Yoko Suda

The c-Jun N-Terminal Kinase Activator Dual Leucine Zipper Kinase Regulates Axon Growth and Neuronal Migration in the Developing Cerebral Cortex

Mammalian corticogenesis substantially depends on migration and axon projection of newborn neurons that are coordinated by a yet unidentified molecular mechanism. Dual leucine zipper kinase (DLK) induces activation of c-Jun N-terminal kinase (JNK), a molecule that regulates morphogenesis in various organisms. We show here, using gene targeting in mice, that DLK is indispensable for establishing axon tracts, especially those originating from neocortical pyramidal neurons of the cerebrum. Direct and quantitative analysis of radial migration of pyramidal neurons using slice culture and a time-lapse imaging system revealed that acceleration around the subplate was affected by DLK gene disruption and by administration of a JNK inhibitor. Phosphorylation of JNK substrates, including c-Jun and doublecortin, and of JNK itself at the activation loop were partially affected in brains of DLK-deficient mouse embryos. These data suggest that DLK plays a significant role in the coordinated regulation of radial migration and axon projection by modulating JNK activity.

Cooperation between Otx1 and Otx2 genes in developmental patterning of rostral brain

Otx1 and Otx2 genes are mouse cognates of a Drosophila head gap gene orthodenticle. The homozygous mutants have previously indicated that Otx2 is essential to development of structures anterior to rhombomere 3, probably reflecting its expression around the early primitive streak stage. Otx2 mutation also exhibits craniofacial defects by haplo-insufficiency. Affected structures correspond to the most anterior and most posterior parts of the Otx2 expression where Otx1 is not, or is only weakly, expressed at the time of brain regionalization. No apparent defects are found in early brain development by the Otx1 mutation, suggesting that the Otx1 and Otx2 functions overlap in the regions where both are expressed. To demonstrate this, the Otx1/Otx2 double heterozygous phenotype was examined in this study. Analyses with molecular markers at 9.5 days post coitus suggested the failure in development of mesencephalon and caudal diencephalon with the expansion of anterior metencephalon. Genes expressed in isthmus exhibited a characteristic lateral stripe normally, although rostrally shifted, except that Fgf8 expression was expanded dorsally. The defects were apparent at the 6-somite stage, but not at the 3-somite stage. Broad Fgf8 expression at the 3-somite stage took place normally, but it did not concentrate into a spot corresponding to future isthmus. The double heterozygous phenotype implicates a previously unsuspected mechanism for development of the mes/metencephalic territory; at the 3- to 6-somite stage Otx1 cooperates with Otx2 to establish the mes/diencephalic domain, allowing for the correct development of isthmus/ rhombomere 1.

Zinc-finger genes Fez and Fez-like function in the establishment of diencephalon subdivisions.

Fez and Fez-like (Fezl) are zinc-finger genes that encode transcriptional repressors expressed in overlapping domains of the forebrain. By generating Fez;Fezl-deficient mice we found that a redundant function of Fez and Fezl is required for the formation of diencephalon subdivisions. The caudal forebrain can be divided into three transverse subdivisions: prethalamus (also called ventral thalamus), thalamus (dorsal thalamus) and pretectum. Fez;Fezl-deficient mice showed a complete loss of prethalamus and a strong reduction of the thalamus at late gestation periods. Genetic marker analyses revealed that during early diencephalon patterning in Fez;Fezl-deficient mice, the rostral diencephalon (prospective prethalamus) did not form and the caudal diencephalon (prospective thalamus and pretectum) expanded rostrally. Fez;Fezl-deficient mice also displayed defects in the formation of the zona limitans intrathalamica (ZLI), which is located on the boundary between the prethalamus and thalamus. Fez and Fezl are expressed in the region rostral to the rostral limit of Irx1 expression, which marks the prospective position of the ZLI. Transgene-mediated misexpression of Fezl or Fez caudal to the ZLI repressed the caudal diencephalon fate and affected the formation of the Shh-expressing ZLI. These data indicate that Fez and Fezl repress the caudal diencephalon fate in the rostral diencephalon, and ZLI formation probably depends on Fez/Fezl-mediated formation of diencephalon subdivisions.

Driven by the same Ig enhancer and SV40 T promoter ras induced lung adenomatous tumors, myc induced pre-B cell lymphomas and SV40 large T gene a variety of tumors in transgenic mice.

