The comparison of a novel continuous cardiac output monitor based on pulse wave transit time and echo Doppler during exercise.

A new technology called estimated continuous cardiac output (esCCO) uses pulse wave transit time (PWTT) obtained from an electrocardiogram and pulse oximeter to measure cardiac output (CO) non-invasively and continuously. This study was performed to evaluate the accuracy of esCCO during exercise testing. We compared esCCO with CO measured by the echo Doppler aortic velocity-time integral (VTIao_CO). The correlation coefficient between esCCO and VTIao_CO was r= 0.87 (n= 72). Bias and precision were 0.33 ± 0.95 L/min and percentage error was 31%. The esCCO could detect change in VTIao_CO larger than 1 L/min with a concordance rate of 88%. In polar plot, 83% of data are within 0.5 L/min, and 100% of data are within 1 L/min. Those results show the acceptable accuracy and trend ability of esCCO. Change in pre-ejection period (PEP) measured by using M-mode of Diagnostic Ultrasound System accounted for approximately half of change in PWTT. This indicates that PEP included in PWTT has an impact on the accuracy of esCCO measurement. In this study, the validity of esCCO during exercise testing was assessed and shown to be acceptable. The result of this study suggests that we can expand its application.

Inhibitory and excitatory subtypes of cochlear nucleus neurons are defined by distinct bHLH transcription factors, Ptf1a and Atoh1.

The cochlear nucleus (CN), which consists of dorsal and ventral cochlear nuclei (DCN and VCN), plays pivotal roles in processing and relaying auditory information to the brain. Although it contains various types of neurons, the origins of the distinct subtypes and their developmental molecular machinery are still elusive. Here we reveal that two basic helix-loop-helix transcription factors play crucial roles in specifying neuron subtypes in the CN. Pancreatic transcription factor 1a (Ptf1a) and atonal homolog 1 (Atoh1) were found to be expressed in discrete dorsolateral regions of the embryonic neuroepithelia of the middle hindbrain (rhombomeres 2-5). Genetic lineage tracing using mice that express Cre recombinase from the Ptf1a locus or under the control of the Atoh1 promoter revealed that inhibitory (GABAergic and glycinergic) or excitatory (glutamatergic) neurons of both DCN and VCN are derived from the Ptf1a- and Atoh1-expressing neuroepithelial regions, respectively. In the Ptf1a or Atoh1 null embryos, production of inhibitory or excitatory neurons, respectively, was severely inhibited in the CN. These findings suggest that inhibitory and excitatory subtypes of CN neurons are defined by Ptf1a and Atoh1, respectively and, furthermore, provide important insights into understanding the machinery of neuron subtype specification in the dorsal hindbrain.

The p21-activated kinase is required for neuronal migration in the cerebral cortex.

The normal formation and function of the mammalian cerebral cortex depend on the positioning of its neurones, which occurs in a highly organized, layer-specific manner. The correct morphology and movement of neurones rely on synchronized regulation of their actin filaments and microtubules. The p21-activated kinase (Pak1), a key cytoskeletal regulator, controls neuronal polarization, elaboration of axons and dendrites, and the formation of dendritic spines. However, its in vivo role in the developing nervous system is unclear. We have utilized in utero electroporation into mouse embryo cortices to reveal that both loss and gain of Pak1 function affect radial migration of projection neurones. Overexpression of hyperactivated Pak1 predominantly caused neurones to arrest in the intermediate zone (IZ) with apparently misoriented and disorganized leading projections. Loss of Pak1 disrupted the morphology of migrating neurones, which accumulated in the IZ and deep cortical layers. Unexpectedly, a significant number of neurones with reduced Pak1 expression aberrantly entered into the normally cell-sparse marginal zone, suggesting their inability to cease migrating that may be due to their impaired dissociation from radial glia. Our findings reveal the in vivo importance of temporal and spatial regulation of the Pak1 kinase during key stages of cortical development.

Reduction of Ptf1a gene dosage causes pancreatic hypoplasia and diabetes in mice.

