Mammalian Neurogenesis Requires Treacle-Plk1 for Precise Control of Spindle Orientation, Mitotic Progression, and Maintenance of Neural Progenitor Cells

The cerebral cortex is a specialized region of the brain that processes cognitive, motor, somatosensory, auditory, and visual functions. Its characteristic architecture and size is dependent upon the number of neurons generated during embryogenesis and has been postulated to be governed by symmetric versus asymmetric cell divisions, which mediate the balance between progenitor cell maintenance and neuron differentiation, respectively. The mechanistic importance of spindle orientation remains controversial, hence there is considerable interest in understanding how neural progenitor cell mitosis is controlled during neurogenesis. We discovered that Treacle, which is encoded by the Tcof1 gene, is a novel centrosome- and kinetochore-associated protein that is critical for spindle fidelity and mitotic progression. Tcof1/Treacle loss-of-function disrupts spindle orientation and cell cycle progression, which perturbs the maintenance, proliferation, and localization of neural progenitors during cortical neurogenesis. Consistent with this, Tcof1+/− mice exhibit reduced brain size as a consequence of defects in neural progenitor maintenance. We determined that Treacle elicits its effect via a direct interaction with Polo-like kinase1 (Plk1), and furthermore we discovered novel in vivo roles for Plk1 in governing mitotic progression and spindle orientation in the developing mammalian cortex. Increased asymmetric cell division, however, did not promote increased neuronal differentiation. Collectively our research has therefore identified Treacle and Plk1 as novel in vivo regulators of spindle fidelity, mitotic progression, and proliferation in the maintenance and localization of neural progenitor cells. Together, Treacle and Plk1 are critically required for proper cortical neurogenesis, which has important implications in the regulation of mammalian brain size and the pathogenesis of congenital neurodevelopmental disorders such as microcephaly

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Prevention of Treacher Collins syndrome craniofacial anomalies in mouse models via maternal antioxidant supplementation

Craniofacial anomalies account for approximately one-third of all birth defects and are a significant cause of infant mortality. Since the majority of the bones, cartilage and connective tissues that comprise the head and face are derived from a multipotent migratory progenitor cell population called the neural crest, craniofacial disorders are typically attributed to defects in neural crest cell development. Treacher Collins syndrome (TCS) is a disorder of craniofacial development and although TCS arises primarily through autosomal dominant mutations in TCOF1, no clear genotype–phenotype correlation has been documented. Here we show that Tcof1 haploinsufficiency results in oxidative stress-induced DNA damage and neuroepithelial cell death. Consistent with this discovery, maternal treatment with antioxidants minimizes cell death in the neuroepithelium and substantially ameliorates or prevents the pathogenesis of craniofacial anomalies in Tcof1(+/−) mice. Thus maternal antioxidant dietary supplementation may provide an avenue for protection against the pathogenesis of TCS and similar neurocristopathies

Dynamic localization of Treacle in mitotic cells.

<p>(A) Immunofluorescence images of wild-type forebrain at E11.5 detecting Treacle (red), the centrosome (γ-tubulin; green) and nuclei (DAPI; blue). Arrowheads and arrows indicate Treacle localization at the centrosome in interphase cells and mitotic cells respectively. (B) Immunofluorescence images of HeLa cells showing the dynamic localization of Treacle (green) during mitosis, particularly at the centrosomes (arrowheads) and kinetochore in prophase, prometaphase and metaphase cells as well as at the midzone during anaphase (arrow) and at the midbody (arrow) in telophase. The microtubular networks are detected by immunostaining with an anti-α-tubulin antibody. (C) Treacle co-localizes with the kinetochore marker, CENP-E. Scale Bars: A and B, 10 µm.</p

Plk1 co-operates in controlling mitotic progression and mitotic spindle orientation.

