XB-ART-34781Dev Dyn January 1, 2007; 236 (1): 192-202.
Zac1 promotes a Müller glial cell fate and interferes with retinal ganglion cell differentiation in Xenopus retina.
The timing of cell cycle exit is tightly linked to cell fate specification in the developing retina. Accordingly, several tumor suppressor genes, which are key regulators of cell cycle exit in cancer cells, play critical roles in retinogenesis. Here we investigated the role of Zac1, a tumor suppressor gene encoding a zinc finger transcription factor, in retinal development. Strikingly, in gain-of-function assays in Xenopus, mouse Zac1 promotes proliferation and apoptosis at an intermediate stage of retinogenesis. Zac1 also influences cell fate decisions, preferentially promoting the differentiation of tumor-like clusters of abnormal neuronal cells in the ganglion cell layer, as well as inducing the formation of supernumerary Müller glial cells at the expense of other cell types. Thus Zac1 has the capacity to influence cell cycle exit, and cell fate specification and differentiation decisions by retinal progenitors, suggesting that further functional studies will uncover new insights into how retinogenesis is regulated.
PubMed ID: 17072860
Article link: Dev Dyn
Genes referenced: casp3 isl1 kcnd2 myc ncam1 nefh nefl nefm plag1
Antibodies: BrdU Ab3 Casp3 Ab4 GABA Ab2 Isl1/2 Ab1 Kcnd2 Ab2 Myc Ab3 Nefm Ab2 Neuronal Ab1 Neuronal Ab5
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|Figure 2. Misexpression of mZac1c in Xenopus retina biases progenitors to acquire retinal ganglion cell (RGC) and M�ller glial cell fates. A: Schematic illustration of a mature stage 40 Xenopus retina. B: Stage 40 control-injected retina showing green fluorescent protein (GFP) epifluorescence (green) of transfected cells. C�F: Stage 40 retina co-injected with expression constructs for GFP and myc-tagged Zac1, showing GFP epifluorescence (green, C,E,F) and immunostaining with anti-myc (red, D,E). F is a high-magnification image of the boxed area in C, showing a representative abnormal cluster of RGC layer (GCL) cells induced by Zac1. G,H: Anti-Zac1 immunostaining (red, G,H) of stage 40 Zac1+GFP (green, H) injected retina. Note that Zac1 antibody does not cross-react with Xenopus Zac1. I: Quantification of GFP+ cells corresponding to each retinal cell class, showing a significant (***P < 0.001) expansion of M�ller glial and cells in the RGC layer at the expense of most other cell types after misexpression of mZac1c. n = number of retinae analyzed, and total cell counts were 1,553 for control and 1,146 for mZac1c. Error bars are SEM. gcl, ganglion cell layer; inl, inner nuclear layer; onl, outer nuclear layer; le, lens; PR, photoreceptors; Am, amacrine cells; Bp, bipolar cells; Mu, M�ller glia; Hc, horizontal cells; RGC, retinal ganglion cells.|
|Figure 4. mZac1c-induced retinal ganglion cell layer (GCL) clusters have a neuronal identity. A�P: Stage 40 green fluorescent protein�Zac1 (GFP+Zac1) -injected retina showing GFP epifluorescence (green, A�C,E�G,I�K,M�O) and immunolabeling with the pan-neuronal markers nerve cell adhesion molecule (NCAM, red, B�D), neurofilament (NF, red, F�H), Xen-1 (red, J�L), and Zn12 (red, N�P). C�D, G�H, K�L, O�P are high-magnification images of the boxed areas in B, F, J, N, respectively. Arrowheads in L and P mark disorganized inner plexiform layer. le, lens; gcl, ganglion cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; onl, outer nuclear layer.|
|Figure 5. mZac1c perturbs the differentiation of retinal ganglion cells (RGCs) while supernumerary M�ller glia are correctly specified. A�T: Immunostaining of stage 40 control�green fluorescent protein (GFP, green, A,E,I,M,Q) and Zac1+GFP (green, B�D,F�H,J�L,N�P,R�T) co-injected retinae with the cell-type specific markers Islet (red, A�H), γ-aminobutyric acid (GABA, red, I�L), and Kv4.2 (red, M�T). E and F�H are high-magnification images of the boxed areas in A and D, respectively. Arrows in E show double GFP/islet positive RGCs, while arrows in G show Zac1-misexpressing cells in the RGC layer (GCL) express low levels of Islet. K,L are high-magnification images of the boxed area in J. Arrow in L indicates that a Zac1-induced GCL cluster does not express the amacrine cell marker GABA, while a Zac1-misexpressing cell in the inner nuclear layer (INL) does express GABA (asterisk, L). Q and R�T are high-magnification images of the boxed areas in M and O, respectively. M,N,Q�T: Arrowheads point to M�ller glia and their radial processes. Note the GFP+ M�ller glial cell clusters induced by Zac1 misexpression in N�O (arrowheads). Red label in N is myc immunoreactivity, indicating Zac1-misexpressing cells. Arrowheads in T show that Zac1-induced M�ller glial cell clusters express Kv4.2. le, lens; gcl, ganglion cell layer; inl, inner nuclear layer; onl, outer nuclear layer.|
|Figure 6. mZac1c promotes the proliferation of retinal progenitors at an intermediate stage of retinogenesis. A�H: Embryos injected with green fluorescent protein (GFP, green, A�C) or co-injected with Zac1+GFP (green, D�F) were exposed to a short bromodeoxyuridine (BrdU) pulse before harvesting at stage 28 (A,D), stage 32 (B,E,G,H) and stage 40 (C,F). Immunolabeling of BrdU is in red (all panels). Inserts in A�F show high-magnification images of GFP/BrdU double positive cells. Arrowheads shows BrdU (red)/GFP (green) double-positive cells in the inner nuclear layer (F) and in a retinal ganglion cell layer (GCL) cluster (the boxed area in G is shown in high magnification in H). I: Quantification of BrdU-labeling index for control and Zac1-misexpressing embryos at each developmental stage. n refers to the number of retinae examined, and total cells counted were as follows: st28: control, 507 and Zac1, 715; st 32: control, 963 and Zac1, 1,280; st 40: control, 1,524 and Zac1, 1,301. Error bars are SEM. **P < 0.01. gcl, ganglion cell layer; inl, inner nuclear layer; onl, outer nuclear layer; vz, ventricular zone.|
|Figure 7. mZac1c promotes apoptosis in Xenopus retina at stage 32. A�F: Stage 32 control green fluorescent protein (GFP, A�C) and Zac1+GFP�injected (D�F) retinae labeled with anti�activated caspase-3 (ac-3, red, A,C,D,F) and GFP epifluorescence (green, B,C,E,F). The insert in F shows a high-magnification image, and arrowheads in F show GFP/ac-3 double positive cells. G�I: Stage 40 Zac1+GFP�injected retinae labeled with ac-3 (red, G,I) and GFP epifluorescence (green, H, I). J: Quantification of apoptotic control and Zac1-misexpressing cells in stage 32 retinae. n refers to the number of retinae examined, and total cells counted were control (n = 2,115) and Zac1 (n = 1,705). Error bars are SEM. *P < 0.05. gcl, ganglion cell layer; inl, inner nuclear layer; onl, outer nuclear layer; vz, ventricular zone.|