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First published online 23 June 2005
doi: 10.1242/dev.01915

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Direct and indirect roles of CNS dorsal midline cells in choroid plexus epithelia formation

Departments of Pathology and Developmental and Cell Biology, University of California, Irvine, D440 Medical Sciences I, Irvine, CA 92697-4800, USA

View larger version (31K): [in a new window]   Fig. 1. (A) Dorsal schematics of the E9.5-10.5 neural tube (left) and E12.5 CNS (right); rostral is towards the top and caudal is towards the bottom. In the neural tube, the dorsal midline (DM, blue) of the hindbrain has a rhomboidal shape owing to unique morphogenetic movements (Alexandre and Wassef, 2003; Awatramani et al., 2003). Choroid plexus (CP, orange) forms at or near the midline in the hindbrain (4th ventricle), diencephalon (3rd ventricle) and dorsomedial telencephalon bilaterally (lateral ventricles). Whether the diencephalic and telencephalic CP are separate or continuous was uncertain. Ovals designate the telencephalon. (B) Schematics of dorsal and medial views of E12.5 forebrain, showing the positions of sections 1-3 on the right. The medial view, adapted from in situ hybridization images (Grove et al., 1998), illustrates one telencephalic hemisphere with overlying diencephalon (gray). The dorsomedial telencephalon (DMT) consists of telencephalic CP epithelium (CPe) and cortical hem (brown). At anterior DMT levels (section 1), these bilateral structures are separated by the choroid plaque and occupy a midline position. After reaching the diencephalon (section 2), the bilateral DMT are dorsomedial rather than midline and contiguous with both telencephalic and diencephalic neuroepithelium. Once past the interventricular foramina (section 3), the DMT loses its diencephalic connection and becomes contiguous solely with other telencephalic structures. (C) Schematics of the transgenic mating strategies used in this study; see text for details. A, anterior; CP, choroid plexus; CPe, choroid plexus epithelium; DM, dorsal midline; DMT, dorsomedial telencephalon; n.e., neuroepithelium; P, posterior.

View larger version (60K): [in a new window]   Fig. 2. Gdf7 activation in DM neuroepithelium at neural tube stages. X-gal stains of Gdf7Cre;R26R embryos (A-D) and coronal sections (E-G), and wild-type Gdf7 in situ hybridization (H); rostral is rightwards in A, towards the top in B-D. (A,B) E9.5 embryos. lacZ is weakly detected in the DM throughout the CNS, including the telencephalon (arrow in A) and hindbrain (arrowheads in B). (C,D) E10.5 embryos. lacZ is more readily detected in the DM of the telencephalon (arrow in C), diencephalon (C) and hindbrain (arrowheads in D). The telencephalic and diencephalic DM domains are separated by weaker staining at the di-telencephalic midline boundary (C). (E,G) E10.5 telencephalon. Labeling is detected in many, but not all, DM neuroepithelial cells in the roof plate (rp). Little to no labeling is seen laterally in the cortical primordia (cx) or radially in the overlying mesenchyme (m) and surface ectoderm (s). (F,H) E10.5 hindbrain. X-gal staining (F) and Gdf7 transcripts (H) are primarily localized to RP neuroepithelium and its junction with the cerebellar anlage (cbl), with little to no expression in surface ectoderm or overlying mesenchyme (s/m). Scale bars: 0.1 mm.

View larger version (88K): [in a new window]   Fig. 3. Gdf7 fate mapping to the diencephalic and myelencephalic CPe. Gdf7 in situ hybridization (A-B,G-H) and X-gal staining (C-F,I-L) on wild-type or Gdf7Cre;R26R coronal cryosections. Sections in B,D,F are 40-60 µm posterior to those in A,C,E, respectively. (A-F) Diencephalon (3rd ventricle). Gdf7 transcripts mark the E12.5 diencephalic midline and adjacent neuroepithelium (A,B). The E12.5 Gdf7 fate map highlights the same regions as Gdf7 in situ hybridization (C,D), as well as the dCPe at E14.5 (E,F). (G-L) Hindbrain (4th ventricle). Gdf7 transcripts are detected in the mCPe (G,H), and most mCPe cells belong to the Gdf7 fate map (I-L). Prominent labeling is also seen at the junctions between mCPe and adjacent neuroepithelium (cerebellum rostrally, myelencephalon caudally), but little labeling is seen elsewhere at these stages. Scale bars: 0.4 mm in G,I,K; 0.2 mm in A-F,H,J,L.

