The zebrafish dyrk1b gene is important for endoderm formation.

Nodal-signaling is required for specification of mesoderm, endoderm, establishing left-right asymmetry, and craniofacial development. Wdr68 is a WD40-repeat domain-containing protein recently shown to be required for endothelin-1 (edn1) expression and subsequent lower jaw development. Previous reports detected the Wdr68 protein in multiprotein complexes containing mammalian members of the dual-specificity tyrosine-regulated kinase (dyrk) family. Here we describe the characterization of the zebrafish dyrk1b homolog. We report the detection of a physical interaction between Dyrk1b and Wdr68. We also found perturbations of nodal signaling in dyrk1b antisense morpholino knockdown (dyrk1b-MO) animals. Specifically, we found reduced expression of lft1 and lft2 (lft1/2) during gastrulation and a near complete loss of the later asymmetric lft1/2 expression domains. Although wdr68-MO animals did not display lft1/2 expression defects during gastrulation, they displayed a near complete loss of the later asymmetric lft1/2 expression domains. While expression of ndr1 was not substantially effected during gastrulation, ndr2 expression was moderately reduced in dyrk1b-MO animals. Analysis of additional downstream components of the nodal signaling pathway in dyrk1b-MO animals revealed modestly expanded expression of the dorsal axial mesoderm marker gsc while the pan-mesodermal marker bik was largely unaffected. The endodermal markers cas and sox17 were also moderately reduced in dyrk1b-MO animals. Notably, and similar to defects previously reported for wdr68 mutant animals, we also found reduced expression of the pharyngeal pouch marker edn1 in dyrk1b-MO animals. Taken together, these data reveal a role for dyrk1b in endoderm formation and craniofacial patterning in the zebrafish.

Blood genomic expression profile for neuronal injury.

This study determined whether stroke and other types of insults produced a gene expression profile in blood that correlated with the presence of neuronal injury. Adult rats were subjected to ischemic stroke, intracerebral hemorrhage, status epilepticus, and insulin-induced hypoglycemia and compared with untouched, sham surgery, and hypoxia animals that had no brain injury. One day later, microarray analyses showed that 117 genes were upregulated and 80 genes were downregulated in mononuclear blood cells of the "injury" (n = 12) compared with the "no injury" (n = 9) animals. A second experiment examined the whole blood genomic response of adult rats after global ischemia and kainate seizures. Animals with no brain injury were compared with those with brain injury documented by TUNEL and PANT staining. One day later, microarray analyses showed that 37 genes were upregulated and 67 genes were downregulated in whole blood of the injury (n = 4) animals compared with the no-injury (n = 4) animals. Quantitative reverse transcription-polymerase chain reaction confirmed that the vesicular monoamine transporter-2 increased 2.3- and 1.6-fold in animals with severe and mild brain injury, respectively, compared with no-injury animals. Vascular tyrosine phosphatase-1 increased 2.0-fold after severe injury compared with no injury. The data support the hypothesis that there is a peripheral blood genomic response to neuronal injury, and that this blood response is associated with a specific blood mRNA gene expression profile that can be used as a marker of the neuronal damage.

Geldanamycin induces heat shock proteins in brain and protects against focal cerebral ischemia.

Geldanamycin (GA), a benzoquinone ansamycin, binds Hsp90 in vitro, releases heat shock factor (HSF1) and induces heat shock proteins (Hsps). Because viral and transgenic overexpression of Hsps protects cells against ischemia in vitro, we hypothesized that GA would protect brain from focal ischemia by inducing Hsps in vivo. Adult male Sprague-Dawley rats were subjected to 2-hour middle cerebral artery occlusions (MCAO) using the suture technique followed by 22-h reperfusions. GA or vehicle was injected into the lateral cerebral ventricles (i.c.v) 24 h before ischemia. Geldanamycin at 1 microg/kg decreased infarct volumes by 55.7% (p < 0.01) and TUNEL-positive cells by 30% in cerebral cortex. GA also improved behavioral outcomes (p < 0.01) and reduced brain edema (p < 0.05). Western blots showed that the 1 microg/kg GA dose induced Hsp70 and Hsp25 protein 8.2-fold and 2.7-fold, respectively, by 48 h following administration. Immunocytochemistry showed that GA induced Hsp70 in neurons and Hsp25 in glia and arteries in cortex, hippocampus, hypothalamus, and other brain regions. GA reduced co-immunoprecipitation of HSF1 with Hsp90 in brain tissue homogenates, promoted HSE-binding of HSF in brain nuclear extracts using gel shift assays, and increased luciferase reporter gene transcription for the Hsp70 promoter in PC12 cells. The data show that geldanamycin protects brain from focal ischemia and that this may be due, at least in part, to geldanamycin stimulation of heat shock gene transcription.

17-beta-estradiol induces heat shock proteins in brain arteries and potentiates ischemic heat shock protein induction in glia and neurons.

Estradiol reduces brain injury from many diseases, including stroke and trauma. To investigate the molecular mechanisms of this protection, the effects of 17-beta-estradiol on heat shock protein (HSP) expression were studied in normal male and female rats and in male gerbils after global ischemia. 17-beta-estradiol was given intraperitoneally (46 or 460 ng/kg, or 4.6 microg/kg) and Western blots performed for HSPs. 17-beta-estradiol increased hemeoxygenase-1, HSP25/27, and HSP70 in the brain of male and female rats. Six hours after the administration of 17-beta-estradiol, hemeoxygenase-1 increased 3.9-fold (460 ng/kg) and 5.4-fold (4.6 microg/kg), HSP25/27 increased 2.1-fold (4.6 microg/kg), and Hsp70 increased 2.3-fold (460 ng/kg). Immunocytochemistry showed that hemeoxygenase-1, HSP25/27,and HSP70 induction was localized to cerebral arteries in male rats, possibly in vascular smooth muscle cells. 17-beta-estradiol was injected intraperitoneally 20 minutes before transient occlusion of both carotids in adult gerbils. Six hours after global cerebral ischemia, 17-beta-estradiol (460 ng/kg) increased levels of hemeoxygenase-1 protein 2.4-fold compared with ischemia alone, and HSP25/27 levels increased 1.8-fold compared with ischemia alone. Hemeoxygenase-1 was induced in striatal oligodendrocytes and hippocampal neurons, and HSP25/27 levels increased in striatal astrocytes and hippocampal neurons. Finally, Western blot analysis confirmed that estrogen induced heat shock factor-1, providing a possible mechanism by which estrogen induces HSPs in brain and other tissues. The induction of HSPs may be an important mechanism for estrogen protection against cerebral ischemia and other types of injury.