The shroom family proteins play broad roles in the morphogenesis of thickened epithelial sheets

Abstract

Thickened epithelial sheets are found in a wide variety of organ systems and the mechanisms governing their morphogenesis remain poorly defined. We show here, through expression patterns and functional studies, that Shroom family proteins are broadly involved in generating thickened epithelial sheets. Through in situ hybridization, we report the temporal and spatial expression patterns of the four Shroom family members during early Xenopus development, from oocytes to tadpole stage embryos. Further, we show that Shroom1 and 2 mRNAs are maternally expressed, while Shroom3 and Shroom4 are zygotic transcripts. In addition, maternal Shroom1 and 2 mRNAs localize in the animal hemisphere of the Xenopus egg and early blastula. During later development, all four Shroom family proteins are broadly expressed in developing epithelial organs, and the epithelial cells that express Shrooms are elongated. Moreover, we show that ectopic expression of Shroom2, like Shroom3, is able to increase cell height and that loss of Shroom2 function results in a failure of cell elongation in the neural epithelium. Together, these data suggest that Shroom family proteins play an important role in the morphogenesis of several different epithelial tissues during development. Developmental Dynamics 238:1480-1491, 2009. (c) 2009 Wiley-Liss, Inc.

Figure 1. RT-PCR for Shroom family genes during early Xenopus development

Reverse transcriptase PCR (RT-PCR) was performed with specific primers of Shroom family genes in unfertilized egg, mid-blastrula (st.8 and 9) early gastrula (st.10) and mid-gastrula (st. 11.5). ODC (ornithine decarboxylase) is used as a control. The weak bands for Shroom3 and 4 in egg and stage 8 are the result of genomic DNA contamination (data not shown).

Figure 2. mRNA localization of Shroom1 and 2 during oogenesis and early development

(A) in situ hybridization against Shroom1 and 2 in unfertilized egg, 1-cell, 2-cell stage and stage 6.5 (B) in situ hybridization against Shroom1 and 2 during oogenesis (C) in situ hybridization against AN2, Vg1 and Histone H3. Animal to the top in all panels.

Figure 3. Expression pattern of Shroom1 in Xenopus

(A) Lateral view of stage 10.5, animal to the top. (B–D) stage 17 (B) Dorsal view, anterior to the left. (C) Lateral view, anterior to the left, dorsal to the top. (D) Transverse cross-section view (So:somite, Noto:notochord), dorsal to the top. The sectioning level is indicated by yellow dotted line in panel C. (E–G) Stage 22 (E) Lateral view, anterior to the left, dorsal to the top (CG: cement gland). (F) Dorsal view of anterior region, anterior to the left (OV: otic vesicle). (G) Cement gland, dorsal to the top. (H–I) Stage 25 (H) Cement gland, dorsal to the top. (I) Lateral view, anterior to the left, dorsal to the top (PN: prenephric tubes and duct). (J–K) Stage 30 (J) Lateral view, anterior to the left, dorsal to the top. (K) High magnification view of anterior region. Yellow arrowheads indicate the otic vesicle (OV) and white arrows indicate the prenephric tubes and duct (PN).

Figure 4. Shroom expression in neural plate

(A) Schematic depicting cross-section view of Xenopus neurula. (B) Shroom1 expresses in the deep layer of neuroepithelial cells. (C) Shroom2 expresses in the deep layer. (D) Shroom3 expresses in the superficial layer. White dashed lines indicate the border between superficial layer and deep layer. White solid lines indicate the border between neural plate and mesodermal tissue.

Figure 5. Expression pattern of Shroom2 in Xenopus

(A) Stage 10.5, lateral view, animal to the top. (B-E) Stage 17 (B) Dorsal view, anterior to the left. (C) Lateral view, anterior to the left, dorsal to the top. (D) Anterior view, dorsal to the top. (E) Transverse cross-section view, dorsal to the top. The sectioning level is indicated by yellow dotted line in panel C. (F–I) Stage 21 (F) Lateral view, anterior to the left, dorsal to the top. (G) Dorsal view, anterior to the left (OV:otic vesicle, PN:prenephric tubes and duct). (H) Ventral view, anterior to the left. (I) Anterior view, dorsal to the top. (J–L) Stage 25 (J) Dorsal view, anterior to the left. (K) Lateral view of anterior region, dorsal to the top. (L) Anterior view, dorsal to the top. (M) Stage 30, lateral view, anterior to the left, dorsal to the top (Noto:notochord). Yellow arrowheads indicate the otic vesicle (OV), white arrows indicate the prenephric tubes and duct (PN) and red arrowhead indicates the notochord (Noto).

