Molecular evidence of keratin and melanosomes in of the Early

Yanhong Pana,1, Wenxia Zhengb, Alison E. Moyerb,2, Jingmai K. O’Connorc, Min Wangc, Xiaoting Zhengd,e, Xiaoli Wangd, Elena R. Schroeterb, Zhonghe Zhouc,1, and Mary H. Schweitzerb,f,1

aKey Laboratory of Economic Stratigraphy and Palaeogeography of the Chinese Academy of Sciences, Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences Nanjing 210008, China; bDepartment of Biological Science, North Carolina State University, Raleigh, NC 27695; cKey Laboratory of Vertebrate Evolution and Human Origins of the Chinese Academy of Sciences, Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences, Beijing 100044, China; dInstitute of Geology and Paleontology, Linyi University, Linyi City, Shandong 276005, China; eShandong Tianyu Museum of Nature, Pingyi, Shandong 273300, China; and fNorth Carolina Museum of Natural Sciences, Raleigh, NC 27601

Contributed by Zhonghe Zhou, October 20, 2016 (sent for review August 10, 2016; reviewed by Dominique G. Homberger and Roger H. Sawyer) Microbodies associated with feathers of both nonavian reported microbodies were embedded. Whether keratinous and early were first identified as bacteria but have been proteins were preserved in these feathers, and the potential ex- reinterpreted as melanosomes. Whereas melanosomes in modern tent of this preservation, has not been explored. feathers are always surrounded by and embedded in keratin, Indeed, if both microbodies and matrix are preserved, tests can melanosomes embedded in keratin in fossils has not been be conducted to chemically characterize the composition of each. If demonstrated. Here we provide multiple independent molecular these bodies are melanosomes, they should be contained within a analyses of both microbodies and the associated matrix recovered keratinous matrix; if they are microbial in origin, this matrix should from feathers of a new specimen of the basal bird Eoconfuciusornis consist of exopolymeric substances secreted by the microbes and from the Jehol Biota of China. Our work represents subsequently mineralized. To distinguish between these alternative the oldest ultrastructural and immunological recognition of avian hypotheses, we applied multiple methods, well-established for beta-keratin from an Early Cretaceous (∼130-Ma) bird. We apply molecular and chemical characterization of modern materials, to immunogold to identify protein epitopes at high resolution, by chicken (Gallus gallus) feathers and to preserved feathers of a new – localizing antibody antigen complexes to specific fossil ultrastructures. specimen of the bird Eoconfuciusornis [Shandong Tianyu Museum Retention of original keratinous proteins in the matrix surrounding of Nature (STM) 7-144] (: Confuciusornithiformes) (Fig. electron-opaque microbodies supports their assignment as mela- 1A) from the 130-Ma horizon of the Huajiying Forma- nosomes and adds to the criteria employable to distinguish melano- tion in Fengning, northern Hebei, China. Our results are consistent somes from microbial bodies. Our work sheds new light on molecular with the retention of original organic components derived from preservation within normally labile tissues preserved in fossils. both keratin and melanin, thus supporting a melanosome origin for these ancient microstructures. keratinous protein | immunogold | ChemiSTEM | melanosome | Early Cretaceous Results Fossil Histology. Scanning electron microscopy (SEM) was eathers and feather-like epidermal structures are now well- used to demonstrate that samples 201 to 205 were preserved as Fdocumented in several groups of nonavian dinosaurs and three-dimensional, thick carbon sheets. SEM images of the fossil basal birds, with the most abundant evidence coming from the Middle feathers (Fig. 1 F–J) were compared with those of G. gallus – -to-Early Cretaceous deposits in northeastern China (1 3). remiges (Fig. 1 B–E). At higher magnifications, much of the Round-to-elongated microbodies associated with these feathers and fossil is composed of 3D microbodies, with some regions feather-like structures were first interpreted as microbes (4, 5), leading to the hypothesis that microbial activity played a key role in Significance the preservation of these normally labile remains (5). This hypothesis was based on and supported by experiments and observations of bacterial decomposition of feather keratins in modern birds (6, 7). We report fossil evidence of feather structural protein (beta- More recently, these bodies were reinterpreted as remnant keratin) from a 130-My-old basal bird (Eoconfuciusornis)from melanosomes (pigment-containing intracellular organelles) (8–11) the famous Early Cretaceous Jehol Biota, which has produced and, subsequently, hypotheses of dinosaurian color (8–13), be- many feathered dinosaurs, early birds, and mammals. Multiple havior (10), habitat (14), and physiology (15) were proposed based independent molecular analyses of both microbodies and asso- upon this reinterpretation. However, melanosomes and microbes ciated matrix recovered from the fossil feathers confirm that overlap completely in size and shape (16–18), and thus these hy- these microbodies are indeed melanosomes. We use trans- potheses are equally plausible. Furthermore, the majority of the mission electron microscopy and immunogold to show localized data presented to support a melanosome origin are based not on binding of antibodies raised against feather protein to matrix the morphology of the bodies themselves but rather on their im- filaments within these ancient feathers. Our work sheds new pressions within a structural matrix (9–15, 19) that is presumed, light on molecular constituents of tissues preserved in fossils. but not demonstrated, to be keratin. Author contributions: Y.P., W.Z., Z.Z., and M.H.S. designed research; Y.P. and W.Z. per- The most rigorous test for the origin of these microbodies is formed research; A.E.M. contributed new reagents/analytic tools; X.Z. and X.W. provided the chemical characterization of both the microbodies and the materials for analysis; Y.P., W.Z., J.K.O., M.W., X.Z., X.W., and E.R.S. analyzed data; and Y.P., matrix in which they were embedded. However, where chemical Z.Z., and M.H.S. wrote the paper. data were presented previously, with few exceptions (20, 21) the Reviewers: D.G.H., Louisiana State University; and R.H.S., University of South Carolina. data were not sufficiently specific to support or eliminate either The authors declare no conflict of interest. – hypothesis (22 26). Whether the melanin-related chemical sig- 1To whom correspondence may be addressed. Email: [email protected], zhouzhonghe@ nals originated from the microbodies or from the surrounding ivpp.ac.cn, or [email protected]. matrix (22–26) could not be determined, with the exception of 2Present address: Department of Biology, Drexel University, Philadelphia, PA 19104. one study by Lindgren et al. (21). No detailed morphological or This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. chemical studies have been conducted on the matrix in which the 1073/pnas.1617168113/-/DCSupplemental.

