The silkmoth chorion: Morphogenesis of surface structures and its relation to synthesis of specific proteins

The morphogenesis of four spatially differentiated surface regions of the silkmoth eggshell (chorion) has been documented and correlated with differing patterns of chorion protein synthesis within the corresponding secretory cells. During the first half of choriogenesis the polygonal pattern of ridges which cover the entire chorion appears. Regional differences in the morphology of developing ridges are not accompanied by significant protein differences, and thus presumably reflect differences in secretory cell behavior and shape. During the second half of choriogenesis expanding domes of the chorion located immediately beneath three-cell junctions of the overlying secretory surface become prominent surface features exclusively in the aeropyle crown region. Domes are composed of a thin lamellar skin and an inner buttressing “filler.” Continued filler deposition appears to cause a ripping of the lamellar skin, transforming the dome into a multiple-pronged crown that overflows with filler. Continued synthesis of lamellar chorion components elongates and strengthens the crowns until they can stand alone without the support of filler. In the aeropyle crown region, synthesis of regionally specific proteins begins in the second half of choriogenesis and accelerates until the final stages, in parallel with dome/crown formation. The more numerous proteins which are common to all regions are synthesized at approximately equal rates within all regions, and their synthesis decelerates toward the end of choriogenesis. Fifteen of the proteins (excluding filler) which are found predominantly in the aeropyle crown region may be necessary but not sufficient for crown formation, since they also occur in the stripe region (1); presumably the secretory cell surfaces mold the same components differently in the two regions. Filler appears to play an important scaffolding role in crown formation. A group of eight aeropyle crown region-specific chorion proteins which compose filler have been identified on two-dimensional gels and shown to be restricted to one of five previously described classes of chorion proteins.     

Phylogenetics of eggshell morphogenesis in Antheraea (lepidoptera: saturniidae): unique origin and repeated reduction of the aeropyle crown

Integrated phylogenetic and developmental analyses should enhance our understanding of morphological evolution and thereby improve systematists' ability to utilize morphological characters, but case studies are few. The eggshell (chorion) of Lepidoptera (Insecta) has proven especially tractable experimentally for such analyses because its morphogenesis proceeds by extracellular assembly of proteins. This study focuses on a morphological novelty, the aeropyle crown, that arises at the end of choriogenesis in the wild silkmoth genus Antheraea. Aeropyle crowns are cylindrical projections, ending in prominent prongs, that surround the openings of breathing tubes (aeropyle channels) traversing the chorion. They occur over the entire egg surface in some species, are localized to a circumferential band in many others, and in some are missing entirely, thus exhibiting variation typical of discrete characters analyzed in morphological phylogenetics. Seeking an integrated developmental-phylogenetic view, we first survey aeropyle crown variation broadly across Antheraea and related genera. We then map these observations onto a robust phylogeny, based on three nuclear genes, to test the adequacy of character codings for aeropyle crown variation and to estimate the frequency and direction of change in those characters. Thirdly, we draw on previous studies of choriogenesis, supplemented by new data on gene expression, to hypothesize developmental-genetic bases for the inferred chorion character transformations. Aeropyle crowns are inferred to arise just once, in the ancestor of Antheraea, but to undergo four or more subsequent reductions without regain, a pattern consistent with Dollo's Law. Spatial distribution shows an analogous trend, though less clear-cut, toward reduction of coverage by aeropyle crowns. These trends suggest either that there is little or no natural selection on the details of the aeropyle crown structure or that evolution toward functional optima is ongoing, although no direct evidence exists for either. Genetic, biochemical, and microscopy studies point to at least two developmental changes underlying the origin of the aeropyle crown, namely, reinitiation of deposition of chorionic lamellae after the end of normal choriogenesis (i.e., heterochrony), and sharply increased production of underlying "filler" proteins that push the nascent final lamellae upward to form the crown (i.e., heteroposy). Identification of a unique putative cis-regulatory element shared by unrelated genes involved in aeropyle crown formation suggests a possible simple mechanism for repeated evolutionary reduction and spatial restriction of aeropyle crowns.     

