Haemocyte-derived SPARC is required for collagen-IV-dependent stability of basal laminae in Drosophila embryos.

SPARC is an evolutionarily conserved collagen-binding extracellular matrix (ECM) glycoprotein whose morphogenetic contribution(s) to embryonic development remain elusive despite decades of research. We have therefore used Drosophila genetics to gain insight into the role of SPARC during embryogenesis. In Drosophila embryos, high levels of SPARC and other basal lamina components (such as network-forming collagen IV, laminin and perlecan) are synthesized and secreted by haemocytes, and assembled into basal laminae. A SPARC mutant was generated by P-element mutagenesis that is embryonic lethal because of multiple developmental defects. Whereas no differences in collagen IV immunostaining were observed in haemocytes between wild-type and SPARC-mutant embryos, collagen IV was not visible in basal laminae of SPARC-mutant embryos. In addition, the laminin network of SPARC-mutant embryos appeared fragmented and discontinuous by late embryogenesis. Transgenic expression of SPARC protein by haemocytes in SPARC-mutant embryos restored collagen IV and laminin continuity in basal laminae. However, transgenic expression of SPARC by neural cells failed to rescue collagen IV in basal laminae, indicating that the presence of collagen IV deposition requires SPARC expression by haemocytes. Our previous finding that haemocyte-derived SPARC protein levels are reduced in collagen-IV-mutant embryos and the observation that collagen-IV-mutant embryos showed a striking phenotypic similarity to SPARC-mutant embryos suggests a mutual dependence between these major basal laminae components during embryogenesis. Patterning defects and impaired condensation of the ventral nerve cord also resulted from the loss SPARC expression prior to haemocyte migration. Hence, SPARC is required for basal lamina maturation and condensation of the ventral nerve cord during Drosophila embryogenesis.

Specific distribution of barrier-relevant ceramides in the emerging epidermis and the periderm/subperiderm during chicken embryogenesis.

During mammalian embryogenesis the emerging epidermis is temporarily covered by an epithelial monolayer, the periderm. In chicken, a second epithelial layer, the subperiderm, located underneath the periderm develops in later embryogenesis. Together the periderm and the subperiderm are referred to as the PSP unit. The cells of the PSP unit are tightly connected by tight junctions (TJ), thereby providing the embryo with an impermeable bilayered diffusion barrier. The emerging epidermis assumes its barrier function by cornification beginning at embryonic day 17 (E17) before at E18 the PSP unit undergoes desquamation. Lipid analysis of both epithelia after their mechanical separation revealed a dramatic increase to about 100-fold values of barrier-relevant ceramides, i.e. those known to essentially contribute to the diffusion barrier of the cornified envelope, in the emerging epidermis between E17 and E19. In contrast, the content of barrier-relevant ceramides in the PSP unit remained at constantly low levels throughout embryogenesis. These data strongly argue in favour of different mechanisms for the barrier function of the two epithelia. TJ in the PSP unit provide the main diffusion barrier protecting the embryo until beginning of desquamation at E18. At this developmental stage the content of cornified envelope-specific ceramides is substantially elevated, thus enabling the epidermis to fulfil its function as the major diffusion barrier after desquamation of the PSP unit. The observation that barrier-relevant ceramides are formed prior to desquamation of the PSP unit points to a precisely regulated sequence in that desquamation does not occur until the lipid-based barrier of the cornified envelope is completed and suggests in addition that these lipids might be essential regulators of the interaction between the PSP unit and the emerging epidermis.

Simultaneous cell death and desquamation of the embryonic diffusion barrier during epidermal development.

The periderm is an epithelial layer covering the emerging epidermis in early embryogenesis of vertebrates. In the chicken embryo, an additional cellular layer, the subperiderm, occurs at later embryonic stages underneath the periderm. The questions arose what is the function of both epithelial layers and, as they are transitory structures, by which mechanism are they removed. By immunocytochemistry, the tight junction (TJ) proteins occludin and claudin-1 were localized in the periderm and in the subperiderm, and sites of close contact between adjacent cells were detected by electron microscopy. Using horseradish peroxidase (HRP) as tracer, these contacts were identified as tight junctions involved in the formation of the embryonic diffusion barrier. This barrier was lost by desquamation at the end of the embryonic period, when the cornified envelope of the emerging epidermis was formed. By TUNEL and DNA ladder assays, we detected simultaneous cell death in the periderm and the subperiderm shortly before hatching. The absence of caspases-3, -6, and -7 activity, key enzymes of apoptosis, and the lack of typical morphological criteria of apoptosis such as cell fragmentation or membrane blebbing point to a special form of programmed cell death (PCD) leading to the desquamation of the embryonic diffusion barrier.

Defective epidermal barrier in neonatal mice lacking the C-terminal region of connexin43.

More than 97% of mice in which the C-terminal region of connexin43 (Cx43) was removed (designated as Cx43K258stop) die shortly after birth due to a defect of the epidermal barrier. The abnormal expression of Cx43K258stop protein in the uppermost layers of the epidermis seems to perturb terminal differentiation of keratinocytes. In contrast to Cx43-deficient mice, neonatal Cx43K258stop hearts show no lethal obstruction of the right ventricular outflow tract, but signs of dilatation. Electrocardiographies of neonatal hearts reveal repolarization abnormalities in 20% of homozygous Cx43K258stop animals. The very rare adult Cx43K258stop mice show a compensation of the epidermal barrier defect but persisting impairment of cardiac function in echocardiography. Female Cx43K258stop mice are infertile due to impaired folliculogenesis. Our results indicate that the C-terminally truncated Cx43K258stop mice lack essential functions of Cx43, although the truncated Cx43 protein can form open gap junctional channels.