Figure 1.
Summary of YAC transgenic mouse lines.
(A) Schematic map of human chromosome 11p13 showing the locations of the PAX6 and ELP4 loci, which are in an antisense orientation relative to each other. The locations of PAX6 promoters (P0, P1, Pa and P4) are indicated by black arrows. PAX6 and ELP4 exons are shown as open rectangles. The breakpoints of the two distal-most aniridia-associated rearrangements are indicated by “SGL” and “SIMO”. Red circles indicate some known PAX6 regulatory elements. The location of the downstream regulatory region (DRR) is shown by red arrows. The schematic maps of YACs Y593 (420 kb) and Y589 (310 kb) are indicated. In transgenic mice, Y593, but not Y589, rescues the mouse Sey phenotype and homozygous Sey lethality. (B) Schematic map of YAC transgenes in different YAC transgenic mouse lines. Construct pDT-1 contained sequences expressing tau-GFP and conferring neomycin (neo) resistance, separated by an internal ribosomal entry site (IRES), terminated with polyadenylation and C2MAZ (pAC2m) sequences and flanked by 5' and 3' homology regions (5'HR and 3'HR). Homologous recombination resulted in the introduction of this construct at the translation initiation site (ATG) in exon 4 of the human PAX6 locus, to produce the YAC Y1123.. The PAX6 transcription unit is indicated by a blue box into which a tau-GFP-IRES-Neomycin cassette, indicated by green box, was inserted. The known hypersensitive sites (HS sites) within the DRR region are shown by red arrows. The position of LoxP sites are indicated by orange triangles. The highly conserved cis-regulatory elements E60, E100 and RB are indicated by purple ellipses. Line DTy54 contains a single-copy of YAC 1123. Line Y223 carries 6 copies of YAC 374 in which a pair of LoxP sites flank a 35 kb genomic region within the DRR. Line Y001 contains a single copy of the truncated YAC 374 lacking the distal-most 20 kb sequences including the known RB enhancer. Line Y001ΔDRR is directly derived from Y001 line by Cre-mediated deletion of the 35 kb DRR region. All YACs have the same sequences at the 5’ end.
Figure 2.
Expression of Pax6 and tau-GFP in DTy54 embryonic forebrain at E12.5.
Double immunofluorescence for Pax6 (red) and GFP (green) on coronal sections taken along the rostrocaudal axis. (A, D, G) In the telencephalon, Pax6 is expressed in the ventricular zone of the cortex (CTX), in the VZ of LGE with very low levels, in a stream of cells migrating towards the basal ventral telencephalon, and in the amygdaloid area (AMY). In the diencephalon, Pax6 is broadly expressed in the pretectum (PT) and prethalamus (PTH) with high expression levels, and in the midline of the thalamus (TH) with relatively low levels. (B, E, H) tau-GFP is expressed in all Pax6 expressing regions. (C, F, I) Direct comparison of tau-GFP and Pax6 expression revealed that tau-GFP expression replicates the known Pax6 pattern with some exceptions. (J) Pax6 single expressing cells are present within the amygdaloid region (arrows). (K) A band of Pax6 only expressing cells is located adjacent to the tau-GFP labeled cellular processes in the prethalamus (arrows). (L) tau-GFP labeled radial glial processes extend into the mantle region of the cortex. LCS, lateral cortical stream; PSPB, pallial subpallial boundary; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence. n=10 DTy54 embryos. Scale bars: A-I, 200µm; J-L, 100 µm.
Figure 3.
In situ hybridization for Pax6 and Tau-GFP on coronal sections of E12.5 DTy54 embryos.
