- Email: [email protected]
LIMK1 in Xenopus retinal development
580
Poster abstracts / Int. J. Devl Neuroscience 24 (2006) 495–603
of brain cell death in a rat pup model of hypoxia/ischemia and hyperoxic resuscitation. Methods: Ischemia is induced by cauterization of the left common carotid artery followed by 90 min of hypoxia (7.8% O2 in nitrogen) and 2 h of hyperoxia (>95% O2 ) in 7 d rat pups. These pups are then treated with subcutaneous homocysteine (75 nmol/kg), for 2 d and then sacrificed. Brains were collected for determination of sulfur containing amino acids and apoptosis in frontal cortex and cerebellum. Results: Increased cell death was found in those animals treated with homocysteine. Discussion: The implication of this finding is that preterm infants subjected to hypoxia stress at birth followed by hyperoxic resuscitation may be at increased risk for brain injury if they have increased amounts of homocysteine, possibly due to poor maternal folic acid status. This confluence of events may be an explanatory mechanism for the cognitive deficits observed in such infants. Keywords: Homocysteine; Hypoxia/ischemia; Hyperoxia; Preterm development doi:10.1016/j.ijdevneu.2006.09.255 [P196] Mst3b, a STE20-related protein kinase, regulates axon outgrowth N. Irwin ∗ , L. Benowitz Children’s Hospital/Harvard Medical School, USA Axon outgrowth is critical for brain development and for recovery following nerve injury. Our prior work demonstrated that inosine, a purine nucleoside, stimulates neuronal sprouting after stroke or spinal cord injury, and leads to improvements in functional recovery. In order to understand the signaling that leads to axon outgrowth, we purified a purine-sensitive kinase from brain and amino acid sequencing demonstrated that it is Mst3, an STE20-related kinase. In many cell types, STE20-like kinases have been implicated in modular signaling pathways that control diverse aspects of cellular function. There are two known isoforms of Mst3 that differ at their amino termini. We show that the neuron-specific isoform, Mst3b, is activated in response to neurotrophins and is critical for axon outgrowth in embryonic rat primary neurons and in PC12 cells. Either expression of a dominant-negative mutant of Mst3b or introduction of Mst3b siRNA blocks axon outgrowth. Inosine stimulates axon outgrowth in cell culture and in vivo, and Mst3b kinase activity measured in vitro; 6-thioguanine, a purine analog that blocks outgrowth, inhibits kinase activity. Thus, Mst3b is a purinesensitive protein kinase that is essential for axon outgrowth. Keywords: Axon outgrowth; Signaling; Inosine
[P197] LIMK1 in Xenopus retinal development J. Hocking 1,∗ , S. McFarlane 1 , H. Funakoshi 2 , T. Nakamura 2 1 University of Calgary, Canada; 2 Osaka University Graduate
School of Medicine, Japan Retinal ganglion cells (RGCs) are the output neurons of the eye, and must elaborate extensive dendritic arbors to receive visual information, and extend long axons to targets in the brain in order to transmit that data. The morphological development of RGCs is directed by both intrinsic and extrinsic signals. We use the frog, Xenopus laevis, as a model for studying these signals. LIM kinase1 (LIMK1) promotes actin polymerization and is a key effecter of Rho GTPase pathways. In vitro, LIMK1 promotes cortical dendrite outgrowth downstream of the BMP type II receptor (Lee-Hoeflich et al., 2004). We have examined the expression of LIMK1 mRNA by in situ hybridization in the developing retina of Xenopus embryos. Expression begins at stage 28, extending through most of the width of the central retina, except for the presumptive photoreceptors. LIMK1 mRNA is excluded from the ciliary margin zone (CMZ), and is in a dorsoventral gradient across the central retina. By stage 30, expression is robust in the forming RGC and inner nuclear layers, and has extended to the dorsal CMZ, but is excluded from the ventral CMZ. At stage 33/34, expression in the central retina has weakened, while the dorsal CMZ is strongly labelled. Interestingly, the ventral retina now shows robust expression in a patch adjacent to the ventral CMZ, as well as expression within the CMZ. This pattern is maintained until at least stage 37/38. While the levels of LIMK1 expression vary across regions of the retina, there is maintained expression within all RGCs from stage 30, just as they first extend axons, until at least stage 37/38, when the axons are almost at their target, and a dendritic arbor has formed. We hypothesize that LIMK1 is an important factor downstream of signalling molecules that regulate RGC dendrite growth. In order to investigate this, we have prepared wildtype and dominant negative (dn) LIMK1 constructs for expression in the developing retina. Acknowledgements Funding was provided by NSERC, AHFMR, and CIHR. Keywords: Dendrite; Xenopus; LIM kinase; Retinal ganglion cell Reference Lee-Hoeflich, S.T., Causing, C.G., Podkowa, M., Zhao, X., Wrana, J.L., Attisano, L., 2004. Activation of LIMK1 by binding to the BMP receptor, BMPRII, regulates BMP-dependent dendritogenesis. EMBO 23, 4792–4801.
