Ptf1a is essential for the differentiation of GABAergic and glycinergic amacrine cells and horizontal cells in the mouse retina

doi:10.1242/dev.02781

QUICK SEARCH:

[advanced]

	Author:		Keyword(s):

Home Help Feedback Subscriptions Archive Search Table of Contents

First published online 14 February 2007
doi: 10.1242/dev.02781

Development 134, 1151-1160 (2007)
Published by The Company of Biologists 2007

This Article

	Summary
	Full Text
	Full Text (PDF)
	Supplementary Material
	Alert me when this article is cited
	Alert me if a correction is posted

Services

	Email this article to a friend
	Similar articles in this journal
	Similar articles in PubMed
	Alert me to new issues of the journal
	Download to citation manager


Citing Articles

	Citing Articles via HighWire
	Citing Articles via Google Scholar

Google Scholar

	Articles by Nakhai, H.
	Articles by Schmid, R. M.
	Search for Related Content

PubMed

	PubMed Citation
	Articles by Nakhai, H.
	Articles by Schmid, R. M.


Social Bookmarking

	What's this?

Ptf1a is essential for the differentiation of GABAergic and glycinergic amacrine cells and horizontal cells in the mouse retina

Hassan Nakhai¹^,*^,, Saadettin Sel²^,*, Jack Favor³, Lidia Mendoza-Torres¹, Friedrich Paulsen⁴, Gernot I. W. Duncker² and Roland M. Schmid¹

¹ II. Medizinische Klinik, Klinikum rechts der Isar, Technische Universtität München, Ismaninger Str. 22, D-81675 Munich, Germany.
² Department of Ophthalmology, Martin-Luther-University Halle-Wittenberg, Ernst-Grube-Strasse 40, D-06120 Halle/Saale, Germany.
³ Institute of Human Genetics, GSF-National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany.
⁴ Department of Anatomy and Cell Biology, Martin-Luther-University Halle-Wittenberg, Grosse Steinstrasse 52, D-06097 Halle/Saale, Germany.

View larger version (82K):
[in this window]
[in a new window]

Fig. 1. Expression pattern of Ptf1a protein during mouse retinogenesis. Ptf1a protein was visualized by immunostaining with an anti-Ptf1a antibody of wild-type retina at E12.5 (A,D), E16.5 (B,E) and P1 (C,F). The boxed areas in A,B,C are shown at higher magnifications in D,E,F, respectively. The expression of Ptf1a protein is first seen at E12.5 in cells scattered in the NBL (D, arrows). As development proceeds, the number of Ptf1a-expressing cells increases and some cells reach out to the innermost zone of the NBL (E,F). Scale bars: 100 µm in A-C; 50 µm in D-F. GCL, ganglion cell layer; NBL, neuroblastic layer.

View larger version (58K):
[in this window]
[in a new window]

Fig. 2. Identification of Ptf1a cell lineage in Ptf1a^+/Cre⁽^ex1⁾ R26R retinae of adult mice. Ptf1a-mediated ß-galactosidase expression is co-localized with markers of specific classes of retinal neurons in the adult Ptf1a^+/Cre⁽^ex1⁾ R26R retina. (A,D,G,J,M,P) Expression of selected markers is shown with Alexa 488 (green). (B,E,H,K,N,Q) Expression of ß-galactosidase is shown by immunostaining with Alexa 568 (red). (C,F,I,L,O,R) In merged images, the yellow color represents co-localization of ß-galactosidase with a specific marker. (C) ß-galactosidase is present in the GCL (arrows) but does not co-localize with Thy1.2. (F) ß-galactosidase coexpression with syntaxin, a general marker of amacrine cells, is evident in INL (arrows). (I) GABA coexpression with ß-galactosidase is demonstrated in GABAergic amacrine (arrowheads, yellow) and displaced amacrine cells (arrows, yellow). (L) Coexpression of ß-galactosidase with glycine transporter 1 (GlyT1) is found in glycinergic amacrine cells (arrows). (O) ß-galactosidase coexpression with calbindin is present in horizontal cells (arrows, yellow). PKC- ${alpha}$ -expressing bipolar interneurons (P, arrowheads) do not co-localize with ß-galactosidase expression (R, inset). Insets in merge images (C,F,I,L,O,R) represent a 2.67-fold enlargement. DAPI staining in E shows the retinal nuclear layers. Scale bars: 50 µm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; OPL, outer plexiform layer.