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Fig. 1. New specimen of Eoconfuciusornis (STM 7-144) collected from the Early Cretaceous lake deposits in Hebei, northern China, and SEM images of as- sociated feather material, compared with SEM images of a black feather from the extant chicken G. gallus.(A) Photograph of the new fossil specimen, indicating sample locations (boxes; not to scale). (B–E) SEM images of a black feather from G. gallus.(B) Low-magnification image. (C–E) High-magnification images of the boxed areas in B.(F–J) SEM images of the feather samples 111 (crural feathers) and 202 (interpreted as the tertials of the left wing) from Eoconfuciusornis.(F) Low-magnification image. (G) High-magnification image of the boxed area in F.(H) Melanosomes are shown located immediately below the structured thin layer, which is indicated with yellow arrowheads. (I and J) High-magnification images of the boxed areas in H. These bodies are embedded within the feathers and not observed on the surface of the feathers. (Scales are as indicated.)

retaining a very thin carbon layer (Fig. 1 F–I) that covers the matrix (Fig. 4 G–I), but were sparsely distributed relative to microbodies (Fig. 1J). This thin layer displays fibrous bundles in binding in modern controls (Fig. 4 D and F–L). However, other arrays that form regular angles (Fig. 1 G and I), a feature pre- structures, including the electron-dense microbodies and needle- viously reported in the epicortex of G. gallus feathers (27) and shaped structures probably of diagenetic origin, were completely also observed in Fig. 1 C and D. unlabeled (Figs. 4E and 5A). No gold particles were visible in the Transmission electron microscopy (TEM) confirms micro- negative controls (omitting the primary antiserum) (Fig. 4 A–C), structural similarity between Eoconfuciusornis and extant similar to results from immunofluorescence. The higher-resolution G. gallus feathers (Fig. 2). The microbodies preserved in Eocon- immunoelectron localization of gold particles generally confirms fuciusornis STM 7-144 are distinct, relatively uniform, elongated, the binding of the anti-feather antiserum visualized in fluores- electron-dense, and embedded in a less electron-dense filamen- cence microscopy, and verifies that these fossil feathers partially tous matrix (Fig. 2 A and B), patterns also observed in extant preserved the molecular structure of the feather–keratin epitopes, feathers (Fig. 2 C and D). Both filaments and microbodies are which remains immunoreactive. more densely packed in the fossils, suggesting some diagenetic alteration perhaps involving compression.

