Hippi is essential for node cilia assembly and Sonic hedgehog signaling - PubMed (original) (raw)

Hippi is essential for node cilia assembly and Sonic hedgehog signaling

Caroline Houde et al. Dev Biol. 2006.

Abstract

Hippi functions as an adapter protein that mediates pro-apoptotic signaling from poly-glutamine-expanded huntingtin, an established cause of Huntington disease, to the extrinsic cell death pathway. To explore other functions of Hippi we generated Hippi knock-out mice. This deletion causes randomization of the embryo turning process and heart looping, which are hallmarks of defective left-right (LR) axis patterning. We report that motile monocilia normally present at the surface of the embryonic node, and proposed to initiate the break in LR symmetry, are absent on Hippi-/- embryos. Furthermore, defects in central nervous system development are observed. The Sonic hedgehog (Shh) pathway is downregulated in the neural tube in the absence of Hippi, which results in failure to establish ventral neural cell fate. Together, these findings demonstrate a dual role for Hippi in cilia assembly and Shh signaling during development, in addition to its proposed role in apoptosis signal transduction in the adult brain under pathogenically stressful conditions.

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Figures

Fig. 1

Hippi targeted deletion. (A) Two targeting vectors were generated resulting in deletion of exon 1, intron A and part of exon 2 of the Hippi locus and insertion of either a neomycin resistance cassette alone (construct 1) or along with the β-galactosidase gene (construct 2). Black boxes are exons, dashed boxes are the inserted cassettes and capital letters indicate introns. (B) Western blot of protein extracts obtained from whole E9.5 embryos and probed with an affinity-purified anti-Hippi antibody showing Hippi expression in Hippi+/+ and Hippi+/−, but no expression in _Hippi_−/− extracts.

Fig. 2

_Hippi_−/− embryos show severe development defects at mid-gestation. (A) Wildtype E9.5 embryo, lateral view. (B) _Hippi_−/− littermate, lateral view, same magnification as in panel A, showing an example of a mild phenotype with incomplete forebrain closure (arrow). (C–E) _Hippi_−/− embryos, ventral, dorsal and lateral views, same magnification as in panel A, showing examples of severe phenotypes. Note retardation in growth and turning, inverted heart looping (dotted line in panel C shows reverse right ventricule looping to the left side), pericardial edema, kinks in the spinal cord, retarded closure of the neural tube in the brain region (arrows), uneven closure of the spinal cord and abnormal cranial flexture (dotted line in panel E). (F) Wildtype E13.5 embryo, ventral view. (G) Ventral view of a _Hippi_−/− embryos at E13.5 exhibiting exencephaly, hypotelorism and abnormal maxillary processes. Insert shows forelimb with at least 6 digits.

Fig. 3

Hippi expression pattern. Whole-mount in situ hybridization shows Hippi mRNA expression pattern. (A–D) From E7.0 to E7.5, Hippi is expressed most strongly in the ectoderm and in the region of the node (arrows). (E, F) At E8.5, expression is detected mainly in the forebrain and the midline (arrow). (G) At E9.5, the highest expression of Hippi is detected in the forebrain, branchial arches and limb buds but also throughout the neural tube. (H) Section though a E9.5 embryo. Note expression in the neuroepithelium (n), mesoderm (m), luminal neural tube (arrowheads) and ventral foregut (arrows) but not in the ectoderm (e). (I) Hippi expression pattern is maintained at E10.5.

Fig. 4

Asymmetrical expression of LR axis patterning genes is lost in _Hippi_−/− embryos. Whole-mount in situ hybridization of E8.0 embryos. (A to C) Nodal is expressed bilaterally in the LPM in the 9 out of 9 _Hippi_−/− embryos observed, with downregulation around the node region. (D, E) Lefty-2 is expressed bilaterally in the 9 out of 9 _Hippi_−/− embryos observed and (F, G) Pitx2 is expressed bilaterally in the 9 out of 9 _Hippi_−/− embryos observed. (H, I) Brachyury is expressed in the midline in both wildtype and _Hippi_−/− embryos. (J, K) Lefty-1 is not expressed in the midline of the 5 out of 5 _Hippi_−/− embryos observed. (L, M) At E7.5–7.75, Hnf3β is expressed in the node and the midline in both wildtype and _Hippi_−/− embryos. For all genes, heterozygotes always stain as the wildtype embryos.

Fig. 5

Node monocilia are absent on _Hippi_−/− embryos. Scanning electron microscopy of the node region of E8.0 embryos. (A) 700× Magnification of the Hippi+/+ node. (B) 11,000× Magnification of the Hippi+/+ node showing monocilia. (C) 700× Magnification of the _Hippi_−/− node showing normal gross structure. (D) 11,000× Magnification of the _Hippi_−/− node. No cilia are present. Scale bars represent 25 μm in panels A and C and 1.7 μm in panels B and D.

Fig. 6

The Sonic hedgehog pathway is downregulated in the neural tube of _Hippi_−/− embryos. Whole-mount in situ hybridization for (A, B) Hnf3β (arrowheads: red for gut, black for neural tube) (C to F) Ptc and (G, H) Shh. All three genes are downregulated in the neural tube of _Hippi_−/− embryos. Panels A to D are E8.5 embryos, panels E to H are E9.5.

Fig. 7

Neural tube ventralization is lost in _Hippi_−/− embryos. Whole-mount in situ hybridization sections with dotted lines highlighting neural tube borders. (A, B) Normal Shh expression in the floorplate (arrowheads) is lost in _Hippi_−/− embryos and remains only in the notochord (arrows). (C, D) Hnf3β expression is absent in _Hippi_−/− embryos floorplate (E, F) Pax6 lateral neural tube expression pattern observed in Hippi+/+ embryos extends to the ventral region (asterisk) in _Hippi_−/− tissue. (G, H) Isl-1 lateral ventral neural tube expression pattern (arrows) is lost in _Hippi_−/− embryos.

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Hippi is essential for node cilia assembly and Sonic hedgehog signaling - PubMed (original) (raw)