QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online December 28, 2004
doi: 10.1242/10.1242/dev.01580

This Article Summary Full Text Full Text (PDF) 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 Spoelgen, R. Articles by Willnow, T. E. Search for Related Content PubMed PubMed Citation Articles by Spoelgen, R. Articles by Willnow, T. E. Social Bookmarking                         What's this?

LRP2/megalin is required for patterning of the ventral telencephalon

Robert Spoelgen^*, Annette Hammes^*, Uwe Anzenberger^*, Dietmar Zechner, Olav M. Andersen, Boris Jerchow and Thomas E. Willnow

Max-Delbrueck-Center for Molecular Medicine, Berlin, 13092, Germany

View larger version (61K): [in a new window]   Fig. 1. Neuroanatomy of megalin-deficient neonates and embryos. (A,B) External appearance of wild-type (A) and megalin–/– neonates (B), highlighting craniofacial abnormalities in the receptor-deficient mouse. (C,D) Coronal H+E stained sections through the rostral brain of E18.5 embryos, depicting enlarged ventricles in the megalin-deficient animal (D) compared with the control (C). (E,F) Horizontal sections through the brain at E18.5, demonstrating a single cerebral hemisphere with a central fused ventricle in the megalin–/– embryo (F) compared with bilateral hemispheres with two lateral ventricles in the control (E). (G-I) Coronal forebrain sections of wild-type (G) and megalin–/– E14.5 embryos (H,I), with abnormalities ranging from enlarged ventricles (H) to exencephalus and a common ventricular system (I). (K-N) Sagittal forebrain sections from wild type (K,L) and megalin-deficient E12.5 embryos (M,N) subjected to H+E staining (K,M) or detection of cell death by TUNEL assay (L,N). megalin–/– embryos suffer from a reduction in neuroepithelial wall thickness (M, arrowhead) compared with controls (K) that coincides with massive apoptosis in the roof of the cerebral cortex (N, arrowhead). (O,P) Lateral aspects of wild-type (O) and megalin–/– (P) E10.5 embryos. Note the reduced size of the telencephalic vesicles in the knockout. (Q,R) Coronal forebrain sections, indicating a decrease in thickness of the ventral neuroepithelium that is most pronounced in the AEP and the MGE of megalin–/– embryos (R) compared with megalin+/+ embryos (Q). (C-M,O-R: H+E staining). AEP, anterior entopeduncular area; MGE, medial ganglionic eminence; tv, telencephalic vesicles.

View larger version (109K): [in a new window]   Fig. 2. Forebrain abnormalities and megalin expression pattern in E14.5 embryos with conditional megalin gene inactivation. (A-D) Immunohistological detection of megalin in yolk sac (A,B) and neuroepithelium (C,D) of E14.5 embryos. megalinlox/lox embryos express the receptor in the yolk sac (A) and the neuroepithelium (C), whereas megalinlox/lox/Meox2tm1(cre)Sor embryos express the receptor in the yolk sac (B) but not in the neuroepithelium (D). (E,F) Exencephalus in E14.5 embryos with ubiquitous (megalin–/–) (E) or with epiblast-specific (megalinlox/lox/Meox2tm1(cre)Sor) megalin gene knockout (F).

View larger version (130K): [in a new window]   Fig. 3. Analysis of cell proliferation and apoptosis in the neural tube of E10.5 embryos. Coronal sections through the rostral ventral neural tube of wild-type (A-C) and megalin knockout embryos (D-F) stained with H+E (A,D), or analyzed for cell proliferation (B,E) (anti-phosphohistone H3 immunofluorescence) or apoptosis (C,F) (TUNEL assay). The number of mitotic cells is reduced in the rostroventral neural tube of the megalin–/– embryo (E, arrowheads) compared with the control (B, arrowheads). No difference is seen in the number of apoptotic nuclei in both genotypes (C,F; arrowheads).

View larger version (115K): [in a new window]   Fig. 4. Shh expression in wild-type and megalin–/– embryos. (A,B) Whole-mount in-situ hybridization (ISH) in E9.5 wild type (A) and megalin-deficient embryos (B), demonstrating an identical expression pattern for Shh in floorplate and ventral prosencephalon. (C,D) Whole-mount ISH for Shh in E10.5 embryos, indicating expression in the ventral telencephalon of the wild type (C, arrowhead) but not in the ventral telencephalic region anterior to the optic recess of megalin–/– embryos (D). (E,F) Frontal head aspects of wild type (E) and megalin–/– E10.5 embryos (F), depicting Shh expression in the AEP (arrowheads) of wild-type but not knockout animals. Expression of Shh in other areas of the forebrain, such as in the basal plate of the diencephalon (bpd) or the zona limitans intrathalamica, is identical in both. (G-K) ISH for Shh on coronal sections of the rostral (G,H) and the caudal (I,K) neural tube. Shh is expressed in the AEP of the rostroventral neural tube of wild-type (G) but not megalin–/– E10.5 embryos (H), while expression in caudal regions of the neural tube is identical in wild-type (I) and knockout tissues (K). bpd, basal plate of the diencephalon; fp, floorplate; or, optic recess; te, telencephalon; vp, ventral prosencephalon; Zli, Zona limitans intrathalamica.