Immunohistochemistry. A polyclonal antiserum raised against ex- tracts of mature modern white chicken feathers (28) was used to test the hypothesis that the exceptional microstructural preserva- tion in these fossil feathers extended to the molecular level. Both immunofluorescence and immunogold labeling support the pres- ence of epitopes in fossil tissues consistent with beta-keratin, a family of proteins not produced by either mammals or microbes but that comprise ∼90% of proteins in extant feathers (28) and are coexpressed with alpha-keratin in the skin, beak, and claw tissues of reptiles and birds (29). These are also referred to as corneous proteins (30).

Specific immunoreactivity to components within the fossil EVOLUTION feathers of Eoconfuciusornis (Fig. 3) was revealed by tissue-spe- cific, although weaker, localization of fluorescence signal (Fig. 3 C and D) in a pattern comparable to extant black chicken feathers used as a positive control (Fig. 3 G and H). No fluorescence was present in any controls, where the primary antiserum was omitted, but all other steps were kept identical (Fig. 3 A, B, E,andF). Furthermore, antibodies to hemoglobin (a protein found in ver- tebrate red blood cells, used as an irrelevant control) and pepti- doglycan (a glycosaminoglycan produced exclusively by bacteria) did not react with the fossil feather sample (Fig. S1), supporting both the persistence of keratinous components and the specificity of the anti-feather antibody. We applied the same anti-feather antiserum (28) to ultrathin Fig. 2. TEM images of the crural feathers sampled in Eoconfuciusornis STM ∼ 7-144 (A and B, sample 111) compared with that of a black feather from sections ( 90-nm) of fossil and modern black feathers. Binding G. gallus (C and D). (A) Low-magnification image. (B) High-magnification image was detected using a secondary antibody conjugated to 12-nm gold of the boxed area in A.(C) Low-magnification image. (D) High-magnification particles. The gold particles were seen associated with fibril bun- imageoftheboxedareainC. Both melanosomes and beta-keratin filaments dles measuring ∼3 nm, consistent with filaments of beta-keratin are more densely distributed in the fossil sample. (Scales are as indicated.)

Pan et al. PNAS | Published online November 21, 2016 | E7901 Downloaded by guest on September 23, 2021 Fig. 3. Immunofluorescence results of fossil feathers (sample 205, propatagium, which is covered dorsally and ventrally by feathers) compared with feathers of G. gallus. (A, C, E,andG) Overlay images showing where the antibodies bind to tissues (green) superimposed on transmitted light images. (B, D, F,andH) Fluorescence images showing only antibody binding, as represented by green fluorescence of the FITC label. (A and B) Negative control corresponding to C and D, where the primary antibody is omitted. (C and D) Eoconfuciusornis feathers exposed to anti-feather antibody demonstrates specific and localized antibody binding, as reflected by green fluorescence signal. (E and F) Negative control, with the primary antibody omitted. (G and H) In situ fluorescence signal corresponding to bound antibodies on the feather tissue of G. gallus. Binding patterns are similar between samples, although the ancient feathers show reduced binding as indicated by less intense fluorescence signal. All data were collected under identical conditions and all images were processed identically. (Scales are as indicated.)

