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146
JOURNAL OF
M.H.
EXPERIMENTAL
SCHWEITZER ET
ZOOLOGY
AL.
(MOL DEV EVOL) 285:146–157 (1999)
Beta-Keratin Specific Immunological Reactivity in
Feather-Like Structures of the Cretaceous
Alvarezsaurid, Shuvuuia deserti
M.H. SCHWEITZER,1,2* J.A. WATT,1 R. AVCI,3 L. KNAPP,4 L. CHIAPPE,5,6
M. NORELL,5,6 AND M. MARSHALL7
1
Department of Biology, Montana State University, Bozeman, Montana 59717
2
Museum of the Rockies, Montana State University, Bozeman, Montana 59717
3
ICAL Facility and Department of Physics, Montana State University,
Bozeman, Montana 59717
4
Department of Biological Sciences, University of South Carolina, Columbia,
South Carolina 29208
5
Department of Ornithology, American Museum of Natural History, New
York, New York 10024
6
Department of Vertebrate Paleontology, American Museum of Natural
History, New York, New York 10024
7
Walther Oncology Center, Indiana University, Indianapolis, Indiana 46202
ABSTRACT
We report small fibrous structures associated with a new specimen of Shuvuuia
deserti, which we hypothesize are remnants of feather-like epidermal appendages. Multiple analyses suggest that these structures are epidermally derived and contain epitopes consistent with βkeratin, a protein expressed only in extant “reptiles” and birds. Morphological, microscopic, mass
spectrometric, and immunohistochemical studies are consistent with the interpretation that these
structures are related to feathers. These data suggest that proteinaceous components may survive
across geological time and support the view that alvarezsaurids (Shuvuuia and its allies) are
either a lineage of birds or are a lineage phylogenetically close to them. J. Exp. Zool. (Mol. Dev.
Evol.) 285:146–157, 1999. © 1999 Wiley-Liss, Inc.
An exceptionally well-preserved skeleton of the
alvarezsaurid Shuvuuia deserti (IGM 100/977) was
recovered in 1993 in dune-derived fluvial sediments
at Ukhaa Tolgod in southwestern Mongolia by the
Mongolian-American Museum Expedition (Dashzeveg et al., ’95, Fig. 1a). Alvarezsaurids (Perle et
al., ’93, ’94; Chiappe et al., ’96; Novas, ’96)1 are
known from over 25 specimens at six localities
throughout Late Cretaceous sediments of the Gobi
Desert. Some of these specimens are complete and
exhibit remarkable structural preservation, including well-preserved skulls and postcrania (Chiappe
et al., ’98). Physical remnants of originally keratinous structures have been reported previously from
other specimens (oviraptorosaurs) collected from
Ukhaa Tolgod (Norell et al., ’95).
During preparation of IGM 100/977, small white
fibers of varying lengths and diameters were observed around the perimeter of the bones (Fig. 1b).
These fibers were organized in small clumps and
often lay nearly perpendicular to the axis of the
bones. The fibers varied in length and width, averaging about 200 µm in diameter. The spatial
orientation and arrangement of the extremely delicate fibers suggested that these might be remnants of feather-like structures. Furthermore,
these fibers are hollow like the rami or barbs of
all modern feathers. Figure 2 shows brightfield
and darkfield micrographs of a 0.5 µm cross section of one of the fibers.
With few and still controversial exceptions
(Chen et al., ’98; Qiang et al., ’98), the appearance of feathers in the fossil record has been unambiguously linked to the presence of birds.
1
The type specimen of Mononykus olecranus is from Bugin Tsav, a
putative Nemegt equivalent. Specimens collected at Ukhaa Tolgod
can be diagnosed as separate, but closely allied taxon, Shuvuuia sp.,
recently described Parvicursor from Barun Goyot beds, consequently
the collective mononykosaur is used here. IGM refers to Institute of
Geology, Mongolia.
Grant sponsor: National Science Foundation; Grant number: EAR
97-53187.
