Rika Kawabata

The micro1 gene is necessary and sufficient for micromere differentiation and mid/hindgut-inducing activity in the sea urchin embryo

In the sea urchin embryo, micromeres have two distinct functions: they differentiate cell autonomously into the skeletogenic mesenchyme cells and act as an organizing center that induces endomesoderm formation. We demonstrated that micro1 controls micromere specification as a transcriptional repressor. Because micro1 is a multicopy gene with at least six polymorphic loci, it has been difficult to consistently block micro1 function by morpholino-mediated knockdown. Here, to block micro1 function, we used an active activator of micro1 consisting of a fusion protein of the VP16 activation domain and the micro1 homeodomain. Embryos injected with mRNA encoding the fusion protein exhibited a phenotype similar to that of micromere-less embryos. To evaluate micro1 function in the micromere, we constructed chimeric embryos composed of animal cap mesomeres and a micromere quartet from embryos injected with the fusion protein mRNA. The chimeras developed into dauerblastulae with no vegetal structures, in which the micromere progeny constituted the blastula wall. We also analyzed the phenotype of chimeras composed of an animal cap and a mesomere expressing micro1. These chimeras developed into pluteus larvae, in which the mesomere descendants ingressed as primary mesenchyme cells and formed a complete set of skeletal rods. The hindgut and a part of the midgut were also generated from host mesomeres. However, the foregut and nonskeletogenic mesoderm were not formed in the larvae. From these observations, we conclude that micro1 is necessary and sufficient for both micromere differentiation and mid/hindgut-inducing activity, and we also suggest that micro1 may not fulfill all micromere functions.

Cortical Gene Expression After a Conditional Knockout of 67 kDa Glutamic Acid Decarboxylase in Parvalbumin Neurons

In the cortex of subjects with schizophrenia, expression of glutamic acid decarboxylase 67 (GAD67), the enzyme primarily responsible for cortical GABA synthesis, is reduced in the subset of GABA neurons that express parvalbumin (PV). This GAD67 deficit is accompanied by lower cortical levels of other GABA-associated transcripts, including GABA transporter-1, PV, brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B, somatostatin, GABAA receptor α1 subunit, and KCNS3 potassium channel subunit mRNAs. In contrast, messenger RNA (mRNA) levels for glutamic acid decarboxylase 65 (GAD65), another enzyme for GABA synthesis, are not altered. We tested the hypothesis that this pattern of GABA-associated transcript levels is secondary to the GAD67 deficit in PV neurons by analyzing cortical levels of these GABA-associated mRNAs in mice with a PV neuron-specific GAD67 knockout. Using in situ hybridization, we found that none of the examined GABA-associated transcripts had lower cortical expression in the knockout mice. In contrast, PV, BDNF, KCNS3, and GAD65 mRNA levels were higher in the homozygous mice. In addition, our behavioral test battery failed to detect a change in sensorimotor gating or working memory, although the homozygous mice exhibited increased spontaneous activities. These findings suggest that reduced GAD67 expression in PV neurons is not an upstream cause of the lower levels of GABA-associated transcripts, or of the characteristic behaviors, in schizophrenia. In PV neuron-specific GAD67 knockout mice, increased levels of PV, BDNF, and KCNS3 mRNAs might be the consequence of increased neuronal activity secondary to lower GABA synthesis, whereas increased GAD65 mRNA might represent a compensatory response to increase GABA synthesis.

Krüppel-like is required for nonskeletogenic mesoderm specification in the sea urchin embryo

The canonical Wnt pathway plays a central role in specifying vegetal cell fate in sea urchin embryos. SpKrl has been cloned as a direct target of nuclear beta-catenin. Using Hemicentrotus pulcherrimus embryos, here we show that HpKrl controls the specification of secondary mesenchyme cells (SMCs) through both cell-autonomous and non-autonomous means. Like SpKrl, HpKrl was activated in both micromere and macromere progenies. To examine the functions of HpKrl in each blastomere, we constructed chimeric embryos composed of blastomeres from control and morpholino-mediated HpKrl-knockdown embryos and analyzed the phenotypes of the chimeras. Micromere-swapping experiments showed that HpKrl is not involved in micromere specification, while micromere-deprivation assays indicated that macromeres require HpKrl for cell-autonomous specification. Transplantation of normal micromeres into a micromere-less host with morpholino revealed that macromeres are able to receive at least some micromere signals regardless of HpKrl function. From these observations, we propose that two distinct pathways of endomesoderm formation exist in macromeres, a Krl-dependent pathway and a Krl-independent pathway. The Krl-independent pathway may correspond to the Delta/Notch signaling pathway via GataE and Gcm. We suggest that Krl may be a downstream component of nuclear beta-catenin required by macromeres for formation of more vegetal tissues, not as a member of the Delta/Notch pathway, but as a parallel effector of the signaling (Krl-dependent pathway).

Expression of Transcripts Selective for GABA Neuron Subpopulations across the Cortical Visuospatial Working Memory Network in the Healthy State and Schizophrenia

Visuospatial working memory (WM), which is impaired in schizophrenia, depends on a distributed network including visual, posterior parietal, and dorsolateral prefrontal cortical regions. Within each region, information processing is differentially regulated by subsets of γ-aminobutyric acid (GABA) neurons that express parvalbumin (PV), somatostatin (SST), or vasoactive intestinal peptide (VIP). In schizophrenia, WM impairments have been associated with alterations of PV and SST neurons in the dorsolateral prefrontal cortex. Here, we quantified transcripts selectively expressed in GABA neuron subsets across four cortical regions in the WM network from comparison and schizophrenia subjects. In comparison subjects, PV mRNA levels declined and SST mRNA levels increased from posterior to anterior regions, whereas VIP mRNA levels were comparable across regions except for the primary visual cortex (V1). In schizophrenia subjects, each transcript in PV and SST neurons exhibited similar alterations across all regions, whereas transcripts in VIP neurons were unaltered in any region except for V1. These findings suggest that the contribution of each GABA neuron subset to inhibitory regulation of local circuitry normally differs across cortical regions of the visuospatial WM network and that in schizophrenia alterations of PV and SST neurons are a shared feature across these regions, whereas VIP neurons are affected only in V1.