ChemiSTEM. We obtained high-resolution elemental maps of ultra- (Fig. 5 A–C) and modern (Fig. 5 E, I,andJ) feathers. Keratin mol- thin sections of fossil feathers using ChemiSTEM technology [FEI ecules incorporate the S-bearing amino acid residue cysteine in rel- Titan G2 series of Cs-corrected STEM (scanning/transmission elec- atively high concentrations (31, 32), supporting the hypothesis that tron microscopy)]. This technology reveals the elemental distribution these antibodies are most likely binding keratin. of nanogold particles bound to antibodies (Fig. 5 B–D and I–K)and We applied the same technology to modern feathers to show that identifies, at high spatial resolution, other elements (e.g., N) that extant melanosomes contain higher concentrations of copper, sulfur, would be expected to be present in organic remains. This technology and calcium (Fig. 6 G–J) than are seen in the keratin matrix. Simi- also identifies and localizes potential diagenetic compounds (e.g., larly, the elements Cu, S, and Ca localize to electron-dense micro- calcium mineral morphs). Antibodies tagged with gold localize to and bodies visualized in the fossil feathers (Fig. 6 A–D). We also note overlap with relatively high concentrations of sulfur in both ancient that the keratinous matrix surrounding melanosomes in modern

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Fig. 4. Immunogold labeling of fossil feathers (sample 201) compared with feathers of G. gallus.(A–C) Negative control of D–F, when the primary antiserum is omitted. (B) High-magnification image of the boxed area in A.(C) High-magnification image of the boxed area in B.(D–I) In situ nanogold immunohis- tochemistry of Eoconfuciusornis feathers exposed to anti-feather antibody. (E) Melanosomes from the same section of D and F; no nanogold particles were seen to specifically bind. (F) High-magnification image of the boxed area in D; arrowheads show binding of the nanogold beads. (G–I) High-angle annular dark-field (HADDF) images show binding of the nanogold beads from the same section of D–F.(H) High-magnification image of the boxed area in G.(I) High- magnification image of H; arrowheads show the filaments at the bead-binding regions. (J–L) In situ nanogold immunohistochemistry of G. gallus feathers exposed to anti-feather antibody. Nanogold particles specifically bind the keratinous tissues but do not bind the melanosomes, visualized in cross-section. (K) High-magnification image of the boxed area in J.(L) High-magnification image of the boxed area in K. (Scales are as indicated.)

feathers (Fig. 6 K and L) contains greater concentrations of carbon ples, immediately below this dense cortex, 3D microbodies were and nitrogen than are seen in fossil feathers (Fig. 6 E and F), in- present. Melanosomes are suspended in a beta-keratin matrix (33), a

dicating a possible diagenetic effect. pattern and distribution similar to what is seen in Eoconfuciusornis. EVOLUTION The fossil feathers also show needle-shaped structures (Fig. 5A) SEM analyses of modern feathers rarely showed melanosomes, as thatarehighinCa(Fig.5F). We propose that these structures are these are obscured by the dense outer feather cortex, and melano- most likely of diagenetic origin (Fig. 5F) and possibly microbially somes herein were not visualized unless this cortex was removed mediated, because they are not found in modern feathers and (27). However, this dense cortex is rarely observed in fossil feathers, their shape is consistent with early diagenetic morphs of calcium- exposing microbodies for easier observation. It is likely that the containing minerals. However, phosphorus is barely measurable, and cortex disintegrated early in diagenesis, as seen in taphonomic ex- does not map to the Ca signal, so a CaPO4 mineral morph can be periments of feather degradation. excluded. The elemental maps obtained from lower-magnification TEM of 90-nm sections taken from Eoconfuciusornis feathers images using environmental scanning electron microscope with en- was comparable to images of modern feathers in that the matrix ergy dispersive spectroscopy (ESEM-EDS) (Fig. S2) indicate a of both samples contained filaments consistent with fibrillar higher concentration of Ca in the fossil feather material, which maps keratin and electron-dense bodies consistent with melanosomes to both the microbodies and the needle-like mineral structures. in size, orientation, and location. However, although suggestive, imaging alone is not conclusive. Discussion Therefore, we hypothesized that the exceptional ultrastructural The smooth and dense outer cortex of the feathers of Eocon- preservation may extend to the molecular level. Capitalizing on fuciusornis was imaged using SEM and compared with that of the specificity and sensitivity of the vertebrate immune response, extant chicken feathers at the same magnification. In both sam- which has been used successfully to characterize other originally

