/. Embryol. exp. Morph. Vol. 29, 3, pp. 529-538, 1973
529
Printed in Great Britain
Competence tests of early
amphibian gastrula tissue containing nuclei of one
species (Rana palustris) and cytoplasm of
another (Rana pipiens)
By SALLY HENNEN 1
From the Department of Biology, Marquette University
SUMMARY
Competence tests of tissue from lethal nucleocytoplasmic hybrids consisting of diploid
R. palustris nuclei in R. pipiens cytoplasm were carried out by grafting pieces of early gastrulae to normal R. palustris early gastrula hosts. In the majority of cases, early gastrula ectoderm of the nucleoplasmic hybrids failed to differentiate further than if left in the intact embryo,
although the grafts healed nicely and survived for several days. Grafts of the dorsal lip of the
blastopore from nucleocytoplasmic hybrids to normal R. palustris hosts induced small
secondary embryos with prominent suckers or sucker Anlage, a characteristic of nucleocytoplasmic hybrid development. These results indicate that the abnormal behaviour of the nucleocytoplasmic hybrids is an inherent property of the tissue, in the sense that contact with normal
host does not alter its expression. Furthermore, the results with dorsal lip grafts suggest that
nucleocytoplasmic hybrid tissue influences the direction in which normal tissue differentiates.
INTRODUCTION
When diploid blastula nuclei are transplanted between Rana pipiens and
Rana palustris, the resulting nucleocytoplasmic hybrids, without exception,
develop abnormally (Hennen, 1965, 1967, 1912a). Thus, transfer of R. palustris
nuclei into enucleated eggs of R. pipiens gives lethal embryos whose development is characterized by macrocephaly. Embryos derived from the reciprocal
transfer are microcephalic. This abnormal behavior is not associated with
cytologically detectable alterations in karyotype and can be corrected by the
addition of a haploid set of chromosomes from the same species as the host
cytoplasm.
Since the nucleocytoplasmic incompatibility between R.pipiens and R.palusths
leads to highly predictable and characteristic abnormalities in the main morphogenetic events of early development (gastrulation and neurulation), we can ask
the following question. Are these abnormalities intrinsic to the tissue or can
their expression be altered when tissue from a nucleocytoplasmic hybrid is
1
Author's address: Department of Biology, Marquette University, Milwaukee, Wisconsin
53233, U.S.A.
530
S. HENNEN
placed in a normal environment? A test of these alternatives by standard grafting experiments (e.g. Spemann, 1938; Moore, 1947,1948; Brachet, 1944; King&
Briggs, 1953) is reported in this communication. The rationale for carrying out
such experiments is given in the Discussion.
MATERIALS AND METHODS
Fertilized controls
The procedures for obtaining and maintaining adult Rana pipiens and Rana
palustris as well as those for procuring gametes and raising embryos have been
previously described (Hennen, 1965, 1912a). The frequency of normal development displayed by fertilized controls was 90% or higher and did not vary significantly from one batch of eggs to another. The enucleation procedure
(Porter, 1939) used to prepare the recipients for nuclear transplantation was
monitored by producing androgenetic haploids. As in previous experiments
(Hennen, 1965, 1972a) this method was 100% effective.
Nuclear transplantation
The procedure for nuclear transplantation, as originally devised by Briggs &
King (1952), is given in detail elsewhere (Hennen, 1963, 1965, 1972a). In the
experiments communicated in this report, all donor nuclei were obtained from
diploid R. palustris blastulae. Nuclei from a single blastula donor were transplanted into both nucleated and enucleated R. pipiens eggs. When R. palustris
nuclei are transplanted into nucleated R. pipiens eggs, the transplanted and host
nuclei fuse to give triploid hybrids which develop into normal tadpoles (Hennen,
1967, 1972 a). Such triploid hybrids served as nuclear transplant controls for
the nucleocytoplasmic hybrids containing diploid R. palustris nuclei in R.
pipiens cytoplasm. Some of the nucleocytoplasmic hybrids were allowed to
develop. Others were sacrificed at early gastrulation for grafting experiments
carried out in the following way.
Grafting experiments
Except where noted, recipient hosts were fertilized JR. palustris controls and
donors were either fertilized R. pipiens controls or nucleocytoplasmic hybrids of
the type just described (i.e. diploid R. palustris nuclei in R. pipiens cytoplasm).
All of the operations except the enzyme treatments were carried out in a Plexiglass hood under sterile conditions. Solutions contained 0-1 % streptomycin and
0-1% penicillin and were passed through Millipore filters. Instruments and
dishes were sterilized in an autoclave prior to use.