<p>(A) Expression of <i>Plk1</i> mRNA detected by <i>in situ</i> hybridization on coronal sections of E10.5–E16.5 embryos. (B) E11.0 mouse embryos were cultured with 50–200 nM BI 2536 for 16 hours. Mitotic cells were analyzed by immunostaining with phospho-Histone H3 (green) and Centrin (red). (C) The number of total pH 3-positive cells and percentage of surface and non-surface mitotic cells were quantified. The nuclei were stained with DAPI (blue). Scale Bars: A, 200 µm; B, 50 µm.</p

Abnormal brain development resulting from Treacle deficiency.

<p>(A) Neurons in E12.5–16.5 embryonic forebrains were visualized by immunofluorescence with anti-MAP2 antibody (green). (B) Quantification of neurons in forebrain. MAP2-positive cells in the wild-type and <i>Tcof1</i>^+/− brain were counted in a unit section of 100 µm width. (C) Immunostaining of cortical layers with anti-Reelin (layer I), anti-Cux2 (layer II, III and IV; asterisk) and anti-FoxP2 (layer V and VI) antibodies and <i>in situ</i> hybridization for <i>Cux2</i> on coronal sections of E18.5 wild-type and <i>Tcof1</i>^+/− mice. To observe the cortical cortex, MAP2-positive neurons (red) are co-stained with Cux2 and FoxP2 (green). Scale Bars: A, 25 µm; C, 100 µm.</p

Essential function of Treacle in mitotic spindle formation and mitotic progression.

<p>(A) PCR analysis of efficacy of <i>TCOF1</i> knockdown in HeLa cells 48 hours after control (siGL2) or <i>TCOF1</i> (si TCOF1) siRNA transfection. (B) Immunostaining of mitotic HeLa cells in control knock-down (siGL2) and <i>TCOF1</i> knock-down (si TCOF1) cultures analyzed with anti-α-tubulin (red) and anti-Treacle (green) antibodies. (C) Mitotic cells in <i>TCOF1</i>knock-down (siTCOF1) and control (siGL2) cultures immunostained with anti-α-tubulin (red) and anti-centrin (green) antibodies. Arrowheads indicate abnormal mitotic spindle and chromosome alignment in <i>TCOF1</i> knock-down cultures. (D) Graph depicting the frequency of mitotic cells labeled via immunostaining with a pH 3 antibody at 6–16 hours post siGL2 or siTCOF1 transfection and the marked delay in mitotic exit exhibited by <i>TCOF1</i> knock-down cells. Scale Bars: B and C, 5 µm.</p

<i>Tcof1</i> heterozygous mutant mice show small brain.

<p>(A) Dorsal view of wild-type (+/+) and <i>Tcof1</i> heterozygous mutant (+/−) brains of P14 mice. (B) Brain weight per body weight and body weight of wild-type (n = 6) and <i>Tcof1</i>^+/− mice (n = 6). (C) Coronal sections of wild-type (+/+) and <i>Tcof1</i> heterozygous (+/−) cerebrum stained with HE. The brain is much smaller, but tissue architecture and morphology is normal. Scale Bars: C, 200 µm.</p

Neural progenitor cells in <i>Tcof1</i>^+/− embryos exhibit mitotic defects.

<p>(A) Immunostaining of E10.5–E16.5 embryonic forebrains using a pH 3 antibody (red). Arrowheads indicate scattered progenitor cells in the ventricular and subventricular zones of the telencephalon in E14.5 <i>Tcof1</i>^+/− embryos. The neuron layers were visualized by immunofluorescence of anti-MAP2 antibody (green). (B) Bar graph depicting average numbers of mitotic cells in the telencephalon of E10.5–E16.5 wild-type and <i>Tcof1</i>^+/− embryos, counted in a unit section of 100 µm width. (C) Bar graph depicting the percentage of apical neural progenitor cells and abnormal scattered neural progenitor cells in the telencephalon of E12.5 and E14.5 wild-type and <i>Tcof1</i>^+/− embryos. (D) Co-labeling of the telencehalon in wild-type and <i>Tcof1</i> mutant embryos with IdU (green) and BrdU (red). S-phase and total cell cycle length were estimated by the number of IdU- and BrdU-positive cells and revealed an increase in total cell cycle length in <i>Tcof1</i>^+/− embryos compared to wild-type littermates. Scale Bars: A and D, 50 µm.</p