View larger version (112K): [in a new window]   Fig. 4. Gdf7 cell lineages in the anterior, but not posterior, telencephalic CPe. X-gal stains (A-F,J-L) and Gdf7 in situ hybridization (G-I) on E11.5-14.5 Gdf7Cre;R26R or wild-type coronal cryosections. Semi-serial sections starting at the choroid plaque level are 120 µm (A-F,J-L) or 40-60 µm apart (G-I). Gdf7 cell lineages localize to the choroid plaque (arrowheads), tCPe (arrows) and cortical hem (brackets) in the anterior DMT (A,D,J). No tCPe or hem labeling is detected in the much-larger posterior DMT (B,C,E,F,K-L). In contrast to nearby diencephalic neuroepithelium (asterisks in H,I), Gdf7 mRNA is not detected in the tCPe or hem (arrows in G-I), which suggests true lineage relationships between the Gdf7-expressing DM cells at neural tube stages and the labeled anterior DMT cells at later stages. Scale bar: 0.2 mm.

View larger version (105K): [in a new window]   Fig. 5. Reduced diencephalic and myelencephalic CPe after late Gdf7-mediated ablation. Ttr in situ hybridization (A-D,I-L), fluorescent TUNEL assays (E,F,M,N; TUNEL green, nuclei blue, red blood cells yellow-orange), and Hematoxylin and Eosin (G,H,O,P; H&E) stains of E12.5-16.5 Gdf7Cre;Gdf7DTA coronal cryosections. (A-H) Diencephalon. The anterior diencephalic midline shows markedly reduced Ttr expression (A,C), increased apoptosis (E) and lack of papillary CP (G) in mutants compared with controls (B,D,F,H). (I-P) Hindbrain. As in the diencephalon, the mutant hindbrain shows reduced Ttr expression (I,K), increased apoptosis (M) and a lack of papillarity (O). Arrows designate CPe or its expected location; arrowheads designate TUNEL-positive cells; broken lines delimit the CPe. Scale bars: 0.4 mm in K,L; 0.1 mm in A-J,M-O.

View larger version (59K): [in a new window]   Fig. 6. Preserved telencephalic CPe after late Gdf7-mediated ablation. Ttr in situ hybridization (A-D), fluorescent TUNEL assays (E-H), and Hematoxylin and Eosin staining (I,J) as in Fig. 5. Anterior sections (A,B) at level of choroid plaque; posterior sections (C,D) at level of anterior diencephalon. Gdf7Cre;Gdf7DTA mutants maintain strong Ttr expression in the E12.5 telencephalon (A,C), in contrast to the diencephalon (arrow in C). Anterior tCPe volume appears slightly reduced (A) compared with normal (B). Significant tCPe apoptosis is not detected in mutant embryos (E,G), except at the CP base (arrowheads) and in the hippocampal mantle (asterisks), as seen in controls (H). The tCPe of E16.5 mutants appears normal histologically (I). Broken lines outline the CP. Scale bars: 0.2 mm.

View larger version (23K): [in a new window]   Fig. 7. Separation of the telencephalic and diencephalic CPe fields. Ttr in situ hybridization on E11.5-12.5 wild-type coronal cryosections. Semi-serial sections spanning the choroid plaque and anterior diencephalon are 40-80 µm apart. (A,B) E11.5 studies. Ttr-expressing tCPe domains are separated by the Ttr-negative choroid plaque (arrow in A). No diencephalic expression is detected at this stage. (C-F) E12.5 studies. Ttr expression commences in the dCPe, which is separated from the tCPe by Ttr-negative diencephalic neuroepithelium (arrowheads in E,F). Ttr-negative choroid plaque continues to separate the bilateral tCPe fields (arrows in D,E). d, dCPe; t, tCPe. Scale bar: 0.4 mm.

View larger version (86K): [in a new window]   Fig. 8. DM apoptosis in normal embryos and after early Gdf7-mediated ablation. Fluorescent TUNEL assays (TUNEL, green; nuclei, blue; red blood cells, yellow-orange) on E9.5-11.5 ACTBCre;Gdf7DTA 10µm coronal cryosections. Semi-serial sections are 20-90 µm (A-F) or 120-250 µm (G-R) apart. (A-F) E9.5 studies. TUNEL labeling localizes to the midline region (neuroepithelium and overlying tissues) in mutants and controls. The increased labeling in mutants is consistent with enhanced apoptosis caused by DTA delivery. (G-L) E10.5 studies. Apoptosis in mutants is no longer elevated, but remains localized to the anterior midline, as seen in controls (G,J). In mutants, significant apoptosis is absent caudally where the posterior tCPe anlagen should reside (H,I). The normal posterior tCPe anlagen also displays negligible apoptosis (K,L). (M-R) E11.5 studies. Similar to the E10.5 findings, apoptosis is present at low levels in anterior midline of mutant embryos (M), while posterior levels show little to no apoptosis (N,O). In normal E11.5 embryos, the anterior tCPe demonstrates significant apoptosis (P), while the posterior tCPe does not (Q,R). Similar findings were seen in at least two embryos from each group/stage combination. Arrowheads designate TUNEL-positive regions; arrows designate the definitive tCPe or its anlagen. Scale bars: in F, 0.1 mm for A-F; in R, 0.1 mm for G-R.

