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First published online 29 August 2007
doi: 10.1242/dev.003095

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Molecularly and temporally separable lineages form the hindbrain roof plate and contribute differentially to the choroid plexus

Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.

View larger version (32K): [in this window] [in a new window]   Fig. 1. Roof plate epithelium in the hindbrain. (A) Cartoon of a dorsal view of the neural tube (E11.5); neuroepithelium of the rhombic lip is dark gray and hindbrain roof plate epithelium (hRPe) is light gray. The rhombic lip in rhombomere 1 (r1) is the cerebellar or upper rhombic lip (URL, white arrowhead); that within r2-r8 is the hindbrain or lower rhombic lip (LRL, black arrowhead). Broken line marked by `b' indicates the level of the idealized transverse section shown in B and the section plane in subsequent figures. Broken line marked by `d' indicates the axial level of idealized transverse section through the spinal neural tube depicted in D. (B) Vertical bracket demarcates the dorsoventral (DV) region of the rhombic lip (RL); horizontal brackets demarcate medially (med) versus laterally (lat) located hRPe (relevant to subsequent figures). Boxed area (c) is further schematized in C. (E) Schematic of cumulative and inducible (temporal) fate mapping. A broadly active promoter (BAP) drives expression of an indicator transgene containing either an FRT- or loxP-flanked stop cassette followed by a sequence encoding a reporter molecule that serves as a lineage tracer. Transcription of the reporter is prevented by the stop cassette (top); excision by Flpe or Cre (or following FlpeERT2-mediated recombination in the presence of tamoxifen), permits reporter transcription (bottom). mb, midbrain; hb, hindbrain; ov, otic vesicle; 4v, fourth ventricle; vz, ventricular zone; mz, post-mitotic mantle zone; ect, epidermal ectoderm; mes, mesenchyme; RPe, roof plate epithelium.

View larger version (42K): [in this window] [in a new window]   Fig. 2. Medial versus lateral roof plate cells differ in tissue organization, proliferative capacity and order of emergence from the rhombic lip. (A,B) Doubly transgenic embryos Egr2::cre (r3/r5 cre allele); RC::PFwe (Cre-responsive ßgal indicator allele) X-gal-stained for ßgal activity. Broken lines demarcate the rhombic lip. (A) At E9.5, hindbrain roof plate epithelium (hRPe) cells arising from different rhombomeres intermix anteriorly. (B) At E11.5, medially located (med) hRPe cells are an admixture from different rhombomeres, whereas laterally located (lat) hRPe cells are compartmentalized with respect to rhombomeric origin. (C) Transverse section through an E11.5 doubly transgenic Wnt1::cre; Cre-responsive ßgal indicator embryo so as to distinguish hRPe cells by ßgal activity (green); DAPI is shown in blue. Broken white line highlights the sharp border in tissue organization observed between laterally and medially located hRPe cells. Asterisk indicates a tear in the tissue, resulting in ectoderm cells situating away from mesenchymal cells. Arrowheads indicate laterally located hRPe that is segregated from overlying mesenchyme and epidermal ectoderm. (D-G) Transverse sections through a doubly transgenic Wnt1::cre; Cre-responsive ßgal indicator embryo, with ßgal expression (green by immunodetection) distinguishing rhombic lip and hRPe from mesenchyme and ectoderm; Ki-67 immunoreactivity is in red. Co-labeling was detected in both medially and laterally located hRPe at E9.5 (arrowheads, D,E), but largely absent in medially and laterally located hRPe at E11.5 (F and arrowheads in G). Inset in G shows proliferation (co-labeling) of rhombic lip cells; arrowhead demarcates non-mitotic laterally located hRPe. (H,I) Doubly transgenic Wnt1::FlpeERT2; R26::FRAP embryos (E11.5) treated for PLAP histochemistry (cells marked by black precipitate). Broken lines demarcate the lower rhombic lip. Induction of recombination by tamoxifen (TAM) administration separates temporal cohorts of Wnt1 (rhombic lip)-derived hRPe. (H) E7.5 TAM administration (E8 recombination in the rhombic lip) resulted in labeled hRPe cells in both medial (med, red arrowheads) and lateral (lat) roof plate locations, albeit a modest number. (I) E9.5 TAM administration (E10 recombination in the rhombic lip) resulted in labeled hRPe cells in only laterally located roof plate. (J) Summary schematic. At E9.5, hRPe cells intermix and are mitotic (yellow). At E11.5, two fields of hRPe are present: (1) cells that are medially located, dispersed and largely non-mitotic that emerged early from the rhombic lip (yellow) and (2) cells that are laterally located, compartmentalized along the anteroposterior (AP) and dorsoventral (DV) axes, are non-mitotic, and that emerged later from the rhombic lip (blue). mb, midbrain; hb, hindbrain; ov, otic vesicle; ect, epidermal ectoderm; mes, mesenchyme; rec, recombination time point; harv, harvest time point; RL, rhombic lip; TAM, tamoxifen.

View larger version (90K): [in this window] [in a new window]   Fig. 3. A sequential change in progenitor-cell gene expression in the rhombic lip distinguishes laterally from medially fated roof plate cells. (A-D) Doubly transgenic Wnt1::cre; Cre-responsive ßgal indicator embryos, with ßgal detected by X-gal staining. (A-C) Dorsal whole-mount views showing ßgal activity throughout the entire hindbrain roof plate epithelium (hRPe; arrows), both medially and laterally. Black broken lines demarcate the rhombic lip. Inset in A shows cre mRNA in the lower rhombic lip at E9.25 (arrowhead); Wnt1 and Wnt1::cre are expressed in dorsal hindbrain neuroepithelium, but not in hRPe, from E8 onwards. (D) Transverse section taken at the rhombomeric level of the white broken line in C, revealing ßgal+ hRPe cells medially (med) and laterally (lat). (E-H) Doubly transgenic Gdf7::cre; R26R embryos, with ßgal detected by X-gal staining. (E-G) Dorsal whole-mount views showing ßgal in laterally (arrows) but not medially located hRPe. Inset in E shows little to no cre mRNA in the rhombic lip at E9.25, with Gdf7 and cre expression starting between E9.25 and E9.5. (H) Transverse section taken at the rhombomeric level of the white dashed line in G, revealing ßgal+ hRPe cells laterally only (arrowheads). Black broken line demarcates the rhombic lip. (I) Schematic of hRPe fields at E11.5. Medially located hRPe (yellow) has a history of Wnt1 expression, whereas laterally located hRPe (blue) has a history of Wnt1 and Gdf7 expression. ov, otic vesicle; hb, hindbrain; med, medial; lat, lateral.

View larger version (116K): [in this window] [in a new window]   Fig. 4. Lateral roof plate derivatives from rhombomere 1 show a temporal lag in molecular fate as compared with those from rhombomeres 2-8. (A-F) Dorsal view of whole embryos processed to detect Kcne2 mRNA (A-C) or Ttr mRNA (D-F). Broken lines demarcate the rhombic lip. Kcne2 or Ttr transcripts were undetectable in the medial hindbrain roof plate epithelium (hRPe) field at all time points, but were readily detectable in the lateral hRPe field, except in those lateral cells derived from rhombomere 1 (r1). (G) Dorsal view of E12.5 doubly transgenic En1::cre; Cre-responsive ßgal indicator embryos stained with X-gal to identify r1-derived cells. White broken lines indicate the levels of transverse sections shown in H-S (h,i,j,k); black broken lines demarcate the rhombic lip. (H-S) Serial transverse sections taken from doubly transgenic En1::cre; Cre-responsive ßgal indicator embryos; one set was processed for Ttr mRNA detection, the other for X-gal detection of ßgal. (H-K) Serial sections reveal that r1-derived ßgal+ hCPe (red arrowheads) lacks Ttr activity at E12.5, whereas caudal (r2-r8-derived) ßgal-negative (ßgal-) hCPe (white arrowheads) is Ttr+. (L-S) By E13.5, all hCPe was Ttr+, whether derived from r1 (red arrowheads, ßgal+) or from r2-r8 (white arrowheads, ßgal-negative). (T) Schematic of three hRPe fields at E11.5: field 1 (yellow) is medially located, and is Ttr- and Kcne2-; field 2 (light blue) is laterally located but caudally derived (r2-r8), and is Ttr+ and Kcne2+ from E9.5 onwards; field 3 (dark blue) is laterally located but rostrally (r1)-derived, and is Ttr+ and Kcne2+ after E12.5 only. CPe, choroid plexus epithelium; hb, hindbrain; ov, otic vesicle.

