p53 protects against genome instability following centriole duplication failure

Centriole function has been difficult to study because of a lack of specific tools that allow persistent and reversible centriole depletion. Here we combined gene targeting with an auxin-inducible degradation system to achieve rapid, titratable, and reversible control of Polo-like kinase 4 (Plk4), a master regulator of centriole biogenesis. Depletion of Plk4 led to a failure of centriole duplication that produced an irreversible cell cycle arrest within a few divisions. This arrest was not a result of a prolonged mitosis, chromosome segregation errors, or cytokinesis failure. Depleting p53 allowed cells that fail centriole duplication to proliferate indefinitely. Washout of auxin and restoration of endogenous Plk4 levels in cells that lack centrioles led to the penetrant formation of de novo centrioles that gained the ability to organize microtubules and duplicate. In summary, we uncover a p53-dependent surveillance mechanism that protects against genome instability by preventing cell growth after centriole duplication failure

A USP28-53BP1-p53-p21 signaling axis arrests growth after centrosome loss or prolonged mitosis

Precise regulation of centrosome number is critical for accurate chromosome segregation and the maintenance of genomic integrity. In nontransformed cells, centrosome loss triggers a p53-dependent surveillance pathway that protects against genome instability by blocking cell growth. However, the mechanism by which p53 is activated in response to centrosome loss remains unknown. Here, we have used genome-wide CRISPR/Cas9 knockout screens to identify a USP28-53BP1-p53-p21 signaling axis at the core of the centrosome surveillance pathway. We show that USP28 and 53BP1 act to stabilize p53 after centrosome loss and demonstrate this function to be independent of their previously characterized role in the DNA damage response. Surprisingly, the USP28-53BP1-p53-p21 signaling pathway is also required to arrest cell growth after a prolonged prometaphase. We therefore propose that centrosome loss or a prolonged mitosis activate a common signaling pathway that acts to prevent the growth of cells that have an increased propensity for mitotic errors

Genome-Wide Analyses Reveal a Role for Peptide Hormones in Planarian Germline Development

Genomic/peptidomic analyses of the planarian Schmidtea mediterranea identifies >200 neuropeptides and uncovers a conserved neuropeptide required for proper maturation and maintenance of the reproductive system

<i>Tryptophan hydroxylase</i> Is Required for Eye Melanogenesis in the Planarian <i>Schmidtea mediterranea</i>

<div><p>Melanins are ubiquitous and biologically important pigments, yet the molecular mechanisms that regulate their synthesis and biochemical composition are not fully understood. Here we present a study that supports a role for serotonin in melanin synthesis in the planarian <i>Schmidtea mediterranea</i>. We characterize the tryptophan hydroxylase (<i>tph</i>) gene, which encodes the rate-limiting enzyme in serotonin synthesis, and demonstrate by RNA interference that <i>tph</i> is essential for melanin production in the pigment cups of the planarian photoreceptors. We exploit this phenotype to investigate the biological function of pigment cups using a quantitative light-avoidance behavioral assay. Planarians lacking eye pigment remain phototactic, indicating that eye pigmentation is not essential for light avoidance in <i>S</i>. <i>mediterranea</i>, though it improves the efficiency of the photophobic response. Finally, we show that the eye pigmentation defect observed in <i>tph</i> knockdown animals can be rescued by injection of either the product of TPH, 5-hydroxytryptophan (5-HTP), or serotonin. Together, these results highlight a role for serotonin in melanogenesis, perhaps as a regulatory signal or as a pigment substrate. To our knowledge, this is the first example of this relationship to be reported outside of mammalian systems.</p></div

Pigment cup and photoreceptors remain intact in <i>tph(RNAi)</i> animals.

<p>(A-B) The planarian visual system. Photoreceptors (magenta) are visualized by immunofluorescence with VC-1 antibody against arrestin, and pigment cup cells (green) are visualized by fluorescent <i>in situ</i> hybridization of <i>tyrosinase (tyr)</i>. (A) depicts a maximum intensity confocal projection, whereas (B) represents a single confocal section. (C) By 10 days post-amputation, control and <i>tph(RNAi)</i> animals regenerate both photoreceptors and pigment cup cells, as indicated by VC-1 staining and <i>tyr</i> mRNA expression. Scale bars 20 μm.</p

Tryptophan derivatives rescue eye pigment in <i>tph</i> knockdowns.

<p>(A) Progress of pigment recovery after injection of 2.5 M 5-HTP is shown at hour-intervals. Eye pigment in <i>tph</i> knockdowns is largely recovered by 3 hr post-injection. (B) <i>tph(RNAi</i>) and control worms were injected with DMSO (n = 3), 100 mM tryptophan (n = 3), 100 mM 5-HTP (n = 5), 250 mM serotonin (n = 5), or 10 mM L-DOPA (n = 3). Images show phenotype at pre-injection and 24 hr time points. Insets highlight the increased pigmentation of control worms injected with 5-HTP vs. DMSO control. Scale bar is 200 μm. (C) Comparison of traditional and hypothesized melanin synthesis pathways.</p

<i>tph</i> is essential for photoreceptor pigmentation after regeneration.

<p>(A) Structure and genomic organization of the <i>tph</i> gene. Top, gene structure of <i>tph</i>; the 5’-untranslated region, the coding region, and the 3’-untranslated region are depicted in yellow, red and blue, respectively. Below, organization of genomic regions (supercontigs) that encode <i>tph</i>. (B-C) Whole-mount <i>in situ</i> hybridization to localize <i>tph</i> expression. The <i>tph</i> gene was expressed in the pigment cups, the peripharyngeal secretory cells (dorsal) and cells within the central and peripheral nervous systems (ventral). (D-E) RNAi-mediated knockdown of <i>tph</i>. In comparison to controls (D), <i>tph</i> knockdowns (E) regenerate pigment cups that appear to lack pigment; 21-day regenerates shown. (F-G) <i>in situ</i> hybridizations to detect <i>tph</i> mRNA levels following RNAi treatment. Relative to controls (F), <i>tph</i> dsRNA-treated animals show dramatically reduced <i>tph</i> mRNA expression; 21-day regenerates shown. Scale bars 200 μm.</p