Roger H. Sawyer

Comparative genomics reveals insights into avian genome evolution and adaptation

Birds are the most species-rich class of tetrapod vertebrates and have wide relevance across many research fields. We explored bird macroevolution using full genomes from 48 avian species representing all major extant clades. The avian genome is principally characterized by its constrained size, which predominantly arose because of lineage-specific erosion of repetitive elements, large segmental deletions, and gene loss. Avian genomes furthermore show a remarkably high degree of evolutionary stasis at the levels of nucleotide sequence, gene synteny, and chromosomal structure. Despite this pattern of conservation, we detected many non-neutral evolutionary changes in protein-coding genes and noncoding regions. These analyses reveal that pan-avian genomic diversity covaries with adaptations to different lifestyles and convergent evolution of traits.

Sequencing three crocodilian genomes to illuminate the evolution of archosaurs and amniotes

The International Crocodilian Genomes Working Group (ICGWG) will sequence and assemble the American alligator (Alligator mississippiensis), saltwater crocodile (Crocodylus porosus) and Indian gharial (Gavialis gangeticus) genomes. The status of these projects and our planned analyses are described.

Avian scale development

Epidermal-dermal tissue interactions during development control not only the morphogenesis of skin derivatives, but also the synthesis of the α- and β-keratins, which are the products of distinct structural genes. The electrophoretic patterns of the s-carboxymethyl (SCM) derivatives of the keratins produced by either embryonic or hatched chick epidermis from normal overlapping scales (scutate and scutellate scales), the radially symmetrical scales (reticulate scales), and the general body integument have been compared with the patterns from corresponding regions of birds homozygous for the gene, scaleless (scsc). We find six polypeptides in the molecular weight range of α-keratin, three β-keratins, and one major polypeptide of an intermediate molecular weight; any of which may or may not be present in a given tissue. Our comparisons show (1) that for some regions of the integument there are inconsistencies between the data obtained from our electrophoretic studies and those obtained from fine structural and X-ray diffraction studies (i.e., some regions which are fine structurally distinct give identical SDS-PAGE patterns); (2) that the biosynthetic differentiation of the overlapping scales is dependent upon specific morphogenetic events, while the biosynthetic differentiation of the reticulate scales is not; and (3) that there is region-specific gene expression for the epidermal keratins of all scaled regions when compared with the keratins of all nonscaled regions of the integument.

Multiple paternity and mating patterns in the American alligator, Alligator mississippiensis

Eggs were sampled from 22 wild American alligator nests from the Rockefeller Wildlife Refuge in south‐west Louisiana, along with the females guarding the nests. Three nests were sampled in 1995 and 19 were sampled in 1997. Females and offspring from all clutches were genotyped using five polymorphic microsatellite loci and the three nests from 1995 were also genotyped using one allozyme locus. Genotypes of the hatchlings were consistent with the guarding females being the mothers of their respective clutches. Multiple paternity was found in seven of the 22 clutches with one being fathered by three males, and the remaining six clutches having genotypes consistent with two males per clutch. Paternal contributions of multiply sired clutches were skewed. Some males sired hatchlings of more than one of the 22 clutches either as one of two sires of a multiple paternity clutch, as the sole sire of two different clutches, or as the sole sire of one clutch and one of two sires of a multiply sired clutch. There was no significant difference between females that had multiple paternity clutches and those that had singly sired clutches with respect to female total length (P = 0.844) and clutch size (P = 0.861). Also, there was no significant correlation between genetic relatedness of nesting females and pairwise nest distances (r2 = 0.003, F1,208 = 0.623, P = 0.431), indicating that females in this sample that nested close to one another were no more related than any two nesting females chosen at random. Eleven mutations were detected among hatchlings at the five loci over the 22 clutches. Most of these mutations (eight of 11) occurred at Amiµ‐17, the only compound microsatellite locus of the five used in this study, corresponding to a mutation rate of 1.7 × 10−3. Finally, most of the mutations (82%) were homoplasious, i.e., mutating to an allelic state already present in this Louisiana population.

Hatchery practices in relation to early embryology of the loggerhead sea turtle, Caretta caretta (Linné)

The developmental biology of the loggerhead sea turtle, Caretta curetta (Linne), was investigated in the field on Ossabaw Island (Georgia), and in the laboratory. Hatching success in wild, untouched nests was 0%; for successfully transplanted nests it was 75.8%; for nests transported to a hatchery tent, 72%; while for those transported to the laboratory and incubated under controlled conditions it was 95%. Mean clutch sizes and incubation times are presented. Gross inspection of unhatched hatchery eggs showed > 90% fertility in the nests. Using laboratory eggs, we find that sea turtle development closely follows that described for the freshwater turtle Chelydra serpentina (Linne). Early fusion of the amnion with the chorionic membrane is evident, and disruption of the organization of these extra-embryonic membranes may be related to decreased viability and the low hatch rates reported for hatchery conditions.

