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First published online April 22, 2004
doi: 10.1242/10.1242/dev.01122

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FKBP8 is a negative regulator of mouse sonic hedgehog signaling in neural tissues

Oleg V. Bulgakov^1,*, Jonathan T. Eggenschwiler^2,*,, Dong-Hyun Hong¹, Kathryn V. Anderson² and Tiansen Li^1,

¹ The Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, MA 02114, USA
² Developmental Biology Program, Sloan Kettering Institute, New York, NY 10021, USA

View larger version (52K): [in a new window]   Fig. 1. FKBP8 is a widely expressed integral membrane protein. (A) Domain structure of murine FKBP8. Numbers on top indicate positions of amino acid residues. N is the N-terminal sequence missing in previous literature; PPI (residues 110-194), TPR (262-329) and the transmembrane domain (M; 381-400) are marked. (B) Immunoblots of mouse tissue homogenates. (C) FKBP8 is solubilized only with a detergent. TGN38, a known integral membrane protein shown here as a control. (D) Left, immunoblotting analyses of in vitro translated FKBP8 in the presence or absence of membranes. Middle, protease protection assay to determine membrane topography. FKBP8 was not protected by intact membranes. By comparison, the ER luminal protein BiP was fully protected. Right, a schematic diagram showing FKBP8 situated outside of the ER lumen. (E) FKBP8 is localized in the mitochondria of cultured fibroblasts (left), as shown by overlapping staining patterns of FKBP8 (red) and MitoTracker (green). Scale bar: 10 µm.

View larger version (62K): [in a new window]   Fig. 2. Neural development defects in mice with targeted disruption of the Fkbp8 gene. (A) (Top panel) targeting strategy. Black boxes, exons; arrowheads, PCR primers. (Bottom panels) Left two panels: PCR screening for targeted ES clones and mouse genotyping. Middle panel: RT-PCR analyses find the mutant transcripts reduced or absent. E1-3, primers spanning exons 1-3; E1-7, primers spanning exons 1-7. GAPDH amplification served as a control. Right panel: immunoblots showing complete ablation of FKBP8 protein. -tubulin is shown as a gel loading control. (B) Developmental defects in the CNS of Fkbp8-/- (knockout) embryos. The caudal neural tube defect (upper left and right) and failure of eye development (upper right and lower left) are apparent. The mutant neural tube is dilated (lower right). Dorsal root ganglia (arrowheads) are missing or disorganized. (C) Apoptosis monitored by immunofluorescence for activated caspase 3. Sections through the caudal neural tube of E10.5 wild type and Fkbp8-/- mutant embryos are shown (apoptotic cells are indicated by white arrows). Quantification (three embryos per genotype and four sections/embryo) showed no statistically significant difference between genotypes in the thoracic or lumbar neural tube (NS, not statistically significant). Scale bars: 100 µm in B; 50 µm in C.

View larger version (61K): [in a new window]   Fig. 3. Embryonic expression pattern of Fkbp8. (A) Whole-mount in situ hybridization in E10 embryos. Fkbp8 appears uniformly expressed. Negative controls included the sense probe and Fkbp8-/- mutants hybridized with the antisense probe. The minimal signal in the mutant further indicated that the mutant Fkbp8 transcript is destabilized. (B) In situ hybridization of sections. Thoracic neural tube is on the right, and lumbar neural tube is on the left. No differences in expression could be detected in the neural tube along the dorsoventral or anteroposterior axis. (C) Immunofluorescence of neural tube sections from E9.5 wild type (+/+) and Fkbp8 mutant (–/–) embryos, stained with the FKBP8 antibody.

View larger version (54K): [in a new window]   Fig. 4. The Fkbp8-/- neural tube is ventralized. Sections of E11.5 wild type and Fkbp8-/- neural tubes were analyzed by in situ hybridization. Sections at caudal levels of the neural tube just anterior to the hindlimbs are shown. Markers of dorsal fates, Wnt1 (A,B) and Gdf7 (data not shown) are not expressed in Fkbp8-/- mutants. In the mutant, dorsal cells adopt lateral fates marked by Pax6 and Dbx1 (D,F), and lateral cells acquire ventral fates marked by Foxa2 (H) and Nkx2.2 (data not shown). Expression of MSX1 and MSX2 in the E10.5 dorsal neural tube, monitored by immunofluorescence with an antibody that recognizes both proteins, is relatively unaffected in Fkbp8-/- mutants despite changes in neural tube morphology (I,J). Scale bars: 65 µm.

