MiCASA is a new method for quantifying cellular organization

While many tools exist for identifying and quantifying individual cell types, few methods are available to assess the relationships between cell types in organs and tissues and how these relationships change during aging or disease states. We present a quantitative method for evaluating cellular organization, using the mouse thymus as a test organ. The thymus is the primary lymphoid organ responsible for generating T cells in vertebrates, and its proper structure and organization is essential for optimal function. Our method, Multitaper Circularly Averaged Spectral Analysis (MiCASA), identifies differences in the tissue-level organization with high sensitivity, including defining a novel type of phenotype by measuring variability as a specific parameter. MiCASA provides a novel and easily implemented quantitative tool for assessing cellular organization

Roles of growth hormone and insulin-like growth factor 1 in mouse postnatal growth

To examine the relationship between growth hormone (GH) and insulin-like growth factor 1 (IGF1) in controlling postnatal growth, we performed a comparative analysis of dwarfing phenotypes manifested in mouse mutants lacking GH receptor, IGF1, or both. This genetic study has provided conclusive evidence demonstrating that GH and IGF1 promote postnatal growth by both independent and common functions, as the growth retardation of double Ghr/Igf1 nullizygotes is more severe than that observed with either class of single mutant. In fact, the body weight of these double-mutant mice is only �17 % of normal and, in absolute magnitude (�5 g), only twice that of the smallest known mammal. Thus, the growth control pathway in which the components of the GH/IGF1 signaling systems participate constitutes the major determinant of body size. To complement this conclusion mainly based on extensive growth curve analyses, we also present details concerning the involvement of the GH/IGF1 axis in linear growth derived by a developmental study of long bone ossification in the mutants. © 2001 Academic Press Key Words: growth; growth rate; growth retardation; growth hormone; growth hormone receptor; insulin-like growt

Cell Cycle-Related Kinase (CCRK) regulates ciliogenesis and Hedgehog signaling in mice

<div><p>The Hedgehog (Hh) signaling pathway plays a key role in cell fate specification, proliferation, and survival during mammalian development. Cells require a small organelle, the primary cilium, to respond properly to Hh signals and the key regulators of Hh signal transduction exhibit dynamic localization to this organelle when the pathway is activated. Here, we investigate the role of Cell Cycle Related kinase (CCRK) in regulation of cilium-dependent Hh signaling in the mouse. Mice mutant for <i>Ccrk</i> exhibit a variety of developmental defects indicative of inappropriate regulation of this pathway. Cell biological, biochemical and genetic analyses indicate that CCRK is required to control the Hedgehog pathway at the level or downstream of Smoothened and upstream of the Gli transcription factors, Gli2 and Gli3. <i>In vitro</i> experiments indicate that <i>Ccrk</i> mutant cells show a greater deficit in response to signaling over long time periods than over short ones. Similar to <i>Chlamydomonas</i> mutants lacking the CCRK homolog, LF2, mouse <i>Ccrk</i> mutant cells show defective regulation of ciliary length and morphology. <i>Ccrk</i> mutant cells exhibit defects in intraflagellar transport (the transport mechanism used to assemble cilia), as well as slowed kinetics of ciliary enrichment of key Hh pathway regulators. Collectively, the data suggest that CCRK positively regulates the kinetics by which ciliary proteins such as Smoothened and Gli2 are imported into the cilium, and that the efficiency of ciliary recruitment allows for potent responses to Hedgehog signaling over long time periods.</p></div

Localization of Hedgehog signaling proteins to <i>Ccrk</i> mutant cilia.

<p>(A) Localization of Hh pathway components to wild-type and <i>Ccrk</i> mutant MEFS under unstimulated or stimulated (200nM SAG for 24 hours) conditions. Note that Gli2 staining (arrowhead) was rarely observed in <i>Ccrk</i> mutant cilia under unstimulated conditions. (B) Ciliary localization of Gli2 and Smo was monitored in terms of frequency and staining intensity as a function of time of SAG exposure (0 to 24 hours). Percentages of cilia with Gli2 or Smo localization were determined from between 100 and 300 cilia per condition and statistical significance was analyzed using Chi-square tests. Staining intensity was measured from between 25 and 40 cilia per condition and statistical significance was analyzed using Student’s t-tests. Error bars represent standard error of the mean. P values from statistical analyses: **. P<0.01; *, p<0.05; ns, not significant.</p

CCRK regulates Gli2 and Gli3 proteins.