Heterochrony and the introduction of novel modes of morphogenesis during the evolution of moth choriogenesis

Summary Choriogenesis in silkmoths (superfamily Bombycoidea) and in a sphingid moth (super-family Sphingoidea) differ in major, but discrete, ways. In silkmoths, the predominant lamellar component assembles early in choriogenesis to form a thin framework. Subsequently, the lamellar framework is modified, first by expansion, and then by densification. Finally, ornate surface structures called aeropyle crowns form in some silkmoths, but they are absent in the species described here. In the sphingid, lamellar framework formation occurs throughout choriogenesis rather than largely during the early stages as in silkmoths. Lamellar densification occurs, but lamellar expansion and aeropyle crown formation do not. An evolutionary model is presented that accounts for the observed morphogenetic differences. Patterns of chorion protein synthesis in the sphingid differ from those in silkmoths in ways that are interpretable in light of the observed morphogenetic differences and the previously postulated functions of the proteins in silkmoths.     

Morphogenesis of silkmoth chorion: sequential modification of an early helicoidal framework through expansion and densification

The silkmoth chorion is a helicoidally layered, fibrous structure which is constructed in four sequential morphogenetic modes, beginning with the assembly of a thin, low density lamellar framework. Subsequently, the framework expands in height by the insertion of additional fiber sheets into the preexisting lamellae. This expansion mode begins farthest from the follicular secretory cells and progresses in reverse. Individual fibers then grow in thickness, presumably through accretion of newly synthesized proteins, and eventually fuse. This third mode, which also begins in the most distant lamellae and proceeds in reverse, is called densification, as it results in an approximately two fold increase in overall chorion density without further lamellar expansion. Finally, lamellogenesis is recapitulated in miniature in a region of the chorion's surface, where very-late-forming lamellae are molded into prominent surface structures, the aeropyle crowns. The densification and especially the expansion modes suggest considerable fluidity in the developing chorion, consistent with its proposed cholesteric liquid crystalline structure. Such a structure is also consistent with numerous deviations from the ideal helicoidal array. These distortions and defects are described and discussed in terms of their possible origin and function.     

Ootaxonomy and eggshell ultrastructure of Phlebotomus sandflies

The eggshell structure of four sandfly species: Phlebotomus perniciosus Newstead, P.perfiliewi Parrot, P.papatasi Scopoli and P.duboscqi Neveu-Lemaire, was examined by scanning and transmission electron microscopy (SEM and TEM). At the TEM level, the eggshell appears to have a homogeneous vitelline envelope and a thick chorion. At SEM level, the eggshell of all species is characterized by the outer chorion forming a series of fifteen to twenty longitudinal sinuous ridges, cross-linked in places to form a pattern of polygons, each line of the chorion consisting of columns arranged in a palisade. The aeropyle region of the egg is described for the first time in phlebotomine sandflies. Specific characters of the eggshell topography are described for distinguishing between these and other species of Phlebotomus.     

Egg surface structure in the annual fishes Simpsonichthys (subgenera Ophthalmolebias and Xenurolebias) and Nematolebias (Teleostei: Cyprinodontiformes: Rivulidae): variability and phylogenetic significance

The surfaces of the egg chorion of nine species of Simpsonichthys , two of Nematolebias and eight other species of rivulids were studied with scanning electron microscopy revealing details of ornamentation that were previously undocumented. Surface structures of eggs of Simpsonichthys of the subgenus Ophthalmolebias are described for the first time: eggs of Simpsonichthys constanciae , Simpsonichthys perpendicularis , Simpsonichthys rosaceus and Simpsonichthys suzarti are ornamented with a very characteristic palm-like structure, that is restricted to these species among the rivulids examined. The surface of the egg chorion of Simpsonichthys bokermanni lacks the well-developed palm-like structure, but has a characteristic conical projection, distinct from the structures of other rivulids examined, that is proposed as homologous to the palm-like structure. Simpsonichthys bokermanni shares with Nematolebias papilliferus and Nematolebias whitei the presence of concentric rows of spiny projections around the micropylar region. Species of Simpsonichthys of the subgenus Ophthalmolebias , Simpsonichthys myersi (of the subgenus Xenurolebias ), and N. whitei share the presence of large rounded protuberances on the surface of the egg chorion. The phylogenetic significance of these features are evaluated and discussed in light of current knowledge about rivulid relationships.     