View larger version (75K): [in a new window]   Fig. 5. Shh expression in megalinlox/lox and megalinlox/lox/Meox2tm1(cre)Sor E10.5 embryos. (A,C) Lateral and (B,D) frontal head aspects of whole-mount in-situ hybridization for Shh in E10.5 embryos, indicating expression in the AEP of megalinlox/lox (A,B; arrowheads) but not megalinlox/lox/Meox2tm1(cre)Sor embryos (C,D, arrowheads). bpd, basal plate of the diencephalon; Zli, zona limitans intrathalamica.

View larger version (127K): [in a new window]   Fig. 6. Co-expression of Shh and megalin in the AEP of E10.5 embryos. In-situ hybridization for Shh (A) and immunohistology for megalin (B) on coronal sections of the rostroventral neural tube of wild-type E10.5 embryos identify co-expression in the AEP (arrowheads). The inset highlights localization of megalin protein on the apical surface of neuroepithelial cells.

View larger version (55K): [in a new window]   Fig. 7. Expression of marker genes of early forebrain development in E10.5 embryos. (A,B) In-situ hybridization (ISH) of frontal head aspects (upper panel) and coronal forebrain sections (lower panel) demonstrate reduced signals for Olig2 (A) and Nkx2.1 (B) in the AEP (arrowheads) but not in the diencephalon of megalin–/– embryos. (C) Expression of Dlx2 is lost in the ventral telencephalon (arrowheads) and significantly reduced in the ventral thalamus region of the diencephalon (asterisks) of megalin–/– embryos as shown by whole-mount ISH. (D) Immunohistology of coronal E10.5 forebrain sections indicate lack of TuJ1 expression in the ventral (arrowheads), but not in the dorsal, telencephalic region of megalin–/– compared with control embryos. (E-H) Using whole-mount ISH, no differences can be seen in the expression patterns of Ptch1 (ventral neural tube, including tel-, di- and mesencephalon) (E), Hnf3b (ventral neural tube, including di- and mesencephalon) (F), or Wnt1 (roof of di- and mesencephalon, isthmus) (G), whereas expression of Pax6 extends from the dorsal into the ventral neural tube of receptor-deficient embryos (H, arrowhead). Also, most knockouts lack the Pax6 signal in the optic vesicle. di, diencephalon; is, isthmus; me, mesencephalon; ov, optic vesicle; te, telencephalon.

View larger version (80K): [in a new window]   Fig. 8. Analysis of Fgf8 and Bmp4 pathways in E9.5 and E10.5 embryos. (A) Anteroventral head aspects of E10.5 embryos, demonstrating a reduction of Fgf8 expression in the ventral telencephalon (asterisk) and an extension from the commissural plate (dotted line) into more dorsal regions of the midline (arrowhead) in megalin-deficient animals. (B) In-situ hybridization of lateral head aspects (left panel) Bmp4 expression in wild types is restricted to the dorsomedial part of the telencephalon and the dorsal midline of the most anterior diencephalon, whereas in megalin mutants the Bmp4 expression domain extends along the midline into more caudal regions of the roof (arrowhead). Coronal sections (right panel) highlight increased Bmp4 signals in the telencephalon and the anterior diencephalon of knockout compared with control embryos. (C) Immunodetection of P-Smad proteins, indicating a signal in E10.5 wild types that is restricted to the dorsal midline of the tel- and diencephalon (arrowheads) but that is significantly enhanced and ventrally expanded (arrowheads) in megalin–/– littermates. P-Smad signals in the spinal cord are identical in both genotypes. (D) At E9.5, the Fgf8 expression domain in megalin–/– animals is reduced in the ventral (asterisk), but extended into the dorsal, region of the telencephalon (arrowhead). (E-G) At E9.5, expression of Bmp4 is increased in the rostral and dorsal telencephalon (arrowheads) of megalin–/– embryos as shown by whole-mount ISH (E, left panel) or coronal dorsal forebrain sections thereof (E, right panel). Increases in Bmp4 message result in enhanced phosphorylation of Smad proteins (F, arrowheads) and induced expression of Bmp4 target gene Msx1 in the rostral dorsal telencephalon (G, arrowheads). Msx1 expression in the hindbrain region is unchanged (asterisks). di, diencephalon; sp, spinal cord; te, telencephalon.

View larger version (13K): [in a new window]   Fig. 9. Megalin mediates binding and cellular catabolism of BMP4. (A) Surface plasmon resonance (SPR) analysis of binding of 0.5 µmol/l BMP4 but not of 0.5 µmol/l BMP5 or WNT1-conditioned medium to purified megalin immobilized on the sensor chip surface (see Materials and methods for details). As a positive control, binding of 1 µmol/l receptor-associated protein (RAP) to megalin was tested. (B) Megalin-expressing BN16 cells mediate uptake and lysosomal degradation of 125I-BMP4. Degradation of BMP4 can be blocked by chloroquine (200 µmol/l) or the receptor-antagonist RAP (100 µg/ml).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter