Lhx2 mediates the activity of Six3 in zebrafish forebrain growth.

The telencephalon shows the greatest degree of size variation in the vertebrate brain. Understanding the genetic cascade that regulates telencephalon growth is crucial to our understanding of how evolution of the normal human brain has supported such a variation in size. Here, we present a simple and quick approach to analyze this cascade that combines caged-mRNA technology and the use of antisense morpholino oligonucleotides in zebrafish embryos. Lhx2, a LIM-homeodomain protein, and Six3s (Six3b and Six3a), another homeodomain proteins, show very similar expression patterns early in forebrain development, and these are known to be involved in the growth of this part of the brain. The telencephalon of six3b and six3a double morphant (six3 morphant) embryos is markedly reduced in size due to impaired cellular proliferation. Head-specific overexpression of Lhx2 by photoactivation of a caged-lhx2 mRNA completely rescued this size reduction, whereas similar head-specific activation of Six3b could not rescue the knockdown effect of lhx2. In the forebrain of medaka embryos, Six3 facilitates cellular proliferation by sequestration of Geminin from Cdt1, a key component in the assembly of the prereplication complex. Our results suggest that Lhx2 may mediate an alternative or parallel pathway for control of cellular proliferation in the developing forebrain via Six3.

Zebrafish gcmb is required for pharyngeal cartilage formation.

The glial cells missing (gcm) gene in Drosophila encodes a GCM-motif transcription factor that functions as a binary switch to select between glial and neuronal cell fates. To understand the function of gcm in vertebrates, we isolated the zebrafish gcmb and analyzed the function of this gene using antisense morpholino oligonucleotides against gcmb mRNA (gcmb-MO) and transgenic overexpression. Zebrafish gcmb is expressed in the pharyngeal arch epithelium and in cells of the macrophage lineage. gcmb-MO-injected larvae show significantly reduced branchial arch cartilages. fgf3-MO-injected larvae display a similar phenotype to that of gcmb-MO-injected larvae with respect to the lack of pharyngeal cartilage formation. In addition, gcmb expression in the pharyngeal arches is down-regulated in fgf3-MO-injected larvae. The gcmb transgenic larvae show a protrusion of the lower jaw and abnormal spatial arrangement of the pharyngeal cartilage elements. These results suggest that gcmb is required for normal pharyngeal cartilage formation in zebrafish and that its expression is dependent on fgf3 activity.

Neural cell adhesion molecule L1 is required for fasciculation and routing of thalamocortical fibres and corticothalamic fibres.

To examine the role of neural cell adhesion molecule L1 in thalamocortical projections, we analysed L1 deficient (L1-/y) mice. Immunohistochemistry of pleiotrophin/HB-GAM, a marker for thalamocortical axons and axonal tracing experiments showed that thalamocortical axons were abnormally and highly fasciculated when they pass through the developing internal capsule. Within the cortex, however, their course was more diffuse. The corticofugal fibres immunoreactive for TAG-1 were also more strongly fasciculated and their number was decreased in L1-/y mice. Furthermore, no TAG-1-positive corticofugal axons reached the dorsal thalamus. These data suggest that L1 plays an important role in the fasciculation and routing of axons connecting between the thalamus and the cortex.

Towards crystallization of hydrophobic myelin glycoproteins: P0 and PASII/PMP22.

The preparation of a pure and homogeneous protein sample at proper concentration is a prerequisite for success when attempting their crystallization for structural determination. The detergents suitable for solubilization particularly of membrane proteins are not always the best for crystallization. Myelin of the peripheral nervous system of vertebrates is the example of a membrane for which neutral or "gentle" detergents are not even strong enough to solubilize its proteins. In contrast, sodium- or lithium-dodecyl sulfate is very effective. We solubilized myelin membrane in 2%(w/v) sodium dodecyl sulfate, followed by chromatographic purification of the hydrophobic myelin glycoproteins P0 and PASII/PMP22, and finally, we have exchanged the sodium dodecyl sulfate bound to protein for other neutral detergents using ceramic hydroxyapatite column. Theoretically, we should easily exchange sodium dodecyl sulfate for any neutral detergent, but for some of them, the solubility of myelin glycoproteins is low. To monitor the potential variability in the secondary structure of glycoproteins, we have used circular dichroism. Sodium dodecyl sulfate seems to be the appropriate detergent for the purpose of purification of very hydrophobic glycoproteins, since it can be easily exchanged for another neutral detergent.

Immuno-viral therapy of brain tumors by combination of viral therapy with cancer vaccination using a replication-conditional HSV.

Here we developed an effective therapeutic approach using a replication-conditional mutant of herpes simplex virus (HSV), G207, for the treatment of metastatic tumors in the immunologically privileged central nervous system. An experimental model of brain metastasis was developed using BALB/c mice that harbored both intracranial (i.c.) and subcutaneous (s.c.) mouse CT26 colon adenocarcinoma tumors. Intratumoral injections of G207 into s.c. tumors elicited cytotoxic T-cell responses not only to HSV but also to a tumor antigen; however, only a limited antitumor effect was observed on metastatic brain tumors. To improve this antitumor effect, G207 was also injected into the brain tumor. After intratumoral injections of G207 into both i.c. and s.c. CT26 tumors, a significant antitumor effect was observed in the metastatic brain tumors. This therapeutic efficacy was absent in athymic mice, indicating that the antitumor effect could be mediated by T cells. Cytotoxic T-cell responses to HSV and the tumor antigen were induced by injections of G207 into i.c. and s.c. CT26 tumors. These results suggest that HSV-infected brain tumors may be efficiently eliminated by the induced anti-HSV T cells as well as by antitumor T cells. Therefore, this strategy of immuno-viral therapy, involving direct viral oncolytic activities and inducing antitumor and antiviral immune responses, may be useful for the treatment of tumors in the immunologically privileged central nervous system.