Evolution and higher-order structure of architectural proteins in silkmoth chorion.

Genomic and cDNA clones have been sequenced that encode the E2 silkmoth chorion protein. E2 assembles with E1 [Regier, J.C. and Pacholski, P. (1985) Proc. Natl. Acad. Sci. USA, 82, 6035-6039] to form the 'filler' that helps mold prominent chorion surface structures called aeropyle crowns. E2 has two distinct domains. The amino terminal domain consists of four alternating stretches of hydrophobic and hydrophilic residues, the first three of which are homologous in sequence to about half of the E1 protein. Comparison of predicted secondary structures provides further support for the localized homology of E2 and E1. The carboxy terminal domain of E2 is much longer, is hydrophilic and consists entirely of multiple tandem copies of a single, variant hexapeptide repeat sequence that is absent from E1. Numbers of hexapeptide repeat sequences differed dramatically in two animals. The types of events required for such variation are discussed. Finally, we have elaborated our earlier model for how E proteins may assemble in vivo to form filler.     

Multiple replication origins are used during Drosophila chorion gene amplification

DNA from Drosophila egg chambers undergoing chorion gene amplification was analyzed using the two-dimensional gel technique of Brewer and Fangman. At stage 10, 34% of DNA molecules from the maximally amplified region of the third chromosome chorion gene cluster contained replication forks or bubbles. These nonlinear forms were intermediates in the process of amplification; they were confined to follicle cells, and were found only within the replicating region during the time of amplification. Multiple origins gave rise to these intermediates, since three separate regions of the third chromosome chorion locus contained replication bubbles. However, initiation was nonrandom; the majority of initiations appeared to occur near the Bgl II site located between the s18 and s15 chorion genes. The P[S6.9] chorion transposon also contained abundant replication intermediates in follicle cells from a transformed line. Initiation within P[S6.9] occurred near two previously defined cis-regulatory elements, one near the same Bgl II site (in the AER-d region) and one near the ACE3 element.     

Gene regulation and evolution in the chorion locus of Bombyx mori. Structural and developmental characterization of four eggshell genes and their flanking DNA regions

We report on the detailed structural and developmental characterization of four chorion genes and a truncated pseudogene located within a 9.5 X 10(3) base chromosomal segment. These genes belong to the A and B multigene families and, like previously characterized moth chorion genes, are arranged in tightly linked pairs, which are divergently transcribed (A/B.L11 and A/B.L12). On the basis of their high degree of sequence divergence, the A genes define two distinct subfamilies, while the more homologous B genes represent different copies of the same gene type. The A.L11 and B.L11 introns are much longer, in each case because of a single inserted DNA segment that is missing from A.L12 or B.L12. The 2.1 X 10(3) base insertion in A.L11 is the first retrovirus-like transposable element characterized in Bombyx mori. The very short 5' flanking sequences of A/B.L11 and A/B.L12 (277 and 276 base-pairs) are distinct as shown by hybridization but both recur in additional chorion gene pairs, forming two respective classes that are expressed during distinctly different developmental periods. The divergently transcribed genes of each pair, which border the same 5' flanking sequence, are expressed co-ordinately, during the same developmental period. Detailed comparisons of the 5' flanking regions, and of the corresponding region of the Drosophila s15-1 chorion gene, revealed numerous, very short sequence elements that are shared. One such element, T-C-A-C-G-T, is also associated with all five sequenced Drosophila chorion genes. Some elements are repeated in a dyad symmetrical pattern, i.e. are associated with each of the two genes in a pair, while others, including T-C-A-C-G-T, occur only once per 5' flanking region, and, if functionally important, would presumably act bi-directionally on both genes of the pair.     

Stable correlation structure among multiple plumage colour traits: can they work as a single signal?

