Craniofacial Ciliopathies Reveal Specific Requirements for GLI Proteins during Development of the Facial Midline

<div><p>Ciliopathies represent a broad class of disorders that affect multiple organ systems. The craniofacial complex is among those most severely affected when primary cilia are not functional. We previously reported that loss of primary cilia on cranial neural crest cells, via a conditional knockout of the intraflagellar transport protein KIF3a, resulted in midfacial widening due to a gain of Hedgehog (HH) activity. Here, we examine the molecular mechanism of how a loss of primary cilia can produce facial phenotypes associated with a gain of HH function. We show that loss of intraflagellar transport proteins (KIF3a or IFT88) caused aberrant GLI processing such that the amount of GLI3FL and GLI2FL was increased, thus skewing the ratio of GLIFL to GLIR in favor of the FL isoform. Genetic addition of GLI3R partially rescued the ciliopathic midfacial widening. Interestingly, despite several previous studies suggesting midfacial development relies heavily on GLI3R activity, the conditional loss of GLI3 alone did not reproduce the ciliopathic phenotype. Only the combined loss of both GLI2 and GLI3 was able to phenocopy the ciliopathic midfacial appearance. Our findings suggest that ciliopathic facial phenotypes are generated via loss of both GLI3R and GLI2R and that this pathology occurs via a de-repression mechanism. Furthermore, these studies suggest a novel role for GLI2R in craniofacial development.</p></div

Table of genotype and phenotype frequency for wild-type, <i>Gli2</i>^<i>fl/fl</i><i>;Gli3</i>^<i>fl/+</i> <i>Wnt1-Cre</i>, <i>Gli2</i>^<i>fl/+</i><i>;Gli3</i>^<i>fl/fl</i><i>;Wnt1-Cre and Gli2</i>^<i>fl/fl</i><i>;Gli3</i>^<i>fl/fl</i><i>;Wnt1-Cre</i>.

<p>Table of genotype and phenotype frequency for wild-type, <i>Gli2</i>^<i>fl/fl</i><i>;Gli3</i>^<i>fl/+</i> <i>Wnt1-Cre</i>, <i>Gli2</i>^<i>fl/+</i><i>;Gli3</i>^<i>fl/fl</i><i>;Wnt1-Cre and Gli2</i>^<i>fl/fl</i><i>;Gli3</i>^<i>fl/fl</i><i>;Wnt1-Cre</i>.</p

GLI isoforms binding to GLI binding regions is aberrant in ciliary mutants.

<p>(A) Schematic diagram of experimental design. (B) Pull-down of GLI3 protein using <i>Ptch</i> oligo containing GBR (n = 3). (C) Whole mount <i>in situ</i> hybridization of <i>Ptch</i> in wild-type, <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre</i> and <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre;Gli3</i>^{<i>Δ699/+</i>} embryos. (D) SUFU pull down by GLI3 in the cytosolic fraction of FNPs from wild-type and <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre</i> embryos. GAPDH was used as a loading control. (E) Nuclear fractionation of SUFU in wild-type and <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre</i> FNPs. Lamin and GAPDH were used as loading control for nuclear and cytosolic fraction, respectively. Scale bars in C = 1000 μm. Inset schematics of facial prominences in A, B, D, E indicate FNP, maxillary prominence (MXP) and mandibular prominence (MNP) were harvested for experiments in A and B, while only the FNP (red) was harvested for experiments in D and E.</p

Schematic of hypothesized model for GLI-dependent facial patterning.

<p>In (A) wild-type, (B) <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre</i>, (C) <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre;Gli3</i>^{<i>Δ699/+</i>}, (D) <i>Kif3a^fl/fl;Wnt1-Cre;ΔNGli2</i> and (E) <i>Gli2^fl/fl; Gli3^fl/fl; Wnt1-Cre</i> embryos, GLI binding on GBRs, overall ratio of GLIFL activator versus GLIR activity, and net GLI activity within the developing FNP are indicated. Red indicates GLIR activity, green indicates GLIA activity, white indicates loss of activity. mxp, maxillary prominence. GLI3R* = GLI3^Δ699R. GLI2A*= ΔNGLI2.</p

Table of genotype and phenotype frequency for wild-type, <i>Ift88</i>^<i>fl/fl</i><i>;Wnt1-Cre and Ift88</i>^<i>fl/fl</i><i>;Wnt1-Cre;Gli3</i>^{<i>Δ699/+</i>}.

<p>Table of genotype and phenotype frequency for wild-type, <i>Ift88</i>^<i>fl/fl</i><i>;Wnt1-Cre and Ift88</i>^<i>fl/fl</i><i>;Wnt1-Cre;Gli3</i>^{<i>Δ699/+</i>}.</p

Loss of anterograde intraflagellar transport proteins in NCCs results in severe midfacial widening.

