) J30 J/m2 u.v. radiation
resulted in these limbs with digit patterns: (A) 43234, (B) 233234, (C) 2234, and (D)
234 (normal). The limb in (B) shows a 'fused' digit 3. Such 'fused' or 'split' digits
are found somewhat more frequently using grafts of cell pellets under the apical
ridge than when using standard cube-shaped grafts of intact polarizing regions.
Magnifications are ~ 6 x .
identification could be made (e.g. the 4 in Fig. 1 a). The summed total is expressed as a percentage of the maximal possible total (all 4's, i.e. three times the
total number of operated limbs in a set) to yield a value of 0 % if there are no
reduplications and 100% if all operations result in 4-reduplications. This
percentage is a measure of the strength of polarizing activity. It has been shown
that attenuation of the polarizing region by a variety of means results in the
progressive loss of extra digits 4, 3, and 2 next to the graft (Smith et al. 1978;
Honig et al 1981; Tickle, 1981; Honig & Hornbruch, 1982). While the exact
digit pattern obtained depends on the site and stage of the host limb, the digit
adjacent to the graft on the anterior margin will depend only, for grafts opposite somites 16/17 in stage 19-22 embryos, on the 'strength' of the polarizing
region.
Cell viability was monitored by the cell spreading assay (Honig et al. 1981)
and by dye exclusion (Paul, 1975) using the inverted compound microscope.
For spreading assays, five to ten pieces of cell pellet were incubated at 38 °C in
HMEM for 24-48 h and the amount of cell spreading was scored with 0-3
plusses. In some cases, portions of treated cell suspensions were incubated as
well. In these cases spreading was the same as that observed for the pieces of
cell pellet. Control untreated cells scored + + + . For dye exclusion, cell
Effects of ultraviolet on avian limb polarizing activity
227
100
20
40
60
80
100
Ultraviolet dose (J/m 2 )
120
Fig. 2. Activity of limb polarizing region cells after irradiation with u.v. light. The
inset shows a semi-logarithmic plot of the data.
suspensions were examined and counted in a haemocytometer after 5-10 min
at room temperature in 0 0 1 % trypan blue (Gibco).
Ultraviolet dose rates were measured using the Blak-Ray detector model
J-225 (Ultra-violet Products Inc., San Gabriel, California) one of which was
kindly loaned by Dr W. Tampion of the Royal Free Hospital Medical School.
RESULTS
Six experiments were performed consisting of a total of 22 separate irradiations and 119 embryonic operations. Each experiment had its own shamirradiated control cells. The pooled data showing the effects of ultraviolet
radiation on the polarizing region cells are presented in Table 1 and Figs. 1 and
2. Increasing doses of u.v. radiation caused attenuation of signalling ability.
The effect on polarizing activity is not all-or-nothing but is graded: at doses
higher than 14 J/m 2 , no 4-reduplications occur and ability to specify extra digit
3 and extra digit 2 is progressively lost. Since control polarizing activity was
85%, the attenuation became significant (P(t) < 0 0 1 ; P(x2) < 0001) at a dose
L. S. HONIG
*
•
•
»
•
Fig. 3. Cultured limb polarizing region cells following irradiation with u.v. light.
Control (A) or irradiated quail cells were plated at 2x 106 cells per 35 mm tissueculture dish and incubated 2 days. Doses were (B) 7 J/m2, (C) 21 J/m2 and (D and E)
42 J/m2 (D is a rare group of spread cells, E is a representative field). Magnifications
are 430 x.
Effects of ultraviolet on avian limb polarizing activity
2
229
2
of 28 J/m and severe at 60-70 J/m . At still higher doses the u.v.-irradiated
cells did not signal. The semi-log plot in the inset to Fig. 2 has a shoulder
and in form is typical of multi-target or multi-hit irradiation effects (e.g. see
Casarett, 1968). There appears to be a threshold dose of ~ 20-30 J/m 2
followed by exponential decline of activity. The D37, or dose required for reduction by a factor of e, is ~ 18 J/m 2 and the extrapolation number, n, is
3-6. A figure of n > 1 implies some sort of cumulative radiation damage is
necessary for complete inactivation of polarizing activity. The n value may indicate that with these small grafts of reaggregated cell pellet there is still a 3-6-fold
excess of polarizing signal (number of cells?) over the minimum necessary to
produce a 4-duplication. (Alternatively one may assume that most grafts have
only that minimum strength, noting the presence of less than 100% activity in
the 0-4 J/m 2 columns of Table 1. In this case the n value would suggest that 3 or
4 hits, at one or more targets, per cell are necessary for loss of polarizing
activity.)
