Lack of centrioles and primary cilia in <i>STIL^−/−</i> mouse embryos

<div><p>Although most animal cells contain centrosomes, consisting of a pair of centrioles, their precise contribution to cell division and embryonic development is unclear. Genetic ablation of <i>STIL</i>, an essential component of the centriole replication machinery in mammalian cells, causes embryonic lethality in mice around mid gestation associated with defective Hedgehog signaling. Here, we describe, by focused ion beam scanning electron microscopy, that <i>STIL^−/−</i> mouse embryos do not contain centrioles or primary cilia, suggesting that these organelles are not essential for mammalian development until mid gestation. We further show that the lack of primary cilia explains the absence of Hedgehog signaling in <i>STIL^−/−</i> cells. Exogenous re-expression of STIL or STIL microcephaly mutants compatible with human survival, induced non-templated, <i>de novo</i> generation of centrioles in <i>STIL^−/−</i> cells. Thus, while the abscence of centrioles is compatible with mammalian gastrulation, lack of centrioles and primary cilia impairs Hedgehog signaling and further embryonic development.</p></div

GCS in hypothalamic neurons regulates neuronal leptin signaling at the plasma membrane.

<p>(A) Stat3 phosphorylation was markedly decreased in Arc sections of <i>Ugcg</i>^{f/f//CamKCreERT2} mice in response to peripheral leptin (5 mg/kg; 45 min) 6 wk p.i. (<i>n</i> = 16–33). Three independent animal groups were analyzed. (B) Serum leptin levels were unchanged 3 wk p.i. and increased prominently 7 wk p.i. in <i>Ugcg</i>^{f/f//CamKCreERT2} mice, reflecting increased body fat mass (<i>n</i> = 12–14). (C) mRNA expression analysis for suppressor of cytokine signaling 3 (SOCS-3) expression in Arc-enriched hypothalamic tissue was carried out 2, 6, and 9 wk p.i. <i>Socs-3</i> expression normalized to the housekeeping gene tubulin was unaltered (<i>n</i> = 3–5). (D) mRNA expression analysis for the long form of the leptin receptor, <i>Leprb</i>, in mediobasal hypothalamus was carried out 6 wk p.i. <i>Leprb</i> expression normalized to the housekeeping gene tubulin was unaltered at that time point (<i>n</i> = 4–5). (E) Immortalized mouse hypothalamic cells (N-41 cells) were analyzed for cell surface expression of ObR. Non-detergent-treated cells were fixed and simultaneously stained with two ObR antibodies. A proximity ligation assay (PLA) indicated quantifiable and unchanged ObR expression on the surface of controls and cells treated with the specific GCS inhibitor NB-DNJ (<i>n</i> = 41–47 cells). PLA principle is depicted on the right side. (F–G) N-41 cells were incubated with either saline or 100 ng/ml leptin (10 min). Close interactions between GCS-derived neuronal gangliosides GD1a/ObR (F) and GM1/ObR (G) were detected by PLA. Leptin treatment dynamically increased the GD1a/ObR and GM1/ObR PLA spots per cell (<i>n</i> = 48–67 cells). (H) Extracts from saline- and leptin-treated N-41 cells were immunoprecipitated with an ObR antibody, lipids were extracted, and GD1a and GM1 were visualized by immune overlay TLC. GD1a and GM1 co-immunoprecipitated (Co-IP) with ObR, which tended to be stronger in leptin-treated cells. Addition of a blocking peptide almost totally abolished ganglioside signals. Gangliosides GD1b and GT1b, expressed in mouse brain tissue, were not co-precipitated with ObR from hypothalamic tissue of <i>Ugcg</i>^f/f mice (5 mg/kg leptin, 45 min). (I) Co-IP showed significantly decreased leptin-induced complex formation between ObR and Jak in NB-DNJ-treated N-41 cells (<i>n</i> = 4). (J) Sustainable Jak phosphorylation could be induced in N-41 cells after 15 min of leptin treatment (0.5 µg/ml). NB-DNJ-treated cells showed a markedly delayed response to leptin. (K) Thirty minutes after leptin treatment, Jak phosphorylation was decreased in NB-DNJ-treated cells (<i>n</i> = 4). *<i>p</i>≤0.05; ***<i>p</i>≤0.001. Means ± SEM.</p

rAAV-mediated <i>Ugcg</i> gene delivery to the hypothalamic Arc ameliorates obesity and hyperleptinemia in <i>Ugcg</i>^{f/f//CamKCreERT2} mice.