Different types of tumors developed in transgenic mice following the introduction of the entire coding region of ras, myc or SV40 large T gene (T) linked to the same regulatory unit, consisting of a human immunoglobulin gene enhancer (Ig) and SV40 early gene promoter (Tp) with a 21‐bp repeat. All the 12 transgenic mice harboring the intact T gene developed a variety of tumors including choroid plexus tumor, B cell lymphoma, histiocytic lymphoma, thymoma and others. This suggests that the Ig/Tp regulatory unit has transcriptional activity in these heterologous tissues. With this regulatory unit, myc gene induced solely pre‐B cell lymphomas (five out of nine mice). Contrary to our expectation, however, the mutated ras gene induced lung adenomatous tumors in six out of eight transgenic mice over the 10‐month observation period; the tumors are histologically comparable to adenocarcinomas in man. The tumors developed as early as 4 weeks after birth and the introduced ras gene was as efficiently expressed in both normal and neoplastic bronchioloalveolar epithelial cells as in normal lymphoid cells. An unidentified secondary event thus appears to be necessary for these ras‐expressing cells to become neoplastic, as observed for myc (Leder et al., 1986). In a variety of tumors induced by Ig/Tp‐T, on the other hand, T gene was expressed only in the tumor cells, but not in normal cells. Thus, derepression of T gene in normal cells appears to be closely related to their malignant change as observed in development of pancreatic acinar cell tumors by the T gene (Ornitz et al., 1985). These results suggest that ras and myc oncogenes penetrate differentially specific types of cells, while the SV40 T gene is tumorigenic in a variety of cell types.

A stream of cells migrating from the caudal telencephalon reveals a link between the amygdala and neocortex

The amygdaloid complex consists of diverse nuclei that belong to distinct functional systems, yet many issues about its development are poorly understood. Here, we identify a stream of migrating cells that form specific amygdaloid nuclei in mice. In utero electroporation showed that this caudal amygdaloid stream (CAS) originated in a unique domain at the caudal telencephalic pole that is contiguous with the dorsal pallium, which was previously thought to generate only neocortical cells. The CAS and the neocortex share mechanisms for specification (transcription factors Tbr1, Lhx2 and Emx1/2) and migration (reelin and Cdk5). Reelin, a critical cue for migration in the neocortex, and Cdk5, which is specifically required for migration along radial glia in the neocortex, were both selectively required for the normal migration of the CAS, but not for that of other amygdaloid nuclei. This is first evidence of a dorsal pallial contribution to the amygdala, demonstrating a developmental and mechanistic link between the amygdala and the neocortex.

Induction of a variety of tumors by c-erbB2 and clonal nature of lymphomas even with the mutated gene (Val659----Glu659).

The c‐erbB2 gene is expressed uniquely in fetal epithelium in vivo and has been suggested to contribute to the development and/or progression of adenocarcinomas in man. In order to assess the oncogenicity of the c‐erbB2 gene in vivo, normal c‐erbB2 and mutant c‐erbB2 encoding glutamic acid instead of valine at position 659 within the transmembrane domain were introduced into mice under the transcriptional regulatory unit of mouse mammary tumor virus long terminal repeat (MMTV‐LTR) or immunoglobulin enhancer‐‐SV40 early gene promoter (Ig/Tp). In transgenic mice with normal c‐erbB2 under MMTV‐LTR, not only adenocarcinomas but also a variety of tumors including B lymphomas were induced at relatively late onset. Induction of pre‐B cell lymphomas with normal c‐erbB2 was also observed using the Ig/Tp regulatory unit within 6‐10 months in some members of one transgenic family among seven lines established. In contrast, with the mutant c‐erbB2 under the Ig/Tp regulatory unit, the lymphoma was induced neonatally in all members of four transgenic families among ten lines obtained. However, the immunoglobulin heavy chain gene rearrangement pattern indicated that even with the mutant c‐erbB2 the induced lymphomas were clonal.

Emx2 and Pax6 Function in Cooperation with Otx2 and Otx1 to Develop Caudal Forebrain Primordium That Includes Future Archipallium