Spreading falls off in a similar manner to polarizing activity, although perhaps
slightly more rapidly as can be seen in Table 1 and Fig. 3. When examined for
trypan blue uptake 1 h after irradiation, at least 95 % of cells exclude dye
even after doses as high as 130 J/m 2 which completely abolishes polarizing
activity. At the very highest dose used (2700 J/m 2 ). 54 % of cells excluded
trypan blue. However, the lower doses of u.v. are ultimately lethal. After culture
for 20 h, fewer cells are able to exclude trypan blue after doses of 28 J/m 2
(61 %), 59 J/m 2 (49 %), 130 J/m 2 (30 %) or 2700 J/m 2 (0 %).
DISCUSSION
Ultraviolet radiation, like y- or X-radiation causes a progressive graded loss
of polarizing activity. There is no evidence for an all-or-nothing effect, for which
behaviour a tendency was observed in the chemical inhibitor study of Honig
et al. (1981) in which 4-reduplications and non-reduplications exceed in number
those of 3- and 2-reduplications. The graded nature of the u.v. results does not
seem to depend on the disaggregated cell assay since the u.v. results are more
graded than were chemical inhibitor grafts done by the same assay (Honig &
Hornbruch, 1982). And the results of Smith et al. (1978) were graded despite
using the conventional polarizing region assay. It is not certain whether the
graded quality in the u.v. and y-radiation experiments might be due to reduction
of each cell to a fraction of its full signal strength (or signalling lifetime, e.g.
Smith, 1980), or whether it might be due to reduction in the number of cells
capable of signalling (as per Tickle, 1981). Spreading and long-term viability
are affected in parallel with polarized activity following u.v. treatment, but
during the initial time period in which signalling occurs (~ 18 h; Smith, 1980),
at least 30-50 % of cells are 'viable' by the dye exclusion test over the dose range
where there is negligible (0-10%) polarizing activity (59-130 J/m 2 ). Reference
230
L. S. HONIG
to the numbers of cells required for signalling (Tickle, 1981), shows that the
u.v. inhibition of polarizing activity is not due simply to cell killing.
The effects of u.v. radiation on signalling occur over the dose range of 20100 J/m 2 . These amounts of radiation are similar to those lethal for cultured
mammalian cells for which clonable cell survival is reduced 90 % at doses of
20-50 J/m 2 (Erickson & Szybalski, 1963). However, the effective u.v. doses in
the chick limb are considerably lower than those doses required to destroy
'anterior determinants' in insect eggs or dorsal organization in amphibian eggs.
To produce 90 % double abdomens in Smittia eggs requires about 2000 J/m 2
(Kalthoff, 1977). While u.v. opacity of the insect chorion is not sufficient to
account for this difference, deep localization of the anterior determinants might
be responsible. Alternatively, the sensitivities may differ because the insect
phenomenon is nucleus-independent (cytoplasmic ribonucleoprotein is the presumed target) while the 20-fold more sensitive chick target might be nuclear.
Similarly, the ultraviolet doses required to impair dorsalization in Rana or
Xenopus eggs are 1000-2000 J/m 2 (Malacinski, Allis & Chung, 1974; Scharf &
Gerhart, 1980): the probable targets are superficial but proteinaceous.