*Correspondence to: M.H. Schweitzer, Dept. of Biology and Museum of the Rockies, Montana State University, Bozeman, MT 59717.
E-mail: mary@iggy.oscs.montana.edu
Received 4 March 1999; Accepted 3 June 1999.
© 1999 WILEY-LISS, INC.
KERATIN IMMUNOREACTIVITY IN CRETACEOUS SPECIMEN
147
Fig. 1. (a) Photograph of Shuvuuia deserti during preparation showing articulated state
and sediments surrounding the specimen. (b) Fibers surrounding the skeleton in situ.
Recent feathers consist of keratin proteins, durable, hydrophobic filament proteins that, like
other structural proteins (collagen, osteocalcin),
are relatively resistant to degradation. Feathers
are not susceptible to digestion by commonly occurring proteolytic enzymes such as trypsin, and
must undergo some type of modification (e.g., disulfide bridge reduction, hydrolysis) for the degradation process to begin (Williams et al., ’90). For
these reasons, keratin-derived structures, including feathers (Davis and Briggs, ’95; Norell et al.,
’95; Chiappe et al., ’98; Schweitzer et al., ’99), are
second only to bones and teeth in appearance in
the fossil record (Logan et al., ’91).
Extant feathers generally consist of a hollow
calumus (root) and rachis (shaft), from which arise
hollow barbs and from these, barbules and barbicels (Lucas and Stettenheim, ’72). Variants of
the basic structure include down feathers, after-
feathers and filoplumes, and the small, unbranched, bristle-like facial feathers that are seen
near the eyes (Lucas and Stettenheim, ’72). The
shape and distribution of these variants are related to their role in aerodynamic function (Parakkal and Alexander, ’72), or insulating and/or
waterproofing capacity. The complexity of feather
structure is reduced by varying degrees in most
birds that have become secondarily flightless, and
is least constrained in ratites, which have been
flightless the longest (McGowan, ’89).
On the molecular level, mature feathers are the
result of the controlled expression of a family of
genes that code for the keratin proteins. Although
there are no “feather” genes, the diverse morphologies of feathers in a single specimen or among
different bird taxa are the result of variation in
timing and expression of a small group of homologous and relatively homogeneous beta keratin
148
M.H. SCHWEITZER ET AL.
Fig. 2. Dark-field (a) and bright-field (b) micrographs of
fiber in cross section. Arrow indicates hollow central region.
For comparison, a cross section of a duck feather barbule is
seen in fluorescent image in Figure 4. Bar = 40 µm.
genes (Gregg et al., ’83; Gregg and Rogers, ’86;
Brush, ’93, ’96).
Although alpha keratin is expressed in the epithelial tissues of all vertebrates (Sawyer et al., ’86;
Shames and Sawyer, ’87), the beta keratin structural proteins are unique to nonavian reptiles
(crocodiles, turtles and lepidosaurs) and birds
among extant vertebrates. Alpha keratins can be
distinguished from the more restricted beta
keratins by a greater molecular weight (40,000–
70,000), and protein filament diameters of 8–14
nm. Beta keratins can be divided into two groups.
The larger (MW 14,000–25,000) are isolated from
scale, claw, and beak tissues. Feather proteins are
smaller (MW ~10,000), as a result of a 50 bp deletion in the original beta keratin gene (Gregg and
Rogers, ’86), although the filament diameter in
both groups is 3–4 nm. In feathers, beta keratin
filaments occur in bundles oriented along the long
axis of the feather, a different pattern than that
observed in other epidermal structures in both
nonavian reptiles and birds (Parakkal and Alexander, ’72). Thus, while both alpha and beta
keratins are essential components of the differentiation of epidermal structures of nonavian reptiles and birds, mature feathers are unique among
epidermal structures in that they consist only of
beta keratin.
to avoid contamination. Treatment of modern controls was identical to that of fossil tissues for all
analyses. All experiments were repeated multiple
times to validate results.
