Negative and positive auto-regulation of BMP expression in early eye development

Abstract

Previous results have shown that Bone Morphogenetic Protein (BMP) signaling is essential for lens specification and differentiation. How BMP signals are regulated in the prospective lens ectoderm is not well defined. To address this issue we have modulated BMP activity in a chicken embryo pre-lens ectoderm explant assay, and also studied transgenic mice, in which the type I BMP receptors, Bmpr1a and Acvr1, are deleted from the prospective lens ectoderm. Our results show that chicken embryo pre-lens ectoderm cells express BMPs and require BMP signaling for lens specification in vitro, and that in vivo inhibition of BMP signals in the mouse prospective lens ectoderm interrupts lens placode formation and prevents lens invagination. Furthermore, our results provide evidence that BMP expression is negatively auto-regulated in the lens-forming ectoderm, decreasing when the tissue is exposed to exogenous BMPs and increasing when BMP signaling is prevented. In addition, eyes lacking BMP receptors in the prospective lens placode develop coloboma in the adjacent wild type optic cup. In these eyes, Bmp7 expression increases in the ventral optic cup and the normal dorsal-ventral gradient of BMP signaling in the optic cup is disrupted. Pax2 becomes undetectable and expression of Sfrp2 increases in the ventral optic cup, suggesting that increased BMP signaling alter their expression, resulting in failure to close the optic fissure. In summary, our results suggest that negative and positive auto-regulation of BMP expression is important to regulate early eye development.

Keywords: Auto-regulation; BMP; Coloboma; Development; Lens; Specification.

Fig. 1

Results of qRT-PCR studies in which Hamburger-Hamilton stage 8 explants of pre-lens ectoderm were cultured in basal medium, or exposed to the BMP antagonist Noggin, or a mixture of BMP4 and BMP7. Expression levels relative to Gapdh are shown for Bmp2, 4 and 7 transcripts. The lower graph shows the basal expression levels of each of the BMP transcripts, relative to chicken Gapdh.

Fig. 2

(A-C) Stage 8 L explants cultured for 48 hr and analyzed by immunohistochemistry. (A) Stage 8 L explants (n=15) cultured alone generated Keratin⁺ and δ-crystallin⁺ cells; characteristic of lens fiber cells. (B) In stage 8 L explants (n=15) cultured together with Noggin, the generation of lens cells was blocked, whereas HuC/D⁺/Keratin⁺ cells, characteristic of olfactory epithelial cells, were increased. (C) In stage 8 L explants (n=15) cultured together with BMP4 and BMP7, the generation of lens cells was blocked, whereas Keratin⁺ epidermal cells were generated.

Fig. 3

Fluorescent in situ hybridization showing the levels of Bmp4 and Bmp7 and immunofluorescent staining of phospho-Smad 1/5/8 (pSmad1/5/8) in wild type (WT) eyes (A-G) and in eyes in which the type I BMP receptors, Bmpr1a and Acvr1 were conditionally deleted in the prospective lens placode (BMPR CKO) (H-N). (A-C) In wild type eyes, Bmp4 expression was detected in the pre-lens ectoderm at E9.5 (A), but not at E10 (B) or 10.5 (C), and was restricted to the dorsal part of the optic vesicle and cup at E10 and E10.5 (B,C). (D-F) In wild type eyes, Bmp7 was expressed in the pre-lens ectoderm at E9.5 (D), decreased at E10 (E), but was not detected at E10.5 (F). Bmp7 expression was detected in a scattered pattern in the optic vesicle at E9.5 and E10, but not detected at E10.5. (H-J) In BMPR CKO eyes, Bmp4 expression was increased in both the presumptive lens ectoderm and optic vesicle. (K-M) In BMPR CKO eyes, Bmp7 expression was up-regulated at E10 (L) and maintained increased at E10.5 (M) in the presumptive lens ectoderm, and was expanded in the ventral optic vesicle (K-M). (G,N) Phospho-Smad1/5/8 enrichment is increased in the ventral optic vesicle at E10 in BMPR CKO eyes compared to wild type. Arrows indicate changes in levels of Bmp4, Bmp7 and pSmad1/5/8 in the lens region. Arrowheads indicate changes in level of Bmp7 and pSmad1/5/8 in the ventral optic vesicle. Abbreviations: BMPR, BMP receptor; CKO: conditional knockout; LP, lens placode; PLP, prospective lens placode; LV, Lens Vesicle; OV, optic vesicle; OC, optic cup.

Fig. 4

Analysis of the optic cup of E12.5 wild type eyes and eyes in which the type I BMP receptors, Bmpr1a and Acvr1 were conditionally deleted in the prospective lens placode (BMPR CKO). (A, B) The extent of coloboma formation in whole embryo heads in the BMPR CKO embryos. Asterisk indicates ventral side of tissue where fissure has not closed in the CKOs. (C, D) Immunostaining (brown staining) for Pax2 was observed at the margins of the optic fissure in wild type eyes (white arrowhead), but undetectable in the BMPR CKO eyes (white arrowhead). (E, F) Sfrp2 transcripts (blue staining) were increased at the margins of the optic fissure in BMPR CKO eyes (black arrowheads).

Fig. 5

Schematic image illustrating the negative and positive regulation of Bmp signaling/expression in wild type eyes and in eyes in which the type I BMP receptors, Bmpr1a and Acvr1 were conditionally deleted in the prospective lens placode. Abbreviations: LP, lens placode; PLP, prospective lens placode; OV, optic vesicle.