Figure 1.
Opposing roles of Kif7 and Sufu in FP development.
(A–D) Nkx2.2 and Olig2 immunofluorescence labeling shows increasing neural tube ventralization in E9.5 Kif7−/−, Sufu−/− and Kif7−/−;Sufu−/− embryos. (E–H') Shh and Foxa2 expression contrasts the FP deficiencies Kif7−/− embryos to ectopic FP induction in Sufu−/− and Kif7−/−;Sufu−/− embryos. Kif7−/−;Sufu−/− and Sufu−/− embryos exhibit similar FP defects, indicating that Kif7 is epistatic to Sufu in FP induction. Scale bar, 25 µm. (I–L) In situ hybridization analysis of Shh RNA expression verifies FP phenotypes. (M) Graphs indicate the number of Olig2+ pMN, Nkx2.2+ p3 and Shh+/Foxa2+ FP cells, represented as the mean ± SEM, n≥4, n.s., not significant.
Figure 2.
Kif7 promotes ectopic FP induction by restricting Sufu in Ptch1 mutants.
(A–D) Nkx2.2 and Olig2 expression shows an increased number of Olig2+ pMN cells by Kif7 inactivation in the Ptch1−/− background. (E–H') Shh and Foxa2 expression shows that Kif7 inactivation abolishes ectopic FP induction in Ptch1−/− embryos, which is restored by reducing one gene dosage of Sufu. Arrowheads indicate the dorsal limit of the neural tube. Scale bar, 25 µm. (I–L) In situ hybridization analysis of Shh RNA expression from the FP in anterior spinal cord sections. (M) Graphs indicate the number of FP cells, represented as the mean ± SEM, n≥5.
Figure 3.
Kif7 alleviates Sufu inhibition of Gli2-dependent FP development.
(A–E) Nkx2.2 and Olig2 expression shows that Kif7 inactivation in Gli2+/− mice results in a ventral shift of the Olig2+ pMN and Nkx2.2+ p3 domains, and reduced formation of p3 cells. The dorsalization and reduced p3 cell specification of Kif7−/−;Gli2+/− embryos is restored to that of Kif7−/− embryos by reducing Sufu gene dosage. (F–J') Shh and Foxa2 expression shows Kif7 inactivation in Gli2+/− embryos leads to FP defects, which is rescued in Kif7−/−;Gli2+/−;Sufu+/− embryos by the reduction in Sufu gene dosage. Scale bar, 25 µm. (K) Graphs indicate the number of FP cells, represented as the mean ± SEM, n = 3. (L–O) Whole mount in situ hybridization of Shh RNA expression from the notochord (Nc) and FP showing that FP defects in Kif7−/−;Gli2+/− embryos are rescued by the reduced Sufu gene dosage of Kif7−/−;Gli2+/−;Sufu+/− embryos. (P) Model of Kif7 and Sufu regulation of Gli2 during FP development: Kif7 restricts the potent inhibitory action of Sufu on Gli2 to promote FP induction.
Figure 4.
Diverse ventral fates form despite the absence of graded Shh signaling when Sufu function is abolished.
(A–D) Nkx2.2 and Pax6 expression shows that Kif7 or Sufu inactivation rescues dorsalization of the Smo−/− neural tube. (E–H) Nkx2.2 and Olig2 expression shows that in Smo−/− embryos, Kif7 inactivation rescues Olig2+ pMN cell specification, but rarely that of Nkx2.2+ p3 cells, while Sufu inactivation leads to robust induction of both cell fates. (I–L') Shh and Foxa2 expression shows that only Sufu inactivation, but not that of Kif7, rescues FP development in Smo−/− embryos. Scale bar, 25 µm. (M) Graphs indicate the number of pMN, p3 and FP cells, represented as the mean ± SEM, n≥4. (N) Western blot analysis shows reduced Gli2 expression in Sufu−/− embryos are restored when Smo is simultaneously inactivated; little to no Gli383kD repressor is expressed in Sufu−/− and Smo−/−;Sufu−/− embryos.
Figure 5.
A model of Kif7 and Sufu regulation of graded Gli activity in ventral cell fate specification.
The concerted actions of Kif7 and Sufu alter the Gli activator to repressor ratio to regulate intracellular Gli activity for the specification of ventral cell fates (pMN, p3 and FP) from naïve neuroepithelial precursor cells. As Shh signaling increases Smo activity, Gli repressor formation and the inhibition of activators become less efficient as denoted (i–iii), while robust Smo activation results in Kif7 antagonizing the inhibition of Gli activators by Sufu for p3 and FP induction (iv).