Programmed Scale Detachment in the Wing of the Pellucid Hawk Moth, Cephonodes hylas: Novel Scale Morphology, Scale Detachment Mechanism, and Wing Transparency.

No scales of most lepidopterans (butterflies and moths) detach from the wings through fluttering. However, in the pellucid hawk moth, Cephonodes hylas, numerous scales detach from a large region of the wing at initial take-off after eclosion; consequently, a large transparent region without scales appears in the wing. Even after this programmed detachment of scales (d-scales), small regions along the wing margin and vein still have scales attached (a-scales). To investigate the scale detachment mechanism, we analyzed the scale detachment process using video photography and examined the morphology of both d- and a-scales using optical and scanning electron microscopy. This study showed that d-scale detachment only occurs through fluttering and that d-scales are obviously morphologically different from a-scales. Although a-scales are morphologically common lepidopteran scales, d-scales have four distinctive features. First, d-scales are much larger than a-scales. Second, the d-scale pedicel, which is the slender base of the scale, is tapered; that of the a-scale is columnar. Third, the socket on the wing surface into which the pedicel is inserted is much smaller for d-scales than a-scales. Fourth, the d-scale socket density is much lower than the a-scale socket density. This novel scale morphology likely helps to facilitate scale detachment through fluttering and, furthermore, increases wing transparency.

Coherent array of branched filamentary scales along the wing margin of a small moth.

In butterflies and moths, the wing margins are fringed with specialized scales that are typically longer than common scales. In the hindwings of some small moths, the posterior margins are fringed with particularly long filamentary scales. Despite the small size of these moth wings, these scales are much longer than those of large moths and butterflies. In the current study, photography of the tethered flight of a small moth, Phthorimaea operculella, revealed a wide array composed of a large number of long filamentary scales. This array did not become disheveled in flight, maintaining a coherent sheet-like structure during wingbeat. Examination of the morphology of individual scales revealed that each filamentary scale consists of a proximal stalk and distal branches. Moreover, not only long scales but also shorter scales of various lengths were found to coexist in each small section of the wing margin. Scale branches were ubiquitously and densely distributed within the scale array to form a mesh-like architecture similar to a nonwoven fabric. We propose that possible mechanical interactions among branched filamentary scales, mediated by these branches, may contribute to maintaining a coherent sheet-like structure of the scale array during wingbeat.

Characterization of an Antiserum against Feather Keratins of the Chick: Its Crossreaction with a Lens Protein, δ-crystallin: (antiserum/feather keratins/δ-crystallin/immunohistochemistry/immunoblotting).

We prepared an antiserum against a fraction of solubilized keratins extracted from down feathers of newly hatched chicks. The specificity of the antiserum was tested by double immunodiffusion, immunofluorescent staining, and immunoblotting after sodium dodecylsulfate-polyacrylamide gel electrophoresis. All the bands except "Fast protein" reacted with the antiserum, suggesting the presence of a common antigenicity through various polypeptides in solubilized feather keratins. Delta-crystallin, which is a lens specific protein, also reacted with the antiserum. The presence of a common antigenicity between δ-crystallin and feather and scale keratins was confirmed by affinity-purification of the antiserum, and its significance is discussed.

Role of integrins in differentiation of chick retinal pigmented epithelial cells in vitro.

When retinal pigmented epithelial cells (PEC) of chick embryos are cultured under appropriate conditions, the phenotype changes to that of lens cells through a process known as transdifferentiation. The first half of the process, characterized by dedifferentiation of PEC, is accompanied by a marked decrease in adhesiveness of PEC to collagen type I- or type IV-coated dishes. To understand the underlying mechanisms of this change, we analyzed the expression of integrins, which are major receptors for extracellular matrix components. Northern blot analysis with cDNA probes for chicken α3, α6, α8, αv, ß1 and ß5 integrin mRNA showed that the genes for all these integrins are transcribed at similar levels in PEC and dedifferentiated PEC (dePEC). Further analysis of ß1 integrin, which is a major component of integrin heterodimers, showed that although the protein amount of ß1 integrin was not changed, its localization at focal contacts seen in PEC was lost in dePEC. When anti-ß1 integrin antibody was added to the PEC culture medium, a decrease of cell-substrate adhesiveness occurred, followed by a gradual change in both morphology and gene expression patterns to ones similar to those of dePEC. These findings suggest that an appropriate distribution of ß1 integrin plays an essential role in maintaining the differentiated state of PEC through cell-substrate adhesion.

Expression of the Retinal Pigmented Epithelial Cell-Specific pP344 Gene during Development of the Chicken Eye and Identification of Its Product: (eye development/retinal pigmented epithelium/gene expression/in situ hybridization).