View larger version (69K): [in this window] [in a new window]   Fig. 5. Production of hindbrain choroid plexus epithelium begins at E9.5, peaks between E11 and E12, and terminates by E14. (A-F) Transverse sections through E9.5-E14.5 hindbrain that has been processed to detect FlpeERT2 mRNA show expression limited to the rhombic lip (vertical brackets in A-C), and absent from the hindbrain roof plate epithelium (hRPe, opposing black arrowheads) and hindbrain choroid plexus epithelium (hCPe). (G-L) Coronal sections processed for PLAP detection (dark precipitate) after harvest from E16.5 doubly transgenic Wnt1::FlpeERT2; R26::FRAP embryos (n=9). Induction of FlpeERT2-mediated recombination by tamoxifen (TAM) administration separates temporal cohorts of Wnt1 (rhombic lip)-derived hCPe. (G) E9.5 TAM administration (recombination at E10 in the rhombic lip) resulted in a few labeled E16.5 hCPe cells. (H-J) TAM at E10.5 (recombination at E11), E11.5 (recombination at E12) or E12.5 (recombination at E13) resulted in numerous labeled cells in the E16.5 hCPe. (K,L) TAM administration at E13.5 (recombination at E14) resulted in few labeled hCPe cells (K), and none were present after administration at E14.5 (L). Insets in G-L reveal no PLAP activity when diluent alone was administered. (M-R) Coronal sections from doubly transgenic Wnt1::cre; Cre-responsive ßgal indicator embryos, with ßgal expression (green by immunodetection) distinguishing hCPe from underlying mesenchyme and vasculature, and red indicating BrdU incorporation, reflecting birthdate (time of single BrdU injection indicated above each panel). BrdU administered at either E9.5 or E14.5 showed very few co-labeled hCPe cells at E16.5 (M,R, respectively), whereas BrdU administered from E10.5 to E13.5 showed numerous co-labeled hCPe cells (N-Q). Insets show higher-power views of the boxed areas. rec, recombination time point; harv, harvest time point; hCP, hindbrain choroid plexus; LRL, lower rhombic lip.

