Interdependence of Bad and Puma during Ionizing-Radiation-Induced Apoptosis

Ionizing radiation (IR)-induced DNA double-strand breaks trigger an extensive cellular signaling response that involves the coordination of hundreds of proteins to regulate DNA repair, cell cycle arrest and apoptotic pathways. The cellular outcome often depends on the level of DNA damage as well as the particular cell type. Proliferating zebrafish embryonic neurons are highly sensitive to IR-induced apoptosis, and both p53 and its transcriptional target puma are essential mediators of the response. The BH3-only protein Puma has previously been reported to activate mitochondrial apoptosis through direct interaction with the pro-apoptotic Bcl-2 family proteins Bax and Bak, thus constituting the role of an “activator” BH3-only protein. This distinguishes it from BH3-only proteins like Bad that are thought to indirectly promote apoptosis through binding to anti-apoptotic Bcl-2 family members, thereby preventing the sequestration of activator BH3-only proteins and allowing them to directly interact with and activate Bax and Bak. We have shown previously that overexpression of the BH3-only protein Bad in zebrafish embryos supports normal embryonic development but greatly sensitizes developing neurons to IR-induced apoptosis. While Bad has previously been shown to play only a minor role in promoting IR-induced apoptosis of T cells in mice, we demonstrate that Bad is essential for robust IR-induced apoptosis in zebrafish embryonic neural tissue. Moreover, we found that both p53 and Puma are required for Bad-mediated radiosensitization in vivo. Our findings show the existence of a hierarchical interdependence between Bad and Puma whereby Bad functions as an essential sensitizer and Puma as an essential activator of IR-induced mitochondrial apoptosis specifically in embryonic neural tissue

p63 Mediates an Apoptotic Response to Pharmacological and Disease-Related ER Stress in the Developing Epidermis

SummaryEndoplasmic reticulum (ER) stress triggers tissue-specific responses that culminate in either cellular adaptation or apoptosis, but the genetic networks distinguishing these responses are not well understood. Here we demonstrate that ER stress induced in the developing zebrafish causes rapid apoptosis in the brain, spinal cord, tail epidermis, lens, and epiphysis. Focusing on the tail epidermis, we uncover an apoptotic response that depends on Puma, but not on p53 or Chop. puma is transcriptionally activated during this ER stress response in a p53-independent manner, and is an essential mediator of epidermal apoptosis. We demonstrate that the p63 transcription factor is upregulated to initiate this apoptotic pathway and directly activates puma transcription in response to ER stress. We also show that a mutation of human Connexin 31, which causes erythrokeratoderma variabilis, induces ER stress and p63-dependent epidermal apoptosis in the zebrafish embryo, thus implicating this pathway in the pathogenesis of inherited disease

Ccdc94 Protects Cells from Ionizing Radiation by Inhibiting the Expression of p53

DNA double-strand breaks (DSBs) represent one of the most deleterious forms of DNA damage to a cell. In cancer therapy, induction of cell death by DNA DSBs by ionizing radiation (IR) and certain chemotherapies is thought to mediate the successful elimination of cancer cells. However, cancer cells often evolve to evade the cytotoxicity induced by DNA DSBs, thereby forming the basis for treatment resistance. As such, a better understanding of the DSB DNA damage response (DSB–DDR) pathway will facilitate the design of more effective strategies to overcome chemo- and radioresistance. To identify novel mechanisms that protect cells from the cytotoxic effects of DNA DSBs, we performed a forward genetic screen in zebrafish for recessive mutations that enhance the IR–induced apoptotic response. Here, we describe radiosensitizing mutation 7 (rs7), which causes a severe sensitivity of zebrafish embryonic neurons to IR–induced apoptosis and is required for the proper development of the central nervous system. The rs7 mutation disrupts the coding sequence of ccdc94, a highly conserved gene that has no previous links to the DSB–DDR pathway. We demonstrate that Ccdc94 is a functional member of the Prp19 complex and that genetic knockdown of core members of this complex causes increased sensitivity to IR–induced apoptosis. We further show that Ccdc94 and the Prp19 complex protect cells from IR–induced apoptosis by repressing the expression of p53 mRNA. In summary, we have identified a new gene regulating a dosage-sensitive response to DNA DSBs during embryonic development. Future studies in human cancer cells will determine whether pharmacological inactivation of CCDC94 reduces the threshold of the cancer cell apoptotic response

T-Lymphoblastic Lymphoma Cells Express High Levels of BCL2, S1P1, and ICAM1, Leading to a Blockade of Tumor Cell Intravasation

The molecular events underlying the progression of T-lymphoblastic lymphoma (T-LBL) to acute T-lymphoblastic leukemia (T-ALL) remain elusive. In our zebrafish model, concomitant overexpression of bcl-2 with Myc accelerated T-LBL onset while inhibiting progression to T-ALL. The T-LBL cells failed to invade the vasculature and showed evidence of increased homotypic cell-cell adhesion and autophagy. Further analysis using clinical biopsy specimens revealed autophagy and increased levels of BCL2, S1P1, and ICAM1 in human T-LBL compared with T-ALL. Inhibition of S1P1 signaling in T-LBL cells led to decreased homotypic adhesion in vitro and increased tumor cell intravasation in vivo. Thus, blockade of intravasation and hematologic dissemination in T-LBL is due to elevated S1P1 signaling, increased expression of ICAM1, and augmented homotypic cell-cell adhesion.Stem Cell and Regenerative Biolog

Neural Crest Migration and Survival Are Susceptible to Morpholino-Induced Artifacts

<div><p>The neural crest (NC) is a stem cell-like embryonic population that is essential for generating and patterning the vertebrate body, including the craniofacial skeleton and peripheral nervous system. Defects in NC development underlie many birth defects and contribute to formation of some of the most malignant cancers in humans, such as melanoma and neuroblastoma. For these reasons, significant research efforts have been expended to identify genes that control NC development, as it is expected to lead to a deeper understanding of the genetic mechanisms controlling vertebrate development and identify new treatments for NC-derived diseases and cancers. However, a number of inconsistencies regarding gene function during NC development have emerged from comparative analyses of gene function between mammalian and non-mammalian systems (chick, frog, zebrafish). This poses a significant barrier to identification of single genes and/or redundant pathways to target in NC diseases. Here, we determine whether technical differences, namely morpholino-based approaches used in non-mammalian systems, could contribute to these discrepancies, by examining the extent to which NC phenotypes in <i>fascin1a (fscn1a)</i> morphant embryos are similar to or different from <i>fscn1a</i> null mutants in zebrafish. Analysis of <i>fscn1a</i> morphants showed that they mimicked early NC phenotypes observed in <i>fscn1a</i> null mutants; however, these embryos also displayed NC migration and derivative phenotypes not observed in null mutants, including accumulation of <i>p53</i>-independent cell death. These data demonstrate that morpholinos can cause seemingly specific NC migration and derivative phenotypes, and thus have likely contributed to the inconsistencies surrounding NC gene function between species. We suggest that comparison of genetic mutants between different species is the most rigorous method for identifying conserved genetic mechanisms controlling NC development and is critical to identify new treatments for NC diseases.</p></div

<i>Fscn1aMO</i> induced <i>tp53</i>-independent cell death in NC cells.

<p><b>(A)</b> Lateral and dorsal cranial views of 28 hpf <i>tp53</i>^<i>zdf1</i> mutant embryos injected with <i>coMO</i> or <i>fscn1aMO</i> and stained with AO. Arrowheads highlight AO-positive cells adjacent to neural tube. <b>(B)</b> Lateral view of 24 hpf <i>Tg(sox10</i>:<i>rfpmb)</i> embryo injected with <i>tp53MO</i> plus <i>fscn1aMO</i> and stained with AO. Numbers correspond to NC streams. Arrows indicate regions of RFP-positive/AO-positive cells. e; eye, nt; neural tube. In all lateral views or dorsal cranial views, anterior is to the left or bottom, respectively. Experiments in this figure were performed independently at least three times with similar results.</p

Late-stage NC-cell migration is disrupted in <i>fscn1a</i>-morphant embryos.

<p>(A-B) Dorsal cranial views of <i>tp53</i>^<i>zdf1</i> embryos injected with <i>coMO</i> or <i>fscn1aMO</i> and analyzed by whole-mount <i>in situ</i> hybridization (ISH) for (A) <i>foxd3</i> mRNA at 10 hpf and (B) <i>sox10</i> mRNA at 15 hpf. (C) Dorsal cranial and lateral views of 26 hpf <i>tp53</i>^<i>zdf1</i> embryos injected with <i>coMO</i> or <i>fscn1aMO</i> and analyzed by whole-mount ISH for <i>dlx2a</i>. Numbers correspond to pharyngeal arches. Arrow denotes reduction in <i>dlx2a</i>-positive cranial NC cells in <i>fscn1a</i> morphants. (D) Lateral views of cranial NC streams in 22, 25, 28 and 36 hpf <i>Tg(sox10</i>:<i>gfp)</i> embryos injected with <i>tp53MO</i> or <i>tp53MO</i> plus <i>fscn1aMO</i>. Numbers correspond to NC streams. Arrows highlight NC cells migrating independently of NC streams in <i>fscn1a</i> morphants. e; eye, ov; otic vesicle. In all panels, anterior is to the left. All experiments in this figure were performed independently at least three times with similar results. All scale bars in this figure = 100 μm.</p

NC-derived tissues form abnormally in <i>fscn1a</i>-morphant embryos.

<p>(A) Lateral and ventral views of 5 dpf <i>tp53</i>^<i>zdf1</i> embryos injected with <i>coMO</i> or <i>fscn1aMO</i> and stained with Alcian blue. Numbers correspond to pharyngeal arches. Asterisk denotes arches that are severely reduced in size or absent. (B) Lateral views of 3 dpf <i>tp53</i>^<i>zdf1</i> embryos analyzed by whole-mount ISH for <i>th</i>. Arrows denote <i>th</i>-positive neurons of sympathetic ganglia. (C) Lateral views of section of the gut in 4 dpf <i>Tg(phox2b</i>:<i>gfp)</i> embryos injected with <i>tp53MO</i> or <i>tp53MO</i> plus <i>fscn1aMO</i>. Arrows denote <i>phox2b</i>-positive enteric neurons. (D) Lateral views of trunk in 3 dpf Tg(<i>ngn1</i>:<i>gfp</i>) embryos injected with <i>tp53MO</i> or <i>tp53MO</i> plus <i>fscn1aMO</i>. Arrows in top panel highlight <i>ngn1</i>-positive NC-derived dorsal root ganglia (drg) and central nervous system (CNS)-derived Rohon-Beard neurons (rb). In lower panel, arrowhead and asterisk indicate misplaced and absent dorsal root ganglia, respectively. drg; dorsal root ganglia, rb; Rohan-Beard neurons, ye; yolk extension. In all panels, anterior is to the left. All experiments in this figure were performed independently at least three times with similar results. All scale bars in this figure = 100 μm.</p