Pan et al. PNAS | Published online November 21, 2016 | E7903 Downloaded by guest on September 23, 2021 Fig. 5. ChemiSTEM data derived from the region immunoreactively binding nanogold particles and needle-shaped structures of the Eoconfuciusornis feathers (sample 205), and compared with feathers of G. gallus.(A) HADDF image of an immunogold-labeled section of the Eoconfuciusornis feather, in- dicating the region selected for elemental mapping. (B–D) Various element distributions for the immunogold-labeled region (arrowhead). (B) Gold (Au) distribution map indicating the localization of nanoparticles. (C) Sulfur map showing the distribution of this element of overlap with antibody-bound Au beads. (D) Nitrogen map suggesting the entire tissue is dominated by N, consistent with a proteinaceous source. (F–H) Element distributions for the needle- shaped structures observed in some regions of the fossil feather. (F) Calcium distribution outlines the shape of the structure, indicating it contains a relatively high Ca level. (G and H) Nitrogen and phosphorus distribution maps, respectively, which are not specifically localized within the needle-shaped structures. (E) HADDF image of an immunogold-labeled section of G. gallus feather, indicating the region selected for elemental mapping. (I–K) Various element distributions for the immunogold-labeled region. (I) Au distribution map. (J) S map. (K) N map. (Scales are as indicated.)

keratinous fossil material (34, 35), we show that antibodies raised the anti-feather antibodies localize to filaments within the ma- against feather proteins (28) react specifically with ancient trix, supporting the hypothesis that the matrix is composed of feathers in in situ experiments. Binding, as visualized by green material consistent with filamentous beta-keratin proteins. No fluorescence signal, is reduced in intensity but comparable in gold labeling was detected in or associated with the microbodies pattern and distribution to that of extant feathers. The decreased embedded in this matrix in either fossil or extant feathers (Fig. intensity of antibody binding in the fossil feather may be due to 4), supporting the specificity of antibody binding to keratinous several factors: (i) greater degradation resulting in a reduced matrix but not other components of the feathers. These experi- number of recognizable epitopes; (ii) some epitopes present in ments produced complementary data that confirm the presence modern feathers (against which the antibody was raised) may not of molecules consistent with feather keratins in the fossil tissues. have been present at this early stage in avian evolution (29, 36); High-resolution elemental mapping (ChemiSTEM) adds addi- or (iii) epitopes may be blocked or masked by infiltration with tional information. This technique showed that regions binding minerals or other factors resulting from diagenetic processes. antibodies complexed to gold also demonstrate a relatively high To confirm the results we observed by immunofluorescence concentration of both sulfur and nitrogen, which would be expected labeling, we applied nanogold (12-nm)-labeled antibodies to in keratinous material. The keratin protein family incorporates high 90-nm sections of fossil feathers and observed localized binding concentrations of amino acids rich in sulfur (31, 32), whereas carbon under TEM, which allows much higher resolution. We show that and nitrogen are common to all amino acids and proteins. However,

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Fig. 6. ChemiSTEM data derived from the microbodies of feather samples from Eoconfuciusornis (A–F), compared with melanosomes of a black feather from G. gallus (G–L). (A) HADDF image showing the selected region for elemental mapping. (B–F) Various element distributions for the selected region. (B) Cu map. (C) S map. (D) Ca map. (E) C map. (F) N map. (G) HADDF image showing the region on the G. gallus feather selected for elemental mapping. (H–L) Various element distributions for the selected region. (H) Cu map. (I) S map. (J) Ca map. (K) C map. (L) N map. (Scales are as indicated.)

the possibility that the sulfur signals might be enlarged by the an- microbodies. It thus confirms that both Cu and S colocalize to EVOLUTION tibodies cannot be fully excluded based on the present experiments. proposed melanin-bearing organelles. Although S is also observed Recently, it has been demonstrated that certain trace ele- in both the fossil feather matrix and the microbodies, Cu is found ments, such as copper, sulfur, and calcium, are present in both only within the melanosomes, indicating little diagenetic movement fossil and modern melanosome-bearing tissues, and it has been and supporting the original distribution of this element. Hence, we suggested that these elements may be used as biomarkers for conclude that the microbodies examined herein are indeed the melanin-derived compounds in fossils (23–25). This is problem- fossilized remains of melanin-bearing organelles and that they atic, however, in that all Cu, S, and Ca are also derived from contain high concentrations of the original trace elements of the bacterially induced decay as well as many diagenetic processes living organisms preserved through geological time. (37, 38). One of the ways to differentiate between melanosomes Another notable trace metal is calcium, which was once thought to and morphologically similar bacteria is to directly correlate, at be chelated by and affect the chemical properties of melanin (39). high resolution, these trace elements to the microstructures but Because of this association, it was proposed that Ca distribution could not the surrounding matrix. However, the resolution of either be used as a proxy for melanin distribution in fossil feathers (23). In ESEM-EDS or synchrotron rapid scanning X-ray fluorescence previous studies, the SRS-XRF maps of Ca in the neck region of a (SRS-XRF) maps is not sufficient to attribute the elements specimen of sanctus showed a strong correlation with specifically to any organelles (25). The high-resolution elemental Cu (23). Our ESEM-EDS maps (Fig. S2) also suggest that the dis- maps achieved by ChemiSTEM technology display high levels of tribution of Ca strongly correlates with diagenetically altered feather copper and sulfur in both the modern melanosomes and ancient structures. However, the high-resolution elemental maps show that