OBJECTIVE: Most pancreatic endocrine cells derive from Ptf1a-expressing progenitor cells. In humans, nonsense mutations in Ptf1a have recently been identified as a cause of permanent neonatal diabetes associated with pancreatic agenesis. The death of Ptf1a-null mice soon after birth has not allowed further insight into the pathogenesis of the disease; it is therefore unclear how much pancreatic endocrine function is dependent on Ptf1a in mammals. This study aims to investigate gene-dosage effects of Ptf1a on pancreas development and function in mice. RESEARCH DESIGN AND METHODS: Combining hypomorphic and null alleles of Ptf1a and Cre-mediated lineage tracing, we followed the cell fate of reduced Ptf1a-expressing progenitors and analyzed pancreas development and function in mice. RESULTS: Reduced Ptf1a dosage resulted in pancreatic hypoplasia and glucose intolerance with insufficient insulin secretion in a dosage-dependent manner. In hypomorphic mutant mice, pancreatic bud size was small and substantial proportions of pancreatic progenitors were misspecified to the common bile duct and duodenal cells. Growth with branching morphogenesis and subsequent exocrine cytodifferentiation was reduced and delayed. Total beta-cell number was decreased, proportion of non-beta islet cells was increased, and alpha-cells were abnormally intermingled with beta-cells. Interestingly, Pdx1 expression was decreased in early pancreatic progenitors but elevated to normal level at the mid-to-late stages of pancreatogenesis. CONCLUSIONS-The dosage of Ptf1a is crucial for pancreas specification, growth, total beta-cell number, islet morphogenesis, and endocrine function. Some neonatal diabetes may be caused by mutation or single nucleotide polymorphisms in the Ptf1a gene that reduce gene expression levels.

Origin of climbing fiber neurons and their developmental dependence on Ptf1a.

Climbing fiber (CF) neurons in the inferior olivary nucleus (ION) extend their axons to Purkinje cells, playing a crucial role in regulating cerebellar function. However, little is known about their precise place of birth and developmental molecular machinery. Here, we describe the origin of the CF neuron lineage and the involvement of Ptf1a (pancreatic transcription factor 1a) in CF neuron development. Ptf1a protein was found to be expressed in a discrete dorsolateral region of the embryonic caudal hindbrain neuroepithelium. Because expression of Ptf1a is not overlapping other transcription factors such as Math1 (mouse atonal homolog 1) and Neurogenin1, which are suggested to define domains within caudal hindbrain neuroepithelium (Landsberg et al., 2005), we named the neuroepithelial region the Ptf1a domain. Analysis of mice that express beta-galactosidase from the Ptf1a locus revealed that CF neurons are derived from the Ptf1a domain. In contrast, retrograde labeling of precerebellar neurons indicated that mossy fiber neurons are not derived from Ptf1a-expressing progenitors. We could observe a detailed migratory path of CF neurons from the Ptf1a domain to the ION during embryogenesis. In Ptf1a null mutants, putative immature CF neurons produced from this domain were unable to migrate or differentiate appropriately, resulting in a failure of ION formation. Apoptotic cells were observed in the mutant hindbrain. Furthermore, the fate of some cells in the Ptf1a lineage were changed to mossy fiber neurons in Ptf1a null mutants. These findings clarify the precise origin of CF neurons and suggest that Ptf1a controls their fate, survival, differentiation, and migration during development.

Neurabin-I is phosphorylated by Cdk5: implications for neuronal morphogenesis and cortical migration.

The correct morphology and migration of neurons, which is essential for the normal development of the nervous system, is enabled by the regulation of their cytoskeletal elements. We reveal that Neurabin-I, a neuronal-specific F-actin-binding protein, has an essential function in the developing forebrain. We show that gain and loss of Neurabin-I expression affect neuronal morphology, neurite outgrowth, and radial migration of differentiating cortical and hippocampal neurons, suggesting that tight regulation of Neurabin-I function is required for normal forebrain development. Importantly, loss of Neurabin-I prevents pyramidal neurons from migrating into the cerebral cortex, indicating its essential role during early stages of corticogenesis. We demonstrate that in neurons Rac1 activation is affected by the expression levels of Neurabin-I. Furthermore, the Cdk5 kinase, a key regulator of neuronal migration and morphology, directly phosphorylates Neurabin-I and controls its association with F-actin. Mutation of the Cdk5 phosphorylation site reduces the phenotypic consequences of Neurabin-I overexpression both in vitro and in vivo, suggesting that Neurabin-I function depends, at least in part, on its phosphorylation status. Together our findings provide new insight into the signaling pathways responsible for controlled changes of the F-actin cytoskeleton that are required for normal development of the forebrain.

Localized activation of p21-activated kinase controls neuronal polarity and morphology.

