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Differential Display of Genes Expressed at the Midbrain – Hindbrain Junction Identifies sprouty2: An FGF8-Inducible Member of a Family of Intracellular FGF Antagonists

https://doi.org/10.1006/mcne.1999.0801Get rights and content

Abstract

Specification and polarization of the midbrain and anterior hindbrain involve planar signals originating from the isthmus. Current evidence suggests that FGF8, expressed at the isthmus, provides this patterning influence. In this study, we have sought to identify novel genes which are involved in the process by which regional identity is imparted to midbrain and anterior hindbrain (rhombomere 1). An enhanced differential display reverse transcription method was used to clone cDNAs derived from transcripts expressed specifically in either rhombomere 1 or midbrain during the period of isthmic patterning activity. This gene expression screen identified 28 differentially expressed cDNAs. A clone upregulated in cDNA derived from rhombomere 1 tissue showed a 91% identity at the nucleotide level to the putative human receptor tyrosine kinase antagonist: sprouty2. In situ hybridization on whole chick embryos showed chick sprouty2 to be expressed initially within the isthmus and rhombomere 1, spatially and temporally coincident with Fgf8 expression. However, at later stages this domain was more extensive than that of Fgf8. Introduction of ligand-coated beads into either midbrain or hindbrain region revealed that sprouty2 could be rapidly induced by FGF8. These data suggest that sprouty2 participates in a negative feedback regulatory loop to modulate the patterning activity of FGF8 at the isthmus.

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      This effect can be seen when Fgf8 is expressed ectopically in caudal forebrain explants from mouse embryos (Liu et al., 1999) or when Fgf8-soaked beads are implanted in the caudal diencephalon or midbrain of chick embryos (Crossley et al., 1996; Martinez et al., 1999). Numerous downstream genes have been identified that are induced by Fgf8, including, but not limited to, members of the Engrailed, Pax, and Sprouty families (Crossley et al., 1996; Lee et al., 1997; Liu et al., 1999; Martinez et al., 1999; Minowada et al., 1999; Chambers et al., 2000; Furthauer et al., 2001). Reciprocally, the continued expression of Engrailed-1 and Engrailed-2 is required to sustain the presence of FGF8 (Liu and Joyner, 2001), and, in the developing chick brain, ectopic expression of Engrailed can shift the di-mesencephalic boundary and transform regions of dorsal diencephalon into tectum (Araki and Nakamura, 1999).

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    1

    Present address: Institute of Psychiatry, King's College London, 1 Windsor Walk, Denmark Hill, London SE5 8AF, UK.

    2

    To whom correspondence should be addressed at the MRC Brain Development Programme, Department of Developmental Neurobiology, King's College London, New Hunt's House, Guy's Campus, London SE1 9RT, UK. E-mail: [email protected].

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