Bullwhip neurons in the retina regulate the size and shape of the eye

Abstract

Bullwhip and mini-bullwhip cells are unconventional types of retinal neurons that utilize the neuropeptides glucagon, glucagon-like peptide 1 (GLP1) and substance P. These cells have been implicated in regulating the proliferation of neural progenitors in the circumferential marginal zone (CMZ) of the chicken retina. The purpose of this study was to investigate the roles of the bullwhip cells in regulating ocular size and shape. We found that intravitreal delivery of colchicine at postnatal day 7 destroys the vast majority (approximately 98%) of the bullwhip and mini-bullwhip cells and their peptidergic terminals that are concentrated in the CMZ near the equator of the eye. Interestingly, colchicine-treatment resulted in excessive ocular growth that involved the expansion of equatorial diameter, but not axial length. Intraocular injections of glucagon completely prevented the equatorial expansion that occurs with colchicine-treatment. In eyes with undamaged retinas, exogenous glucagon suppressed equatorial eye growth, whereas glucagon receptor antagonists caused excessive equatorial growth. Furthermore, visual stimuli that increase or decrease rates of ocular growth caused a down- or up-regulation, respectively, of the immediate early gene Egr1 in the bullwhip cells; indicating that the activity of the bullwhip cells is regulated by growth-guiding visual cues. We found that the glucagon receptor was expressed by cells in the fibrous and cartilaginous sclera in equatorial regions of the eye. Taken together, these findings suggest that glucagon peptide released from the terminals of the bullwhip and mini-bullwhip cells regulates the growth of the equatorial sclera in a vision-dependent manner. Although the bullwhip and mini-bullwhip cells are not abundant, less than 1000 cells per retina, their influence on the development of the eye is substantial and includes vision-guided ocular growth.

Hedgehog-signaling stimulates the formation of proliferating Müller glia-derived progenitor cells in the retina

“…Chickens were anesthetized and intraocular injections were performed as described previously (Fischer et al, 2008(Fischer et al, , 2009a. Injected compounds included NMDA (6.4 or 154 µg/dose; Sigma-Aldrich), FGF2 (250 ng/dose; Sigma-Aldrich), IGF1 (400 ng/dose; R&D Systems), rhShh (500 ng/dose; R&D Systems), KAAD-cyclopamine (1 µg/dose; Selleck Chemicals; CAS 306387-90-6), 5E1 anti-Shh (20 µl/dose; Developmental Studies Hybridoma Bank), Gli antagonist GANT58 (1 µg/dose; Selleck Chemicals), Gli antagonist GANT61 (1 µg/dose; Tocris Bioscience), smoothened agonist (SAG) (500 ng/dose; EMD Millipore; CAS 364590-63-6), DAPT {N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester; 865 ng/ dose; Sigma-Aldrich}, UO126 (1.7 μg/dose; Calbiochem) and 5-bromo-2′-deoxyuridine (BrdU; 2 µg/dose; Sigma-Aldrich).…”

“…Digital photomicroscopy was performed as described in previous studies (Fischer et al, 2008Ghai et al, 2009Ghai et al, , 2010. Central retina was defined as the region within a 3 mm radius of the posterior pole of the eye, and peripheral retina was defined as an annular region between 3 mm and 0.5 mm from the CMZ.…”

Glucocorticoid receptors in the retina, Müller glia and the formation of Müller glia-derived progenitors

“…Chickens were anesthetized and intraocular injections were performed as described previously (Fischer et al, 2008(Fischer et al, , 2009a. Injected compounds included NMDA (6.4 or 154 μg/dose; Sigma-Aldrich), dexamethasone (100-200 ng/dose; Sigma-Aldrich), CpdA (500 ng/dose; Millipore), aldosterone (200 ng/dose; Sigma-Aldrich), RU486 (1 μg/dose; SigmaAldrich), FGF2 (200 ng/dose; Sigma-Aldrich) and BrdU (1 μg/dose; Sigma-Aldrich).…”

“…Tissues were fixed, sectioned and immunolabeled as described previously (Fischer et al, 2008(Fischer et al, , 2009b. Working dilutions and sources of primary and secondary antibodies used in this study are listed in supplementary material Table S2.…”

“…Digital photomicroscopy was performed as described in previous studies (Fischer et al, 2008Ghai et al, 2009Ghai et al, , 2010. Similar to previous studies (Fischer et al, 2009a(Fischer et al, ,b, 2010Ghai et al, 2009), immunofluorescence was quantified using ImagePro 6.2 (Media Cybernetics).…”