View larger version (55K): [in a new window]   Fig. 5. Delayed eye development in Fkbp8-/- mutant embryos. (A) At E11.5, Fkbp8-/- mutant eyes are smaller, have a thinner retinoblast layer and exhibit a hypo-pigmented pigment epithelium in the ventral region. (B) PAX2 (red) and PAX6 (green) immunofluorescence staining of optic vesicles at E10.5. PAX2 is ectopically expressed in the dorsal regions of the optic cup (white arrowheads) in the mutant. PAX6 expression appears comparable with wild type in the dorsal retina and in the lens primordium (white arrows), although lens morphogenesis is clearly delayed. (C) At E11.5, PAX6 expression is apparently reduced in the mutant retinoblast layer and lens. Background staining in the lens capsule and skin is due to non-specific binding of the secondary antibody. Scale bar: 30 µm in A,C; 50 µm in B.

View larger version (59K): [in a new window]   Fig. 6. Loss of Fkbp8 partially restores CNS development in the Shh-/- mutant. Wild-type (A), Shh-/- (B) and Shh-/-;Fkbp8-/- (C) littermates from an E12.5 litter are shown. Phenotype of the Shh-/- single mutant includes growth retardation, failure of limb outgrowth, severe holoprosencephaly (white arrow) and a single ventrally positioned eye. This phenotype is partially rescued in the Shh-/-;Fkbp8-/- embryo (note rescue of cyclopia and holoprosencephaly, white arrow). Other features, such as limb outgrowth defects (arrowhead), are not rescued. Scale bar: 1 mm.

View larger version (84K): [in a new window]   Fig. 7. SHH-independent dorsoventral neural patterning. E11.5 neural tube sections were assayed for expression of Pax3 (A-D), Pax6 (E-H), Dbx1 (I-L), Isl2 (M-P), Nkx2.2 (Q-T) and Foxa2 (U-X) in wild-type (A,E,I,M,Q,U), Shh-/- (B,F,J,N,R,V), Fkbp8-/- (C,G,K,O,S,W) and Shh-/-;Fkbp8-/- (D,H,L,P,T,X) embryos. Dorsal and lateral cell fates (marked by Pax3 and Pax6, respectively) are excluded from the ventral neural tube of wild-type and Fkbp8-/- embryos but were expressed across the ventral midline of Shh-/- mutants. Ventral repression of these cell fates was restored in the Shh-/-;Fkbp8-/- double mutant. Note the specification in Shh-/-;Fkbp8-/- mutants of lateral and ventral cell types such as Dbx1+ V0 interneuron progenitors, Isl2+ motoneurons (arrows in M,O,P), Nkx2.2+ V3 interneuron progenitors, and Foxa2+ floorplate. These cell types are absent in E11.5 Shh-/- mutants. In addition to motoneurons, dorsal root ganglia (DRG, arrowheads in M-O) also express Isl2. DRG, derived from the dorsally specified neural crest, are observed in wild-type and Shh-/- mutant embryos but are displaced dorsally or missing altogether in Fkbp8-/- and Shh-/-;Fkbp8-/- mutants, consistent with disruption of dorsal fates in these embryos. Scale bars: 65 µm.

View larger version (88K): [in a new window]   Fig. 8. Neural-specific effects of the Fkbp8 mutation on hedgehog signaling. E11.5 neural tube sections stained for Gli3 (A-D), Ptch1 (E-H), Gli1 (I-L), Pax1 (M-P) and Shh (Q-T) expression. In the Shh-/- ventral neural tube, Gli3 is ectopically expressed (B), whereas Ptch1 and Gli1 fail to be expressed (F,J). SHH is also required for expression of Ptch1, Gli1 and Pax1 in sclerotomal tissues (arrowheads in E,I). In Fkbp8-/- and Shh-/-;Fkbp8-/- embryos, Gli3 expression is repressed ventrally, whereas Ptch1 and Gli1 are expressed in the neural tube in dorsally expanded domains. Note that Gli1 is not expressed in ventral neural tube regions of Fkbp8-/- and Shh-/-;Fkbp8-/- embryos nor in the wild-type floor plate. Sclerotomal expression of Ptch1, Gli1, and Pax1 is not rescued in Shh-/-;Fkbp8-/- mutants (H,L,P). Shh is expressed in the gut of all four mutants (insets in Q-T) but not in the notochord or floorplate of Shh-/- mutants (R). The expanded floorplates of Fkbp8-/- and Shh-/-;Fkbp8-/- mutants express Shh, but notochord expression (arrows in Q,S) is not seen in the Shh-/-;Fkbp8-/- mutant. Scale bars: 65 µm.

View larger version (56K): [in a new window]   Fig. 9. Expression of hedgehog family members in Shh-/-; Fkbp8-/- mutants. (A) Neither Indian hedgehog (Ihh) nor desert hedgehog (Dhh) expression is detected in the vicinity of the E11.5 Shh-/-; Fkbp8-/- double mutant neural tube by in situ hybridization, although their endogenous expression (in the gut and gonad, respectively) is readily detected. (B) Hedgehog proteins are monitored using the monoclonal antibody 5E1. No staining is found within or around the E10.5 double mutant neural tube, although SHH protein is clearly detected in the notochord and floor plate of the wild-type embryo. Scale bars: 65 µm.

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