View larger version (80K):
[in this window]
[in a new window]

Fig. 3. Morphological comparison of Ptf1a^+/Cre⁽^ex1⁾ R26R and Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R retinae. (A,E) To confirm the absence of Ptf1a protein in Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R retinae, retinal sections from E18.5 embryos were immunostained with an antibody against Ptf1a (arrows). (E) No immunoreactivity can be detected in Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R retina. (B,C,F,G) X-Gal staining of Ptf1a^+/Cre⁽^ex1⁾ R26R and Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R retinae at E18.5. Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R retina shows morphological change in the inner retina (F) compared with the Ptf1a^+/Cre⁽^ex1⁾ R26R retina (B). Higher magnification of boxed areas in B and F are shown in C and G, respectively. At E18.5, the GCL and IPL are formed in Ptf1a^+/Cre⁽^ex1⁾ R26R retina (B,C). ß- galactosidase-expressing cells are primarily restricted to the inner zone of the NBL (C, arrows). By contrast, in Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R retina (F,G), the GCL and NBL are fused and the IPL is lost. The ß-galactosidase-positive cells are scattered in the inner retina (G, arrows). In both Ptf1a^+/Cre⁽^ex1⁾ R26R and Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R retinae some isolated cells are found in the outermost zone of the NBL. (B,C,F,G) Background is stained by neutral red. (D,H) Images are taken in ApoTome mode (Zeiss) for the purpose of 3D reconstruction showing immunohistochemistry for N-cadherin (red) with a DAPI counterstain (blue). (D) In Ptf1a^+/Cre⁽^ex1⁾ R26R retina, N-cadherin stains GCL and IPL. (H) By contrast, N-cadherin-positive processes are strongly reduced in Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R, resulting in loss of the IPL. Scale bars: 50 µm in A,C,E,G; 100 µm in B,F; 10 µm in D,H. GCL, ganglion cell layer; IPL, inner plexiform layer; NBL, neuroblastic layer; rpe, retinal pigment epithelium.

View larger version (115K):
[in this window]
[in a new window]

Fig. 4. Effect of Ptf1a absence on generation of retinal cell types at E18.5. Retinal sections were prepared from Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R and control Ptf1a^+/Cre⁽^ex1⁾ R26R embryos at E18.5. Co-immunostaining for Pax6 (green, A) and ß-galactosidase (red, B) shows the expression of both genes in the inner NBL of Ptf1a^+/Cre⁽^ex1⁾ R26R retina (C, inset). In Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R retina, Pax6 is expressed at a low level in the NBL (D), whereas ß-galactosidase-positive cells are in the GCL (E). The merged image shows no coexpression of Pax6 and ß-galactosidase (F). Immunohistochemistry on cryosections from Ptf1a^+/Cre⁽^ex1⁾ R26R retina shows the expression of Hu/D antigen in the GCL (green, G) and ß-galactosidase in the inner NBL (red, H). ß-galactosidase-positive cells do not coexpress Hu/D (I, inset). In Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R retina, both Hu/D (J) and ß-galactosidase (K) are expressed in the GCL. Merged image shows the coexpression of Hu/D and ß-galactosidase in the GCL (L, inset, arrows). Insets in C,I and L represent a 2.67x enlargement. Background was stained by DAPI (blue). Scale bars: 50 µm. GCL, ganglion cell layer; NBL, neuroblastic layer.

View larger version (30K):
[in this window]
[in a new window]

Fig. 5. Identification of transdifferentiated precursor cells in Ptf1a-deficient retina at E18.5. (A-F) Sections from E18.5 retinae of Ptf1a^+/Cre⁽^ex1⁾ Z/EG and Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ Z/EG were double-immunostained with anti-GFP and anti-Brn3 antibodies. (A,D) Ptf1a-mediated GFP expression is present in Ptf1a^+/Cre⁽^ex1⁾ Z/EG and Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ Z/EG retinae (green). (B,E) Ganglion cells are marked with Brn3 (red). (C,F) Yellow in merged images represents co-localization of GFP with Brn3. GFP coexpression with Brn3 is not present in Ptf1a^+/Cre⁽^ex1⁾ Z/EG retina (C). By contrast, in about one-third of Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ Z/EG retinal cells, GFP is coexpressed with Brn3 (F, arrows, inset). (G,H) Quantification of Brn3-positive cells in wild-type (wt) and Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ (Cre/Cre) retinae (G) and of GFP and GFP/Brn3-positive cells in Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ Z/EG retinae (H). Each histogram represents the mean±SD for four retinae (Number of Brn3-positive cells in wt are 28.5±2.08 and in Cre/Cre are 40.75±3.40. In Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ Z/EG retinae, the number of GFP-positive cells are 27.2±1.70 and GFP/Brn3-positive cells are 9.2±3.59). Positive cells were scored in an area that was above a line of 200 µm in length parallel to the retinal pigment epithelium. Scale bars: 50 µm. GCL, ganglion cell layer; NBL, neuroblastic layer.

