By comparison of the skull, mandible, and teeth of an adult female Mesoplodon densirostris (the o... more By comparison of the skull, mandible, and teeth of an adult female Mesoplodon densirostris (the only adult female of this species known to science) to those of males, sexual dimorphism is found as follows: 1) the partial but meager filling of the mesorostral groove in the female as opposed to complete filling for 90 per cent of its length in adult males; 2) smaller development of the female rostrum in width and depth, but 3) greater development in length; 4) much smaller development of the female tooth, alveolus, and dental pulpit; and 5) failure of the tooth to erupt in the adult female. Comparison of the skull, mandible, and teeth of the adult female to those of a subadult female revealed the following differences regarded here as potentially developmental: 1) moderate development of the vomer and mesethmoid in the mesorostral groove of the subadult as compared to the extensive thickening of these bones in the adult so as to fill the mesorostral groove proximally; 2) differences in rostral form as revealed by cross-sectional profiles and rostral measurements, probably resulting from development described above; 3) meager development of bony overlays of the maxillary, palatine, and pterygoid in the orbital and auditory regions of the subadult as compared to their extensive development and thickening in the adult which is reflected in 4) the smaller size of the fossae in the orbital region of the adult; 5) rounded posterior profile of the brainease of the subadult compared to its straight, angular character in the adult; 6) greater ize of the temporal fossa in the subadult; 7) open pulp cavity of the tooth of he subadult as compared to its complete closure in the adult. In addition, the ympanic bulla of the subadult is larger relative to skull length than that of the dult, but asymmetry is greater in the adult. The relative anteroposterior position f the maxillary and premaxillary foramina was found to vary in Mesoplodon ensirostris , making it unreliable as a taxonomic character.
The photoreceptor connecting cilium bears a unique transmembrane assemblage which stably links ce... more The photoreceptor connecting cilium bears a unique transmembrane assemblage which stably links cell surface glycoconjugates with the underlying axonemal cytoskeleton. Structural similarities between the photoreceptor connecting cilium and the transition zone of motile cilia suggests that this assemblage may also be present in motile cilia. Using a subcellular fraction enriched in detergent-extracted photoreceptor axonemes, three high molecular mass glycoconjugates (425, 600, and 700 kD) were previously identified as potential components of the assemblage. Through oligosaccharide characterization and binding of a specific monoclonal antibody, we have verified the localization of the 425 kD glycoconjugate to the transmembrane assemblage. Binding of the lectin peanut agglutinin (PNA) to the 425 kD glycoconjugate on nitrocellulose blots, and to isolated detergent-extracted axonemes, was assessed following treatment with the enzymes neuraminidase and O-glycanase. Changes in binding to the 425 kD glycoconjugate precisely paralleled changes in binding to intact axonemes, supporting the hypothesis that the 425 kD glycoconjugate is a component of the transmembrane assemblage. Furthermore, the results suggest that the 425 kD glycoconjugate contains sialated galactose-N-acetylgalactosamine oligosaccharides which are O-linked to the protein backbone. To directly assess the distribution of the 425 kD glycoconjugate, we produced a monoclonal antibody directed against this glycoconjugate. The antibody, K26, recognizes only the 425 kD on transblots of the axoneme fraction. K26 immunoreactivity of intact axonemes is identical to that seen by PNA staining. K26 staining of isolated photoreceptors and whole retina is uniquely localized to the region of the connecting cilium. Thus, in the photoreceptor, the 425 kD is not only a component of the transmembrane assemblage but is also completely restricted to the connecting cilium. Based on morphological similarities, the photoreceptor connecting cilium is thought to be homologous to the transition zone of the motile cilium. As such, we have stained oviduct epithelium with the K26 monoclonal antibody. Immunoreactivity is restricted to the region of the transition zone at the base of motile cilia.
The photoreceptor outer segment (OS), a well-defined sensory cilium, provides an important contex... more The photoreceptor outer segment (OS), a well-defined sensory cilium, provides an important context for the study of intraflagellar transport (IFT). The early phases of OS development involve successive events that are common to virtually all cilia. Additionally, intense protein trafficking occurs through the cilium and relies on IFT to maintain proper cellular morphology and optimize the photosensitive function. In the past decade, progress has been made in the characterization of photoreceptor OS trafficking in murine and amphibian models. Recently, powerful and cost-effective molecular tools and techniques for zebrafish have opened new opportunities to study photoreceptor IFT. Studies using zebrafish take advantage of its rapid embryogenesis to characterize the early events involved in photoreceptor ciliogenesis and OS assembly. In this overview, we describe phenotypes associated with knockdown strategies or genetic mutations of IFT components in zebrafish and detail a general experimental approach that has enabled us to study the function of the two anterograde IFT motors, KIF17 and kinesin II, in zebrafish cone photoreceptors.