During early cleavage, prospective hosts were manually dejellied and treated
for 5 min with a solution containing 2 % cysteine-HCl, 0-2 % papain, and
0-2 % alpha chymotrypsin (3 x crystalline, Worthington), at pH 8 on a shaker.
Following this treatment the embryos were washed with several changes of
Competence tests of amphibian tissue
531
20% Steinberg's (1957) solution. Exposure to these enzymes considerably
weakens the vitelline membrane, thus facilitating its removal with forceps prior
to grafting. Donor embryos received no treatment but the vitelline membranes
were removed in such a way that any ensuing minor damage to the embryo's
surface coat did not occur in areas destined for donor grafts. In several cases
each donor supplied tissue for more than one graft.
Demembranated hosts and donors were rinsed in sterile Steinberg's solution
and placed in 60 mm Petri dishes containing Steinberg's solution over a base of
2% agar. Various areas as indicated in the results section were removed from
the hosts and replaced with donor grafts by cutting the tissue with a very fine
pair of scissors (Trident v 38559BR, Aloe Division of W. Curtain). After preparing the hosts, donor grafts of appropriate size were fitted in place where they
subsequently healed without the aid of glass rods or bridges.
When the grafts had healed, the embryos were transferred to fresh agar
bottom Petri dishes containing 10 % Steinberg's solution with antibiotics. Instead
of maintaining their spherical form, demembranated gastrulae flatten in such a
way that they resemble fat pancakes. Consequently invagination may be retarded (Spemann, 1938). This difficulty can be alleviated by positioning the
embryos in such a way that the developing blastopore faces the agar during
gastrulation.
RESULTS
In a series of preliminary experiments, different types of grafting combinations were attempted to see which ones would be the most successful. Presumptive ventral ectoderm, neural ectoderm, and chordamesoderm (dorsal lip)
were grafted either heterotopically or orthotopically between normal R. pipiens
and R. palustris controls. Since embryonic cells of R. palustris lack the high
concentration of melanin found in R. pipiens, grafted material is easily distinguished from host tissue. Fig. 1 depicts three control recipients of orthotopically grafted presumptive neural plates. Heterotopic grafts of presumptive
ventral ectoderm to presumptive neural plate and presumptive sense plate are
illustrated in Fig. 2. The subsequent development of these grafted embryos was
essentially normal until stage 21 of the Shumway (1940) series. Thereafter some
of the grafted material showed signs of being absorbed, a phenomenon described
by Moore (1947) and attributed to a tissue incompatibility between the two
species. However, as Moore pointed out, this drawback is a minor one since the
tissue has had ample time to differentiate. Not all of the interspecific control
grafts and none of the intraspecific control grafts were absorbed; such graft
animals were quite normal at the end of embryonic development.
Orthotopic grafts of the dorsal lip (organizer) were more difficult to carry out
and gave less successful results. In contrast, heterotopic grafts of the dorsal lip
to presumptive ventral ectoderm at the endodermal border yielded respectable
secondary embryos (Fig. 3).
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1
S. HENNEN
2
533
Competence tests of amphibian tissue
Table 1. Grafts of normal diploid Rana pipiens tissue to normal R. palustris hosts
Fate of graft
Donor
source*
Host
sitef
No. of
grafts
VE
VE
NP
DLP
DPL
SP
NP
NP
DLP
VE
8
7
2
4
6
No
differentiation
1
Differentiation
slightly
Normal
abnormal differentiation
6
6
2
4t
* VE = presumptive ventral ectoderm. SP = presumptive sense plate. NP = presumptive
neural plate. DLP = dorsal lip.
| Differentiation of secondary embryo similar to primary embryo of host.
Following these preliminary studies, several series of experimental grafts
were carried out with nucleocytoplasmic hybrids. Each series was accompanied
by control grafts of stage 10 R. pipiens donors to stage 10 R. palustris recipient
hosts. The results of the controls grafts are summarized in Table 1. All 27 grafts
healed nicely and 18 went on to differentiate normally. Five grafted embryos
showed slight reductions in the size of axial structures. In only four embryos did
the grafted material fail to differentiate.
Experimental grafts
Nucleocytoplasmic hybrids for grafting experiments were obtained by transplanting diploid R. palustris nuclei into enucleated R. pipiens eggs. The development of 43 nucleocytoplasmic hybrids not sacrificed for grafting is summarized
Fig. 1. Orthotopic grafts of presumptive neural plates between fertilized R. pipiens
(dark) and R. palustris (light) controls. The dorsal side of each embryo is at the left.
Scale line = 1 mm.