The fibers associated with this specimen of
Shuvuuia were removed from the sediment matrix by saturating both with 100% ethanol. Once
isolated, the fibers were allowed to infiltrate overnight or for several days at 4°C in LR White (hard
grade) embedding medium (Ted Pella) designed
for immunohistochemical studies. The infiltrate
was removed, fresh medium applied, and allowed
to polymerize overnight at 60°C. Sections were cut
with dedicated glass knives to a thickness of 0.5–
1.0 µm using a Sorvall Ultramicrotome, and
mounted to glass slides coated with a gelatin adhesive. Sectioning was difficult in some cases because the sample tended to pull away from the
embedding material. Samples of duck feathers
were infiltrated and polymerized in tandem with
the fibers and sectioned with glass knives kept
separate from those used on the ancient material. Tissues were not fixed in either case because
it was felt that chemical treatment of the ancient
material should be kept to a minimum to avoid
alteration of any protein epitopes possibly preserved.
EXPERIMENTAL PROCEDURES
Tissue handling and preparation
At no time were any of the samples handled
directly, and instruments used in analyses were
either new and dedicated or sterilized before use
Scanning electron microscopy
Scanning electron microscopy (SEM) was performed on the ancient fibers associated with IGM
100/977 and compared with feathers from extant
birds. Unembedded sections of all specimens were
placed on double-sided, carbon tape as whole
KERATIN IMMUNOREACTIVITY IN CRETACEOUS SPECIMEN
mounts, coated with gold-palladium (10 Å) and
visualized using a JEOL 6100 scanning EM at
10–15 kV.
Transmission electron microscropy
Ancient and modern samples were infiltrated
and polymerized in LR White, as described. Ultrathin sections of approximately 90–95 nm were
collected on formvar-coated slot grids, counterstained with uranyl acetate and lead citrate, then
viewed using a Zeiss EM10C transmission electron microscope at 60–80 kV.
149
the amount of specific antibodies in the serum
was used to control for spurious binding due to
concentration effects. Other controls are outlined
in Schweitzer et al. (’99). Coverslips were applied
to the sections using Vectashield mounting medium (Vector) and viewed using a BioRad DVC
250 confocal microscope equipped with an argonkrypton laser and a Photonics cooled color integrating CCD camera. All data were integrated
over 1/8 second, and images were captured using
NIH Image software.
Immunochemistry
Time of flight secondary ion mass
spectroscopy (TOF-SIMS)
Antisera used in this study were developed by
O’Guin et al. (’82) to distinguish between alpha
and beta keratins. The specificity of these antisera have been demonstrated by double diffusion
assays, indirect immunofluorescence, and immunoblot analyses (O’Guin et al., ’82; Knapp et al., ’91).
This specificity is further supported by our own experiments preabsorbing the sera against minced
feathers. Following this incubation, preabsorbed
antisera were incubated with the test samples,
and resulting immunofluorescence was almost
eliminated. All incubation steps were performed
at 4°C and separated by at least three 15-min
PBS washes. All sections were initially treated
with 1 mg/ml sodium borohydride (NaBH4) to expose epitopes and reduce autofluorescence. Specimens were then incubated for 1 hr in 4% normal
goat serum to reduce nonspecific staining, then
incubated with either rabbit anti-avian alpha
keratin (1:100) or rabbit anti-avian beta keratin
(1:50) at 4°C overnight. Sections were then incubated sequentially with biotinylated goat anti-rabbit IgG (1:200 for 1 hr, Vector) and conjugated
Avidin-CY5 (1:1000, Jackson ImmunoResearch) or
Avidin-DCS conjugated to fluorescein isothiocyanate (FITC) (1:1000, Vector) for 1.5–2.0 hr.