Pan et al. PNAS | Published online November 21, 2016 | E7905 Downloaded by guest on September 23, 2021 Ca signal is much weaker in melanosomes of extant feathers and S205, which were selected for molecular analysis, were washed successively compared with that in the microbodies of fossil feathers and, al- in 75% (vol/vol) ethanol and E-pure water and ultrasonically cleaned to avoid though Cu is localized within proposed melanosomes in the fossil, potential contaminants. The samples were then dried under a biological hood, Ca maps to both microbodies and the needle-shaped structures and the surface structure was documented using SEM. Subsequently, the – samples were put into 50% HF for 3 to 5 d to dissolve the silicon minerals of within the feather keratin matrix. These Ca-concentrated needle- the matrix and washed with E-pure water three times. Approximately 1- to shaped structures are absent in extant feathers. Thus, we hy- 2-mm3 samples were selected to be dehydrated in two changes of 70% ethanol pothesize that precipitation of calcium, possibly mediated by for 30 min each, followed by one change of a mixture of LR White (medium- microbes during the fossilization process, facilitated ultrastructural grade) and 70% ethanol (2:1) for 1 h, followed by another two changes of and molecular preservation of the feathers by stabilizing them at 100% LR White for 1 h each. The process was carried out at room temperature the molecular level before they could completely degrade (5, 40). (RT). Then the samples were moved to gelatin capsules, filled with 100% LR It has long been known that the association with mineral sub- White, covered to exclude oxygen, and allowed to polymerize for 24 h at strates greatly enhances the preservation potential of biomolecules 60 °C. A Leica EM UC6 ultramicrotome with a DiATOME 45° knife was used to (35). Furthermore, it has been proposed that calcium may in- cut 200-nm sections for immunohistochemistry analysis and 90-nm sections for TEM observations, immunogold tests, and ChemiSTEM analysis. Extant feath- corporate into molecular fragments, conferring stability, and that ers were treated in the same way but sectioned with a different diamond this process may be microbially mediated (35). Because the sur- knife. Neither fossils nor extant feathers were fixed, and only the extant rounding sediments of this Eoconfuciusornis specimen consist feathers were stained with uranyl acetate (5 min) and Reynold’sleadcitrate mainly of Al silicates, with little or no calcium detected, the source (8 min) before the TEM morphological observation. All experiments were of the calcium observed in these feathers remains unknown. repeated multiple times to validate results. Molecular dating techniques suggest that feather beta-keratin diverged from “feather-like” beta-keratins about 143 Ma (29). If SEM. The dried samples were mounted on stubs with double-sided carbon tape these molecular dates are correct, feathers of and (S111 and S112), and one sample from an extant chicken feather was coated with ∼ were constitutively distinct from those of extant bird 10 nm of gold/palladium for 30 s. The SEM-EDS data were collected from un- coated S102, S201, S203, S204, and S205. Samples were imaged with a Leo 1530 VP feathers, and thus feather structure preceded modern feather analytical scanning electron microscope controlled by JEOL InTouchScope version proteins. Molecular data show that only in the Early Cretaceous 1.05A software. Two detectors were used for taking SEM images: (i)anormal did feather beta-keratins begin to diversify to their modern forms. secondary electron detector, used at high vacuum and 1 to 5 keV, and (ii)avar- These molecular changes conferred greater elasticity to feathers iable-pressure secondary electron detector, used at low vacuum and 10 to 20 keV. than is seen in materials composed of other types of beta-keratin (29), thus optimizing the material properties of feathers for flight TEM. The 90-nm sections were mounted on carbon-coated copper grids. (41). The diversification of feather beta-keratin genes coincided Samples were stained with 5% methanolic uranyl acetate (5 min), washed in with and perhaps facilitated the radiation of modern birds. E-pure water, stained with Reynold’s lead citrate (8 min), and washed in Another factor that may contribute to the unique biomaterial E-pure water. Sections were imaged using a JEM-2100 transmission electron properties of feathers is the epidermal differentiation cysteine-rich microscope (AATC) from Nanjing Forestry University and a JEM-2100Plus transmission electron microscope from the State Key Laboratory of Lake protein (42). This molecule has the same gene structure as beta- Science and Environment, Nanjing Institute of Geography and Limnology, keratins, and is coexpressed with them at certain stages of develop- Chinese Academy of Sciences, at 80 to 100 keV. ment in most keratinized tissues of living birds. However, it is only in feathers that this protein continues to be detected throughout life Immunohistochemistry. For the production of anti-feather antibody, see (42). The greatly elevated cysteine levels in this protein facilitate Moyeretal.