Retinal neurons regulate proliferation of postnatal progenitors and Müller glia in the rat retina via TGFβ signaling

Abstract

Development 3016 glia and progenitors. The results of our experiments support a model in which TGFβ2, primarily derived from retinal neurons, inhibits proliferation of retinal progenitors and glia at the end of retinogenesis. Materials and methods Animals and injectionsAll animals used in this study were treated according to guidelines of the University of Washington IACUC. Long Evans rats were purchased from Charles River Laboratories. For P5.5 intraocular injections, animals were anesthetized by hypothermia, and their eyelids opened with iridectomy scissors. Proparacaine topical anesthetic was applied, followed by intraocular injection nasal to the cornea using a 30.5 G needle and Hamilton syringe. For P10 intraocular injections, P10/P11 animals were anesthetized with ketamine/xylazine and injected as described for P5.5 animals. Factors used for intraocular injection experiments include 40 nmoles SB-431542 in dimethylsulfoxide (Sigma) mTGFβRII-hFc (R&D Systems), mouse-anti-TGFβ blocking antibody (MAB1835, R&D Systems) and rhEGF (R&D Systems). BrdU injections were performed intraperitoneally using a sterile 30.5 G needle and 1 ml syringe. For the birthdating study, pups were weighed and given three injections of BrdU (50 mg/kg) over 9 hours on postnatal day 4, 6, 8, 10 or 12, sacrificed at P15 by CO 2 overanesthesia and processed for BrdU immunohistochemistry.

Epidermal growth factor receptor expression regulates proliferation in the postnatal rat retina

“…It is known that retinal progenitors and M€ uller glia have similar gene expression profiles (Blackshaw et al, 2004), and that Muller glia are among the last cells generated in the developing retina (Turner and Cepko, 1987). While recent evidence indicates that the cessation of proliferation during development of the retina involves a TGFb2 mediated signal (Close et al, 2005), the present results support the hypothesis that regulation of the EGFR also plays a role in the maintenance of glial mitotic quiescence in the mature retina. The end of retinal histogenesis is apparently mediated by a combination of at least two factors; in the first postnatal week, TGFb2, released by neurons within the retina, inhibits the proliferation of the progenitors (prior to P7) and the M€ uller glia (after P7); in the second postnatal week, and thereafter, M€ uller glia are maintained in a mitotically quiescent state by the down-regulation of EGFR.…”

“…Intraocular injections were performed as described previously (Close et al, 2005) for rats at postnatal day 10 (P10). For injections made in animals from postnatal day 14 to adulthood, rats were anesthetized using ketamine/ xylazine and proparacaine topical anesthetic was applied to the eyes prior to injection.…”

“…Western blots were performed as previously described (Close et al, 2005), using the following antibodies: sheep anti-EGFR (1:1,000; Upstate), mouse anti-p27 (1:1,000; Transduction Labs), mouse anti-b-Actin (1:5,000; Abcam), goat antimouse horseradish peroxidase (HRP) conjugate (1:20,000; BioRad), and rabbit anti-sheep HRP conjugate (1:20,000). Bands were quantified by scanning the blots.…”

“…This regulation is achieved through the action of both cytostatic and mitotic signaling molecules. For example, we have recently shown that transforming growth factor b 2 (TGFb2) is expressed in the postnatal retina, and inhibits progenitor and glial proliferation in vitro and in vivo (Close et al, 2005). Fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor a (TGFa), transforming growth factor b 3 (TGFb3), and sonic hedgehog (Shh) have all previously been shown to promote retinal progenitor and/or glial proliferation in vitro (Anchan and Reh, 1995;Anchan et al, 1991;Jensen and Wallace, 1997;Levine et al, 1997;Lillien and Cepko, 1992;Moshiri and Reh, 2004;Wang et al, 2002).…”

Control of Retinal Development by Tumor Suppressor Genes

“…We found that Zac1 is required to induce expression of transforming growth factor II (TGF II) in the retina (Ma et al 2007a). Notably, TGF II is also a known tumor suppressor gene, and was shown to negatively regulate the proliferation of retinal progenitor cells and Müller glia (Close et al 2005). Interestingly, previous reports have indicated that TGF regulates p27 Kip1 expression (Ravitz and Wenner 1997), whereas Zac1 does not regulate p27 Kip1 expression (Ma et al 2007a).…”