Sections of duck feather were processed in an
identical manner to the positive controls. Negative controls of both duck feather and ancient fiber were identical to the above, with substitution
of the anti-keratin primary with the nonrelevant
rabbit anti-human ACTH (adrenocorticotropin
hormone, 1:50, to control for nonspecific Ig binding) or normal rabbit sera (1:50, to control for
nonspecific reaction with serum components unrelated to the presence of primary antibodies) in
place of the specific primary antisera. Incubation
with sera preabsorbed against feathers to reduce
A Charles Evans and Associates (now PHIEvans) TRIFT 1 system was used to collect the
TOF-SIMS spectra. Primary ions were produced
by a gallium gun with an average energy of 15
keV. A gallium pulse of width ~1 ns bombarded
the sample with a repetition rate of 10 kHz. The
ion bean was rastered over the 80 × 80 µm area,
and total acquisition time for each sample was 5
min. The gallium primary ions generate low energy ions (<1 eV) of molecular fragments (secondary ions), which are accelerated to ~3000 eV. Only
a small portion of the desorbed fragments are
charged, and these are the source of mass data.
Masses of the fragments are determined by accurate measurements of the time of flight of these
ions from the sample to the detector.
Small fragments (~200–400 µm diameter) of
IGM 100/977 fibers were pressed against clean
gold-coated, indium foil using a specially cleaned
glass slide. A fragment of feather of approximately
the same size as the ancient sample was treated
in an identical manner and used as a positive control. Grains of sand from the sediments surrounding the fiber, a section of duck feather rachis and
solubilized human keratin (sigma) were analyzed
as controls. However mass resolution, defined as
mass divided by mass uncertainty, is highly matrix dependent; therefore patterns and distributions of peaks vary greatly across samples that
exhibit different matrices.
The assignments of protein fragments to TOFSIMS masses have been well described in the literature (e.g., Mantus et al., ’93; Lhoest and
Gastner, ’98; Lhoest and Gastner, pers. comm.),
and the high mass resolution identifies each peak
uniquely and with certainty. The areas under the
peaks corresponding to amino acid fragments are
normalized to the integrated C2H3+ peak area (265
counts/sec) (Galuska, ’94; pers. comm.).
Antisera
150
M.H. SCHWEITZER ET AL.
RESULTS
The morphology, orientation, and association of
the fibers with the skeletal remains of IGM 100/
977 suggested an organic rather than geological
source. Two obvious sources of contamination that
might give rise to structures of similar gross appearance include plant root structures or fungal hyphae. However, gross microscopic study of the fibers
was not consistent with either plant material (no
cell walls visualized) or fungi (no segmentation,
branching, or evidence of spores or fruiting bodies).
Fibers were embedded, sectioned, and subjected to
digestion with cellulase, an enzyme that degrades
plant material by specifically digesting the cellulose in plant cell walls. After a 24-hr incubation with
the enzyme, no alteration of the microstructure of
the fibers was visualized, although the cell walls of
extant plant material treated in parallel were degraded. Additionally, 1-µm sections of the fibers subjected to staining with cotton blue, a common
screening agent for fungi, were negative.
Scanning electron micrographs of the fibers
were then compared with similar images of barbs
from duck contour feathers and specialized head
feathers of Cathartes aura, the turkey vulture.
Figure 3A and B shows a comparison of one of
the fibers associated with Shuvuuia and that of
the vulture. The shafts of both specimens are
rounded, lacking the longitudinal furrow typical
of flight feathers. Additionally, there are no obvious barbs or barbules visualized in either sample.
The vulture feather possesses a series of platelike scales near the base (point of insertion into
the skin), and a slight flattening of the feather
shape is visualized. This pattern is similar to that
seen in the Shuvuuia fibers. Figure 3C, at higher
magnifications, shows a smooth outer surface on
the vulture feather, which covers a more rugose
inner structure, possibly pith. Again, a similar pattern can be seen in the fiber from Shuvuuia (Fig.
3D). Finally, very small hair-like projections were
visualized on the fiber surface (Fig. 3F, arrow) and
compared with like structures projecting from the
barbule of a duck flight feather (Fig. 3E). The similarity of these two projecting structures at high
magnifications is notable.
Since keratin microfilaments are of a known and
consistent diameter, ultrathin sections (~90–95
nm) of the ancient sample and duck feather were
visualized using transmission electron microscopy.
The duck feather shows the microfilaments, either singly or in bundles, embedded in what appears to be an amorphous protein matrix (Fig. 4A).