In order to understand the transdifferentiation of the retinal pigmented epithelial cells (PECs) into the lens cells at the level of gene expression, a gene, tentatively called pP344 gene, was studied, because its expression appeared to be closely related with the differentiated state of PECs. We analyzed pP344 gene expression during chicken eye development by RT-PCR and in situ hybridization and also characterized the pP344 protein using antipeptide antibodies. In addition to the previous observation that the transcript of pP344 gene is limited to the pigmented epithelium and not detected in the melanocytes, we show here that the transcript is limited to retinal PECs and is never observed in iris or ciliary PECs. The time course of expression level showed two peaks; the first peak occurred around the 10th day similarly to the expression of melanosome-related genes, while the second peak occurred just after hatching when PECs had completely differentiated, suggesting that pP344 gene may be related to the function of fully differentiated PECs. Antisynthetic peptide antibodies detected pP344 protein in the culture medium of the PECs but not within the cells. Thus, we concluded that pP344 gene is specifically expressed by the retinal PEC and its product is a secreted protein.

Immunological Relationships among Embryonic and Adult Chicken Pepsinogens: A Study with Monoclonal and Polyclonal Antibodies: (chicken pepsinogens/development/antibodies/immunocytochemistry/immunoblotting).

To investigate the immunological relationships of pepsinogen isozymes present in embryonic and adult chicken proventriculi, we obtained monoclonal and polyclonal antibodies to these pepsinogens. Zymograms and immunoblots demonstrated that monoclonal antibody Y37 reacted with both embryonic and slow-migrating adult pepsinogens, while polyclonal antibodies against embryonic pepsinogen and fast-migrating adult pepsinogen were specific for these respective antigens. Shift from embryonic to adult-type pepsinogen occurred at about the time of hatching and the localizations of embryonic and adult-type pepsinogens within proventricular gland cells were found to differ by the indirect immunofluorescence method. Results with these antibodies revealed the immunological relations of these pepsinogens and the unique properties of embryonic chicken pepsinogen.

NaX sodium channel is expressed in non-myelinating Schwann cells and alveolar type II cells in mice.

Na(x) is an extracellular sodium-level-sensitive sodium channel expressed in the circumventricular organs in the brain, essential loci for the sodium-level homeostasis in body fluids. Here, we examined the localization of Na(x) throughout the visceral organs at the cellular level. In visceral organs including lung, heart, intestine, bladder, kidney and tongue, a subset of Schwann cells within the peripheral nerve trunks were highly positive for Na(x). An electron microscopic study indicated that these Na(x)-positive cells were non-myelinating Schwann cells. In the lung, Na(x)-positive signals were also observed in the alveolar type II cells, which actively absorb sodium and water to aid gas exchange through the alveolar surface. It was thus suggested that the Na(x) sodium channel is involved in controlling the local extracellular sodium level through sodium absorption activity.

Abnormal epithelial cell polarity and ectopic epidermal growth factor receptor (EGFR) expression induced in Emx2 KO embryonic gonads.

The gonadal primordium first emerges as a thickening of the embryonic coelomic epithelium, which has been thought to migrate mediodorsally to form the primitive gonad. However, the early gonadal development remains poorly understood. Mice lacking the paired-like homeobox gene Emx2 display gonadal dysgenesis. Interestingly, the knockout (KO) embryonic gonads develop an unusual surface accompanied by aberrant tight junction assembly. Morphological and in vitro cell fate mapping studies showed an apparent decrease in the number of the gonadal epithelial cells migrated to mesenchymal compartment in the KO, suggesting that polarized cell division and subsequent cell migration are affected. Microarray analyses of the epithelial cells revealed significant up-regulation of Egfr in the KO, indicating that Emx2 suppresses Egfr gene expression. This genetic correlation between the two genes was reproduced with cultured M15 cells derived from mesonephric epithelial cells. Epidermal growth factor receptor signaling was recently shown to regulate tight junction assembly through sarcoma viral oncogene homolog tyrosine phosphorylation. We show through Emx2 KO analyses that sarcoma viral oncogene homolog tyrosine phosphorylation, epidermal growth factor receptor tyrosine phosphorylation, and Egfr expression are up-regulated in the embryonic gonad. Our results strongly suggest that Emx2 is required for regulation of tight junction assembly and allowing migration of the gonadal epithelia to the mesenchyme, which are possibly mediated by suppression of Egfr expression.

Sodium-level-sensitive sodium channel Na(x) is expressed in glial laminate processes in the sensory circumventricular organs.

Na(x) is an atypical sodium channel that is assumed to be a descendant of the voltage-gated sodium channel family. Our recent studies on the Na(x)-gene-targeting mouse revealed that Na(x) channel is localized to the circumventricular organs (CVOs), the central loci for the salt and water homeostasis in mammals, where the Na(x) channel serves as a sodium-level sensor of the body fluid. To understand the cellular mechanism by which the information sensed by Na(x) channels is transferred to the activity of the organs, we dissected the subcellular localization of Na(x) in the present study. Double-immunostaining and immunoelectron microscopic analyses revealed that Na(x) is exclusively localized to perineuronal lamellate processes extended from ependymal cells and astrocytes in the organs. In addition, glial cells isolated from the subfornical organ, one of the CVOs, were sensitive to an increase in the extracellular sodium level, as analyzed by an ion-imaging method. These results suggest that glial cells bearing the Na(x) channel are the first to sense a physiological increase in the level of sodium in the body fluid, and they regulate the neural activity of the CVOs by enveloping neurons. Close communication between inexcitable glial cells and excitable neural cells thus appears to be the basis of the central control of the salt homeostasis.