Adjacent 10μm coronal sections were hybridized with a probe for Pax6 (A,C,E,G) or tau-GFP (B,D,F,H). (A-H) Pax6 and tau-GFP expression throughout caudal to rostral levels of the forebrain. (G-I) Direct comparison of Pax6 and tau-GFP transcripts in the ventral telencephalon. Image in G is colour-inverted and superimposed on that in H to produce the merged image in I. Arrow indicates the PSPB. Strong tau-GFP expression was expanded into the subpallial side of the PSPB, while Pax6 expression levels dropped sharply at the PSPB. In addition, only Pax6 mRNA but not tau-GFP mRNA was expressed in the postmitotic cells in the basolateral telencephalon (asterisk in G). LCS, lateral cortical stream. n=8 DTy54 embryos. Scale bars: 200µm.
Figure 4.
Pax6 is expressed in a subpopulation of tau-GFP heavily labeled LCS in DTy54 transgenic mouse line at both E12.5 and E15.5.
(A) Pax6 is expressed in the tau-GFP labeled LCS cells at E12.5 (arrows). (B) Higher magnification of boxed area in A revealed that at the ventricular zone of the PSPB, tau-GFP heavily expressing cells are located adjacent to the Pax6 positive cell populations, along with a small subset of cells expressing both tau-GFP and Pax6 (arrows). (C) Pax6 is expressed in the tau-GFP labeled LCS cells at E15.5 (arrows). (D) Higher magnification of boxed area in C further revealed that a subset of tau-GFP labeled LCS cells also express Pax6 (arrows). n=10 DTy54 embryos.
Figure 5.
Some tau-GFP labelled LCS cells co-express Tbr2 and Tbr1 in DTy54 transgenic mice.
(A) Tbr2 and tau-GFP expression at E12.5. (B) Higher magnification of boxed area in A revealing that Tbr2 positive cells are juxtaposed to the most strongly tau-GFP labeled cells. (C) Tbr2 and tau-GFP expression at E15.5. (D) Higher magnification of boxed area in C further revealed that a subset of tau-GFP labeled LCS cells also expressed Tbr2 (arrows). (E) Tbr1 is not expressed in the tau-GFP labeled LCS cells at E12.5. (F) Higher magnification of boxed area in E revealed that Tbr1 positive cells are distinct to the tau-GFP strongly labeled LCS cells. (G) A subset of tau-GFP labeled LCS cells co-expressed Tbr1 at E15.5 (arrows). (H) Higher magnification of boxed area in G further revealed that a subset of tau-GFP labeled LCS cells, which seem to arise from the mantle region of the pallium, co-expresses Tbr1 (arrows). n=10 DTy54 embryos.
Figure 6.
Gsh2 is expressed in a subpopulation of tau-GFP heavily labeled LCS cells in DTy54 transgenic mouse line at both E12.5 and E15.5.
(A) The tau-GFP heavily labeled cell populations overlapped the Gsh2 positive domain in the VZ of the subpallium at E12.5. (B) Higher magnification of boxed area in A revealed that a subset of tau-GFP labeled LCS cells co-expressed Gsh2 (e.g. arrows). (C) A stream of Gsh2 expressing cells are present within the tau-GFP labeled LCS at E15.5. (D) Higher magnification of boxed area in C further revealed that a subset of tau-GFP labeled LCS cells co-expresses Gsh2 (e.g. arrows). n=10 DTy54 embryos.
Figure 7.
Double immunostaining for Pax6 and tau-GFP on coronal sections at caudal to rostral levels through the forebrain from E12.5 Y223 embryos.
(A, D, G) Pax6 expression pattern is reflected by red fluorescence. (B, E, H) Tau-GFP is expressed in all Pax6 expressing regions. Tau-GFP heavy labeling of cellular processes can be seen in both the telencephalon and the diencephalon. (C, F, I) Direct comparison of tau-GFP and Pax6 expression revealed that tau-GFP expression replicates the known Pax6 pattern with some exceptions. (J) Pax6 and tau-GFP protein are co-localized in the pretectum (PT). (K) A group of cells only expressing Pax6 and not tau-GFP is intermingled with the tau-GFP labeled cellular processes in the prethalamus. (L) tau-GFP labeled radial glial processes extend into the mantle region of the cortex. (M) tau-GFP highly labeled cells are present on the subpallial side of the PSPB. (N) A subset of tau-GFP labeled migratory cells co-expresses Pax6. (O) Pax6 single expressing cells are present within the amygdaloid (AMY) region. Abbreviations as in Figure 2. n=10 Y223 embryos. Scale bars: A-C, 200µm; D-O, 100 µm.