(28). intramolecular cross-linking, and contribute to the stability and re- The autofluorescence immune-labeling protocol was modified from sistance of feathers to degradation. Further molecular recovery of Schweitzer et al. (45). The 200-nm sections were mounted on six-well Teflon- ancient feather materials may allow direct testing of these hypotheses. coated slides, with about 10 sections in each well, and dried overnight at 42 °C. Sections were etched with 25 μg/mL proteinase K in 1× PBS at 37 °C for 15 min, Conclusion followed by 0.5 M EDTA (pH 8.0) (30 min) and last with NaBH4 (twice for 10 min The multiple independent analyses used in this study provide each). Incubations were interrupted by two 5-min washes in PBS. Following these steps to accomplish epitope retrieval and quenching of autofluorescence, 4% strong evidence for the retention of original and phylogenetically normal goat serum in PBS was applied to occupy nonspecific binding sites and significant protein components in Eoconfuciusornis STM 7-144, prevent spurious binding. Sections were incubated in primary antibody (poly- and are consistent with the identification of melanosomes as well as clonal rabbit anti-feather, 1:200; polyclonal rabbit anti-hemoglobin, 1:200; keratin filaments in these ancient (∼130-Ma)tissues.Thisisthe monoclonal mouse anti-peptidoglycan, 1:200) in primary dilution buffer over- oldest report of ultrastructure consistent with beta-keratin from an night at 4 °C. All sections were then incubated with secondary antibody [bio- Early Cretaceous bird feather. Identifying keratin is key to ruling tinylated goat anti-rabbit IgG (H+L) (antibody recognizes both heavy and light out a microbial source for microbodies identified in fossils. Both chains)] (Vector Laboratories; BA-1000) diluted 1:500 for rabbit primary anti- + the depositional context and mode of preservation of this bird are bodies, and biotinylated goat anti-mouse IgG (H L) (Vector Laboratories; similar to those of numerous other Early Cretaceous Jehol birds BA-9200) diluted 1:500 for monoclonal mouse anti-peptidoglycan, for 2 h at RT. – Sections were then incubated with fluorescein avidin D [fluorescein isothiocyanate (and dinosaurs) and Jurassic Yanliao dinosaurs in China (1 3, 10, (FITC); Vector Laboratories; A-2001] for 1 h at RT. All incubations were interrupted 11, 13, 15). This similarity suggests that other fossils preserved at by sequential washes (twice for 10 min each) in PBS/Tween 20 followed by two these localities could provide additional sources of molecular in- 10-min rinses in PBS. Finally, sections were mounted with Vectashield mounting formation. More importantly, our study incorporating specific an- media (Vector Laboratories; H-1000) and coverslips were applied. Sections were tibody reactivity in this Early Cretaceous specimen demonstrates examined with a Zeiss Axioskop 2 Plus biological microscope and captured using the benefit of testing other fossil tissue samples with antisera an AxioCam MRc 5 (Zeiss) with 10× ocular magnification on the Axioskop 2 Plus generated against specific protein epitopes. Such tests have the in the AxioVision software package (version 4.7.0.0). potential not only to distinguish between keratinous feathers and The postembedding TEM immunogold-labeling protocol was modified from the skin-derived collagen fibers (43, 44) but also to characterize the methods shared in the protocol database from IHCWORLD Life Science Products & Services (www.ihcworld.com/_protocols/em/post_immunoem_embed812.htm). The molecular composition of early feathers and elucidate steps leading 90-nm sections were collected on carbon-coated nickel grids (EMS; CFT200-NI). The to the evolution, at the molecular level, of modern bird feathers. grids were incubated in PBS/Tween 20 for 10 min. Four percent normal donkey serum in PBS was applied to occupy nonspecific binding sites and prevent spurious Materials and Methods binding for 1 h at RT. The grids were incubated in primary antibody (polyclonal Eight samples (S102, S111, S112, S201, S202, S203, S204, and S205) removed rabbit anti-feather, 1:20) in primary dilution buffer for 3 h at RT and then washed from STM 7-144 were used in this study. Samples S102, S111, and S112 were with PBS/Tween 20, 10 times for 2 min each. All grids were then incubated with directly observed by SEM without coating. Samples S201, S202, S203, S204, secondary antibody [12-nm Colloidal Gold AffiniPure Donkey Anti-Rabbit

