Axon guidance proteins are critical for the correct wiring of the nervous system during development. Several axon guidance cues and their family members have been well characterized. More unidentified axon guidance cues are assumed to participate in the formation of the extremely complex nervous system. We identified a secreted protein, draxin, that shares no homology with known guidance cues. Draxin inhibited or repelled neurite outgrowth from dorsal spinal cord and cortical explants in vitro. Ectopically expressed draxin inhibited growth or caused misrouting of chick spinal cord commissural axons in vivo. draxin knockout mice showed defasciculation of spinal cord commissural axons and absence of all forebrain commissures. Thus, draxin is a previously unknown chemorepulsive axon guidance molecule required for the development of spinal cord and forebrain commissures.

Background: Leucine-rich repeats (LRRs) are highly versatile and evolvable protein-ligand interaction motifs found in a large number of proteins with diverse functions, including innate immunity and nervous system development. Here we catalogue all of the extracellular LRR (eLRR) proteins in worms, flies, mice and humans. We use convergent evidence from several transmembrane-prediction and motif-detection programs, including a customised algorithm, LRRscan, to identify eLRR proteins, and a hierarchical clustering method based on TribeMCL to establish their evolutionary relationships.

The mes-metencephalic boundary (isthmus) has been suggested to act as an organizer in the development of the optic tectum. Pax-5 was cloned as a candidate for regulator of the organizing center. Isthmus-specific expression of Pax-5 and analogy with the genetic cascade in Drosophila suggest that Pax-5 may be at a higher hierarchical position in the gene regulation cascade of tectum development. To examine this possibility, a gain-of-function experiment on Pax-5 was carried out. In ovo electroporation on E2 chick brain with the eucaryotic expression vector that encodes chick Pax-5 cDNA was used. Not only was a considerable amount of Pax-5 expressed ectopically in the transfected brain, but irregular bulging of the neuroepithelium was induced in the diencephalon and mesencephalon. At Pax-5 misexpressing sites, uptake of BrdU was increased. Histological examination of E7 transfected brain revealed that Pax-5 caused transdifferentiation of diencephalon into the tectum-like structure. In the bulges of the E7 mesencephalon, differentiation of laminar structure was repressed when compared to the normal side. In transfected embryos, En-2, Wnt-1 and Fgf8 were up-regulated ectopically, and Otx2 was down-regulated in the diencephalon to mesencephalon. Moreover, Ephrin-A2, which is expressed specifically in the tectum with a gradient highest at the caudal end, is suggested to be involved in pathfinding of the retinal fibers, and was induced in the bulges. When the mouse Fgf8 expression vector was electroporated, Pax-5 and chick Fgf8 were also induced ectopically. These results suggest that Pax-5, together with Fgf8, hold a higher position in the genetic hierarchy of the isthmus organizing center and regulate its activity.

Otx2 is expressed in the mesencephalon and prosencephalon, and Gbx2 is expressed in the rhombencephalon around stage 10. Loss-of-function studies of these genes in mice have revealed that Otx2 is indispensable for the development of the anterior brain segment, and that Gbx2 is required for the development of the isthmus. We carried out gain-of-function experiments of these genes in chick embryos with a newly developed gene transfer system, in ovo electroporation. When Otx2 was ectopically expressed caudally beyond the midbrain-hindbrain boundary (MHB), the alar plate of the metencephalon differentiated into the optic tectum instead of differentiating into the cerebellum. On the other hand, when Gbx2 was ectopically expressed at the mesencephalon, the caudal limit of the tectum shifted rostrally. We looked at the effects of misexpression on the isthmus- and tectum-related molecules. Otx2 and Gbx2 interacted to repress each other's expression. Ectopic Otx2 and Gbx2 repressed endogenous expression of Fgf8 in the isthmus, but induced Fgf8 expression at the interface between Otx2 and Gbx2 expression. Thus, it is suggested that interaction between Otx2 and Gbx2 determines the site of Fgf8 expression and the posterior limit of the tectum.

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Three classes of signaling molecule, VG1, WNT and BMP, play crucial roles in axis formation in the chick embryo. Although VG1 and WNT signals have a pivotal function in inducing the primitive streak and Hensen's node in the embryo midline, their action is complemented by that of BMP antagonists that protect the prospective axial tissue from the inhibitory influence of BMPs secreted from the periphery. We have previously reported that a secreted factor, chick Tsukushi (TSK), is expressed in the primitive streak and Hensen's node, where it works as a BMP antagonist. Here, we describe a new crucial function for TSK in promoting formation of the primitive streak and Hensen's node by positively regulating VG1 activity. We provide evidence that TSK directly binds VG1 in vitro, and that TSK and VG1 functionally interact in axis formation, as shown by biological assays performed in chick and Xenopus embryos. Furthermore, we show that alternative splicing of TSK RNA leads to the formation of two isoforms (TSKA, originally designated as TSK, and TSKB) that differ in their C-terminal region. Biochemical and biological assays indicate that TSKB is a much weaker BMP antagonist than TSKA, although both isoforms efficiently interact with VG1. Remarkably, although both TSKA and TSKB are expressed throughout the early extending primitive streak, their expression patterns diverge during gastrulation. TSKA expression concentrates in Hensen's node, a well-known source of anti-BMP signals, whereas TSKB accumulates in the middle primitive streak (MPS), a region known to work as a node-inducing center where VG1 expression is also specifically localized. Lossof-function experiments demonstrate that TSKB, but not TSKA, function is required in the MPS for induction of Hensen's node. Taken together, these results indicate that TSK isoforms play a crucial role in chick axis formation by locally modulating VG1 and BMP activities during gastrulation.

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