View larger version (118K): [in this window] [in a new window]   Fig. 6. Expression of activated Notch1 results in overproduction of hindbrain choroid plexus epithelium but not of other rhombic lip lineages. (A) Schematic of a transverse section through the hindbrain (hb), with the boxed area demarcating the region of the rhombic lip (RL) shown in B-D. (B-D) Detection of Notch1, Gdf7 and Math1 mRNA on E11.5 hindbrain serial sections. Notch1 is expressed in the rhombic lip, but not in the hindbrain roof plate epithelium (hRPe); Gdf7 is expressed in the distal tip of the rhombic lip and into the hRPe; and Math1 is expressed in the rhombic lip ventral to the Gdf7 expression territory. (E-J,L) Comparison of singly transgenic R26::stop-Notch1-ICD::IRES-nGFP (E,G,I) and doubly transgenic Gdf7::cre; R26::stop-Notch1-ICD::IRES-nGFP (F,H,J,L) brains. (E,F) Posterior (pos) whole-mount (wm) view of P7 brains processed for Ttr mRNA detection [hindbrain choroid plexus epithelium (hCPe) marker] showing enlarged hCPe in doubly transgenic tissue (F,H,J,L). (G,H) Sagittal sections showing enlarged postnatal day 0 (P0) hCPe (red boxes) and abnormal cerebellar architecture in doubly but not singly transgenic animals. Inset in G shows Ttr+ hCPe on a serial section to that shown in G. (I,J) Hematoxylin and Eosin staining at P7 shows abnormal cytoarchitecture of hCP in doubly transgenic tissues. (K) P0 doubly transgenic Wnt1::Flpe; Flpe-responsive nßgal indicator distinguishing hCPe (green by immunodetection of ßgal) from underlying mesenchyme and vasculature. BrdU was injected into animals at P0, and tissue was harvested 2 hours later. BrdU immunodetection is shown in red. No co-labeled cells were found in wild-type hCPe, as shown. (L) Similar BrdU injection into doubly transgenic Gdf7::cre; R26::stop-Notch1-ICD::IRES-nGFP animals showed numerous co-labeled cells (arrowheads; GFP positivity identifying hCPe cells and expression of Notch1-ICD, and red indicating BrdU incorporation as a measure of proliferation). (M-V) Comparison of singly transgenic R26::stop-Notch1-ICD::IRES-nGFP and doubly transgenic Math1::cre; R26::stop-Notch1-ICD::IRES-nGFP brains. Coronal sections show no differences in size, relative cell density (Barhl1 expression at P0) or proliferation (Ki-67 immunodetection at P5 shown in red; U,V) of Math1-derived rhombic lip lineages, including mossy fiber neurons of the pontine gray nucleus (PGN, M-P), cerebellar granule cell precursors (CGC, white arrowheads, Q-V) and granule cells of the cochlear nucleus (CNGC, red arrowheads, Q,R). GFP positivity (immunodetection shown in green) reflects expression of the Notch1-ICD transgene in PGN (O,P) and CGC (S,T). White boxes in M-N demarcate regions of the PGN shown in O,P. DAPI is shown in blue. 4v, fourth ventricle; bs, brainstem; cb, cerebellum; CP, choroid plexus; hb, hindbrain; mz, post-mitotic mantle zone; RPe, roof plate epithelium; vz, ventricular zone.

View larger version (33K): [in this window] [in a new window]   Fig. 7. Summary schematic of distinct subdivisions in the hindbrain roof plate, their differential contribution to the hindbrain choroid plexus and differential responses among rhombic lip lineages to activated Notch1. Wnt1 mRNA in the rhombic lip is graded, with the highest expressers giving rise to hindbrain roof plate epithelium (hRPe) beginning at E8. These cells emerge from the rhombic lip and occupy the entire hRPe by E9.5 (yellow); they intermix in the anteroposterior (AP) and dorsoventral (DV) dimensions and are highly proliferative. Beginning at E9.25-E9.5, Gdf7 expression starts in the dorsal-most progenitor cells in the rhombic lip and these progenitors give rise to hRPe (dark blue) that is compartmentalized in both the AP and DV dimensions, settles laterally and is non-mitotic immediately upon emerging from the rhombic lip. The medially located (yellow) hRPe cells have a history of Wnt1 expression, whereas the laterally located (dark blue) hRPe cells have a history of both Wnt1 and Gdf7 expression. Caudally (r2-r8)-derived lateral hRPe (light blue) expresses Ttr and Kcne2 from E9.5 onwards, whereas expression in rostrally (r1)-derived cells only occurs after E12.5. Beginning at E12.5, laterally located hRPe cells (light and dark blue) undergo cell shape changes to form the hindbrain choroid plexus epithelium (hCPe) (not drawn to scale). Medially located hRPe cells (yellow) do not appear to contribute to the hCPe. From E12.5 through to E14, Gdf7+ progenitors within the rhombic lip probably generate hCPe cells directly, without seeming to transition through an hRPe intermediate. At E14, the production interval for hCPe ceases. Ligand-independent activation of the Notch1 signaling pathway in the Gdf7 lineage (the hRPe and hCPe lineage) results in overproliferation of hCPe. 4v, fourth ventricle; hCP, hindbrain choroid plexus; mes, mesenchymal cells.