Figure 6. Expression pattern of Shroom3 in Xenopus

(A–B) Stage 10.5 (A) lateral view, animal to the top. (B) Sagittal-section view, animal to the top. (C–E) Stage 17 (C) Dorsal view, anterior to the left. (D) Anterior view, dorsal to the top. (E) Transverse cross-section (NP: neural plate, NC: neural crest), dorsal to the top. The sectioning level is indicated by yellow dotted line in panel C. (F–G) Stage 23 (F) Lateral view, dorsal to the top (OV: otic vesicle, LP: lateral line placode, Ol: olfactory placode, CG: cement gland, Pr: Proctodeum). (G) Ventral view, anterior to the left. (H–J) Stage 25 (H) Dorsal view, anterior to the left. (I) Anterior view, dorsal to the top. (J) Posterior view, dorsal to the top. (K–L) Stage 30 (K) Lateral view, anterior to the left, dorsal to the top. (L–N) Head views of K (L) Lateral view, anterior to the left, dorsal to the top (M) Dorsal view, anterior to the top (PN: prenephric tubes and duct). Yellow star indicates the brain. (N) Ventral view, anterior to the top, He: heart.

Figure 7. Expression pattern of Shroom4 in Xenopus

(A–B) Stage 10.5 (A) Dorsal view, animal to the top (B) Sagittal-section view, animal to the top. (C–G) Stage 17 (C) Dorsal view, anterior to left. (D) Anterior view, dorsal to top. (E) Transverse cross-section view, dorsal to the top. The sectioning level is indicated by yellow dotted line in panel C. (F–G) Dorsal part (F) and ventral part (G) of E, dorsal to the top (H–K) Stage 21 (H) Lateral view, anterior to the left, dorsal to the top (Som:somite). (I) Anterior view, dorsal to the top (J) Dorsal view, anterior to the left (K) Lateral view of most anterior region, dorsal to the top. Black arrowhead indicates the boundary cells of the cement gland. (L–M) Stage 30 (L) Lateral view, anterior to the left, dorsal to the top (OV: otic vesicle, PN:prenephric tubes and duct). (M) Most posterior region of L. Red arrowhead indicates the most posterior of somatic region.

Fiugre 8. Cell shapes in Shroom expressing tissues

(A) Schematic depicting the sections of embryo imaged for B through G. (B) Lateral line placode, (C) Proctodeum, (D) Cement gland (phalloidin) and (E) Otic vesicle. green is α-tubulin, red is propidium iodide in panel B, C, E. green is phalloidin in panel D. (F–G) Fluorescence images of in situ hybridization against Shroom3, DIC: differential interference contrast image, α-tubulin: image stained with α-tubulin antibody, NBT/BCIP: NBT/BCIT precipitate following in situ hybridization by Shroom3 probe. It was detected by confocal microscopy (F) otic vesicle (G) lateral line placode. Scale bar = 50 µm.

Figure 9. Shroom proteins controls cell heightening

(A–C) Ectopic Shroom2 induces cell heightening in naïve epithelial cells. (A) Control (B) Shroom2 expressing cell (green: γ-tubulin, red: myc-Shroom2) (C) Graph of apicobasal cell height of control and Shroom2 expressing cells (mean ± s.e.m; ctl, n=16;Shroom2, n=15). (D–H) Ectopic Shroom2 expression induces cell heightening in epidermal cells. (D) Control, (E) Shroom2 expressing and (F) Shroom3 expressing epidermis (green: α-tubulin, red: myc-tagged Shroom2 or 3). (G) Graph of apicobasal cell height in Shroom2 expressing epidermal cells (mean ± s.e.m: ctl, n=25;Shroom2, n=25). (H) Graph of apicobasal cell height in Shroom3 expressing epidermal cells (mean ± s.e.m: ctl, n=39;Shroom3, n=40). Scale bar = 20 µm. Yellow arrow bars are showing the way to measure the cell height for control cells (neighboring cells) and pink arrow bars are showing that for Shroom expressing cells. (I–L) Loss of function assay for Shroom2 in neural plate. Transverse cross-section view of neural plate (green: α-tubulin, red: propidium iodide). (I) One side-Shroom2 mismatched morpholino (MM) injected embryo. (J) One side-Shroom2 morpholino (MO) injected embryo. The high magnification views of control side cells (K) and Shroom2 MO injected side cells (L). Scale bar = 50 µm.