In the ancient sample, microfilaments are seen
localized to “pockets” of an electron dense mineral matrix (Fig. 4B). Like the duck feather, however, the microfilaments in the ancient sample are
either seen singly or in bundles, and are of a diameter consistent with beta keratin (Parakkal
and Alexander, ’72).
Structural observations were supplemented with
immunohistochemical studies. Antibody recognition
of small, three-dimensional regions of proteins,
called epitopes (3–5 amino acid residues; Child and
Pollard, ’92), is highly specific and as such is routinely used in medical diagnostics or embryological
or ontogenetic studies to localize specific protein expression in tissues. Because of its specificity, antibody-antigen recognition does not depend on the
presence of total protein, and can be used to identify protein fragments (Child and Pollard, ’92). It is
much more plausible that very small fragments of
proteins may be preserved across geological time
than total proteins. This, coupled with the ability
to localize protein signal to specific tissues, makes
immunohistochemical techniques promising for use
with ancient specimens.
When sections of Shuvuuia fibers were incubated with primary antibodies raised against extant avian alpha and beta keratins (O’Guin et al.,
’82), and compared with sections of duck feather,
treated in tandem with the ancient specimens,
both the modern and ancient samples show very
slight reactivity with the alpha keratin antibodies (Fig. 5a, b), which may be the result of “edge
effect,” or nonspecific binding of antibodies to
roughened edges of sectioned material. However,
tissue reaction to beta keratin antibodies is significantly greater in both samples, indicating
strong reactivity of both the ancient and modern
samples to these specific antibodies (Fig. 5c, d).
When sections were incubated with either normal
(nonimmunized) sera (containing antibodies, but
none specific for beta keratin) or with an antiserum against a non-relevant protein such as human anti-ACTH (adrenocorticotropin hormone) at
the same dilutions as the test sera, reaction above
background was not noted in either specimen (Fig.
5e, f). When beta keratin antisera were first incubated with minced feathers to reduce the concentration of specific antibodies, and then exposed
to fiber sections as described, the fluorescent staining was reduced to background, indicating that
the reaction to the beta keratin antibodies is specific and not artifact (Fig. 5g).
Finally, to verify that amino acid residues consistent with proteinaceous components were
KERATIN IMMUNOREACTIVITY IN CRETACEOUS SPECIMEN
151
Fig. 3. Scanning electron micrographs of Shuvuuia fiber
in comparison with extant samples. (A) Low magnification
showing the cylindrical shape and lack of branching beyond
the base of turkey vulture bristle. (B) IGM 100/977 at the
same magnification shows a similar overall morphology. Note
also in both specimens the plate-like structures near one end.
Higher magnification of vulture feather (C) and IGM 100/
977 (D) shows both structures to have a smooth outer covering, and exposes a rugose, or fibrous interior, consistent with
pith. (E) Small, hair-like projection on the surface of a rachis
from a duck feather (F). Similar projection is seen on the
Shuvuuia fiber. This projection from the duck feather (G) and
IGM 100/977 (H) is magnified, once again demonstrating overall similarity of structure.
present in these fibers, unembedded samples of
the fiber were subjected to time of flight secondary ion mass spectroscopy (TOF-SIMS). This
method was chosen for a number of reasons, including sensitivity (detection to submonolayers),
minimal requirement for sample size, and the ability to resolve molecules that differ by as little as
5–10 milliatomic mass units (amu). Most important was the ability to apply this method to wholesample analyses, which eliminated the need to
subject the very small fibers to chemical extraction techniques which could introduce contamination or cause loss of material through the various
steps involved in extraction and purification. Table
152
M.H. SCHWEITZER ET AL.
Fig. 4. Transmission electron micrographs of 95-nm sections of duck feather (A) and Shuvuuia fiber (B). In (A) thin
filaments or filament bundles are embedded in a protein matrix that appears amorphous at this magnification, but which
is actually an extension of the keratin molecules. Note a single
protein filament of ~3 nm in diameter. In (B), filaments or
bundles of filaments of similar diameter are seen more randomly oriented. Single filaments measure 3–4 nm in diameter. Lower magnifications (not shown) reveal these filaments
to be encased in small “pockets” of electron dense mineral
matrix. Original scope magnification: A, 40,000; B, 50,000.