Fig. 2. Heterotopic control grafts or presumptive ventral ectoderm to presumptive
neural plate (left) and presumptive sense plate (right).
Fig. 3. Grafts of R. pipiens dorsal lip to ventral R. palustris. The grafts have invaginated and induced the formation of secondary embryos (arrows). Scale line = 1 mm.
Fig. 4. Grafts of presumptive ectoderm to the sucker forming region of R. palustris.
Top: donor graft from nucleocytoplasmic hybrid. Note large sucker. Bottom: donor
graft from R. pipiens control.
Fig. 5. Grafts of early gastrula ectoderm to various cephalic regions of/?, palustris.
Top: donor graft from nucleocytoplasmic hybrid. Note poor differentiation and
deterioration of grafted material.
Fig. 6. Grafts of nucleocytoplasmic hybrid dorsal lips to ventral R. palustris. The
grafts have invaginated and produced small secondary embryos. Arrows point to
the sucker Anlage of the secondary embryos. Note poor differentiation of primary
host axis.
534
S. HENNEN
Table 2. Development of nuclear transplant embryos
(Rana palustris nuclear donors, R. pipiens recipients)
Development f
Arrested - abnormal
Blast.
Total Normal Number* and
transfers cleavage observed Gast.
Recipient
eggs
Nucleated (triploid
Hybrids)
Enucleated (nucleocytoplasmic hybrids)
32
24
24
4
Post Normal
neur. tadpoles
Neur.
1
1
(75%)
118
87
18
(75%)
43
(74%)
10
19
14
(23%)
(44%)
(33%)
* Number observed = total normally cleaved blastula minus those used for grafting
experiments.
•f- % = percentage of number observed.
Table 3. Grafts of nucleocytoplasmic hybrid tissue to normal Rana palustris hosts
Fate of graft
Take but
Donor*
source
Host*
site
No. of
grafts
take
VE
VE
VE
NP
DLP
DLP
VE
SP
NP
NP
DLP
VE
10
15
11
2
2
11
—
1
—
1
—
1
Not
No
Abnormal
idifferentiation differentiation
8
13
10
1
1
3
* See footnote *, Table 1.
f Embryos were microcephalic.
% Development of primary andsecondary embryo abnormal.
2
1
It
It
n
in Table 2. As found in previous experiments (Hennen, 1967, 1972#), some of
these embryos are arrested during gastrulation, some die at neurula stages after
forming wide neural plates and folds, and some from early postneurulae with
extremely large anterior axial structures of which the large suckers are the most
pronounced. In these experiments a higher proportion of the nucleocytoplasmic
hybrids developed to postneurula stages (tail-bud) than was observed previously.
The data in Table 2 show also that the majority of triploid hybrid blastulae, as
expected, formed normal tadpoles.
In all of the experiments shortly to be described, donor tissue from nucleocytoplasmic hybrids at stage 10 was grafted to normal stage 10 R. palustris
Competence tests of amphibian tissue
535
hosts. Thus, all nuclei of the grafted embryos contained a R. palustris genome
but those in the graft had been interacting with R. pipiens cytoplasm since the
onset of development. Repeated attempts with the reciprocal graft (nucleocytoplasmic hybrid host, R. palustris donor) failed. Although the grafts healed, the
hosts inexplicably exogastrulated.
When tissues from nucleocytoplasmic hybrids were grafted to R. palustris
hosts most of the grafts healed as well as those in control grafts. After healing,
the grafts either failed to differentiate or they differentiated abnormally. Fig. 4
illustrates the development of a grafted embryo containing the presumptive
ventral ectoderm grafted into its sucker region. The sucker is proportionately
larger than that of the corresponding grafted control embryo picture below it.
The overwhelming majority (21/25) of grafts to presumptive ectoderm, or sense
plate, however, failed to show any significant differentiation (Table 3). Since
these grafts remained on the surface of the embryo they could be observed
throughout development. They showed the same peculiar wrinkling that is
observed in the ectoderm of whole nucleocytoplasmic hybrids during gastrulation. Although these grafts remained intact for several days, they eventually
lost their integrity when their viable hosts were in tail-bud stages (Fig. 5). This
was approximately the same time at which the most advanced nucleocytoplasmic
hybrids died.
Similarly, all but one graft of nucleocytoplasmic hybrid tissue to the presumptive neural plate of the R. palustris host failed to differentiate. Only one
grafted embryo of this type formed a neural tube. This embryo subsequently
developed poorly differentiated axial structures.
