Immediate effects of maternal separation on the development of interneurons derived from medial ganglionic eminence in the neonatal mouse hippocampus.

Aversive experiences, including maternal separation (MS), have been known as a risk for abnormal hippocampus development. Given that impairment of GABA inhibitory system is known as one of the common features of the abnormal hippocampal development induced by MS, we examined whether the MS on 4-day-old (P4) mice for 24 hr abolishes the interneuron development. We observed that the MS reduced the volume of dorsal hippocampus on P14 as long-term effects. In addition, the MS decreased the number of parvalbumin (PV)-positive interneuron on P14 and P28 in the dorsal hippocampus. We further examined the immediate effects of MS by measuring the percentage of glutamic acid decarboxylase (GAD) 67-positive interneurons among the immature interneurons derived from medial ganglionic eminence (MGE) progenitors marked in nkx2.1cre;ß-geo EGFP mice. During normal development from P4 to P5, the percentage of GAD67-positive interneurons among the MGE-derived interneurons in the dorsal hippocampus was significantly increased from 42.29% to 70.73% in the stratum pyramidale of the CA1 and increased from 46.4% to 56.99% in the stratum pyramidale of the CA2/3 region. However, the increase was not observed on P5 among the mice treated with the MS. These results suggest that the maturation of interneurons was suppressed by the MS. The suppressed maturation of interneurons may be one of the causes of the reduced volume of the hippocampus and PV+ interneurons observed in the hippocampus on P14 and P28 as a consequence of the MS during neonatal stage.

Coactosin accelerates cell dynamism by promoting actin polymerization.

During development, cells dynamically move or extend their processes, which are achieved by actin dynamics. In the present study, we paid attention to Coactosin, an actin binding protein, and studied its role in actin dynamics. Coactosin was associated with actin and Capping protein in neural crest cells and N1E-115 neuroblastoma cells. Accumulation of Coactosin to cellular processes and its association with actin filaments prompted us to reveal the effect of Coactosin on cell migration. Coactosin overexpression induced cellular processes in cultured neural crest cells. In contrast, knock-down of Coactosin resulted in disruption of actin polymerization and of neural crest cell migration. Importantly, Coactosin was recruited to lamellipodia and filopodia in response to Rac signaling, and mutated Coactosin that cannot bind to F-actin did not react to Rac signaling, nor support neural crest cell migration. It was also shown that deprivation of Rac signaling from neural crest cells by dominant negative Rac1 (DN-Rac1) interfered with neural crest cell migration, and that co-transfection of DN-Rac1 and Coactosin restored neural crest cell migration. From these results we have concluded that Coactosin functions downstream of Rac signaling and that it is involved in neurite extension and neural crest cell migration by actively participating in actin polymerization.

Secreted factor FAM3C (ILEI) is involved in retinal laminar formation.

FAM3C is a secreted factor, which is involved in the epithelial to mesenchymal transition. In transcriptome profiling of the mouse retina using microarray, we found that FAM3C is highly expressed in the retina. FAM3C is expressed in the ganglion cell layer (GCL) of the retina. To explore the function of FAM3C in retinal development, we cloned Xenopus FAM3C (XFAM3C), and performed Xenopus gain- and loss-of-function analysis. Overexpression of XFAM3C resulted in retinal laminar disorganization and an increase in eye size. Loss of function experiments of XFAM3C using antisense morpholino oligonucleotides caused photoreceptor cell dislocation. Cellular differentiation was not affected by either gain- or loss-of-function experiments of XFAM3C. These findings suggest that FAM3C is involved in retinal laminar formation processes in vertebrates.

Expression of chick Coactosin in cells in morphogenetic movement.

Coactosin is a 17 kDa actin binding protein that belongs to the actin depolymerizing factor/cofilin homology family. Coactosin inhibits barbed-end capping of actin filament, and is involved in actin polymerization. Coactosin is expressed in cephalic and trunk neural crest cells, cranial ganglia and dorsal root ganglia. Coactosin is also expressed in the cells that are forming mesonephric duct, and endodermal cells. Immunocytochemistry with anti-Coactosin antibody shows that Coactosin is localized in the cytoplasm, and associated with actin stress fibers in cultured neural crest cells. Coactosin is also expressed in the axon of oculomotor nerve and trigeminal nerve. In the growth cone of the oculomotor nerve axons, both Coactosin mRNA and protein were localized, which is indicative of the role of Coactosin in pathfinding of the growth cone. Coactosin is expressed in those that require dynamic and highly coordinated regulation of actin cytoskeleton, that is, neural crest cells, cells in the tip of the mesonephros, endodermal cells and axons.

