G2 acquisition by transcription-independent mechanism at the zebrafish midblastula transition.

Following fertilization of many animal embryos, rapid synchronous cleavage divisions give way to longer, asynchronous cell cycles at the midblastula transition (MBT). The cell cycle changes at the MBT, including the addition of gap phases and checkpoint controls, are accompanied by activation of the zygotic genome and the onset of cell motility. Whereas the biochemical changes accompanying the MBT in the vertebrate embryo have been extensively documented, the cellular events are not well understood. We show that cell cycle remodeling during the zebrafish MBT includes the transcription-independent acquisition of a G2 phase that is essential for preventing entry into mitosis before S-phase completion in cycles 11-13. We provide evidence from high-resolution imaging that inhibition of Cdc25a and Cdk1 activity, but not Cdk2 activity, is essential for cell cycle lengthening and asynchrony between cycles 9 and 12. We demonstrate that lengthening is not required for initiation of zygotic transcription. Our results are consistent with findings from Drosophila and Xenopus that indicate the central importance of G2 addition in checkpoint establishment, and point to similar mechanisms governing the MBT in diverse species.

Regulation and function of foxe3 during early zebrafish development.

In this article, we investigate the expression, regulation, and function of the zebrafish forkhead gene foxe3. In wild type embryos, foxe3 is first expressed in a crescent-shaped area at the anterior end of the prechordal plate, corresponding to the polster. At later stages, the hatching gland, the lens, and the anterior pituitary express this gene. Using morpholinos against the zinc finger Kruppel-like factor 4 (KLF4) we show that foxe3 is regulated differently in the polster and in the lens. In the absence of KLF4, expression of foxe3 in the polster is not activated, whereas in the lens placode the expression of KLF4 is not required for the transcription of foxe3. The expression of foxe3 is also regulated by the hedgehog and nodal signaling pathways. foxe3 expression is altered in the hedgehog pathway mutants iguana and you-too and the nodal pathway mutant cyclops. foxe3 function is necessary for the execution of lens-specific gene expression and lens morphogenesis, as the knockdown of foxe3 results in a loss of platelet-derived growth factor receptor alpha (pdgfralpha) expression and in the vacuolization of the lens.

Zebrafish cdc25a is expressed during early development and limiting for post-blastoderm cell cycle progression.

Cdc25 phosphatases are required for eukaryotic cell cycle progression. To investigate mechanisms governing spatiotemporal dynamics of cell cycle progression during vertebrate development, we isolated two cdc25 genes from the zebrafish, Danio rerio, cdc25a, and cdc25d. We propose that Zebrafish cdc25a is the zebrafish orthologue of the tetrapod Cdc25A genes, while cdc25d is of indeterminate origin. We show that both genes have proliferation promoting activity, but that only cdc25d can complement a Schizosaccharomyces pombe loss of function cdc25 mutation. We present expression data demonstrating that cdc25d expression is very limited during early development, while cdc25a is widely expressed and consistent with the mitotic activity in previously identified mitotic domains of the post-blastoderm zebrafish embryo. Finally, we show that cdc25a can accelerate the entry of post-blastoderm cells into mitosis, suggesting that levels of cdc25a are rate limiting for cell cycle progression during gastrulation.

A zebrafish LMO4 ortholog limits the size of the forebrain and eyes through negative regulation of six3b and rx3.

The Six3 and Rx3 homeodomain proteins are essential for the specification and proliferation of forebrain and retinal precursor cells of the vertebrate brain, and the regulatory networks that control their expression are beginning to be elucidated. We identify the zebrafish lmo4b gene as a negative regulator of forebrain growth that acts via restriction of six3 and rx3 expression during early segmentation stages. Loss of lmo4b by morpholino knockdown results in enlargement of the presumptive telencephalon and optic vesicles and an expansion of the post-gastrula expression domains of six3 and rx3. Overexpression of lmo4b by mRNA injection causes complementary phenotypes, including a reduction in the amount of anterior neural tissue, especially in the telencephalic, optic and hypothalamic primordia, and a dosage-sensitive reduction in six3 and rx3 expression. We suggest that lmo4b activity is required at the neural boundary to restrict six3b expression, and later within the neural plate to for attenuation of rx3 expression independently of its effect on six3 transcription. We propose that lmo4b has an essential role in forebrain development as a modulator of six3 and rx3 expression, and thus indirectly influences neural cell fate commitment, cell proliferation and tissue growth in the anterior CNS.

Modulation of zebrafish pitx3 expression in the primordia of the pituitary, lens, olfactory epithelium and cranial ganglia by hedgehog and nodal signaling.

In this article we report the isolation of a novel zebrafish gene, pitx3, which plays an important role in the formation of several placode-derived structures. In wildtype embryos, pitx3 is first expressed in a crescent-shaped area in the anterior end of the embryo. At later stages, the primordia of the anterior pituitary, the lens, the olfactory sensory epithelium, and cranial ganglia express this gene. Pitx3 is not expressed in the more posterior preplacodal region that gives rise to the epibranchial, otic, and lateral line placodes. The dynamics of pitx3 in the anterior region of wildtype embryos suggests that pitx3 expression marks a common step in the formation of the pituitary, lens, olfactory placode as well as the trigeminal placode. Analysis of pitx3 expression in mutants lacking the hedgehog or nodal function demonstrates the differential dependence of pitx3 expression in these structures on nodal and hedgehog signaling. While the lens and trigeminal placodes express pitx3 in the absence of hedgehog and nodal signaling, there is no expression of pitx3 in the anteriormost ectoderm adjacent to the neural plate from which the anterior pituitary would derive. In mutants with impaired hedgehog signaling, the lens placode frequently extends into more anterior ventral regions of the embryo.

Intervention to reduce the incidence of methicillin-resistant Staphylococcus aureus skin infections in a correctional facility in Georgia.

BACKGROUND AND OBJECTIVE: In August 2001, a cluster of MRSA skin infections was detected in a correctional facility. An investigation was conducted to determine its cause and to prevent further MRSA infections. DESIGN: Case-control study. SETTING: A 200-bed detention center. PATIENTS: A case was defined as a detainee with a skin lesion from which MRSA was cultured from July 24 through December 31, 2001. Case-patients were identified by review of laboratory culture results and by skin lesion screening through point-prevalence survey and admission examination. Controls were randomly selected from an alphabetized list of detainees. INTERVENTION: Medical staff implemented measures to improve skin disease screening, personal hygiene, wound care, and antimicrobial therapy. RESULTS: Sixteen cases were identified: 11, 5, and 0 in the preintervention, peri-intervention, and postintervention periods, respectively. Seven case-patients and 19 controls were included in the case-control study. On multivariable analysis, working as a dormitory orderly (OR, 9.8; CI95, 0.74-638; P = .10) and a stay of longer than 36 days (OR, 6.9; CI95, 0.65-128.2; P = .14) were the strongest predictors for MRSA skin infection. The preintervention, peri-intervention, and postintervention MRSA infection rates were 11.6, 8.8, and 0 per 10,000 detainee-days, respectively. The rate of MRSA skin infections declined significantly between both the preintervention and peri-intervention periods and the postintervention period (P < .01 for both comparisons). CONCLUSIONS: MRSA skin disease can become an emergent problem in a correctional facility. Interventions targeted at skin disease screening, appropriate antimicrobial treatment, and hygiene may decrease the risk of acquiring MRSA infection in correctional facilities.

HIDden targets of microRNAs for growth control.

How is the size of an animal determined? Why is it that humans grow larger than mice? Certainly, one of the most astonishing features of animal development is that every animal of a given species, and its organs and appendages, grow to approximately the same size. Surprisingly little is known about the biology of tissue growth and size control. Recent advances in Drosophila research have implicated a microRNA as an important regulator of animal size. These studies reveal an unexpected layer of size regulation in higher animals.

Drosophila mixed lineage kinase/slipper, a missing biochemical link in Drosophila JNK signaling.

Mixed lineage kinases (MLKs) belong to the family of mitogen activated protein kinase kinase kinase (MAPKKK) and cause neuronal cell death mediated through c-Jun, N-terminal kinase (JNK) pathway. Recently, genetic studies in Drosophila revealed the presence of an MLK termed slipper (slpr). However, its biochemical features like physiological substrate, role in different MAPK pathways and developmental and tissue-specific expression pattern were not reported. Here, we report cDNA cloning, expression analysis and biochemical characterization of a Drosophila mixed lineage kinase (dMLK) that is also known as slipper. The protein structure analysis of dMLK/slipper revealed, in addition to the conserved domains, a stretch of glutamine in the amino terminus and an asparagine-threonine stretch at the carboxy-terminus. In situ hybridization and reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that dMLK is expressed in early embryonic stages, adult brain and thorax. Ectopic expression of dMLK either in Drosophila S2 or in mammalian HEK293 cells leads to activation of JNK, p38 and extracellular signal regulated kinase (ERK) pathways. Further, dMLK directly phosphorylates Hep, dMKK4 and also their mammalian counterparts, MKK7 and SEK1, in an in vitro kinase assay. Taken together, our results provide for the first time a comprehensive expression profile and new biochemical insight of dMLK/slipper.

Activation of the Drosophila MLK by ceramide reveals TNF-alpha and ceramide as agonists of mammalian MLK3.

Mixed lineage kinases (MLKs) are MAPKKK members that activate JNK and reportedly lead to cell death. However, the agonist(s) that regulate MLK activity remain unknown. Here, we demonstrate ceramide as the activator of Drosophila MLK (dMLK) and identify ceramide and TNF-alpha as agonists of mammalian MLK3. dMLK and MLK3 are activated by a ceramide analog and bacterial sphingomyelinase in vivo, whereas a low nanomolar concentration of natural ceramide activates them in vitro. Specific inhibition of dMLK and MLK3 significantly attenuates activation of JNK by ceramide in vivo without affecting ceramide-induced p38 or ERK activation. In addition, TNF-alpha also activates MLK3 and evidently leads to JNK activation in vivo. Thus, the ceramide serves as a common agonist of dMLK and MLK3, and MLK3 contributes to JNK activation induced by TNF-alpha.

Dynamic expression and regulation by Fgf8 and Pou2 of the zebrafish LIM-only gene, lmo4.

We report the expression of zebrafish lmo4 during the first 48 h of development. Like its murine ortholog, lmo4 is expressed in somitic mesoderm, branchial arches, otic vesicles, and limb (pectoral fin) buds. In addition, however, we report zebrafish lmo4 expression in the developing eye, cardiovascular tissue, and the neural plate and telencephalon. We demonstrate that expression in the rostral hindbrain requires acerebellar (ace/fgf8) and spiel ohne grenzen (spg/pou2) activity.

Dynamic expression and regulation by Fgf8 and Pou2 of the zebrafish LIM-only gene, lmo4.

We report the expression of zebrafish lmo4 during the first 48 h of development. Like its murine ortholog, lmo4 is expressed in somitic mesoderm, branchial arches, otic vesicles, and limb (pectoral fin) buds. In addition, however, we report zebrafish lmo4 expression in the developing eye, cardiovascular tissue, and the neural plate and telencephalon. We demonstrate that expression in the rostral hindbrain requires acerebellar (ace/fgf8) and spiel ohne grenzen (spg/pou2) activity.