Although nucleocytoplasmic hybrid tissue expressed little capacity to respond
to stimuli from R. palustris hosts, it was capable of inducing a small degree of
differentiation on the part of host tissue. This was evident in grafts of nucleocytoplasmic dorsal lips to the presumptive belly region of R. palustris hosts. In
7 out of 10 cases a small secondary embryo was induced (Table 3). These grafted
embryos had two consistent characteristics. The most prominent feature of the
secondary embryo was the sucker Anlage (Fig. 6). The primary (host) embryo was
always more poorly differentiated than that of control grafts, suggesting that the
hybrid tissue in some way interfered with normal host development.
DISCUSSION
Two well-known features of early amphibian gastrulae were established several
years ago (review by Spemann, 1938). At the onset of gastrulation the entire
ectoderm has the capacity to form central nervous system and epidermis;
presumptive neural and epidermal tissue can be exchanged with no effect on
subsequent differentiation. On the other hand, the material which forms the
dorsal lip of the blastopore is already programmed to form the organizing region
of the chordamesoderm. When an additional dorsal lip is grafted to the ventral
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S. HENNEN
mesoblast or into the blastocoel of an early gastrula, a complete secondary
embryo is induced.
These characteristics of amphibian gastrulae have led to numerous attempts to
' rescue' tissue from lethal amphibian hybrids whose block in development occurs
at the beginning of gastrulation (review by Baltzer, 1952; Brachet, 1957; Briggs
& King, 1959). The results with lethal Rana hybrid grafted tissue have varied.
In some instances there was no beneficial effect on the host on the grafted
tissue (King & Briggs, 1953; Brachet, 1954). In other cases grafted material
from lethal hybrids survived and differentiated in normal hosts (Brachet, 1944;
Moore, 1947, 1948). In the latter case, such hybrids presumably lack the ability
to synthesize material required for further morphogenesis; this material would
diffuse from contiguous normal cells of the host into the graft, which consequently survives and differentiates (Brachet, 1957).
Since grafted tissue from some lethal hybrids which do not gastrulate can be
rescued, there was every reason to expect that tissue from nucleocytoplasmic
hybrids (R. palustris nuclei, R.pipiens cytoplasm), most of which develop to late
gastrula stages and beyond, would also survive and differentiate further when
grafted to normal hosts of the same species from which the original donor
nuclei were obtained.
However, early gastrula ectoderm of nucleocytoplasmic hybrids failed to
show any significant development when grafted to normal R. palustris hosts,
although the grafted material healed nicely and survived for several days.
Only 4 of 38 grafts exhibited substantial differentiation. In two of these, the
graft was in the sense plate, which subsequently developed large suckers, a
characteristic of nucleocytoplasmic hybrid development. Grafts of the dorsal lip
from nucleocytoplasmic hybrids to normal R. palustris hosts induced the formation of tiny secondary embryos in 7 of 11 cases. The most prominent feature of
these secondary embryos was the sucker Anlage.
These results indicate that the incompatibility between R. palustris nuclei and
R. pipiens cytoplasm responsible for the abnormal development of these nucleocytoplasmic hybrids is an intrinsic one in the sense that it cannot be compensated
for by material derived from the normal host. In fact, the results with dorsal-lip
grafts suggest that nucleocytoplasmic hybrid tissue may influence the development of normal tissue.
The failure of nucleocytoplasmic hybrid tissue to undergo extensive differentiation when grafted to normal hosts could mean that either prior to or subsequent
to grafting, the nuclei, as a consequence of interacting with the foreign cytoplasm, undergo stable changes affecting their developmental capacity. While
this possibility cannot be ruled out, it seems unlikely for the following reason.
Under suitable experimental conditions (Hennen, 1970), nuclei from nucleocytoplasmic hybrids in late gastrula or early neurular stages can promote
completely normal development when transplanted into enucleated eggs of
their own species (Hennen, \912b).
Competence tests of amphibian tissue
537
Why grafted tissues from some lethal Rana hybrids derive benefit from host
tissue and others do not is difficult to explain. One possibility, and one which
is not easily verified because of lack of documentation in the literature, is that
the survival and differentiation of the graft may depend on its initial size. If
there are specific components essential for morphogenesis which the hybrid
cells cannot produce, but can obtain by diffusion from the normal cells of the
host (Brachet, 1957; Briggs & King, 1959), presumably there would be a limit
in the distance over which this diffusion could take place (Crick, 1970). The
grafts in the experiments reported here were square and judged, in retrospect,
to be large although they were not measured. Whether or not smaller grafts or
ones with different dimensions would show more extensive differentiation
remains to be worked out.
This work was supported by grant GB 27357 from the National Science Foundation.
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(Received 31 July 1972, revised 4 December 1972)