The presence of multiple distinct ornamental traits in the same species is frequently explained by context‐specificity and different information content. However, the expression of multiple ornaments is often correlated, and such traits may therefore function as a single, integrated signal. Delayed use of an integrated signal relative to production requires temporal stability in integration, which has seldom been examined. We used autumn and spring reflectance data from the breast, breast stripe, and crown of great tits (Parus major) to assess the stability and mating implications of colour signal integration, as well as the repeatability of any integrated colour trait and its correlation with condition during moult. We found high levels of stability between seasons, years, sexes, and ages in the correlation patterns of colour measures across the three plumage areas. The first principal component colour axis described joint variation of ultraviolet (UV) reflectance on the crown and the breast stripe, thereby representing an among‐trait UV chroma axis. However, only breast yellow chroma showed condition‐dependence, whereas temporally consistent and significant assortative mating was restricted to crown UV chroma. Our results therefore do not support the idea that the overall UV chroma of the breast stripe and the crown is special in condition‐dependence and repeatability, or that it plays a specific role in mutual sexual selection as an integrated signal. The results show that stable association between display traits is an existing phenomenon. They also indicate that, even in the presence of correlated traits, functional trait integration among these requires further scrutiny. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 114 , 92–108.     

Target sequences for hunchback in a control region conferring Ultrabithorax expression boundaries.

Boundaries of Ultrabithorax expression are mediated by long-range repression acting through the PBX or ABX control region. We show here that either of these control regions confers an early band of beta-galactosidase expression which is restricted along the anteroposterior axis of the blastoderm embryo. This band is succeeded by a stripe pattern with very similar anteroposterior limits. Dissection of the PBX control region demonstrates that the two patterns are conferred by distinct cis-regulatory sequences contained within separate PBX subfragments. We find several binding sites for hunchback protein within both PBX subfragments. Zygotic hunchback function is required to prevent ectopic PBX expression. Moreover, the PBX pattern is completely suppressed in embryos containing uniformly distributed maternal hunchback protein. Our results strongly suggest that hunchback protein directly binds to the PBX control region and acts as a repressor to specify the boundary positions of the PBX pattern.     

Amplification of a chorion gene cluster in Drosophila is subject to multiple cis-regulatory elements and to long-range position effects

We have used P-element transformation to study cis-acting elements involved in the control of amplification of the third chromosome chorion gene cluster (66D12-15) in Drosophila melanogaster. To reduce position effects large fragments (5.7 to 12 kb; kb = 10(3) bases) of chorion DNA and the 7.2 kb ry+ fragment were used to "buffer" these putative elements from sequences at the insertion site. Nevertheless, even the longest constructs were profoundly affected by the insertion sites and showed amplification levels ranging from undetectable to higher than in the endogenous locus. Any amplification was tissue and temporally correct and extended into the neighboring ry+ sequences. Analysis of amplification levels at various points along two constructs bearing the same 10 kb chorion insert in opposite orientations showed maximal levels occurring at one end of the chorion fragment, irrespective of whether that end was buffered at the middle of the transposon or exposed close to the insertion site. The maximally amplifying region encompasses the amplification control element (ACE), which has been shown to be necessary for amplification, in agreement with its putative role as a replication origin. We have additionally identified amplification-enhancing elements present elsewhere in the 10 kb chorion fragment, which are needed for attainment of high copy number. These elements, distinct from the ACE, have been only coarsely localized within two 2.25 to 2.3 kb regions. Some interesting sequence similarities between these two regions and the ACE element are pointed out.     

Conservation of human and mouse basonuclins as a guide to important features of the protein

The nucleotide sequence of mouse basonuclin has been determined from its cDNA by PCR and compared with the previously known sequence of human basonuclin. Overall, there is 88% identity in the encoded amino acid sequences, but some regions have been much more conserved than others. Zinc fingers 2 and 6, the region containing the nuclear localization signal and the region containing the serine stripe encode identical amino acid sequences in the two species, but differ by numerous silent nucleotide substitutions, suggesting that these regions are likely to be important for the functions of the protein common to the two species. Similarly, zinc fingers 1 and 5 diverge at only a single amino acid residue. In contrast, other regions of the sequence have diverged considerably, such as zinc fingers 3 and 4. The region adjacent to the N-terminus is very divergent and this aids in locating the translation start site. The highly conserved regions are likely to be essential for the common function of the proteins, and the more divergent regions may be either unconstrained or adapted to different requirements in the two species.     