Avian scale development: X. Dermal induction of tissue-specific keratins in extraembryonic ectoderm

Epidermal-dermal tissue interactions regulate morphogenesis and tissue-specific keratinization of avian skin appendages. The morphogenesis of scutate scales differs from that of reticulate scales, and the keratin polypeptides of their epidermal surfaces are also different. Do the inductive cues which initiate morphogenesis of these scales also establish the tissue-specific keratin patterns of the epidermis, or does the control of tissue-specific keratinization occur at later stages of development? Unlike feathers, scutate and reticulate scales can be easily separated into their epidermal and dermal components late in development when the major events of morphogenesis have been completed and keratinization will begin. Using a common responding tissue (chorionic epithelium) in combination with scutate and reticulate scale dermises, we find that these embryonic dermises, which have completed morphogeneis, can direct tissue-specific stratification and keratinization. In other words, once a scale dermis has acquired its form, through normal morphogenesis, it is no longer able to initiate morphogenesis of that scale, but it can direct tissue-specific stratification and keratinization of a foreign ectodermal epithelium, which itself has not undergone scale morphogenesis.

Avian feather development: relationships between morphogenesis and keratinization.

Morphogenesis and expression of the alpha and beta keratin polypeptides are controlled by epidermal-dermal interactions during development of avian skin derivatives. We have examined the relationship between morphogenesis of the embryonic feather and expression of the feather alpha and beta keratins by routine histology, indirect-immunofluorescence, and SDS-PAGE. Initially beta keratins are expressed only in the feather sheath. Following barb ridge morphogenesis beta keratins can be detected in the barb ridge, coincident with the differentiation of barb ridge cells into eight distinct morphological types. Beta keratinization occurs in gradients; from feather apex to base, and from periphery of the barb ridge to the interior. The onset of beta keratinization in the barb ridges is paralleled by an increase in the major feather beta keratin polypeptides, as detected by SDS-PAGE. The alpha keratins are present in both the periderm and feather sheath at early stages of feather development, but become greatly reduced after hatching, when the down feather emerges from the sheath.

Avian scale development: XI. Initial appearance of the dermal defect in scaleless skin☆

The chicken mutant, scaleless, is characterized by the total absence of scutate scales. Previous experiments have shown that the scaleless defect is expressed by the epidermal cells while the dermal cells are able to participate in normal scale morphogenesis. However, in association with 14- to 16-day scaleless dermis, normal epidermis or the simple ectoderm of the chorion failed to develop scutate scale epidermis with its characteristic beta stratum. Thus the question arises: since the scaleless dermis starts out functioning normally, when does it become defective? Heterogenetic, heterotopic associations have been performed between 7.5-day to 11.5-day scaleless dermis and a neutral responding tissue, the midventral apteric epidermis, from 10.5-day normal embryos. The results show that up until 9.5 day of incubation the scaleless dermis is able to give instructions for normal scutate scale formation, if combined with normal epidermis. However, after 9.5 days, the scaleless dermis is not able to induce scale formation in normal apteric epidermis. Thus, the functional defect of the scaleless dermis occurs during the time (9 to 10 days of incubation) when epidermal placodes appear in normal embryos. From the present data, at least two explanations are possible. Either the scaleless epidermis is unable to respond to the placode inducing properties being provided by the scaleless dermis and because an epidermal placode does not form the scaleless dermis becomes defective, or the scaleless epidermis does not provide some earlier cue necessary for the scaleless dermis to acquire its placode inducing capabilities.

Avian scale development: Effect of the scaleless gene on morphogenesis and histogenesis

Abstract It has been found that morphogenesis of the reticulate scales on the footpads of the scaleless mutant chicken is abnormal. This abnormal morphogenesis is responsible for the unusual appearance of the surface of the scaleless footpads. In contrast, histogenesis of the epidermis of the mutant reticulate scales is typical of that seen for the epidermis of normal reticulate scales. Recombination experiments between the mutant scale dermis (at 16 days of incubation) and 8-day chorionic epithelium show that the mutant dermis, at this stage of development, is unable to induce normal morphogenesis; yet it is able to induce normal histogenesis of the epidermis. These results differ from those of previous experiments in which it was shown that the dermis of mutant scutellate scales (from 16-day scaleless embryos) was unable to induce normal histogenesis as a result of an earlier defect in morphogenesis. Differences in the action of the scaleless gene upon reticulate and scutellate scales are dicussed in relation to embryogenesis of the two types of scales. A possible site of action of the scaleless gene is also discussed.

Keratinization of the outer surface of the avian scutate scale: interrelationship of alpha and beta keratin filaments in a cornifying tissue.

SummaryThe outer surface of adult Gallus domesticus scutate scale was studied as a model for epidermal cornification involving accumulation of both alpha and beta keratins. Electron-microscopic analysis demonstrated that the basal cells of the adult epidermis contained abundant lipid droplets and that filament bundles and desmosomes were distributed throughout the cell layers. Indirect immunofluorescence microscopy and double-labeling immunogold-electron microscopy confirmed that the stratum germinativum contained alpha keratin but not beta keratin. Beta keratins were first detected in the stratum intermedium and were always found intermingled with filament bundles of alpha keratin. As the differentiating cells moved into the outer regions of the stratum intermedium and the stratum corneum, the large mixed keratin filament bundles labeled increasingly more with beta keratin antiserum and relatively less so with alpha keratin antiserum. Sodium dodecyl sulfate-polyacrylamide gel analysis of vertical layers of the outer surface of the scutate scale confirmed that cells having reached the outermost layers of stratum corneum had preferentially lost alpha keratin. The mixed bundles of alpha and beta keratin filaments were closely associated with desmosomes in the lower stratum intermedium and with electron-dense aggregates in the cytoplasm of cells in the outer stratum intermedium. Using anti-desmosomal serum it was shown that these cytoplasmic plaques were desmosomes.