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First published online 27 July 2004
doi: 10.1242/dev.01285

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Bmp7 regulates branching morphogenesis of the lacrimal gland by promoting mesenchymal proliferation and condensation

Charlotte Dean^*,, Masataka Ito^*,, Helen P. Makarenkova^*,, Sonya C. Faber and Richard A. Lang^¶

Divisions of Developmental Biology and Ophthalmology, Children's Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA

View larger version (92K): [in a new window]   Fig. 4. Inhibition of Bmps in explant cultures suppresses new bud formation. Lacrimal gland explants that are untreated (A,D) or treated with noggin (B,C) or follistatin (E) at 100 ng/ml. Inhibition of Bmps (B,C,E) causes a dramatic decrease in bud number and an increase in bud size. All explants are shown at the same magnification. (F) Quantification of the decrease in bud number at E17.5 + 48 hours of noggin treatment. (G) Quantification of the decrease in bud number at E16.5 + 48 hours of follistatin treatment. (H-K) Lacrimal gland explants from P6 5.0-lacZ, Bmp7m1Rob/m1Rob mice either untreated (H,I) or treated with noggin for 48 hours (J,K). H and J are taken at t=0 hours; I and K at t=48 hours where the glands have been stained with X-gal. (L) Quantification of the decrease in bud number at E16.5 + 48 hours of noggin treatment of P6 5.0-lacZ, Bmp7m1Rob/m1Rob lacrimal glands.

View larger version (44K): [in a new window]   Fig. 5. Bmp7 induces the formation of epithelial branches in explant culture but does not suppress Fgf10-mediated epithelial bud elongation. (A) E15.5 whole lacrimal gland explants treated with 100 ng/ml Bmp7 for 48 hours contained more epithelial branches than control explants. The error bars are standard errors. The significance value of P<0.05 is calculated according to the Mann-Whitney U test. (B) Left column, epithelial explants adjacent to Fgf10-soaked beads at t=0 hours; right column, the same explants at t=48 hours. Epithelial explants treated with Bmp7 resemble control cultures, whereas those treated with Bmp4 show suppression of the epithelial growth and elongation that is normally observed in response to Fgf10.

View larger version (13K): [in a new window]   Fig. 3. Bmp7 is required for lacrimal gland budding. Quantification of the number of acini (A) and total gland length (B) in control (red lines) and Bmp7m1Rob/m1Rob mice (blue lines) at different embryonic stages. Error bars represent standard error.

View larger version (131K): [in a new window]   Fig. 1. Bmp7 is expressed in developing lacrimal gland. All preparations are stained with X-gal. (A,C,G,H) The dermis overlying the developing lacrimal gland has been removed. (D,E,I,K,L-M) Paraffin wax-embedded sections counterstained with nuclear Fast Red. (A) E13.0, Bmp7lacZ/+ mouse. The region of mesenchyme through which the lacrimal gland will grow does not stain for Bmp7lacZ expression (broken yellow lines). (B) Section through eye region of E13.5 Bmp7lacZ/+ embryo showing Bmp7-positive presumptive eyelid mesenchyme (asterisks) and periorbital mesenchyme (arrow). The section plane shown does not correspond to the Bmp7lacZ-negative region in A. (C) Whole-mount Bmp7lacZ/+, P6 5.0-lacZ embryo at E14.5 showing epithelial bud extending into Bmp7lacZ-positive mesenchyme (arrows). (D) Section through E14.5 Bmp7lacZ/+ lacrimal gland showing lacrimal bud (lb) and mass of mesenchymal cells (mes). The epithelium of the lacrimal bud is negative for Bmp7lacZ expression; the mesenchyme is positive (blue X-gal staining). (E) As in D, but at higher magnification to show X-gal-negative epithelium (ep) and positive mesenchyme (mes). (F) The eye region of E15.5 Bmp7lacZ/+ embryo showing lacrimal bud (lb) extending into Bmp7lacZ-expressing mesenchyme (arrows). (G) As in C but at E15.5. (H) Whole-mount Bmp7lacZ/+ embryo at E17.0 when secondary branching is apparent. The highest levels of lacZ expression are observed in the condensing mesenchyme surrounding the tips of the epithelial buds (red arrows) and the lacrimal duct (ld). (I) Section through E17.5 Bmp7lacZ/+ lacrimal gland showing Bmp7lacZ expression throughout mesenchyme (surrounded by broken line) in some cells of the ductal (ld) and bud epithelium (ep) and in mesenchyme adjacent to epithelial bud tips (arrowheads). (J) Dissected whole-mount preparation of E17.5 Bmp7lacZ lacrimal gland showing general low level Bmp7lacZ expression in mesenchyme and higher levels adjacent to epithelial bud tips. (K) Higher magnification of the lacrimal duct shown in I to indicate Bmp7lacZ-expressing epithelial and mesenchymal cells. (L) Higher magnification of extended epithelial region in Bmp7lacZ lacrimal gland showing distribution of X-gal-positive cells. (M) Section through terminal Bmp7lacZ/+ epithelial bud showing X-gal-positive condensing mesenchyme (arrowheads). (N) Whole-mount Bmp7lacZ/+ lacrimal gland at E18.5. (O) E18.5 P6 5.0-lacZ lacrimal gland to show contrasting pattern of epithelial expression.