In the developing forebrain, neuronal polarization is a stepwise and initially reversible process that underlies correct migration and axon specification. Many aspects of cytoskeletal changes that accompany polarization are currently molecularly undefined and thus poorly understood. Here we reveal that the p21-activated kinase (Pak1) is essential for the specification of an axon and dendrites. In hippocampal neurons, activation of Pak1 is spatially restricted to the immature axon despite its uniform presence in all neurites. Hyperactivation of Pak1 at the membrane of all neurites or loss of Pak1 expression disrupts both neuronal morphology and the distinction between an axon and dendrites. We reveal that Pak1 acts on polarity in a kinase-dependent manner, by affecting the F-actin and microtubule cytoskeleton at least in part through Rac1 and cofilin. Our data are the first to demonstrate the importance of localized Pak1 kinase activation for neuronal polarization and differentiation.

Ptf1a, a bHLH transcriptional gene, defines GABAergic neuronal fates in cerebellum.

The molecular machinery governing glutamatergic-GABAergic neuronal subtype specification is unclear. Here we describe a cerebellar mutant, cerebelless, which lacks the entire cerebellar cortex in adults. The primary defect of the mutant brains was a specific inhibition of GABAergic neuron production from the cerebellar ventricular zone (VZ), resulting in secondary and complete loss of external germinal layer, pontine, and olivary nuclei during development. We identified the responsible gene, Ptf1a, whose expression was lost in the cerebellar VZ but was maintained in the pancreas in cerebelless. Lineage tracing revealed that two types of neural precursors exist in the cerebellar VZ: Ptf1a-expressing and -nonexpressing precursors, which generate GABAergic and glutamatergic neurons, respectively. Introduction of Ptf1a into glutamatergic neuron precursors in the dorsal telencephalon generated GABAergic neurons with representative morphological and migratory features. Our results suggest that Ptf1a is involved in driving neural precursors to differentiate into GABAergic neurons in the cerebellum.

Involvement of a Rac activator,P-Rex1, in neurotrophin-derived signaling and neuronal migration.

Rho-family GTPases play key roles in regulating cytoskeletal reorganization, contributing to many aspects of nervous system development. Their activities are known to be regulated by guanine nucleotide exchange factors (GEFs), in response to various extracellular cues. P-Rex1, a GEF for Rac, has been mainly investigated in neutrophils, in which this molecule contributes to reactive oxygen species formation. However, its role in the nervous system is essentially unknown. Here we describe the expression profile and a physiological function of P-Rex1 in nervous system development. In situ hybridization revealed that P-Rex1 is dynamically expressed in a variety of cells in the developing mouse brain, including some cortical and DRG neurons. In migrating neurons in the intermediate zone, P-Rex1 protein was found to localize in the leading process and adjacent cytoplasmic region. When transfected in pheochromocytoma PC12 cells, P-Rex1 can be activated by NGF, causing an increase in GTP-bound Rac1 and cell motility. Deletion analyses suggested roles for distinct domains of this molecule. Experiments using a P-Rex1 mutant lacking the Dbl-homology domain, a dominant-negative-like form, and small interfering RNA showed that endogenous P-Rex1 was involved in cell migration of PC12 cells and primary cultured neurons from the embryonic day 14 cerebral cortices, induced by extracellular stimuli (NGF, BDNF, and epidermal growth factor). Furthermore, in utero electroporation of the mutant protein into the embryonic cerebral cortex perturbed radial neuronal migration. These findings suggest that P-Rex1, which is expressed in a variety of cell types, is activated by extracellular cues such as neurotrophins and contributes to neuronal migration in the developing nervous system.

Roles of STEF/Tiam1, guanine nucleotide exchange factors for Rac1, in regulation of growth cone morphology.

Rho family GTPases are suggested to be pivotal for growth cone behavior, but regulation of their activities in response to environmental cues remains elusive. Here, we describe roles of STEF and Tiam1, guanine nucleotide exchange factors for Rac1, in neurite growth and growth cone remodeling. We reveal that, in primary hippocampal neurons, STEF/Tiam1 are localized within growth cones and essential for formation of growth cone lamellipodia, eventually contributing to neurite growth. Furthermore, experiments using a dominant-negative form demonstrate that STEF/Tiam1 mediate extracellular laminin signals to activate Rac1, promoting neurite growth in N1E-115 neuroblastoma cells. STEF/Tiam1 are revealed to mediate Cdc42 signal to activate Rac1 during lamellipodial formation. We also show that RhoA inhibits the STEF/Tiam1-Rac1 pathway. These data are used to propose a model that extracellular and intracellular information is integrated by STEF/Tiam1 to modulate the balance of Rho GTPase activities in the growth cone and, consequently, to control growth cone behavior.