1 shows the amino acid fragments identified in a
fragment of the Shuvuuia fiber by this technique.
The numbers in parentheses next to the chemical
formulae show the percent intensities relative to
C2H3+ peak, which allows a relative quantification
of the organic fragments. Figure 6 shows a portion of the positive ion spectra from which the
table is constructed. Data are consistent with the
interpretation that amino acids are present in this
fiber sample. However, because this sample is not
purified, is highly degraded, and contains inorganic components (minerals), it is not possible to
determine amino acid ratios. Additionally, TOFSIMS is highly matrix dependent, and unless two
samples have similar matrices, it is not possible
to compare them.
epidermally derived structures which are similar
to feathers on a molecular level. These fibers are
seen to be hollow, both grossly and in microscopic
cross section, which is consistent with feather
structure. However, they are morphologically dissimilar to modern flight or contour feathers in that
they show no evidence of the complex branching
patterns characteristic of all modern bird body
feathers. This may be a diagenetic effect, thus
structural similarity to the highly specialized and
derived head feathers of the vulture may be only
superficial. Alternatively, this may reflect the
original structural pattern of these fibers. If reflective of original structure, these fibers suggest
an intriguing insight into the evolution of feathers, and may support the idea that the morphological evolution of feathers followed that seen in
ontogenetic patterns, where the earliest feather
structures to arise from placodes resemble small
simple hollow filaments (Brush, ’99). Additionally,
this would provide support for the hypothesis that
feathers as an evolutionary novelty did not arise
for flight.
Although the data from immunological studies
suggest that the antiserum raised against avian
beta keratins specifically recognizes components
within the ancient fiber, the fibers have been in-
DISCUSSION
Multiple lines of evidence suggest that the small
fibers found in association with IGM 100/977 contain organic components that are consistent with
degradation products of the protein beta keratin.
Since this protein is only produced by epidermal
cells of nonavian reptiles and birds, and since
feathers are the only epidermal structures in modern taxa that consist of only beta keratin, it is
hypothesized that these fibers may represent
KERATIN IMMUNOREACTIVITY IN CRETACEOUS SPECIMEN
153
Fig. 5. Immunohistochemical results. (a) Shuvuuia and
(b) duck feather exposed to avian anti-alpha keratin primary
antisera at a 1:100 dilution. (c) Shuvuuia and (d) duck feather
exposed to avian anti-beta keratin primary antisera, at a dilution of 1:50. (e) Shuvuuia and (f) duck feather exposed to
normal (nonimmunized) rabbit sera at 1:50 dilution. (g)
Shuvuuia exposed to avian anti-beta keratin antisera (1:50
dilution) which had been previously incubated repeatedly with
minced feather to significantly reduce the concentration of
antibodies specific to beta keratin. The signal is reduced to
background, demonstrating that the antibodies binding to the
fiber are blocked by exposure to beta keratin, thus supporting the specificity of these antibodies for beta keratin proteins. For all panels, intensity of fluorescent signal is directly
correlated with the degree of antibody binding to epitopes in
the exposed tissues. For all images, bar = 20 µm.
filtrated with minerals. Therefore, one must entertain the possibility that this recognition arises
from nonspecific interaction between the antisera
and the mineral. If this were the case, the control
sera should demonstrate equal reactivity, which
is not the case. Another alternative is that characteristics of the protein epitopes were preserved
intact by the fossilization process in a manner
similar to molecular imprinting (Mosbach and
Ramstrom, ’96; Huaiquiu et al., ’99), without preserving the molecules themselves—although it is
difficult to understand how this could occur in
mineral with sufficient accuracy for antigenic recognition to be maintained. Finally, although these
mass spectrometry data are not sufficient to conclude that identified amino acids are endogenous
154
M.H. SCHWEITZER ET AL.