E7906 | www.pnas.org/cgi/doi/10.1073/pnas.1617168113 Pan et al. Downloaded by guest on September 23, 2021 IgG (H+L), 1:20; Jackson ImmunoResearch; 711-205-152] for 1 h. The grids were ACKNOWLEDGMENTS. We thank technician Ms. Chunzhao Wang of the PNAS PLUS rinsed with PBS/Tween 20, 10 times for 2 min each and in E-pure water three State Key Laboratory of Palaeobiology and Stratigraphy (LPS) of Nanjing In- times for 30 s each, and dried with filter paper. The sections were observed stitute of Geology and Palaeontology, Chinese Academy of Sciences, Research using the FEI Titan G2 80-300 electron microscope at the Analytical In- Scholars Chuanzhen Zhou and Yang Liu of the Analytical Instrumentation strumentation Facility (AIF) of North Carolina State University. Facility (AIF) of North Carolina State University, Dr. Lulu Cong of the Analyt- ical Center of Nanjing Institute of Geography & Limnology, Chinese Academy of Sciences, and Dr. Lingfei Ding and Dr. Yang Jing of the Advanced Analysis ChemiSTEM. Sections (90-nm) were collected on carbon-coated nickel grids Test Center of Nanjing Forestry University for technical assistance. We also (EMS; CFT200-NI) and analyzed with an FEI Titan G2 80-300 electron micro- benefited from discussions with Dr. Johan Lindgren and Prof. Franz Fürsich. scope at the AIF of North Carolina State University. This work was performed in part at the AIF at North Carolina State University, The FEI Titan G2 series of Cs-corrected scanning/transmission electron micro- which is supported by the State of North Carolina and the National Science scopes with the revolutionary ChemiSTEM technology is applied in this study. This Foundation (Award ECCS-1542015). The AIF is a member of the North Caro- new technology greatly enhances EDX (energy dispersive X-ray spectroscopy) de- lina Research Triangle Nanotechnology Network, a site in the National Nano- tection sensitivity due to a number of innovations in the system architecture, in- technology Coordinated Infrastructure. The research was supported by the cluding the X-FEG (a high-brightness Schottky field emission gun source), Super-X Strategic Priority Research Program of the Chinese Academy of Sciences EDX detector system (four windowless silicon drift detectors with shutters integrated (XDB18030501), National Natural Science Foundation of China (41472009 deeply into the objective lens), and high-speed electronics capable of 100,000 and 91514302), Open-Lab Grants of the LPS and National Science Founda- spectra readouts. This new system architecture provides many performance ben- tion USA (INSPIRE Program EAR-1344198 to M.H.S., W.Z., and E.R.S., and efits, such as improved light element detection, better sample tilt response, faster DGE01252376 to A.E.M.), as well as funds from the David and Lucile Packard mapping, and atomic chemical mapping capabilities of crystalline structures. Foundation (to M.H.S.).

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