Functional analysis of chick ONT1 reveals distinguishable activities among olfactomedin-related signaling factors.

The Olfactomedin family is a relatively new class of extracellular proteins. Two family members have been shown to play roles in the early development of ectodermal tissues: Noelin enhances neural crest generation in chick and Tiarin promotes dorsal neural specification in Xenopus. In this study, we introduce a novel member of the Olfactomedin family, ONT1. In the early chick embryo, ONT1 expression first appears at Hensen's node and subsequently in the axial and paraxial mesoderm. When the neural tube closes, strong expression of ONT1 is transiently found in the roof plate region from the rostral midbrain to the hindbrain. Overexpression of ONT1 in these regions prolongs the generation of neural crest cells in a manner similar to that of Noelin. Interestingly, ONT1 and Noelin have opposing effects on the expression of the migrating neural crest marker HNK-1 in the chick: they, respectively, cause suppression and ectopic induction of this marker. Differential activities among Olfactomedin-related factors are further examined in Xenopus. Microinjection of ONT1 mRNA into the Xenopus embryo expands the expression domain of the neural crest marker FoxD3 at the neurula stage whereas overexpression of Tiarin or Noelin suppresses FoxD3. ONT1 exhibits no dorsalizing effects on the Xenopus neural tube, which contrasts with the strong dorsalizing activity seen for Tiarin. Thus, distinct Olfactomedin-related factors evoke qualitatively different phenotypes even in the same experimental systems, suggesting that Olfactomedin family uses multiple response systems to mediate its signals in embryogenesis.

Gain- and loss-of-function in chick embryos by electroporation.

It remained very difficult to manipulate gene expression in chick embryos until the advent of in ovo electroporation which enabled the induction of both gain-of-function, and recently loss-of-function, of a gene of interest at a specific developmental stage. Gain-of-function by electroporation is so effective that it has become widely adopted in developmental studies in the chick. Recently, it became possible to induce loss-of-function by introducing an siRNA expression vector by electroporation. In this review, the methods of electroporation for gain-of-function and for loss-of-function by siRNA are discussed.

Otx2 is involved in the regional specification of the developing retinal pigment epithelium by preventing the expression of sox2 and fgf8, factors that induce neural retina differentiation.

The retinal pigment epithelium (RPE) shares its developmental origin with the neural retina (NR). When RPE development is disrupted, cells in the presumptive RPE region abnormally differentiate into NR-like cells. Therefore, the prevention of NR differentiation in the presumptive RPE area seems to be essential for regionalizing the RPE during eye development. However, its molecular mechanisms are not fully understood. In this study, we conducted a functional inhibition of a transcription factor Otx2, which is required for RPE development, using early chick embryos. The functional inhibition of Otx2 in chick eyes, using a recombinant gene encoding a dominant negative form of Otx2, caused the outer layer of the optic cup (the region forming the RPE, when embryos normally develop) to abnormally form an ectopic NR. In that ectopic NR, the characteristics of the RPE did not appear and NR markers were ectopically expressed. Intriguingly, the repression of Otx2 function also caused the ectopic expression of Fgf8 and Sox2 in the outer layer of the optic cup (the presumptive RPE region of normally developing eyes). These two factors are known to be capable of inducing NR cell differentiation in the presumptive RPE region, and are not expressed in the normally developing RPE region. Here, we suggest that Otx2 prevents the presumptive RPE region from forming the NR by repressing the expression of both Fgf8 and Sox2 which induce the NR cell fate.

Isthmus organizer for midbrain and hindbrain development.