TABLE 1. Amino acid fragments identified in a fragment of the Shuvuuia fiber
AA
ser
gly
pro
ala
cys
val
leu
glu
asp
thr
phe
lys
met
gly-gly
ala-gly
ala-ala
1
2
Positive ion fragments (% intensity relative to C2H3)
CH3O (18.7)
CH4N (146.2)
C4H8N (3.1)
C2H6N (25.8)
CH3S (4.4)
C4H10N (23.1)
C5H12N (35.9)
C2H3O (50.0)
C2H6N (23.9)
C2H5O (14.6)1
C6H5 (8.8)2
C4H11N (8.3)
C3H7S (14.1)
C4H7N2O2 (3.0)
C5H9N2O2 (1.1)
C6H11N2O2 (0.7)
C2H6NO (0.9)
C2H4N (14.9)
C4H6N (2.7)
CH2N (12.2)
C4H7N2O2 (3.0)
C3H6N (18.5)
C3H8N (15.7)
C3H8NO (0.4)
C7H7 (6.1)**
C5H10N (3.9)
C2H5S (1.7)
C3H5O (9.5)
C8H10N (0.9)
The R group of thr C2H5O+ (CH3=+CH-OH) cannot be differentiated from the environmental contaminants +CH2-CH2-OH or CH3-O-+CH2.
Hydrocarbon fragments are common to most surfaces and can be due to environmental contaminants.
to the specimen, their identification in and localization to whole sample analyses of the ancient
fibers supports the possibility that these fibers are
remnants of the original epidermal structures.
In addition, clusters of nitrogen-containing saturated hydrocarbons were identified in IGM 100/
977 (Fig. 6b). Peaks shown in the upper panel are
uniquely identified; however the peaks of greater
mass in the lower panel are labeled as to best fit.
These molecules may arise from protein degradation and subsequent condensation reactions (Collins et al., ’98).
As mentioned above, TOF-SIMS does not allow
us to state conclusively that all amino acid fragments are endogenous to the sample, but the presence of amino acid fragments, peptide linked
fragments, and some high molecular weight molecules does support the hypothesis that some original molecular components remain in these fibers.
This study supports several conclusions: (1) It
is the first report of feather-like structures preserved in three dimensions, not as impressions,
from an eolian depositional environment. (2) The
identification of epidermally derived structures
that are molecularly consistent with feathers in
so ancient a lineage may shed light on the evolution of feather structure, flight and endothermy
in Cretaceous organisms. (3) It supports the hypothesis that informative protein components may
be identified across geological time spans. (4) It
advances the possibility of using immunohistochemical techniques to identify protein fragments and organic components in other fossil
specimens, such as the exceptionally well-pre-
served specimens of Sinosauropteryx (Chen et al.,
’98), Caudipteryx (Qiang et al., ’98) and Protoarchaeopteryx (Qiang et al., ’98), and demonstrates
the utility of this method in cases where the number of samples is extremely limited. (5) It establishes the use of TOF-SIMS as an effective method
for molecular mass analysis of organic components
of fossils without destruction or in cases where
very little material is available. Finally, this research suggests further study into the nature of
the antigens to which the antisera bind. If the
antigen is not organic but a geologically derived
molecular mimic, it may be possible to obtain usable molecular information even though the original organic structures are no longer present.
The occurrence of feathers in association with
alvarezsarid specimens is congruent with the hypothesis of avian relationships of this lineage
(Perle et al., ’93; Chiappe et al., ’96; Novas, ’96;
Forster et al., ’98). Nevertheless, since feathers
have been documented in nonavian maniraptoriforms (Qiang et al., ’98), their presence in Shuvuuia does not provide any additional evidence in
support of such a phylogenetic hypothesis. The phylogenetic placement of the Alvarezsauridae is controversial. Alvarezsaurids are highly specialized
creatures and the rapid rate of new discoveries of
early birds and non-avian maniraptoriforms has
kept phylogenetic hypotheses about this segment
of theropod evolution highly dynamic (Fig. 7). If
alvarezsaurids are regarded as nonavian theropods,
the identification of the structures surrounding the
skeleton of Shuvuuia adds additional evidence to
the idea that feathers were common to most, or
all, nonavian maniraptoriforms.