View larger version (44K):
[in this window]
[in a new window]

Fig. 6. Effect of Ptf1a absence on the generation and formation of different cell types in retinal explants. Phenotypic comparison of retinal explants from Ptf1a^+/Cre⁽^ex1⁾ R26R and Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R mice after 12 days in culture (DIC). Retinae from two siblings were dissected at E18.5 and were maintained in culture as whole retinae without destroying their cellular integrity for 12 days. Retinal explants were cryosectioned and immunolabeled with antibodies to cell-specific markers of all major classes of retinal cells. Immunofluorescence analysis of Ptf1a^+/Cre⁽^ex1⁾ R26R and Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R retinal explants at E18.5+12DIC with Thy1.2 (A,B) Hu/D (C,D), GABA (E,F), Gly-T1 (G,H), syntaxin (I,J), calbindin (K,L), PKC ${alpha}$ (M,N) and recoverin (O,P). (Q,R) Expression of ß-galactosidase. (S,T) Results of the TUNEL assay. (A) In Ptf1a^+/Cre⁽^ex1⁾ R26R retinal explant, Thy1.2 is expressed in the GCL (green, arrows). (B) In Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R retinal explant, Thy1.2-positive cells are located in the innermost zone of the INL and do not form a distinct cell layer (green, arrow). (C) In Ptf1a^+/Cre⁽^ex1⁾ R26R retinal explant, Hu/D is expressed in the GCL (arrowhead) and INL (arrow). (D) In Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R retinal explant, Hu/D positive cells are strongly reduced (arrows). (E) In Ptf1a^+/Cre⁽^ex1⁾ R26R retinal explant, GABA is expressed in amacrine (arrows) and displaced amacrine cells (asterisks). (F) By contrast, GABA-positive cells (asterisk) are virtually missing in Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R retinal explants. (G) Glycinergic amacrine cells express GlyT1 in Ptf1a^+/Cre⁽^ex1⁾ R26R retinal explant (arrows). (H) GlyT1-positive cells are not found in Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R retinal explants. (I) Syntaxin is found in the inner plexiform (IPL) and in amacrine cells (arrows, green) of Ptf1a^+Cre⁽^ex1⁾ R26R retinal explant. (J) By contrast, the IPL is missing in retinal explant from Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R embryos and the number of amacrine cells (arrows) is strongly reduced. (K) Calbindin is expressed in the OPL, horizontal cells (asterisk), amacrine cells (arrows) and displaced amacrine cells (arrows) of the Ptf1a^+/Cre⁽^ex1⁾ R26R retinal explant. (L) In Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R retinal explant only reduced number of calbindin-positive cells (arrows) can be detected. (M) Retinal explant from Ptf1a^+/Cre⁽^ex1⁾ R26R mice expresses PKC- ${alpha}$ in bipolar cells (arrows, green). (N) In retinal explant from Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R, there are few PKC- ${alpha}$ labeled cells (arrows, green), which do not form a cell layer and show a disarrayed structure. Both Ptf1a^+/Cre⁽^ex1⁾ R26R and Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R retinal explants are positive for recoverin in the ONL (O,P, green). Cells expressing ß-galactosidase (arrows) are significantly reduced in retinal explant of Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R (R) in comparison with Ptf1a^+/Cre⁽^ex1⁾ R26R (Q). In Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ R26R retinal explant (S, arrows), compared with Ptf1a^+/Cre⁽^ex1⁾ R26R retinal explant (T, arrows), the number of apoptotic nuclei is significantly increased in both INL and ONL. In A-P and S,T the background was stained by DAPI (blue), showing the location of nuclei. Scale bar: 50 µm. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.

View larger version (64K):
[in this window]
[in a new window]

Fig. 7. Amacrine- and horizontal cell-specific factors are downregulated in Ptf1a-deficient retina. (A) Quantification of differentially expressed genes in Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ retina by real-time PCR at E18.5. Real-time PCR was performed on cDNA samples prepared from four independent retinae of wild type and Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ each. Mean values of relative gene ER (see Materials and methods) for wild-type and Ptf1a knockout retinae are shown for each gene. Differences in ER between wild-type and Ptf1a knockout retinae were statistically significant for Ptf1a, Gad1 and Lim1 (P<0.001). (B,C) Immunostaining of horizontal cells in mice retina. Retinal sections from E18.5 of Ptf1a^+/Cre⁽^ex1⁾ and Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ embryos were immunolabeled with an antibody to Lim1. (B) Arrows indicate Lim1 immunopositive cells in Ptf1a^+/Cre⁽^ex1⁾ retina. In Ptf1a^Cre⁽^ex1⁾^/Cre⁽^ex1⁾ retina, no Lim1-positive cells are detectable (C). Scale bar: 50 µm.

View larger version (20K):
[in this window]
[in a new window]

Fig. 8. Schematic depicting the role of Ptf1a in retinal cell development. During retinogenesis, expression of Neurod1 and Neurod4 initiates specification of RPCs to amacrine precursor cells, after which they migrate to the innermost zone of the NBL. Ptf1a induces amacrine cell precursors to differentiate into GABAergic and glycinergic amacrine cells. In addition, Ptf1a is essential in the specification of horizontal precursor cells to horizontal cells.

CiteULike

Complore

Connotea

Del.icio.us Add to Digg

Digg

Technorati

Twitter What's this?