Figure 8.
Double immunostaining for Pax6 and tau-GFP on coronal sections at caudal to rostral levels through the forebrain from E12.5 Y001 embryos.
(A, D, G) Pax6 expression is reflected by red fluorescence. (B, E, H) In the diencephalon, tau-GFP is only expressed in the prethalamus (PTH). The tau-GFP expression in the cortex is dramatically reduced in comparison with the endogenous Pax6 expression. tau-GFP highly labeled cells are present in the ventral telencephalon, (C, F, I) Direct comparison of tau-GFP and Pax6 expression revealed that tau-GFP expression only exhibits a partial Pax6 pattern, with some notable discrepancies. (J) tau-GFP expression in the pretectum (PT) is lost. (K) A band of Pax6 only expressing cell is located adjacent to the tau-GFP labeled cellular processes in the prethalamus. (L) The expression of tau-GFP in the cortex (CTX) is very weak. (M) tau-GFP heavily labeled cells are present in the mantle region of the ventral telencephalon. (N) A subset of tau-GFP labeled migratory cells co-expresses Pax6. (O) Pax6 single expressing cells are present within the amygdaloid (AMY) region. Abbreviations as in Figure 2. n=8 Y001 embryos Scale bars: A-I, 200µm; J-O, 100 µm.
Figure 9.
Double immunostaining for Pax6 and tau-GFP on coronal sections at caudal to rostral levels through the forebrain from E12.5 Y001ΔDRR embryos.
(A, D, G) Pax6 expression is reflected by red fluorescence. (B, E, H) In the diencephalon, tau-GFP is only expressed in the prethalamus. The tau-GFP expression in the cortex is dramatically reduced in comparison with the endogenous Pax6 expression. tau-GFP highly labeled cells are present in the ventral telencephalon. (C, F, I) Direct comparison of tau-GFP and Pax6 expression revealed that tau-GFP expression only exhibits a partial Pax6 pattern, with some notable discrepancies. (J) Tau-GFP expression in the pretectum (PT) and the thalamus is lost. (K) tau-GFP expression in the prethalamus (PTH) is weak compared with the endogenous Pax6 expression. Tau-GFP labeling of cellular processes still can be clearly seen. (L) The expression of tau-GFP in the cortex (CTX) is very weak. (M) tau-GFP heavily labeled cells are present in the mantle region of the ventral telencephalon. (N) A subset of tau-GFP labeled migratory cells co-expresses Pax6. (O) Pax6 single expressing cells are present within the amygdaloid (AMY) region. Abbreviations as in Figure 2. n=8 Y001ΔDRR embryos. Scale bars: A-I, 200µm; J-O, 100 µm.
Figure 10.
Double immunostaining for Pax6 and tau-GFP on coronal sections through the eye from E12.5 embryos of YAC transgenic mouse lines.
(A, D, G, J) Pax6 is expressed in retinal pigmented epithelium (rpe), neural retina (nr) and lens. (B, C) In the DTy54 line, tau-GFP expression replicates the Pax6 pattern in all tissues of the eye. (E, F) In the Y223 line, tau-GFP expression matches Pax6 expression pattern in all eye tissues. (H, I) In the Y001 line, tau-GFP expression is restricted in the retina and is only present in some retinal ganglion cells (RGCs) that strongly express Pax6. (K, L) In the Y001ΔDRR line, Cre-mediated deletion of the DRR completely abolishes tau-GFP expression in the eye. (B, E, H) tau-GFP labeling of the axonal projections of RGCs can be seen in the optic nerve (on). n>8 YAC transgenic embryos per line. Scale bars: 100µm.