Transport of opsin from photoreceptor inner to outer segments has been assumed to occur via the c... more Transport of opsin from photoreceptor inner to outer segments has been assumed to occur via the connecting cilium, the only permanent structural connection between these two regions. However, in prior work, little or no immunoreactive opsin has been detected in the cilium, despite the high rate of transport of this protein. This suggests that immune epitopes are masked during passage through the cilium or that opsin is transported via an extra-ciliary route. In this study, we stained the photoreceptors of Xenopus laevis with well-characterized monoclonal antibodies directed at the N-terminal, C-terminal, and 5-6 loop regions of bovine opsin. This was done on isolated retinas incubated in vitro under conditions that support rapid disc assembly, to insure that opsin transport to forming discs was occurring at the time of fixation. Five MAbs that gave robust staining of Xenopus rod inner segment/rod outer segment preparations with the light microscope were utilized for electron microscopic studies on LR White embedded or cryo-ultrathin sections. Four of these stained outer segment discs and inner segment vesicles and plasma membrane. However, no significant staining of the connecting cilium was found. Furthermore, freeze-fractured mouse photoreceptors prepared by the 'fracture-label' technique showed extensive labelling of membrane compartments but lacked staining of the connecting cilium. Isolated retinas incubated under conditions that support robust rod disc synthesis contained many finger-like and vesicular projections of the apical inner segment plasma membrane and inner segment vesicles extending into them. Rod outer segment nascent discs usually made close contact with the inner segment. Both the vesicular profiles associated with the inner segment plasma membrane and the basal discs extending to the inner segment were heavily stained with all four anti-opsin antibodies. This suggests an alternate route for bulk transport of opsin to newly forming discs that involves direct transfer from apical inner segment plasma membrane to nascent discs.
Advances in Experimental Medicine and Biology, 2003
During early development of rod and cone photoreceptors, the outer segment (OS) assembles on the ... more During early development of rod and cone photoreceptors, the outer segment (OS) assembles on the apical cell surface by an elaborate process of membrane and protein transport followed by membrane reorganization to form the highly organized stack of membrane disks found ...
By comparison of the skull, mandible, and teeth of an adult female Mesoplodon densirostris (the o... more By comparison of the skull, mandible, and teeth of an adult female Mesoplodon densirostris (the only adult female of this species known to science) to those of males, sexual dimorphism is found as follows: 1) the partial but meager filling of the mesorostral groove in the female as opposed to complete filling for 90 per cent of its length in adult males; 2) smaller development of the female rostrum in width and depth, but 3) greater development in length; 4) much smaller development of the female tooth, alveolus, and dental pulpit; and 5) failure of the tooth to erupt in the adult female. Comparison of the skull, mandible, and teeth of the adult female to those of a subadult female revealed the following differences regarded here as potentially developmental: 1) moderate development of the vomer and mesethmoid in the mesorostral groove of the subadult as compared to the extensive thickening of these bones in the adult so as to fill the mesorostral groove proximally; 2) differences in rostral form as revealed by cross-sectional profiles and rostral measurements, probably resulting from development described above; 3) meager development of bony overlays of the maxillary, palatine, and pterygoid in the orbital and auditory regions of the subadult as compared to their extensive development and thickening in the adult which is reflected in 4) the smaller size of the fossae in the orbital region of the adult; 5) rounded posterior profile of the brainease of the subadult compared to its straight, angular character in the adult; 6) greater ize of the temporal fossa in the subadult; 7) open pulp cavity of the tooth of he subadult as compared to its complete closure in the adult. In addition, the ympanic bulla of the subadult is larger relative to skull length than that of the dult, but asymmetry is greater in the adult. The relative anteroposterior position f the maxillary and premaxillary foramina was found to vary in Mesoplodon ensirostris , making it unreliable as a taxonomic character.
