Tubby-like protein 3 (TULP3) regulates patterning in the mouse embryo through inhibition of Hedgehog signaling

Abstract

Tubby-like protein 3 (TULP3) is required for proper embryonic development in mice. Disruption of mouse Tulp3 results in morphological defects in the embryonic craniofacial regions, the spinal neural tube and the limbs. Here, we show that TULP3 functions as a novel negative regulator of Sonic hedgehog (Shh) signaling in the mouse. In Tulp3 mutants, ventral cell types in the lumbar neural tube, which acquire their identities in response to Shh signaling, are ectopically specified at the expense of dorsal cell types. Genetic epistasis experiments show that this ventralized phenotype occurs independently of Shh and the transmembrane protein Smoothened, but it is dependent on the transcription factor Gli2. The ventralized phenotype is also dependent on the kinesin II subunit Kif3A, which is required for intraflagellar transport and ciliogenesis. In addition, TULP3 is required for proper Shh-dependent limb patterning and for maintaining the correct balance between differentiation and proliferation in the neural tube. Finally, the localization of TULP3 to the tips of primary cilia raises the possibility that it regulates the Hedgehog pathway within this structure.

Figure 1.

The Tulp3 mutant phenotype. (A) E13.5 wild-type and Tulp3 mutants. The mutants exhibited neural tube closure defects (white arrowheads), eye defects (black arrow) and abnormal anterior outgrowth in the limbs (black arrowhead). (B) Alcian blue staining of E13.5 forelimbs revealed an additional anterior digit in the mutant limb (arrowhead). (C) E10.5 posterior neural tube sections from wild-type and Tulp3 embryos stained for Pax7, Dbx2, Msx1/2, Pax6, Nkx6.1, HB9, Olig2, FoxA2 and Brn2. Sections were counterstained with DAPI (blue). Scale bars are 50 µm.

Figure 2.

Expression of Shh and Shh pathway components. (A) In situ hybridization for Patched1, Gli1, Gli2 and Gli3 in E10.5 wild-type and Tulp3 mutant posterior neural tube sections. The Shh targets Ptch1 and Gli1 were ectopically expressed in dorsal regions, and Gli3 expression was dorsally restricted in Tulp3 mutants. (B) Immunostaining for Shh (red) and Pax6 (green) in wild-type and Tulp3 mutants (shown at levels of the hindlimb and flank). Dorsal restriction of Pax6 was seen at both levels of the mutant neural tube, whereas expansion of the Shh+ domain was seen only at more anterior levels (flank). Scale bars are 50 µm.

Figure 3.

Expression of Sox9, Hoxd12, Ptch1, Gli1 and Shh in limb buds. Expression in both forelimb and hindlimb buds is shown (anterior to the left). E12.5 Tulp3 mutant limbs show an increased number of digit rays as outlined by Sox9 expression, and disruption of A–P polarity as indicated by expansion of Hoxd12 expression into the anterior mesenchyme (arrowheads). In E11.5 mutant limb buds, both Ptch1 and Gli1 were expanded anteriorly in forelimb buds and ectopically expressed in a separate domain in the anterior mesenchyme of hindlimb buds (arrowheads). In mutant limb buds, weak Shh expression was detected in an ectopic anterior domain (arrowheads) separate from its normal domain in the posterior mesenchyme. In addition, Shh expression in the posterior domain of mutant forelimb buds was slightly expanded anteriorly (asterisk).

Figure 4.

Neural tube patterning in Shh/Tulp3 and Gli2/Tulp double mutants. Sections through the neural tubes (lumbar level) of E10.5 wild-type, Tulp3^−/−, Shh^−/−, Gli2^−/−, Shh^−/−Tulp3^−/− and Gli2^−/−Tulp3^−/− mutants stained for markers of dorsal/lateral (Pax7 and Pax6) and ventral (HB9, Nkx2.2, FoxA2) neural cell types, which are inhibited or induced by Shh signaling, respectively. Sections were counterstained with DAPI (blue).

Figure 5.

Subcellular localization of TULP3. (A) TULP3 staining (red) is seen at the tips of cilia labeled with α-acetylated alpha tubulin (green) in NIH3T3 fibroblasts and wild-type pMEFs, but not in pMEFs derived from embryos mutant for the tm1Jng1 or hhkr alleles of Tulp3. Cilia tips are indicated in top panels by yellow arrowheads. Nonspecific pericentrosomal staining is indicated by blue arrowheads. (B) Double labeling of γ-tubulin and TULP3 in wild-type and Tulp3 mutant pMEFs. Tip staining is indicated by a yellow arrowhead. (C) TULP3 immunostaining of unusually long cilia (left) or rare double cilia (right) from wild-type pMEFs. (D) TULP3 and Gli2 show partial colocalization at the tips of wild-type cilia (yellow arrowheads). (E) TULP3 (red) and acetylated alpha tubulin (green) immunostaining of a wild-type pMEF showing TULP3 staining in the nucleus (labeled with DAPI). (F) Western blotting of pMEF extracts reveals a truncated form of TULP3 (TULP3 mut) in the Tm1jng allele and very low levels of TULP3 in the hhkr allele. (G) TULP3 was found in nuclear and cytoplasmic factions from E9.5 embryos. HDAC2, which is enriched in nuclear fractions, and β-tubulin, which is limited to the cytoplasmic fractions, serve as controls. The slower migrating species (asterisk) in the nuclear fractions appears to be nonspecific, as it is also seen in Tulp3 mutant nuclear extracts (not shown). Scale bars are 1 µm in A–D and 3 µm in E.

Figure 6.

Neural tube patterning in Smo/Tulp3 and Kif3a/Tulp3 double mutants. Sections through posterior (lumbar) neural tubes of E9.5 wild-type, Tulp3^−/−, Smo^−/−, Kif3a^−/−, Smo^−/−Tulp3^−/− and Kif3a^−/−Tulp3^−/− mutants stained for Pax6, Nkx6.1, Olig2, Nkx2.2 and FoxA2.

Figure 7.

TULP3 is required for proper neural differentiation. (A) Neural tube sections from E10.5 wild-type, Tulp3^−/−, Gli2^−/− and Gli2^−/−Tulp3^−/− mutants stained for Tuj1 and Sox1. (B) Sox1+ progenitor and p27Kip1+ postmitotic zones in the ventral region of the E10.5 wild-type and Tulp3 mutant posterior neural tubes. Sox1 was weakly expressed by cells in the ventricular zone of Tulp3 mutants. (C) E10.5 wild-type and Tulp3 mutant sections stained with α-active caspase 3 and α-phosho-Histone H3 (pHH3) antibodies. (D) Percentage of pHH3+ cells in the ventral and dorsal halves of neural tube sections. Differences between genotypes for the ventral halves were significant (**P < 0.001, Student’s t-test). Differences for the dorsal halves were not significant (n.s., P > 0.2). (E) The number of apoptotic cells, marked by active caspase 3 staining, was comparable between genotypes. (F) Expression of Neurog2, Hes5, cyclin D1 and cyclin D2 in posterior neural tube sections from E9.5 wild-type and Tulp3 mutants. Scale bars are 50 µm in A, C and F, and 20 µm in B.