View larger version (37K): [in a new window]   Fig. 9. Loss of all CPe, including posterior tCPe, after early Gdf7-mediated ablation. Ttr in situ hybridization and Hematoxylin and Eosin stains on E12.5 and E13.5 coronal cryosections (A-D,G,H) or paraffin sections (E,F), and Ttr qRT-PCR analysis (I) on E12.5 ACTBCre;Gdf7DTA embryos and controls. (A,B) Hindbrain. The mutant hindbrain is open and lacks Ttr expression (arrows in A). (C-I) Forebrain. In the closed mutant forebrain, no Ttr expression (C) or tissue resembling CP (F,H) is detectable at E12.5 (E) or E13.5 (G). (I) Ttr qRT-PCR. Ttr transcript levels in the dorsal forebrain are two to three orders of magnitude lower in the three E12.5 mutants relative to three littermate controls after normalizing to 18S rRNA (0.004±0.003) or Cyclophilin A (0.003±0.001). Arrows designate the expected sites of CPe; d, dCPe; m, mCPe; t, tCPe. Scale bars: 0.2 mm. See Materials and methods for additional qRT-PCR details.

View larger version (112K): [in a new window]   Fig. 10. Loss of high-level Bmp signaling after early Gdf7-mediated ablation. In situ hybridization on E10.5 and E12.5 ACTBCre;Gdf7DTA coronal cryosections. Semi-serial sections in G-L are 60-120 µm apart. (A-F) Bmp studies. Gdf7 is no longer detected in mutants (A), consistent with ablation. In controls, Bmp4 (D) and Bmp6 levels (F) are higher in the bilateral tCPe anlagen (arrowheads) and lower at the midline (arrows). After ablation, Bmp4 and Bmp6 levels are reduced and confined to a small midline domain (C,E). Epidermal/mesenchymal Bmp4 expression is maintained after ablation (C). (G-N) Msx1 studies. Msx1 is expressed in the tCPe anlagen at E10.5 (L) and in the definitive tCPe at E12.5 (N). After ablation, Msx1 expression in the neuroepithelium is dramatically reduced at E10.5 (G,I,K) and E12.5 (M). In situ hybridization of sections adjacent to those in A and M demonstrate well-preserved mRNA. Broken lines outline the neuroepithelium; arrows indicate the midline; arrowheads indicate tCPe or its anlagen. Scale bars: 0.1 mm.

View larger version (31K): [in a new window]   Fig. 11. DM-CPe relationships. (A) Summary of the Gdf7 fate map and ablation studies; color-coding as in Fig. 1, with blue denoting DM or CPe domains with apparent lineage relationships to DM cells. Many mCPe and dCPe cells are derived directly from DM cells. In the telencephalon, DM cell lineages contribute to the anterior tCPe domain, but not to the posterior tCPe (orange). Late Gdf7-mediated ablation results in mCPe and dCPe reduction, but preserved posterior tCPe, consistent with ablation after CPe induction. By contrast, early ablation of Gdf7-expressing DM cells causes near total loss of CPe, including the posterior tCPe. (B) The distinct anterior and posterior tCPe domains; dorsal and medial views, as in Fig. 1. The anterior domain contains DM cell lineages and exhibits marked apoptosis, while the larger posterior domain lacks these features. The two domains are separated by a cryptic boundary located lateral to the di-telencephalic midline boundary. A similar domain structure applies to the adjacent cortical hem. (C) Model of DM-dependent tCPe induction. DM cells are required to establish high-level Bmp signaling in the tCPe anlagen (red). In the anterior domain, both cell-autonomous and non-cell-autonomous mechanisms are involved, whereas posterior domain establishment is non-autonomous. In both domains, homeogenetic mechanisms may be involved (see Discussion for details). Following its establishment, high-level Bmp signaling is then responsible for locally inducing the tCPe (orange) (Hebert et al., 2002).

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