<p>(A) Double immunofluorescence showed that Cre activity, indicated by beta galactosidase staining (b-gal), was targeted to Arc neurons expressing the long form of the ObR, as indicated by PStat3 staining in leptin-injected <i>R26R/Ugcg</i>^{f/+//CamKCreERT2} mice (5 mg/kg leptin, 120 min). (B) Stereotactic delivery of rAA viruses encoding <i>Ugcg</i> and <i>lacZ</i> to the Arc of <i>Ugcg</i>^{f/f//CamKCreERT2} mice resulted in a significant amelioration in body weight increase compared to rAAV-Empty/lacZ-injected <i>Ugcg</i>^{f/f//CamKCreERT2} mice (<i>n</i> = 6–8). (C) Serum leptin tended to be lower in rAAV-Ugcg/lacZ-injected <i>Ugcg</i>^{f/f//CamKCreERT2} mice 8 wk p.i. (<i>n</i> = 6–8). (D–F) Targeting of rAAV Ugcg/lacZ- and rAAV Empty/lacZ-injected animals that were included in the analyses. At the end of the experiments, brains were removed and stained for X-Gal to indicate vector delivery. Red marks depict exemplarily areas of strong X-Gal staining in animals considered as Arc targeted. Depicted are areas between bregma −1.9 (D), bregma −2.1 (E), and bregma −2.3 (F). (G) Restored ganglioside biosynthesis in the Arc of rAAV-Ugcg-injected <i>Ugcg</i>^{f/f//CamKCreERT2} mice, as shown by GD1a immunofluorescence 8 wk p.i. Scale bar: 18 µm. *<i>p</i>≤0.05. Means ± SEM.</p

POMC and NPY neurons of <i>Ugcg</i>^{f/f//CamKCreERT2} mice are less responsive to leptin.

<p>(A) Leptin engages POMC neurons in the Arc of control (<i>Ugcg</i>^f/f) mice and <i>Ugcg</i>^{f/f//CamKCreERT2} mice 1–2 wk p.i., as indicated by elevated c-Fos. This response was decreased in Ugcg^{f/f//CamKCreERT2} mice 6 wk p.i. (B) Elevated leptin-induced PStat3 levels in POMC neurons of <i>Ugcg</i>^f/f mice and <i>Ugcg</i>^{f/f///CamKCreERT2} mice 1–2 wk p.i. This response was blunted in Ugcg^{f/f//CamKCreERT2} mice 6 wk p.i. (C) Leptin slightly decreased the activity of NPY neurons in <i>Ugcg</i>^f/f mice and <i>Ugcg</i>^{f/f///CamKCreERT2} mice 1–2 wk p.i. This was not detected in <i>Ugcg</i>^{f/f///CamKCreERT2} mice 6 wk p.i. (D) Unlike 1–2 wk p.i., leptin did not elevate PStat3 in NPY neurons of <i>Ugcg</i>^{f/f///CamKCreERT2} mice 6 wk p.i. Datasets for each time point were acquired individually, and quantification contains normalized data from two (1–2 wk p.i.; <i>n</i> = 4–11) or three (6 wk p.i.; <i>n</i> = 18–27) independent animal groups. Immunofluorescence and image acquisition for each dataset (treated and untreated controls and knockouts) were performed simultaneously. Scale bar: 20 µm; *<i>p</i>≤0.05; **<i>p</i>≤0.01; ***<i>p</i>≤0.001. Means ± SEM.</p

<i>Ugcg</i>^{f/f//CamKCreERT2} mice develop progressive obesity.

