Lengsin expression and function during zebrafish lens formation - PubMed (original) (raw)

Lengsin expression and function during zebrafish lens formation

Rachel L Harding et al. Exp Eye Res. 2008 May.

Abstract

A zebrafish ortholog of human lengsin was identified by EST analysis of an adult lens cDNA library. During zebrafish development, lengsin transcription is first detected at 24 h post-fertilization (hpf). Immunolocalization, using polyclonal antiserum generated against a Lengsin bacterial fusion protein, detects lens-specific protein in whole-mount embryos at 30 hpf. Lengsin expression in zebrafish follows the temporal expression of the alphaA- alphaB1- and betaB1-crystallin proteins in the lens. At 72 hpf, Lengsin is localized to a subpopulation of differentiating secondary fiber cells, while no expression is detected in the lens epithelial cells or central lens fibers. In the adult lens, Lengsin is restricted to a narrow band of cortical fibers and co-localizes with actin at the lateral faces of these interdigitating cells. Stable transgenic lines, using a 3 kb lengsin genomic fragment to regulate EGFP expression, recapitulate the Lengsin temporal and spatial expression patterns. Lengsin function in zebrafish lens formation was examined by antisense morpholino-mediated translation and mRNA splice inhibition. At 72 hpf, the lengsin morphant lenses are reduced in size and exhibit separations within the cortex due to defects in secondary fiber morphogenesis. The location of the morphant lens defects correlates with the Lengsin protein localization at this age. These results demonstrate Lengsin is required for proper fiber cell differentiation by playing roles in either cell elongation or the establishment of cell interactions.

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Figures

Figure 1

Lengsin domain organization and gene expression. (A) The zebrafish, human, mouse and chick Lengsin proteins include a GS (Glutamine Synthetase) homology region composed of the conserved β-GRASP (zebrafish amino acids 248-335) and GS catalytic domains (zebrafish amino acids 346-593). The amino acid sequence within the zebrafish GS homology region is 55%, 54% and 61% identical to this region of sequence in the human, mouse and chick proteins, respectively. In addition to a carboxyl-terminal domain (C-ter) of approximately 75 amino acids, Lengsin proteins possess an amino-terminal domain (N-terminal) whose length varies between species. The numbers of amino acids within the N-terminal domains are shown within the black bars. The calculated molecular weights (MW, in Daltons) for Lengsin from each species are shown in the column on the right. (B) RT-PCR of whole embryos and adult tissues was used to determine the temporal and spatial lengsin expression patterns. Zygotic lengsin transcription is first detected at 24 hpf (591 bp product). In adult zebrafish tissues (lower panel), lengsin is restricted to the lens, while transcripts are not detected in the other eye tissues, the brain, caudal fin or internal organs, β-actin transcripts (lower rows in each panel) were amplified from each sample as a control. Abbreviations: ant seg, anterior segment minus lens; post seg, posterior segment minus retina; int org, internal organs.

Figure 2

Lengsin protein expression during development. Whole-mount embryos were immunolabeled with polyclonal anti-Lengsin (green signal) and monoclonal antibody zl-1 (red signal), a lens-specific marker in zebrafish. At 24 hpf (Panel A), only zl-1 is detected in the differentiating lens cells. At 30 hpf (Panel B), Lengsin expression initiates in the central lens (arrow), while zl-1 is localized at the lens periphery (arrowheads). At 36 hpf (Panel C), Lengsin is expressed in many fiber cells (arrow). The central fibers lack Lengsin protein (asterisk) and zl-1 remains concentrated at the lens periphery near the epithelial cell bases (arrowheads) at this time. Lengsin becomes progressively restricted to a subpopulation of elongated peripheral lens cells between 42–72 hpf (Panels D-F; arrows in Panels E and F). Scale bar in Panel A (25 μm) applies to all panels.

Figure 3

Crystallin protein expression precedes Lengsin during zebrafish lens formation. Polyclonal antisera were used to detect zebrafish αA-, αB1- and βB1-crystallin in whole-mount embryos at 24 and 48 hpf (Panels A–D and E–H, respectively). At 24 hpf, αA-crystallin is barely detectable (Panel A, arrow), while αB1- and (βB1-crystallin are strongly expressed (arrows in Panels B and C, respectively) and Lengsin (Panel D) is not detected. At 48 hpf, these three crystallin proteins (Panels E–G) and Lengsin (Panel H) are detected at high levels in the lens (arrows). At 24 and 48 hpf, αA-crystallin, βB1-crystallin and Lengsin are restricted to the lens, while αB1-crystallin is also expressed in the retina and other parts of the CNS (compare Panels A, C, D, E, G and H to B and F). The lenses in some of the immunolabeled embryos appear yellow because the red channel gain was increased to highlight the extralenticular embryonic tissues. Scale bars represent 50 μm (Panels A–D) and 200 μm (Panels E–H).

