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First published online 20 September 2006
doi: 10.1242/dev.02563

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Shroom2 (APXL) regulates melanosome biogenesis and localization in the retinal pigment epithelium

Pamela D. Fairbank¹, Chanjae Lee¹, Avegiyel Ellis^1,*, Jeffrey D. Hildebrand², Jeffrey M. Gross^1,3 and John B. Wallingford^1,3,

¹ Section of Molecular Cell and Developmental Biology, Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712, USA.
² Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
³ Institute for Neuroscience, University of Texas, Austin, TX 78712, USA.

View larger version (37K): [in a new window]   Fig. 1. Shroom2 induces apical pigment accumulation but not apical constriction in epithelial cells. (A) Shroom2 shares three regions of protein similarity with Shroom3, an N-terminal PDZ domain and two ASD domains. (B) The blastula assay. Xenopus blastomeres at the cleavage stages undergo cell division without transcription (Newport and Kirschner, 1982), and through asymmetric divisions, give rise to both outer, polarized epithelial cells and inner, non-polarized cells (Chalmers et al., 2003). mRNA (red) injected in individual blastomeres at the four-cell stage is distributed to polarized (grey) and non-polarized (white) cells. (C,c') Control Xenopus embryos with normal levels of apical pigment. (D,d') Embryos injected at the four-cell with 1 ng of Shroom3 or Shroom2. (E,e') Dramatic apical pigment accumulation. (F) Shroom3 expression results in apical constriction and a reduction of surface area, while Shroom2 expression had no effect on cell surface area. (G-I) Actin localization is limited to cell junctions in control (G) cells, while Shroom3-injected embryos (H) had high levels of ectopic actin accumulation at their apical surfaces. Shroom3-injected cells accumulate only modest amounts of apical actin (I). Actin accumulation is indicated by red arrows.

View larger version (21K): [in a new window]   Fig. 2. Assays of Shroom2 activity. (A-C) Shroom2 activity was measured by blind-scoring of pigment accumulation in injected embryos. Panels A-C show representative embryos displaying normal, mild and severe phenotypes for the scoring shown in D-F. (D) Shroom2 (576-882) blocked the ability of ectopic Shroom2 to induce apical pigment accumulation in a dose-dependent manner. Shroom2 (0.4 ng) was injected alone or in combination with 0.8 ng or 1.6 ng of Shroom2(576-882) in one blastomeres at the four-cell stage. (E) Shroom2 does not function through the Rap1B GTPase. Shroom2 (0.75 ng) was injected alone or in combination with 2 ng of Rap1B-N17 or RapGAP into one blastomere at the four-cell stage. (F) Shroom2 acts upstream of Rab27a as co-expression of either dominant-negative Rab27a-L130P or Rab27a-A152P inhibited Shroom2 activity. Shroom2 (0.4 ng) was injected alone or in combination with 1.2 ng of Rab27a-L130P or Rab27a-A152P into one blastomere at the four-cell stage.

View larger version (65K): [in a new window]   Fig. 3. Shroom2 is expressed in the eye. (A,B) In situ hybridization reveals Shroom2 mRNA is expressed in the developing eye. (A,a') Shroom2 is expressed in the early eye field of stage 25 embryos, as well as the brain and ear. (B) Stage 40 embryos have Shroom2 mRNA expression restricted to the RPE.

View larger version (98K): [in a new window]   Fig. 4. Mosaic expression of a dominant-negative fragment of Shroom2 disrupts eye pigmentation. (A) Lipofection enables mosaic expression of dominant-negative Shroom2 specifically in the Xenopus eye. Constructs are lipofected into the eye field at stage 18 and grown to stage 42. All constructs are mixed at a ratio of 1 µg plasmid/3 µl DOTAP and pulsed in 10 nl volumes. (B,b') Lipofection of membrane GFP alone results in normal eye morphology and pigmentation (melanin in the RPE obscures GFP fluorescence in b'). (C,c') Embryos lipofected with membrane GFP and Shroom2(576-882) showed patchy hypopigmentation in areas where GFP was localized.

View larger version (41K): [in a new window]   Fig. 5. Morpholino knock-down of Shroom2 results in severe RPE hypopigmentation. (A) RT-PCR confirmation of morpholino efficacy. Normally spliced Shroom2 mRNA was absent from 80 ng Shroom2-SPL-MO injected embryos. (B) Shroom2-SPL-MO or Shroom2-5MM-MO (80 ng) was injected into two animal blastomeres at the four-cell stage and embryos were grown to stage 42. (C,D) Shroom2-SPL-MO injected embryos show severe reduced RPE pigmentation (E,e') when compared with stage-matched uninjected or Shroom-5MM-MO embryos (C-d'). (F) Measurement of pixel density in the eye indicates no significant difference in pigmentation between uninjected and Shroom2-5MM-MO-injected embryos at stage 39, but a drastic difference between pixel density between controls and Shroom2-SPL-MO eyes.

View larger version (111K): [in a new window]   Fig. 6. Retinal lamination and RPE structure are disrupted in Shroom2-SPL-MO morphants. (A,D) Histological analysis indicated that the most severely affected Shroom2-SPL-MO morphants had prominent retinal lamination defects and increased numbers of pyknotic nuclei in their retinas when compared with stage-matched 5 bp-MM MO-injected controls. (B,E) Less severely affected morphants showed significantly better retinal lamination, as demonstrated here via actin localization in optical sections. Additionally, phalloidin staining revealed the regular appearance of the RPE in control embryos (B,b') in contrast to that in Shroom2-SPL-MO morphants (E,e') in which the RPE invaded the retina with numerous apical protrusions. (C,F) Further examination with TEM highlights these apical protrusions and also indicates that melanosome morphology and distribution are disrupted in Shroom2-SPL-MO morphants. Melanosomes appear clumpy and are not limited to the apical cell surface. Red lines delimit apical (left) and basal (right) surfaces of the RPE.

View larger version (88K): [in a new window]   Fig. 7. Ultrastructural analysis of Shroom2 RPE. (A,B) High magnification TEM allowed further analysis of Shroom2-SPL-MO morphant melanosome morphology. (A) Control RPE contained smooth-edged melanosomes of varying size and black because of melanin accumulation. (B,b') Two representative regions of Shroom2-SPL-MO morphant RPE. Melanosome number was similar to controls; however, melanosome morphology was severely abnormal. Melanosomes contained numerous vesicular structures, most of which appeared to have not coalesced into a single structure resulting in a `bunch of grapes' morphology. (C) Measurement of relative melanosome area of melanosomes in Shroom2-SPL-MO morphants and controls, indicated that the melanosome aggregates of Shroom2 morphants were almost 90% larger in area than controls. (D) Shroom2-SPL-MO morphant melanosomes were also found to have highly irregular surfaces, with a relative roundness value of over 1.7 and compared with a value of 1.36 for control melanosomes (a perfect circle has a roundness of 1.00).

View larger version (73K): [in a new window]   Fig. 8. Shroom2 expression induces the apical accumulation of -tubulin in epithelial cells. (A-c') Both Shroom2 and Shroom3 expression in naïve blastomeres was found to trigger dramatic -tubulin accumulation at the apical cell surface of epithelial cells. (B-c') -Tubulin accumulation overlapped entirely with regions of pigment accumulation and had the same intensity in both Shroom2 and Shroom3-expressing embryos (1 ng injected for either mRNA). (D-f') Shroom2 expression (red) in naïve blastomeres did not co-localize with accumulated -tubulin (green), however, but localized at the cell surface basal to -tubulin.

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