<p>Both female (A) and male (B) <i>Ugcg</i>^{f/f//CamKCreERT2} mice showed a progressive increase in body weight after tamoxifen induction (<i>n</i> = 6–9). (C) <i>Ugcg</i>^{f/f//CamKCreERT2} mice were larger than <i>Ugcg</i>^f/f littermates (16 wk p.i.), and body fat mass was prominently elevated. (D) Enlarged adipocytes in <i>Ugcg</i>^{f/f//CamKCreERT2} mice 9 wk p.i. (E) Increased weight of epigonadal WAT 9 wk p.i. in <i>Ugcg</i>^{f/f//CamKCreERT2} mice (<i>n</i> = 4–5). (F) NMR analysis revealed significant and progressive accumulation of body fat mass in <i>Ugcg</i>^{f/f//CamKCreERT2} mice (<i>n</i> = 9–10). *<i>p</i>≤0.05; **<i>p</i>≤0.01;***<i>p</i>≤0.001. Means ± SEM.</p

Hypothalamic neurons of <i>Ugcg</i>^{f/f//CamKCreERT2} mice are less responsive to peripheral leptin.

<p>(A–C) Brains of leptin-stimulated mice were analyzed for neuronal activity indicated by c-Fos immunofluorescence. Detailed pictures in the upper lane indicate regions of the Arc that are outlined in overview pictures (frames). Arrowheads mark c-Fos-positive neurons located in the VMH. Axis indicators were included indicating the medial (m) and ventral (v) axes. (A) <i>Ugcg</i>^{f/f//CamKCreERT2} mice showed leptin-induced neuronal activation comparable to <i>Ugcg</i>^f/f mice in the Arc 1–2 wk p.i. (B) Leptin response in the Arc was decreased in nonobese <i>Ugcg</i>^{f/f//CamKCreERT2} mice weight-matched to controls 3–4 weeks p.i. (C) Decreased c-Fos staining in the Arc was also observed in obese leptin-induced <i>Ugcg</i>^{f/f//CamKCreERT2} mice 6 wk p.i. The percentage of c-Fos-positive neurons per Arc section was depicted as values normalized to saline-injected <i>Ugcg</i>^f/f mice (<i>n</i> = 14–22 sections). Depicted sections are located between bregma levels −1.5 to −1.8. Quantification contains data from bregma levels −1.4 to −2.3. (D–F) <i>Ugcg</i>^{f/f//CamKCreERT2} mice retained leptin responsiveness in the VMH, as elevated c-Fos after leptin stimulation indicated (<i>n</i> = 8–20 sections). Quantification contains data from bregma levels −1.4 to −2.0. Datasets for each time point were acquired individually. Two (1–2 and 3–4 wk) or three (6 wk) independent animal groups were analyzed. Immunofluorescence and image acquisition for each dataset (treated and untreated controls and knockouts) were performed simultaneously. Scale bar: 75 µm; 3^rdv, 3^rd ventricle; *<i>p</i>≤0.05; **<i>p</i>≤0.01; ***<i>p</i>≤0.001. Means ± SEM.</p

Normal ultrastructure in ganglioside-depleted neurons.

<p>(A) Major pathway for biosynthesis of GSL including gangliosides in the brain. (B) X-Gal staining in brains of <i>R26R/Ugcg</i>^{f/+//CamKCreERT2} reporter mice revealed strong Cre activity in the hypothalamic Arc. GD1a immunofluorescence visualized ganglioside depletion in the Arc of <i>Ugcg</i>^{f/f//CamKCreERT2} mice 6 wk p.i. Scale bar: 75 µm. (C) Ceramide levels were not significantly altered in hippocampus of <i>Ugcg</i>^{f/f//CamKCreERT2} mice. Quantification from densitometry analysis of TLC results is depicted (<i>n</i> = 3). (D) Neurons in the Arc of <i>Ugcg</i>^{f/f//CamKCreERT2} mice showed normal ultrastructural morphology of plasma membrane (pm), nucleus (N), mitochondria (M), endoplasmic reticulum (ER), golgi (G), projections (P), and myelin sheaths (my) 6 and 12 wk p.i. Scale bar: 2 µm. 3^rdv, third ventricle.</p

Proposed model for GCS-derived ganglioside GD1a and GM1 regulation of hypothalamic leptin signaling and energy homeostasis.

<p>(A) GCS-derived gangliosides form complexes with ObR, thereby facilitating leptin-dependent Jak and Stat3 phosphorylation, and formation of PIP3. These pathways are crucial contributors to regulation of energy homeostasis. (B) In obese <i>Ugcg</i>^{f/f//CamKCreERT2} mice, ObR signal transduction is abolished in GCS-depleted neurons.</p