<p>(A-C) Frontal view, (D-F) palatal view and (G-I) Safranin-O staining of transverse sections from e15.5 (A, D, G) wild-type, (B, E, H) <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre</i> and (C, F, I) <i>Ift88</i>^<i>fl/fl</i><i>;Wnt1-Cre</i> heads. <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre</i> and <i>Ift88</i>^<i>fl/fl</i><i>;Wnt1-Cre</i> have severe facial widening (B, C; dotted black lines), bilateral cleft of the secondary palate (E, F; black arrows) and duplication of the nasal septum (H, I; dotted white lines). (J) Quantitative measurements of the distance between nasal pits on e13.5 embryos show midfacial widening in both <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre</i> (n = 8) and <i>Ift88</i>^<i>fl/fl</i><i>;Wnt1-Cre</i> (n = 3) is significant, relative to wild-type embryos (n = 12). Statistical analysis was performed by student <i>t</i>-test (<i>*P<</i>0.05). Scale bars = 1000 μm.</p

Table of genotype and phenotype frequency for wild-type, <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre and Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre;Gli3</i>^{<i>Δ699/+</i>}.

<p>Table of genotype and phenotype frequency for wild-type, <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre and Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre;Gli3</i>^{<i>Δ699/+</i>}.</p

Genetic addition of GLI3R partially rescues <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre</i> phenotype.

<p>(A-C) Frontal view, (D-F) palatal view and (G-I) Safranin-O staining of transverse sections from e15.5 (A, D, G) wild-type, (B, E, H) <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre</i> and (C, F, I) <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre;Gli3</i>^{<i>Δ699/+</i>} heads. <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre;Gli3</i>^{<i>Δ699/+</i>} showed a significant reduction in the internasal width relative to <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre</i> (compare B, C; dotted black line) and reduction in patency of bilateral cleft secondary palate (compare E, F; dotted black lines). The duplicated nasal septum of the <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre</i> was restored to a singular cartilaginous element in <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre;Gli3</i>^{<i>Δ699/+</i>} (compare H and I; dotted yellow lines). (J) Western blot analysis of GLI3 expression in wild-type, <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre</i> and <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre;Gli3</i>^{<i>Δ699/+</i>} facial prominences. Asterisk denotes expression of GLI3^Δ699R. GAPDH was used as the loading control. (K) Quantitative analysis of Western blot in (J) by ImageJ (n = 3). GLI3FL to GLI3R ratio is significantly increased in <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre</i> compared to wild type. However, the ratio is significantly reduced in <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre;Gli3</i>^{<i>Δ699/+</i>} compared to <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre</i>. Statistical analysis was performed by student <i>t</i>-test (<i>*P<</i>0.01), with three separate Western blots. (L-N) Phospho-Histone H3 (pHH3) staining of FNP mesenchyme in e11.5 embryos. (O) Quantitative measurement of pHH3 positive cells in the FNP of wild-type (n = 3; 7 consecutive, 8μm sections), <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre</i> (n = 3; 7 consecutive 8μm sections) and <i>Kif3a</i>^<i>fl/fl</i><i>;Wnt1-Cre;Gli3</i>^{<i>Δ699/+</i>} (n = 3; 12 consecutive, 8μm sections) embryos. Statistical analysis was performed by student <i>t</i>-test (<i>*P</i><0.05). Scale bars = 1000 μm. Inset schematic of facial prominences in J indicate FNP, maxillary prominence (MXP) and mandibular prominence (MNP) (red) were harvested for the experiment.</p

SLO BK Potassium Channels Couple Gap Junctions to Inhibition of Calcium Signaling in Olfactory Neuron Diversification

<div><p>The <i>C</i>. <i>elegans</i> AWC olfactory neuron pair communicates to specify asymmetric subtypes AWC^OFF and AWC^ON in a stochastic manner. Intercellular communication between AWC and other neurons in a transient NSY-5 gap junction network antagonizes voltage-activated calcium channels, UNC-2 (CaV2) and EGL-19 (CaV1), in the AWC^ON cell, but how calcium signaling is downregulated by NSY-5 is only partly understood. Here, we show that voltage- and calcium-activated SLO BK potassium channels mediate gap junction signaling to inhibit calcium pathways for asymmetric AWC differentiation. Activation of vertebrate SLO-1 channels causes transient membrane hyperpolarization, which makes it an important negative feedback system for calcium entry through voltage-activated calcium channels. Consistent with the physiological roles of SLO-1, our genetic results suggest that <i>slo-1</i> BK channels act downstream of NSY-5 gap junctions to inhibit calcium channel-mediated signaling in the specification of AWC^ON. We also show for the first time that <i>slo-2</i> BK channels are important for AWC asymmetry and act redundantly with <i>slo-1</i> to inhibit calcium signaling. In addition, <i>nsy-5</i>-dependent asymmetric expression of <i>slo-1</i> and <i>slo-2</i> in the AWC^ON neuron is necessary and sufficient for AWC asymmetry. SLO-1 and SLO-2 localize close to UNC-2 and EGL-19 in AWC, suggesting a role of possible functional coupling between SLO BK channels and voltage-activated calcium channels in AWC asymmetry. Furthermore, <i>slo-1</i> and <i>slo-2</i> regulate the localization of synaptic markers, UNC-2 and RAB-3, in AWC neurons to control AWC asymmetry. We also identify the requirement of <i>bkip-1</i>, which encodes a previously identified auxiliary subunit of SLO-1, for <i>slo-1</i> and <i>slo-2</i> function in AWC asymmetry. Together, these results provide an unprecedented molecular link between gap junctions and calcium pathways for terminal differentiation of olfactory neurons.</p></div