One of the central issues in developmental neurobiology is how the forebrain is organized ontogenetically. The traditional view is that the anterior neuroectoderm first develops into mesencephalic and prosencephalic vesicles; the latter vesicle subsequently develops into the diencephalon and secondary prosencephalon, of which dorsal parts protrude to generate the telencephalon. The diencephalon yields the pretectum, thalamus, and prethalamus, and the telencephalon produces the archipallium, neopallium, and ganglionic eminences. By identifying cell descendants that once expressed Emx2 with use of the Cre knock-in mutant into the Emx2 locus and analyzing phenotypes of double mutants between Emx2 and Otx2/Otx1 and between Emx2 and Pax6, we propose that at the 3-6 somite stage, the anterior neuroectoderm develops into three primordia: midbrain, caudal forebrain, and rostral forebrain. The caudal forebrain primordium generates not only the pretectum, thalamus, and prethalamus but also the archipallium, cortical hem, choroid plexus, choroidal roof, and eminentia thalami. The primordium corresponds to the Emx2- or Pax6-positive region at the 3-6 somite stage that most probably does not include the future neopallium or commissural plate. Otx2 and Otx1 that are expressed in the entire future forebrain and midbrain cooperate with this Emx2 and Pax6 expression in the development of the caudal forebrain primordium; Emx2 and Pax6 functions are redundant. In the embryonic day 9.5 Emx2-/-Pax6-/- double mutant, the caudal forebrain remained unspecified and subsequently transformed into tectum in a mirror image of the endogenous one.

Otx1 function overlaps with Otx2 in development of mouse forebrain and midbrain

Background: We previously reported that the homozygous mutation of Otx2 gene, a mouse cognate of the Drosophila head gap gene orthodenticle, causes failure in the development of the rostral head anterior to rhombomere 3, which may correspond to earlier Otx2 expression in cells destined for the anterior mesoendoderm. At the same time, the Otx2 heterozygous mutation displayed a phenotype characterized as otocephaly, probably related to expression in the anterior neuroectoderm at the subsequent pharyngula stage. Defects were characteristic in the most anterior and posterior regions of Otx2 expression where Otx1, another mouse cognate of orthodenticle, is not or weakly expressed. They were not found in the region where Otx1 is expressed.

Env-derived gp55 gene of Friend spleen focus-forming virus specifically induces neoplastic proliferation of erythroid progenitor cells.

A group of retroviruses carrying truncated viral genes has recently been suggested as the cause of new patterns of diseases. One such virus is the replication defective component of the Friend murine leukemia virus (F‐MuLV) complex, called Friend spleen focus forming virus (F‐SFFV). This virus induces erythroblastosis, and a virion envelope‐related glycoprotein, gp55, encoded by F‐SFFV has been suggested as the pathogenic gene. The role of the gp55 gene is, however, yet unclear in the apparently multistep erythroleukemogenesis. By separately producing transgenic mice harboring the whole F‐SFFV DNA, the gp55 gene alone under the control of the retroviral long terminal repeat (LTR) and the gp55 gene under the control of cytoplasmic beta actin transcriptional regulatory unit, we show here that the gp55 gene is capable of inducing neoplastic proliferation of erythroid progenitor cells specifically in the absence of helper virus and other F‐SFFV sequences. Under the control of the viral LTR the gp55 expression was detected only in leukemic tissues, but under the control of cytoplasmic beta‐actin regulatory sequences, the gp55 was also expressed in a variety of normal tissues including preleukemic normal spleens. The development of erythroleukemia was suppressed under the genetic background of C57B1/6 mouse (resistant to F‐MuLV; Fv‐2rr), and required additional events even under the background of DDD mouse (susceptible to F‐MuLV; Fv‐2ss). The p53 and Spi‐1 genes were frequently aberrant in transplanted tumors and cell lines derived from them, but were not in primary leukemic spleens.

Evolutionary constraint on Otx2 neuroectoderm enhancers-deep conservation from skate to mouse and unique divergence in teleost

Otx2 is a paired type homeobox gene that plays essential roles in each step and site of head development in vertebrates. In the mouse, Otx2 expression in the anterior neuroectoderm is regulated primarily by two distinct enhancers: anterior neuroectoderm (AN) and forebrain/midbrain (FM) enhancers at 92 kb and 75 kb 5′of the Otx2 locus, respectively. The AN enhancer has activity in the entire anterior neuroectoderm at headfold and early somite stages, whereas the FM enhancer is subsequently active in the future caudal forebrain and midbrain ectoderm. In tetrapods, both AN and FM enhancers are conserved, whereas the AN region is missing in teleosts, despite overt Otx2 expression in the anterior neuroectoderm. Here, we show that zebrafish and fugu FM regions drive expression not only in the forebrain and midbrain but also in the anterior neuroectoderm at headfold stage. The analysis of coelacanth and skate genomic Otx2 orthologues suggests that the utilization of the two enhancers, AN and FM, is an ancestral condition. In contrast, the AN enhancer has been specifically lost in the teleost lineage with a compensatory establishment of AN activity within the FM enhancer. Furthermore, the AN activity in the fish FM enhancer was established by recruiting upstream factors different from those that direct the tetrapod AN enhancer, yet zebrafish FM enhancer is active in both mouse and zebrafish anterior neuroectoderm at the headfold stage.