KERATIN IMMUNOREACTIVITY IN CRETACEOUS SPECIMEN
155
Fig. 6. (a) Positive ion spectra of
Shuvuuia in the region from 0 to 200
amu (top panel). Note the inorganic
components that dominate the spectra in the low mass regions (0–50
amu). Additionally, nitrogen-containing hydrocarbons (e.g., 130 amu) are
identified in the fiber. Middle panel
is expansion of the spectra in the region of 60–130 amu. Peaks representing some of the amino acid
fragments in this sample are as labeled. Bottom panel is a further expansion of the region 71.85–72.20
amu. Resolution of this peak is unambiguous. (b) Positive ion spectra
in the region from 165 to 300 amu
(top panel) identifies additional organic components in the fiber in the
higher mass regions, showing clusters of nitrogen-containing saturated
hydrocarbons. We hypothesize that
these are derived from nitrogen-containing parent molecules because
they are separated by CH2 units and
contain a single amino group. Bottom
panel shows the region between 350
and 500 amu. These large peaks may
correspond to parent molecules alluded to above. Molecules containing
one nitrogen are even in mass, while
odd masses contain two nitrogen
groups (e.g., mass 183 with formula
C11H23N2, mass 200 is C14H18N).
156
M.H. SCHWEITZER ET AL.
with feathers can be identified in nonavian
theropods, the phylogenetic proximity of these lineages to birds is robustly supported. (2) The morphological evolution of feathers may be further
elucidated if it can be shown that the first epidermal structures to appear were simple structures
that could not have functioned in flight and that
flight feathers resulted from selection for increasingly complex forms. If beta-keratin specific epidermal structures that were unable to be classified
morphologically as feathers are identified, it would
imply that the molecular mechanism and expression of feather proteins existed before feathers
arose as morphologically distinct structures in
their modern form. (3) The only organisms today
that possess epidermally derived body coverings
are those with basal metabolic rates significantly
above ectothermy. Demonstration of such a body
covering in nonavian theropods implies that they
likewise possessed a metabolic strategy above
ectothermy. Further application of immunological
techniques to suitable fossil specimens, such as
use of monoclonal antibodies, phage display libraries, or epitope mapping techniques may be employed to aid in the elucidation of phylogenetic
hypotheses (Collins et al., ’91; Lowenstein and
Scheuenstuhl, ’91; Lowenstein, ’93).
ACKNOWLEDGMENTS
This research was funded by donations from Dr.
G. Ellis. We thank Dr. C. Paden, in whose lab the
immunohistochemical studies were done. In addition, we thank N. Equall, D. Gustafsen, C.
Horner, and A. Hageston for technical expertise
and J. Clark and A. Davidson for their help identifying samples. Finally, our gratitude goes to S.
Pincus, C. Forster, J. Starkey, M. Tientze, A.
Brush, B.A. Stankiewicz, R. Sawyer, K. Padian
and M. Collins for their patience, critiques, reviews, ideas, support and helpful discussions.
Fig. 7. Possible alternatives for phylogenetic placement
of the Alverezsauridae (Shuvuuia and its relatives). (A)
Alvarezsaurids are considered to be birds, as the sister-taxon
to all birds other than Archaeopteryx (Perle et al., ’93; Chiappe
et al., ’96; Novas, ’96). (B) Alvarezsaurids are regarded to be
the sister-taxon to birds (Chiappe, ’99). (C) Alvarezsaurids
are placed in a more basal position within coelurosaurian
theropods (Novas, ’96; Sereno, ’97).
The presence of feathers in nonavian theropods
is significant from evolutionary and physiological
perspectives for several reasons: (1) Feathers have
been used to uniquely identify birds; however, if
integumentary structures molecularly consistent
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