Tectonic, a novel regulator of the Hedgehog pathway required for both activation and inhibition

Abstract

We report the identification of a novel protein that participates in Hedgehog-mediated patterning of the neural tube. This protein, named Tectonic, is the founding member of a previously undescribed family of evolutionarily conserved secreted and transmembrane proteins. During neural tube development, mouse Tectonic is required for formation of the most ventral cell types and for full Hedgehog (Hh) pathway activation. Epistasis analyses reveal that Tectonic modulates Hh signal transduction downstream of Smoothened (Smo) and Rab23. Interestingly, characterization of Tectonic Shh and Tectonic Smo double mutants indicates that Tectonic plays an additional role in repressing Hh pathway activity.

Figure 1.

Tectonic is expressed in domains of Hh signaling, and is essential for embryonic development. (A) The mouse Tectonic gene is comprised of 13 exons on chromosome 5. The gene trap consists of a strong splice acceptor (SA) followed by an ORF encoding a transmembrane domain (TM) and βGEO. The gene trap also includes an IRES and PLAP coding sequence followed by a polyadenylation sequence (pA). (B) RT–PCR analysis of Tectonic gene expression in E11.5 wild-type, heterozygous, and homozygous mutant embryos. Primers are specific for the Tectonic coding sequence 3′ to the gene trap (Tect), the βGEO transcript, and G3PD. Included is a –RT control using G3PD-specific primers. (C) Northern blot analysis of Tectonic and βGEO expression in wild-type, heterozygous, and mutant embryos. (D–F) β-Galactosidase staining of Tectonic heterozygotes. (D) Lateral and distal views of late headfold stage embryos, demonstrating restricted Tectonic expression in the node (arrow). (E) Tectonic is expressed in the ventral epithelium of the node, as revealed in a transverse section through the node of a six-somite stage embryo. (F) At E9.5, Tectonic is expressed in the neural tube, gut epithelium (arrow), notochord, and somites (arrowhead), as seen both in whole-mount and transverse section. (G) E10.5 Tectonic mutants exhibit reduced telencephalon size and holoprosencephaly (arrow).

Figure 2.

Tectonic is required for Hh-mediated patterning of the ventral neural tube. (A) Hematoxylin-and-eosin-stained transverse sections of E9.5 embryos. Tectonic mutants lack a histologically distinct floorplate (arrow). (B–H) In situ hybridization of E9.5 whole-mount embryos (F), or transverse sections of E9.5 (B) or E10.5 (C–E,G,H) embryos. (B) Shh, a marker of the floorplate, is not expressed in the Tectonic mutant neural tube. However, Tectonic mutants express Shh normally in the notochord and gut epithelium. (C) Similarly, Sim1, a marker of V3 interneurons, is not expressed in the Tectonic mutant neural tube. (D) Expression of Dbx1, a marker of V0 interneuron precursors, is expressed in Tectonic mutants. (E) Expression of Irx3, a gene normally expressed dorsal to the pMN domain, is expanded almost to the ventral midline of Tectonic mutants. (F) Gli1, a general transcriptional target of Hh signaling, is broadly diminished in Tectonic mutants. (G) Similarly, Gli1 expression is reduced in the neural tubes of Tectonic mutants. (H) Ptch, another general Hh transcriptional target, is also down-regulated in the Tectonic mutant neural tube.

Figure 3.

Tectonic is epistatic to Ptch and Rab23. (A) Lateral views of E9.5 littermates. Ptch mutants display a characteristic open neural tube and defective turning whereas normal turning is largely restored in Tectonic Ptch double mutants. (B–D) Transverse sections of E9.5 embryos stained for expression of Pax6 in red and, in green, FoxA2 (B), Nkx2.2 (C), or Islet1/2 (D). Nuclei are visualized with DAPI staining (blue). (B) Tectonic mutants lack floorplate expression of FoxA2 and show expanded Pax6 expression. Conversely, Ptch mutants display expanded FoxA2 expression and reduced Pax6 expression. Tectonic Ptch double mutants closely resemble Tectonic single mutants. (C) Tectonic mutants lack Nkx2.2 expression, a marker of the p3 domain, whereas Ptch mutants display expanded Nkx2.2 expression. Tectonic Ptch double mutants exhibit a loss of Nkx2.2 expression identical to that of Tectonic single mutants. (D) Motor neuron expression of Islet1/2 is reduced in most (n = 4/5) Tectonic mutants, expanded in Ptch mutants, and reduced in Tectonic Ptch double mutants. (E,F) Transverse sections of E10.5 embryos. (E) Similar to Ptch mutants, Rab23 mutants exhibit an expansion of FoxA2 (green) and a dorsal shift in expression of Olig2, a marker of motor neuron precursors (red). In contrast, Tectonic Rab23 double mutants resemble Tectonic mutants. (F) Expression of the dorsal markers Pax3 and Pax6 is shifted dorsally in Rab23 mutants, but not in Tectonic Rab23 double mutants. (G) Gli1 in situ hybridization of transverse sections of E9.5 neural tubes. Whereas Gli1 is normally expressed in a dorsoventral gradient, in Ptch mutants, Gli1 is widely up-regulated and expressed ectopically in the dorsal neural tube. In Tectonic Ptch double mutants, Gli1 is expressed at a uniform low level throughout the dorsoventral extent of the neural tube.

Figure 4.

In addition to its role in mediating high levels of Hh signaling, Tectonic functions to repress low levels of Hh pathway activation. (A) Lateral view of E10.5 embryos. Shh mutants are one-third the size of littermates and show severely diminished forebrains. Tectonic Shh double mutants are larger than Shh single mutants and develop markedly larger telencephalons (arrow). (B–D) In situ hybridization of transverse sections of E10.5 embryos. (B) In Shh mutants, Dbx1 expression, a marker of the p0 domain, is reduced to a very few cells at the ventral midline. Tectonic Shh double mutants exhibit increased Dbx1 expression relative to Shh single mutants. (C) Dbx2, a marker of the p0 and p1 domains, is markedly reduced or not expressed in Shh mutants. In contrast, Tectonic Shh double mutants express Dbx2 robustly at the ventral midline. (D) Tectonic Shh double mutants display higher levels of Ptch expression than do Shh single mutants. (E) Levels of Hh pathway activity within the developing ventral neural tube are translated into distinct fates, including floorplate (FP) and five neural precursor domains (p3–p0) at defined dorsoventral positions. In Tectonic mutants, neural fates that require the highest levels of Hh signaling are lost, represented as a rightward shift in the Hh pathway activity curve. Disruption of Shh function causes severe reduction of the p0 and p1 domains and loss of more ventral fates. In contrast, loss of both Shh and Tectonic function results in increased Hh pathway activity and restored p0 and p1 development, revealing an inhibitory role for Tectonic in the Hh pathway.