The effects of u.v. radiation may be compared to those of ionizing irradiation
(Smith et al. 1978; Smith, 1982). Both agencies affect cell spreading and polarizing activity in roughly coordinated fashion. Comparison of the effective
amounts of radiation of the two types may yield information about the cell
target and can be performed in several ways. On the basis of pure energy
absorption, disregarding differences in quality of energy, it is readily calculable
from nucleic acid absorption coefficients, cell DNA content and volume that
1 J/m 2 of u.v. irradiation delivers about 10 Gy ( = 1000 rad = 105 erg/cm3) of
energy to DNA and 50 Gy to the cell. (Considering the number of DNA lesions
empirically observed by Paterson, 1978, i.e. ~ 1 hit/pg/rad X-rays and ~ 6000
hit/pg/J/m 2 u.v., one may calculate that 1 J/m 2 is equivalent to ~ 6000 rad =
60 Gy ionizing radiation which figure is similar to the immediately previous
figure.) An alternative basis for comparison of u.v. and y-radiation is provided
by study of the biological effects such as those effects on cell proliferation. For
example with human D98/AG cells, survival curves of colony-forming capacity
(Erickson & Szybalski, 1963) show that 1 J/m 2 is equivalent to about 0-3 Gy
which contrasts greatly with the values above. The two orders of magnitude
discrepancy in equivalent y-radiation doses reflects the potent lethality of
ionizing radiation. Examination of u.v. sensitivity curves of quail polarizing
activity shows that the D 37 is about 18 J/m 2 u.v. while for X-rays it is approximately 580 Gy (Smith, 1982). Hence, for polarizing activity, 1 J/m 2 is equivalent to about 32 Gy. Thus u.v. and X-radiation abolish polarizing activity at
equivalent nucleic acid dosages, not at equivalent lethal doses.
For ionizing radiation, much greater doses are needed to eliminate positional
signalling than to abolish cell survival while for u.v. radiation comparable doses
are needed. The above analysis suggests a nuclear role in positional signalling,
Effects of ultraviolet on avian limb polarizing activity
231
which may be contrasted with observations that cell proliferation (Smith, 1982)
and DNA synthesis (Honig et al. 1981) are not required. Recent experiments
designed to elucidate this matter have used cytochalasin B-induced enucleation
of polarizing region cells: but neither cytoplasts nor karyoplasts appeared
capable of signalling (Honig, 19816). If the polarizing region signal was a
cytoplasm-localized metabolic function or a cell surface function, the cytoplasts
were expected to signal. They did not but neither did the karyoplasts which
essentially contained only cell nuclei. These data, which suggest a requirement
for the cell nucleus, the data from biochemical inhibition studies (Honig et al.
1981; Honig & Hornbruch, 1982) and the u.v. studies presented here are consistent with the possibility that nuclear transcription is an essential element of
chick limb positional signalling.
I thank Dr J. C. Smith for comments and acknowledge with thanks the support of the
Anna Fuller Fund (Fellowship no. 487), the Medical Research Council of Great Britain, and
NIH grants DE-02848, DE-07006 and HL-28325.
REFERENCES
P. N. & BRYANT, P. J. (1977). Participation of lethally irradiated imaginal disc tissue
in pattern regulation in Drosophila. Devi Biol. 60, 298-304.
CASARETT, A. P. (1968). Radiation Biology, pp. 368. Englewood Cliffs, NJ.: Prentice-Hall.
ERICKSON, R. L. & SZYBALSKI, W. (1963). Molecular radiobiology of human cell lines. IV.
Variation in ultraviolet light and X-ray sensitivity during the division cycle. Rad. Res. 18,
200-212.
FALLON, J. F. & CROSBY, G. M. (1977). Polarizing zone activity in limb buds of amniotes.
In Vertebrate Limb and Somite Morphogenesis (ed. D. A. Ede, J. R. Hinchliffe & M. Balls),
pp. 55-69. Cambridge: Cambridge University Press.
HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of
the chick embryo. /. Morph. 88, 49-92.
HOLDER, N., BRYANT, S. V. & TANK, P. W. (1979). Interactions between irradiated and
unirradiated tissues during supernumerary limb formation in the newt. / . exp. Zool. 208,
303-309.
HONIG, L. S. (1981 a). Positional signal transmission in the developing chick limb. Nature,
Lond. 291, 72-73.
HONIG, L. S. (1981 b). Role of the cell nucleus for positional signalling in the embryonic chick
limb bud. /. Cell Biol. 91, Ala.
