XB-ART-34409Development 2006 Oct 01;13320:4109-18. doi: 10.1242/dev.02563.
Show Gene links Show Anatomy links
Shroom2 (APXL) regulates melanosome biogenesis and localization in the retinal pigment epithelium.
Shroom family proteins have been implicated in the control of the actin cytoskeleton, but so far only a single family member has been studied in the context of developing embryos. Here, we show that the Shroom-family protein, Shroom2 (previously known as APXL) is both necessary and sufficient to govern the localization of pigment granules at the apical surface of epithelial cells. In Xenopus embryos that lack Shroom2 function, we observed defects in pigmentation of the eye that stem from failure of melanosomes to mature and to associate with the apical cell surface. Ectopic expression of Shroom2 in naïve epithelial cells facilitates apical pigment accumulation, and this activity specifically requires the Rab27a GTPase. Most interestingly, we find that Shroom2, like Shroom3 (previously called Shroom), is sufficient to induce a dramatic apical accumulation of the microtubule-nucleating protein gamma-tubulin at the apical surfaces of naïve epithelial cells. Together, our data identify Shroom2 as a central regulator of RPE pigmentation, and suggest that, despite their diverse biological roles, Shroom family proteins share a common activity. Finally, because the locus encoding human SHROOM2 lies within the critical region for two distinct forms of ocular albinism, it is possible that SHROOM2 mutations may be a contributing factor in these human visual system disorders.
PubMed ID: 16987870
Article link: Development
Species referenced: Xenopus
Genes referenced: actl6a rab27a rap1b rap1gap rpe shroom2 shroom3 tubg1
Morpholinos: shroom2 MO1
Article Images: [+] show captions
|shroom2 (shroom family member 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, anterior view, dorsal up.|
|shroom2 (shroom family member 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, dorsal up, anterior left, head region only.|
|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.|
|Shroom2 induces apical pigment accumulation but not apical constriction in epithelial cells. (A) Shroom2 shares three regions of protein similarity wi|
|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.|
|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.|
|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.|
|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.|
|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).|
|Fig. 8. Shroom2 expression induces the apical accumulation ofγ -tubulin in epithelial cells. (A-c′) Both Shroom2 and Shroom3 expression in nae blastomeres was found to trigger dramaticγ -tubulin accumulation at the apical cell surface of epithelial cells. (B-c′) γ-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 nae blastomeres did not co-localize with accumulated γ-tubulin (green), however, but localized at the cell surface basal to γ-tubulin.|