Figure 4

Lengsin immunolocalization in the embryonic lens. Frozen eye sections from 48 hpf (Panels A and B) and 72 hpf (Panels C–F) embryos were immunolabeled using the Lengsin antiserum. Lengsin is restricted to the lens and is not detected in the retina or other eye tissues at 48 hpf (Panel A, arrow). The Lengsin immunolocalization pattern changes dramatically between 48 hpf (Panel B) and 72 hpf (Panel C) with a shift from the proximal lens fibers (Panel B, asterisks) to the distal lens fibers and lens equator (Panel C, arrows and asterisks, respectively). Lengsin is also detected in the lens transition zone at 72 hpf (Panel C, arrowheads). In the growing lens at 72 hpf, Lengsin expression initiates at the base of the lens transition zone (Panel D, arrow), but is not detected in either the lateral or central epithelial cells (arrowheads in Panels D and E, respectively). Lengsin is not detected in the more central nuclei-containing fibers (Panel D, asterisks). Compared to Lengsin, αB1-crystallin is expressed in both fiber and epithelial cells at 72 hpf (Panel F, arrows). Scale bars represent 50 μm (Panel A) or 25 μm (Panels B–F).

Figure 5

Lengsin localization in the adult lens. Phalloidin-AF488 staining demonstrates filamentous Actin is concentrated at the lateral fiber cell membranes and membranes of the epithelial cells (Panel A, arrows and arrowheads, respectively). The lateral processes of the fiber cells are characterized by high levels of Actin (Panel B, arrows). Cross sectional profiles of the adult lens cortical fibers, immunolabeled using the Lengsin antiserum (Panel C), reveal Lengsin (green signal, arrows) is localized within a subpopulation of cortical fibers separated from the epithelial cell layer (nuclei stained blue, arrowheads) by fibers lacking detectable Lengsin (asterisks). Double-immunolabeled lens sections reveal Lengsin (green) is co-localized with Actin (red) at the lateral margins of the fibers (Panel D, Actin; Panel E, Lengsin; Panel F, Merge). In contrast to Lengsin, αB1-crystallin localizes around the entire fiber cell membrane (Panel G, Actin; Panel H, αB1-crystallin; Panel I, Merge). Scale bars represent 20 μm (Panels A and B), 50 μm (Panel C), 30 μm (Panels D–F) or 15 μm (Panels G–I).

Figure 6

Lengsin transgene expression during zebrafish lens formation. Two different stable transgenic lines were generated using lengsin 3 kb genomic sequences to regulate expression of either EGFP [Tg(lgs:EGFP)nt20, Panels A–D] or EGFP with a nuclear localization signal [Tg(lgs:EGFP-NLS)nt21, Panels E–I]. Embryo whole-mounts (Panels A–H) and a frozen tissue section (Panel I) are shown. EGFP expression was not detected from either construct by epifluorescent illumination of whole embryos at 24 hpf (Panels A and E). However, EGFP is clearly detected and restricted to the lens at 36 hpf (Panel B, arrow). Between 48–72 hpf, the lens EGFP intensity increases as the fiber cells fill with the fluorescent protein (Panels C and D, arrows). EGFP-NLS is detected in a few nuclei at 36 hpf (Panel F, arrows) with the number of EGFP-positive nuclei increasing between 48–72 hpf (Panels G and H, arrows). The frozen section (Panel I) demonstrates the elongating fiber cell nuclei are EGFP-positive in the Tg(lgs:EGFP-NLS)nt21 fish at 72 hpf (arrows). Scale bars represent 50 μm (Panels A, D, E, G–I) and 25 μm (Panels B, C and F).

Figure 7

Histology of lengsin morphant lens. Plastic resin-embedded eye sections from 72 hpf uninjected control (uiC; Panels A and E), mismatch morpholino-injected control (mmC; Panels B and F) and lengsin morphant embryos (morfA, Panels C and G; morfB, Panels D and H) were stained with methylene blue and azure II. The control lenses display a single layer of epithelial cells and nuclei of the differentiating secondary fibers can be visualized in the transition zone (Panels E and F, arrowheads and arrows, respectively). Small cortical separations (short arrows) are present in some control lenses (Panels A, B, E and F). The lengsin morphant lenses (Panels C, D, G and H) exhibit large separations (asterisks) between cortical fibers and the overlying epithelial cells in the region near the lens equator and distal to the differentiating secondary fiber nuclei. The morphant lens is also reduced in size and lacks the concentric central fiber cell arrangement displayed by the controls (compare Panels G and H to Panels E and F). The lenses shown in Panels C, D, G and H are from different lengsin morphants. Abbreviations: PL, photoreceptor layer; INL, inner nuclear layer; GCL, ganglion cell layer; uiC, uninjected control; mmC, mismatch control morpholino. The scale bars in Panels A and E (50 μm) also apply to Panels B–D and F–H, respectively.

Figure 8

Transmission electron microscopy of lengsin morphant lens. Serial sections of uninjected control and lengsin morphant (morfA) lens were stained with uranyl acetate and lead citrate. Control lens section images (Panels A and B) were taken in the lens transition zone (Panel A, asterisks denote nuclei) and at the interface between the central epithelial cells and underlying fiber cells (Panel B, asterisk and arrows, respectively). Panels C and D show the lengsin morphant lens transition zone of two different fish. In Panel C, a large gap (asterisk) is located just distal to the secondary fiber nucleus (double arrowheads). The fiber cell end appears to curve around the perimeter of the tissue gap (arrow). Panel D demonstrates morphant lens secondary fibers are shrunken and retracted from the adjacent fiber cells (arrows). Electron-dense vacuoles are evident in some morphant lens fibers (Panel E, asterisks). The lengsin morphant fiber cell separations in the area adjacent to the central epithelial cells are less distinct compared to the control (compare Panel F to B, arrows). Scale bars represent 2 μm (Panels A, D and F), 1 μm (Panel B) and 3 μm (Panels C and E).

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Lengsin expression and function during zebrafish lens formation - PubMed (original) (raw)