HONIG, L. S. & HORNBRUCH, A. (1982). Biochemical inhibition of positional signalling in the
avian limb bud. Differentiation 21, 50-55.
HONIG, L. S., SMITH, J. C , HORNBRUCH, A. & WOLPERT, L. (1981). Effects of biochemical
inhibitors on positional signalling in the chick limb bud. /. Embryol. exp. Morph. 62,
203-216.
KALTHOFF, K. (1977). Analysis of a cytoplasmic determinant in an insect egg. In Cell Interaction in Development (ed. M. Karkinen-Jaaskelainen, L. Saxen & L. Weiss), pp. 5-21.
London: Academic Press.
MACCABE, A. B., GASSELING, M. T. & SAUNDERS, J. W., Jr. (1973). Spatiotemporal distribution of mechanisms that control outgrowth and anteroposterior polarization of the limb
bud in the chick embryo. Mech. Age Dev. 2, 1-12.
MACCABE, J. A. & PARKER, B. W. (1976). Polarizing activity in the developing limb of the
Syrian hamster. /. exp. Zool. 195, 311-317.
MADEN, M. (1979). The role of irradiated tissue during pattern formation in the regenerating
limb. / . Embryol. exp. Morph. 50, 235-242.
ADLER,
232
L. S. HONIG
G. M., ALLIS, C. D. & CHUNG, H. M. (1974). Correction of developmental
abnormalities resulting from localized ultra-violet irradiation of an amphibian egg.
J. exp. Zool 189, 249-254.
PAUL, J. (1975). Cell and Tissue Culture, p. 368. Edinburgh: Churchill Livingstone.
PATERSON, M. C. (1978). Use of purified lesion-recognizing enzymes to monitor DNA repair
in vivo. Adv. Rad. Biol. 7, 1-53.
SAUNDERS, J. W., Jr. (1972). Developmental control of three-dimensional polarity in the
avian limb. Ann. N. Y. Acad. Set 193, 29-42.
SAUNDERS, J. W., Jr. & GASSELING, M. T. (1968). Ectodermal-mesenchymal interactions in
the origin of limb symmetry. In Epithelial and Mesenchymal Interactions (ed. R. Fleischmajer & R. E. Billingham), pp. 78-97. Baltimore: Williams and Wilkins.
SAUNDERS, J. W., Jr., GASSELING, M. T. & CAIRNS, J. M. (1959). The differentiation of prospective thigh mesoderm grafted beneath the apical ectodermal ridge of the wing bud in
the chick embryo. Devi Biol. 1, 281-301.
SCHARF, S. R. & GERHART, J. C. (1980). Determination of the dorsal-ventral axis in eggs of
Xenopus laevis: complete rescue of UV-impaired eggs by oblique orientation before first
cleavage. Devi Biol. 79, 181-198.
SMITH, J. C. (1980). The time required for positional signalling in the chick wing bud.
J. Embryol. exp. Morph. 60, 321-328.
SMITH, J. C. (1982). The effects of high doses of y-radiation on positional signalling in the
chick limb bud: radiation causes a decrease in cell number. Submitted to Differentiation.
SMITH, J. C , TICKLE, C. & WOLPERT, L. (1978). Attenuation of positional signalling in the
chick limb by high doses of y-radiation. Nature, Lond. 272, 612-613.
SUMMERBELL, D. (1979). The zone of polarizing activity: evidence for a role in normal chick
limb morphogenesis. /. Embryol. exp. Morph. 50, 217-233.
TICKLE, C. (1981). The number of polarizing region cells required to specify additional digits
in the developing chick wing. Nature, Lond. 289, 295-298.
TICKLE, C , SHELLSWELL, G., CRAWLEY, A. & WOLPERT, L. (1976). Positional signalling by
mouse limb polarizing region in the chick wing bud. Nature, Lond. 259, 396-397.
TICKLE, C , SUMMERBELL, D. & WOLPERT, L. (1975). Positional signalling and specification
of digits in chick limb morphogenesis. Nature, Lond. 254, 199-202.
MALACINSKI,
{Received 10 March 1982, revised 19 April 1982)