doi:10.1016/j.ijdevneu.2006.09.256 doi:10.1016/j.ijdevneu.2006.09.257
Poster abstracts / Int. J. Devl Neuroscience 24 (2006) 495–603
of brain cell death in a rat pup model of hypoxia/ischemia and hyperoxic resuscitation. Methods: Ischemia is induced by cauterization of the left common carotid artery followed by 90 min of hypoxia (7.8% O2 in nitrogen) and 2 h of hyperoxia (>95% O2 ) in 7 d rat pups. These pups are then treated with subcutaneous homocysteine (75 nmol/kg), for 2 d and then sacrificed. Brains were collected for determination of sulfur containing amino acids and apoptosis in frontal cortex and cerebellum. Results: Increased cell death was found in those animals treated with homocysteine. Discussion: The implication of this finding is that preterm infants subjected to hypoxia stress at birth followed by hyperoxic resuscitation may be at increased risk for brain injury if they have increased amounts of homocysteine, possibly due to poor maternal folic acid status. This confluence of events may be an explanatory mechanism for the cognitive deficits observed in such infants. Keywords: Homocysteine; Hypoxia/ischemia; Hyperoxia; Preterm development doi:10.1016/j.ijdevneu.2006.09.255 [P196] Mst3b, a STE20-related protein kinase, regulates axon outgrowth N. Irwin ∗ , L. Benowitz Children’s Hospital/Harvard Medical School, USA Axon outgrowth is critical for brain development and for recovery following nerve injury. Our prior work demonstrated that inosine, a purine nucleoside, stimulates neuronal sprouting after stroke or spinal cord injury, and leads to improvements in functional recovery. In order to understand the signaling that leads to axon outgrowth, we purified a purine-sensitive kinase from brain and amino acid sequencing demonstrated that it is Mst3, an STE20-related kinase. In many cell types, STE20-like kinases have been implicated in modular signaling pathways that control diverse aspects of cellular function. There are two known isoforms of Mst3 that differ at their amino termini. We show that the neuron-specific isoform, Mst3b, is activated in response to neurotrophins and is critical for axon outgrowth in embryonic rat primary neurons and in PC12 cells. Either expression of a dominant-negative mutant of Mst3b or introduction of Mst3b siRNA blocks axon outgrowth. Inosine stimulates axon outgrowth in cell culture and in vivo, and Mst3b kinase activity measured in vitro; 6-thioguanine, a purine analog that blocks outgrowth, inhibits kinase activity. Thus, Mst3b is a purinesensitive protein kinase that is essential for axon outgrowth. Keywords: Axon outgrowth; Signaling; Inosine
[P197] LIMK1 in Xenopus retinal development J. Hocking 1,∗ , S. McFarlane 1 , H. Funakoshi 2 , T. Nakamura 2 1 University of Calgary, Canada; 2 Osaka University Graduate
School of Medicine, Japan Retinal ganglion cells (RGCs) are the output neurons of the eye, and must elaborate extensive dendritic arbors to receive visual information, and extend long axons to targets in the brain in order to transmit that data. The morphological development of RGCs is directed by both intrinsic and extrinsic signals. We use the frog, Xenopus laevis, as a model for studying these signals. LIM kinase1 (LIMK1) promotes actin polymerization and is a key effecter of Rho GTPase pathways. In vitro, LIMK1 promotes cortical dendrite outgrowth downstream of the BMP type II receptor (Lee-Hoeflich et al., 2004). We have examined the expression of LIMK1 mRNA by in situ hybridization in the developing retina of Xenopus embryos. Expression begins at stage 28, extending through most of the width of the central retina, except for the presumptive photoreceptors. LIMK1 mRNA is excluded from the ciliary margin zone (CMZ), and is in a dorsoventral gradient across the central retina. By stage 30, expression is robust in the forming RGC and inner nuclear layers, and has extended to the dorsal CMZ, but is excluded from the ventral CMZ. At stage 33/34, expression in the central retina has weakened, while the dorsal CMZ is strongly labelled. Interestingly, the ventral retina now shows robust expression in a patch adjacent to the ventral CMZ, as well as expression within the CMZ. This pattern is maintained until at least stage 37/38. While the levels of LIMK1 expression vary across regions of the retina, there is maintained expression within all RGCs from stage 30, just as they first extend axons, until at least stage 37/38, when the axons are almost at their target, and a dendritic arbor has formed. We hypothesize that LIMK1 is an important factor downstream of signalling molecules that regulate RGC dendrite growth. In order to investigate this, we have prepared wildtype and dominant negative (dn) LIMK1 constructs for expression in the developing retina. Acknowledgements Funding was provided by NSERC, AHFMR, and CIHR. Keywords: Dendrite; Xenopus; LIM kinase; Retinal ganglion cell Reference Lee-Hoeflich, S.T., Causing, C.G., Podkowa, M., Zhao, X., Wrana, J.L., Attisano, L., 2004. Activation of LIMK1 by binding to the BMP receptor, BMPRII, regulates BMP-dependent dendritogenesis. EMBO 23, 4792–4801.
doi:10.1016/j.ijdevneu.2006.09.256 doi:10.1016/j.ijdevneu.2006.09.257