Classical transplantation studies showed that the isthmus has an organizing activity upon the tectum and cerebellum. Since Fgf8 is expressed in the isthmus and mimics functionally isthmic grafts, it is accepted that Fgf8 plays pivotal role in the isthmic organizing activity. The fate of brain vesicles is determined by the combinations of transcription factors. The neural tube region where Otx2, Pax2, and En1 are expressed early on acquires midbrain identity. Pax3/7 forces the midbrain to differentiate into tectum. En1/2, Pax2/5, and Fgf8 form a positive feedback loop for their expression, thus misexpression of one of these molecules turns on the loop and forces presumptive diencephalon to differentiate into tectum. The isthmic organizer signal, Fgf8, stabilizes or changes the expression of the transcription factors in mid/hindbrain region. A strong Fgf8 signal activates the Ras-ERK signaling pathway, which in turn activates Irx2 in a rostrodorsal part of the hindbrain, and forces this tissue to differentiate into cerebellum.

Gene silencing in chick embryos with a vector-based small interfering RNA system.

In this paper, the use of vector-based RNA interference (RNAi) to specifically interfere with gene expression in chick embryos is reported. In ovo electroporation was carried out to transfer a small interfering RNA (siRNA) expression vector into chick embryos. En2 was chosen for the target gene because the family gene, En1, is expressed in a similar pattern. Four sets of 19-mer sequences were designed with the En2 open reading frame region connected to a sequence of short hairpin RNA (shRNA), which exerts siRNA effects after being transcribed, and inserted into pSilencer U6-1.0 vector. En2 and En1 expression were suppressed by the siRNA whose sequence completely matched En2 and En1. Suppression occurred when the siRNA sequence differed by up to two nucleotides from the target sequence. The sequence that differed by four nucleotides from the target gene did not show siRNA effects. One set that completely matched the En2 target did not show siRNA effects, which may be due to location of the siRNA in the target gene. Thus, multiple sets of shRNA must be prepared if we are to consider. This system will greatly contribute to the analysis of function of genes of interest, because the target gene can be silenced in a locally and temporally desired manner.

Role of Lmx1b and Wnt1 in mesencephalon and metencephalon development.

The isthmus is the organizing center for the tectum and cerebellum. Fgf8 and Wnt1 are secreted molecules expressed around the isthmus. The function of Fgf8 has been well analyzed, and now accepted as the most important organizing signal. Involvement of Wnt1 in the isthmic organizing activity was suggested by analysis of Wnt1 knockout mice. But its role in isthmic organizing activity is still obscure. Recently, it has been shown that Lmx1b is expressed in the isthmic region and that it may occupy higher hierarchical position in the gene expression cascade in the isthmus. We have carried out misexpression experiment of Lmx1b and Wnt1, and considered their role in the isthmic organizing activity. Lmx1b or Wnt1 misexpression caused expansion of the tectum and cerebellum. Fgf8 was repressed in a cells that misexpress Lmx1b, but Fgf8 expression was induced around Lmx1b-misexpressing cells. As Lmx1b induced Wnt1 and Wnt1 induced Fgf8 expression in turn, Wnt1 may be involved in non cell-autonomous induction of Fgf8 expression by Lmx1b. Wnt1 could not induce Lmx1b expression so that Lmx1b may be put at the higher hierarchical position than Wnt1 in gene expression cascade in the isthmus. We have examined the relationship among isthmus related genes, and discuss the mechanism of the formation and maintenance of isthmic organizing activity.

Possible role of Hes5 for the rostrocaudal polarity formation of the tectum.

The alar plate of the mesencephalon differentiates into the optic tectum. Retinal fibers project to the tectum topographically in a retinotopic manner. Engrailed (En) is responsible for the tectum polarity formation and regionalization. Former study indicated the presence of the molecule whose expression is repressed by En and that represses the isthmus-related gene expression. To isolate such molecules, we constructed a subtracted library between cDNA population of the normal rostral mesencephalon and of the rostral mesencephalon that misexpresses En2. From the library, we isolated cHes5, a chicken homolog of Drosophila hairy/Enhancer of split. cHes5 begins to be expressed in the rostral part of the E2 mesencephalon, and spreads to caudal mesencephalon by E3. To our expectation, cHes5 expression was repressed by En2. Furthermore, misexpression of cHes5 in the mesencephalon inhibited expression of ephrinA2, a marker of caudal mesencephalon. An active repressor form of Hes5, which is a chimeric molecule of Hes5 and repressor domain of En2, showed a similar but more severe phenotype. The results indicate that Hes5 is regulated by En and is responsible for rostral identity of mesencephalon by repressing ephrinA2.