The photoreceptor connecting cilium bears a unique transmembrane assemblage which stably links ce... more The photoreceptor connecting cilium bears a unique transmembrane assemblage which stably links cell surface glycoconjugates with the underlying axonemal cytoskeleton. Structural similarities between the photoreceptor connecting cilium and the transition zone of motile cilia suggests that this assemblage may also be present in motile cilia. Using a subcellular fraction enriched in detergent-extracted photoreceptor axonemes, three high molecular mass glycoconjugates (425, 600, and 700 kD) were previously identified as potential components of the assemblage. Through oligosaccharide characterization and binding of a specific monoclonal antibody, we have verified the localization of the 425 kD glycoconjugate to the transmembrane assemblage. Binding of the lectin peanut agglutinin (PNA) to the 425 kD glycoconjugate on nitrocellulose blots, and to isolated detergent-extracted axonemes, was assessed following treatment with the enzymes neuraminidase and O-glycanase. Changes in binding to the 425 kD glycoconjugate precisely paralleled changes in binding to intact axonemes, supporting the hypothesis that the 425 kD glycoconjugate is a component of the transmembrane assemblage. Furthermore, the results suggest that the 425 kD glycoconjugate contains sialated galactose-N-acetylgalactosamine oligosaccharides which are O-linked to the protein backbone. To directly assess the distribution of the 425 kD glycoconjugate, we produced a monoclonal antibody directed against this glycoconjugate. The antibody, K26, recognizes only the 425 kD on transblots of the axoneme fraction. K26 immunoreactivity of intact axonemes is identical to that seen by PNA staining. K26 staining of isolated photoreceptors and whole retina is uniquely localized to the region of the connecting cilium. Thus, in the photoreceptor, the 425 kD is not only a component of the transmembrane assemblage but is also completely restricted to the connecting cilium. Based on morphological similarities, the photoreceptor connecting cilium is thought to be homologous to the transition zone of the motile cilium. As such, we have stained oviduct epithelium with the K26 monoclonal antibody. Immunoreactivity is restricted to the region of the transition zone at the base of motile cilia.
The photoreceptor outer segment (OS), a well-defined sensory cilium, provides an important contex... more The photoreceptor outer segment (OS), a well-defined sensory cilium, provides an important context for the study of intraflagellar transport (IFT). The early phases of OS development involve successive events that are common to virtually all cilia. Additionally, intense protein trafficking occurs through the cilium and relies on IFT to maintain proper cellular morphology and optimize the photosensitive function. In the past decade, progress has been made in the characterization of photoreceptor OS trafficking in murine and amphibian models. Recently, powerful and cost-effective molecular tools and techniques for zebrafish have opened new opportunities to study photoreceptor IFT. Studies using zebrafish take advantage of its rapid embryogenesis to characterize the early events involved in photoreceptor ciliogenesis and OS assembly. In this overview, we describe phenotypes associated with knockdown strategies or genetic mutations of IFT components in zebrafish and detail a general experimental approach that has enabled us to study the function of the two anterograde IFT motors, KIF17 and kinesin II, in zebrafish cone photoreceptors.
Transport of opsin from photoreceptor inner to outer segments has been assumed to occur via the c... more Transport of opsin from photoreceptor inner to outer segments has been assumed to occur via the connecting cilium, the only permanent structural connection between these two regions. However, in prior work, little or no immunoreactive opsin has been detected in the cilium, despite the high rate of transport of this protein. This suggests that immune epitopes are masked during passage through the cilium or that opsin is transported via an extra-ciliary route. In this study, we stained the photoreceptors of Xenopus laevis with well-characterized monoclonal antibodies directed at the N-terminal, C-terminal, and 5-6 loop regions of bovine opsin. This was done on isolated retinas incubated in vitro under conditions that support rapid disc assembly, to insure that opsin transport to forming discs was occurring at the time of fixation. Five MAbs that gave robust staining of Xenopus rod inner segment/rod outer segment preparations with the light microscope were utilized for electron microscopic studies on LR White embedded or cryo-ultrathin sections. Four of these stained outer segment discs and inner segment vesicles and plasma membrane. However, no significant staining of the connecting cilium was found. Furthermore, freeze-fractured mouse photoreceptors prepared by the 'fracture-label' technique showed extensive labelling of membrane compartments but lacked staining of the connecting cilium. Isolated retinas incubated under conditions that support robust rod disc synthesis contained many finger-like and vesicular projections of the apical inner segment plasma membrane and inner segment vesicles extending into them. Rod outer segment nascent discs usually made close contact with the inner segment. Both the vesicular profiles associated with the inner segment plasma membrane and the basal discs extending to the inner segment were heavily stained with all four anti-opsin antibodies. This suggests an alternate route for bulk transport of opsin to newly forming discs that involves direct transfer from apical inner segment plasma membrane to nascent discs.
Advances in Experimental Medicine and Biology, 2003
During early development of rod and cone photoreceptors, the outer segment (OS) assembles on the ... more During early development of rod and cone photoreceptors, the outer segment (OS) assembles on the apical cell surface by an elaborate process of membrane and protein transport followed by membrane reorganization to form the highly organized stack of membrane disks found ...
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Papers by Joseph C Besharse