Steroid metabolism by cells from human chorion laeve isolated on Percoll gradients

Collagenase-dispersed cells from human chorion laeve were examined on Percoll gradients. The 3 beta-hydroxysteroid dehydrogenase (a trophoblast marker) and steroid sulfatase activities of the cells were measured and a system was developed to isolate enriched preparations of the trophoblast cells. No cells were found to sediment at Percoll concentrations greater than 50%, and using continuous gradients of Percoll there appeared to be cells with different 3 beta-hydroxysteroid dehydrogenase (3 beta HSD): steroid sulfatase ratios sedimenting in different regions of the gradient. Cells with a high ratio were found in the denser region of the gradient. Continuous gradients provided inadequate separations of distinct populations of cells, thus to obtain a more reproducible system to isolate cells, discontinuous gradients of Percoll were studied. A discontinuous gradient composed of 5, 20, 40, and 60% Percoll was developed and three bands of cells were found sedimenting at the 20, 40 and 60% interfaces, respectively. The number and appearance of cells at the 20 and 60% interfaces varied from tissue to tissue. In contrast, the cells sedimenting at the 40% interface were less variable, a substantial number was found to be present in every tissue studied, they were similar in appearance to the trophoblast cells and had high 3 beta HSD:sulfatase ratios.     

Localization of acetylcholine receptors by means of horseradish peroxidase-alpha-bungarotoxin during formation and development of the neuromuscular junction in the chick embryo

The localization of acetylcholine receptors (AChR) in the surface of developing myogenic cells of the chick embryo anterior and posterior latissimus dorsi muscles in relation to the process of innervation has been studied at the ultrastructural level utilizing a horseradish peroxidase-alpha-bungarotoxin conjugate. Localized concentrations of AChR were found in small regions 0.1-0.4 micron in width on the surface of myogenic cells of 10- to 14-d-old muscles. Surface specializations consisting of an external coating of extraneous material and an internal accumulation of dense material are associated with the plasma membrane in the regions of AChR concentration. As the muscle fibers are innervated, reactive surface patches are found at the region of contact of the growing nerve fiber and the surface of myotubes or their fusing myoblasts. After the establishment of contact, the patches of reaction product become more numerous and coextensive within the region of the neuromuscular junction and its immediate surroundings forming a dense continuous deposit on the postsynaptic sarcolemma. Activity becomes increasingly restricted to the site of the neuromuscular junction as the embryos approach hatching. At all stages, specializations external and internal to the plasmalemma are found at regions of high density of AChR, suggesting that they play a role in the maintenance of a higher concentration of receptors at these sites. These specializations also occur at the region of initial synaptic contact, indicating that they might be recognized by the nerve and represent preferred sites of innervation. Innervation appears to exert a stabilizing influence on the area of high AChR concentration in contact with the nerve and to induce a further increase in the AChR density of this site while the number of AChR in the remaining portions of the muscle surface declines.     

Organization of regionally expressed silkmoth chorion genes.

We described the organization of two silkmoth chorion genes, called E1 and E2, whose expression is largely restricted in time to the very late period of choriogenesis and in space to one of two major subpopulations of follicle cells. Using E1 and E2 clone cDNAs as probes, we showed that gene copy numbers per haploid genome remain constant throughout silkmoth development despite major changes in total DNA content per nucleus. Furthermore, gene copy numbers are the same in both cellular regions of the choriogenic follicle despite differences in nuclear size and levels of E gene expression. Southern analysis indicated between two and four copies each for E1 and E2 genes. Analysis of chromosomal clones showed that single copies of E1 and E2 are separated by about 7.5 kilobases and are transcribed from the same DNA strand. Two distinct pairs of cloned E1 and E2 genes were characterized. No other chorion genes were in their immediate vicinity.