View larger version (61K): [in a new window]   Fig. 2. Bmp7 is required for normal development of the lacrimal gland. All preparations are stained with X-gal. (A) E13.5 P6 5.0-lacZ mouse and (B) Bmp7m1Rob/m1Rob, P6 5.0-lacZ mouse both showing normal formation of the primary lacrimal bud. (C) Wild-type E19.5 P6 5.0-lacZ mouse showing normal lacrimal gland branching pattern. The gland is composed of a small intra-orbital lobe (io) (blue arrowhead) and a large exorbital lobe (xo). (D-H) Lacrimal glands in E19.5 Bmp7m1Rob/m1Rob, P6 5.0-lacZ mice showing the position of the intraorbital lobe (blue arrowheads), misplaced buds and branches (purple arrowheads) and in some cases (E,F) loss of lobe distinctions. The degree to which the lacrimal gland was affected did not correlate with the degree of micropthalmia (mi). (H) Lacrimal gland dissected from E19.5 Bmp7m1Rob/m1Rob, P6 5.0-lacZ mouse (black arrowheads indicate the extent of gland mesenchyme).

View larger version (92K): [in a new window]   Fig. 6. Bmp7 induces mesenchymal aggregation and proliferation. Mesenchyme from E15.5 lacrimal glands was isolated and cultured for 48 hours in either defined medium (A,D,F,H) or with 100 ng/ml Bmp7 (B,E,G,I). (A,B) Cultures labeled with the nuclear dye Oligreen. Bmp7 treatment results in a striking aggregation of the cells (B). (C) Treatment of lacrimal gland mesenchyme with Bmp7 for 24 hours results in a dose-dependent increase in proliferation as indicated by the increased number of BrdU-labeled cells. Mesenchyme cultures were immunolabeled for connexin 43 (D,E), cadherins (F,G) or -smooth muscle actin (H,I). (D-G) Immunoreactivity is shown in green, while nuclei are counterstained blue with Hoechst. (H,I) Immunoreactivity is shown in red, the nuclei in green. In all three cases, an increase in immunoreactivity was observed in Bmp7-treated cultures and associated with aggregated cells. (J,K) Confocal images of whole, wild-type, explanted lacrimal glands labeled for SMA (red) and Pax6 (green) after growth in the absence (J) or presence (K) of 100 ng/ml Bmp7. Bmp7 increased the level of expression of SMA.

View larger version (46K): [in a new window]   Fig. 7. Bmp7-null lacrimal glands show reduced cadherin and SMA expression as well as reduced proliferation. Sections of wild-type (A,C,E) and Bmp7m1Rob/m1Rob (B,D,F) Day of birth lacrimal glands. (A,B) Pax6 (red) and pan-cadherin (green) immunolabeling. (C-F) Pax6 (red) and SMA (green) immunolabeling. Comparing A with B, it can be seen that the level of cadherin immunoreactivity is greatly reduced in the absence of Bmp7. (C,D) SMA-positive cells (white arrowheads) are fewer in number and stain at lower intensity in the absence of Bmp7. (E,F) Higher magnification of wild-type (E) and Bmp7m1Rob/m1Rob sections, showing reduced number of SMA-positive cells and their reduced labeling intensity. (G) Graphs showing that, compared with wild-type (red bars), the Bmp7m1Rob/m1Rob lacrimal gland (blue bars) shows a reduced number of Ki67 immunoreactive cells in both mesenchyme and epithelium. Error bars indicate s.e.m. and significance values are calculated according to Student's t-test. Day of birth glands were analyzed. (H,I) In situ hybridization performed on lacrimal glands from wild-type (H) and Bmp7m1Rob/m1Rob (I) E17.5 embryos using a probe for Fgf10.

View larger version (109K): [in a new window]   Fig. 8. A model describing Bmp7 function in lacrimal gland development. We propose that in the lacrimal gland, through stimulation of mesenchymal proliferation and aggregation, Bmp7 promotes formation of signaling centers and, as a result, branching morphogenesis (A). We also suggest that the pattern of lobe formation in the lacrimal gland can be explained in part by the combined action of Fgf10 and Bmp7 where Fgf10 is a stimulus for bud elongation and Bmp7 for branching (A,B). During the initial 48 hours of lacrimal gland development, we suggest that the primary bud extends and does not branch because the epithelium is exposed only to Fgf10 (A,B, zone A). By contrast, we propose that upon entering the defined region of Bmp7-expressing mesenchyme, epithelium will branch because it is exposed to Bmp7 (A,B, zone C). It is possible that this regulation of branching is mediated not by the absolute presence or absence of Bmp7, but by different Bmp7 levels.