<p>(A) Gli2 and Gli3 western blotting in E9.5 embryonic extracts; β-actin serves as a loading control. Full-length Gli2 (Gli2-FL) showed a second, faster migrating band (Gli2*) in <i>Ccrk</i> mutants, suggesting differential posttranslational modification. FL-Gli3 levels were significantly increased, whereas processed Gli3 repressor (Gli3-Rep) levels were somewhat reduced in <i>Ccrk</i> mutant extracts. (B) Extracts from wild-type and <i>Ccrk</i> mutant embryos were incubated alone, with addition of lambda phosphatase, or with lambda phosphatase plus the phosphatase inhibitor Na₃OV₄ and analyzed by Gli2 western blotting. The results suggest that the faster migrating species (Gli2*) from <i>Ccrk</i> mutants in (A) represents a dephosphorylated state. Similar results were observed in three separate experiments. (C) Quantitation of Gli3-FL, Gli3-Rep, and Gli3-FL/Gli3-Rep ratios, normalized to β-actin. 2 samples/genotype were analyzed. Error bars represent standard error of the mean (SEM). P values from Student’s t-tests are shown.</p

<i>Ccrk</i> mutants show pleiotropic embryonic phenotypes.

<p>(A) The targeting strategy for generation of a <i>Ccrk</i> null allele. Cre-mediated recombination was used to generate the null allele (<i>Ccrk^KO</i>) from the floxed allele. (B) PCR confirmed mutagenesis and western blotting indicated that <i>Ccrk</i>^<i>KO</i> is a null allele. (C) Morphological features of embryonic day (E) 9.5 (b,d) and 12.5 (f) <i>Ccrk</i> mutants, such as exencephaly. Yellow arrows in c,d indicate the lack of the midbrain furrow in <i>Ccrk</i> mutants (d); black arrowheads in e,f highlight the eye defects in <i>Ccrk</i> mutants (f). (D) Limb skeletal defects, such as preaxial polydactyly (arrow) and achondroplasia of the radius and ulna (arrowhead).</p

<i>Ccrk</i> mutant fibroblasts show diminished Hh pathway activity.

<p>(A) qPCR results for <i>Gli1</i> expression (normalized to <i>β-actin</i>) in wild-type and <i>Ccrk</i> mutant primary mouse embryonic fibroblasts (MEFs) stimulated with increasing concentrations of Smoothened agonist (SAG). (B) Normalized luciferase activity in immortalized wild-type and <i>Ccrk</i> mutant MEFs transfected with 8x Gli-binding site/firefly luciferase, as well as constitutive renilla luciferase, constructs and stimulated with SAG. (C, D) Normalized results of qPCR for <i>Gli1</i> (C) and <i>Ptch1</i> (D) expression in immortalized MEFs stably transfected with pcDNA3.1 (mock), wild-type CCRK, or K33R (kinase dead) mutant CCRK constructs in the presence or absence of 200 nM SAG. Error bars represent standard error of the mean. Data obtained from 3 biological replicates per condition. P values from Student’s t-tests: **, p<0.01; *, p<0.05; ns, not significant.</p

Regulation of ciliary morphology and length by CCRK.

<p>(A) Primary cilia in the E9.5 <i>Ccrk</i> mutant and wild-type neuroepithelium assayed by scanning electron microscopy and Arl13b/gamma-tubulin staining. (B) Primary cilia in <i>Ccrk</i> mutant and wild-type MEFs stained with antibodies against acetylated α-tubulin, Arl13b, and IFT88. Scale bars in A and B are 1 μm. (C) Distribution of ciliary length in <i>Ccrk</i> mutant and control (<i>Ccrk</i>^<i>+/-</i>) MEFs binned in 0.25 μm groups. Lengths were measured from Arl13b and γ-tubulin-stained cilia. Average lengths and standard deviation for distributions are shown.</p

Ciliary phenotypes and IFT assays in wild-type and <i>Ccrk</i> mutant IMCD3 cells harboring an IFT reporter.

<p>(A) <i>Ccrk</i> mutant IMCD3 cells (expressing an IFT88::YFP construct)were generated by CRISPR/Cas9 mutagenesis. A clone with a homozygous 67 bp insertion (causing a frame shift and multiple nonsense codons downstream) was identified and analyzed further. (B) Scanning and transmission electron microscopic analysis of wild-type and <i>Ccrk</i> mutant IMCD3 cilia. The mutant cilia showed swelling at their distal ends. (C) IFT88::YFP expression in wild-type and <i>Ccrk</i> mutant IMCD cells. The <i>Ccrk</i> mutant cells uniformly showed accumulation of IFT88::YFP at their distal ends. Transfection of the mutant cells with a wild-type <i>Ccrk</i> expression construct rescued this phenotype. Kymographs from live imaging of IFT88::YFP particle movement in wild-type and <i>Ccrk</i> mutant cilia are shown (distal cilia ends at the top, time increases from left to right). Anterograde transport occasionally showed abrupt changes in speed in the mutants. Rates (D) and frequencies (E) of anterograde and retrograde IFT particle movement were determined from the kymographs. Speed quantitation was determined from 235 and 61 particles (anterograde) and 74 and 25 particles (retrograde) in wild-type and <i>Ccrk</i> mutant cilia, respectively. Frequency quantitation was determined from 45 and 64 cilia movies from wild-type and <i>Ccrk</i> mutant cells, respectively. Error bars represent standard error of the mean. Statistical analysis represents results from Student’s t-test. Scale bars in B are 1 μm.</p