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Current evidence suggests that the anterior segment of the vertebrate hindbrain, rhombomere 1, gives rise to the entire cerebellum. It is situated where two distinct developmental patterning mechanisms converge: graded signalling from an organising centre (the isthmus) located at the midbrain/hindbrain boundary confronts segmentation of the hindbrain. The unique developmental fate of rhombomere 1 is reflected by it being the only hindbrain segment in which no Hox genes are expressed. In this study we show that ectopic FGF8 protein, a candidate for the isthmic organising activity, is able to induce and repress gene expression within the hindbrain in a manner appropriate to rhombomere 1. Using a heterotopic, heterospecific grafting strategy we demonstrate that rhombomere 1 is able to express Hox genes but that both isthmic tissue and FGF8 inhibit their expression. Inhibition of FGF8 function in vivo shows that it is responsible for defining the anterior limit of Hox gene expression within the developing brain and thereby specifies the extent of the r1 territory. Previous studies have suggested that a retinoid morphogen gradient determines the axial limit of expression of individual Hox genes within the hindbrain. We propose a model whereby activation by retinoids is antagonised by inhibition by FGF8 in the anterior hindbrain to set aside the territory from which the cerebellum will develop.

The midbrain-hindbrain interface gives rise to a boundary of particular importance in CNS development as it forms a local signalling centre, the proper functioning of which is essential for the formation of tectum and cerebellum. Positioning of the mid-hindbrain boundary (MHB) within the neuroepithelium is dependent on the interface of Otx2 and Gbx2 expression domains, yet in the absence of either or both of these genes, organiser genes are still expressed, suggesting that other, as yet unknown mechanisms are also involved in MHB establishment. Here, we present evidence for a role for Notch signalling in stabilising cell lineage restriction and regulating organiser gene expression at the MHB. Experimental interference with Notch signalling in the chick embryo disrupts MHB formation, including downregulation of the organiser signal Fgf8. Ectopic activation of Notch signalling in cells of the anterior hindbrain results in an exclusion of those cells from rhombomeres 1 and 2, and in a simultaneous clustering along the anterior and posterior boundaries of this area, suggesting that Notch signalling influences cell sorting. These cells ectopically express the boundary marker Fgf3. In agreement with a role for Notch signalling in cell sorting, anterior hindbrain cells with activated Notch signalling segregate from normal cells in an aggregation assay. Finally, misexpression of the Notch modulator Lfng or the Notch ligand Ser1 across the MHB leads to a shift in boundary position and loss of restriction of Fgf8 to the MHB. We propose that differential Notch signalling stabilises the MHB through regulating cell sorting and specifying boundary cell fate.

The midbrain–hindbrain boundary (MHB) acts as an organiser/signalling centre to pattern tectal and cerebellar compartments. Cells in adjacent compartments must be distinct from each other for boundary formation to occur at the interface. Here we have identified the leucine-rich repeat (LRR) neuronal 1 (Lrrn1) protein as a key regulator of this process in chick. The Lrrn family is orthologous to the Drosophila tartan/capricious (trn/caps) family. Differential expression of trn/caps promotes an affinity difference and boundary formation between adjacent compartments in a number of contexts; for example, in the wing, leg and eye imaginal discs. Here we show that Lrrn1 is expressed in midbrain cells but not in anterior hindbrain cells. Lrrn1 is down-regulated in the anterior hindbrain by the organiser signalling molecule FGF8, thereby creating a differential affinity between these two compartments. Lrrn1 is required for the formation of MHB — loss of function leads to a loss of the morphological constriction and loss of Fgf8. Cells overexpressing Lrrn1 violate the boundary and result in a loss of cell restriction between midbrain and hindbrain compartments. Lrrn1 also regulates the glycosyltransferase Lunatic Fringe, a modulator of Notch signalling, maintaining its expression in midbrain cells which is instrumental in MHB boundary formation. Thus, Lrrn1 provides a link between cell affinity/compartment segregation, and cell signalling to specify boundary cell fate., Research highlights ►Lrrn1 is expressed in midbrain cells but not in anterior hindbrain cells. ►Lrrn1 is down-regulated in anterior hindbrain by FGF8. ►Lrrn1 creates a differential affinity between midbrain and hindbrain compartments. ►This forms a sharp compartment boundary at the midbrain–hindbrain interface. ►Lrrn1 is required for formation of the mid–hindbrain boundary.

Formation of the trochlear nerve within the anterior hindbrain provides a model system to study a simple axonal projection within the vertebrate central nervous system. We show that trochlear motor neurons are born within the isthmic organiser and also immediately posterior to it in anterior rhombomere 1. Axons of the most anterior cells follow a dorsal projection, which circumnavigates the isthmus, while those of more posterior trochlear neurons project anterodorsally to enter the isthmus. Once within the isthmus, axons form large fascicles that extend to a dorsal exit point. We investigated the possibility that the projection of trochlear axons towards the isthmus and their subsequent growth within that tissue might depend upon chemoattraction. We demonstrate that both isthmic tissue and Fgf8 protein are attractants for trochlear axons in vitro, while ectopic Fgf8 causes turning of these axons away from their normal routes in vivo. Both inhibition of FGF receptor activation and inhibition of Fgf8 function in vitro affect formation of the trochlear projection within explants in a manner consistent with a guidance function of Fgf8 during trochlear axon navigation.

RESUME Abstracts / Resumes 645s / Resumes 645 © 2000 NRC Canada Roles of FGF signalling in regional specification of midbrain and anterior hindbrain Ivor Mason, David Chambers, Carol Irving, Huma Shamim, and Jennifer Walshe MRC Brain Development Programme, Centre for Developmental Neurobiology, King’s College London, New Hunt’s House, Guy’s Campus, London SE1 9RT, U.K. Axial pattern is established along the vertebrate neural tube by both vertical signals from adjacent mesoderm and planar signals from organising centres. One of the best-characterised of the latter is the midbrain-hindbrain boundary, or isthmus. Tissue grafting studies have established that the isthmus is the source of signals that impart regional identity to midbrain and anterior hindbrain. Heterotopic transplants of isthmic tissue into anterior midbrain respecify it to develop posterior midbrain morphological and molecular characteristics, grafts into posterior forebrain convert that tissue to an ectopic midbrain, while grafts into posterior hindbrain cause the development of ectopic cerebellar structures. Fibroblast growth factor 8 (Fgf8) is expressed at the isthmus in all vertebrate classes. We, and others, have shown that ectopic introduction of Fgf8 in the avian embryo is sufficient to cause anterior midbrain to be converted to a posterior character and posterior forebrain to develop as midbrain. Much of this presentation will be devoted to the roles of isthmic Fgf8 in patterning anterior hindbrain; previous reports have suggested that Fgf8 does not participatein this process. Part of the fate of the most anterior hindbrain rhombomere (r), r1, is to give rise to the cerebellum. Data will be presented to show that Fgf8 functions to establish and pattern the r1 territory, thereby setting aside tissue to form the cerebellar anlage. A differential display PCR analysis to identify r1-specific genes has identified a member of a family of intracellular antagonists of Fgf signalling, sprouty2. sprouty2 is rapidly inducible by Fgf8 and its expression domain suggests that it functions as a long range antagonist of the Fgf8 signal within the anterior hindbrain. Finally, the results of studies on regeneration of isthmic tissue will be presented which provide evidence that Fgf8 and the isthmic organiser are induced and (or) maintained by a specific interaction between midbrain and r1 involving a diffusible signal.