Genetic subdivision of the tectum and cerebellum into functionally related regions based on differential sensitivity to engrailed proteins

The genetic pathways that partition the developing nervous system into functional systems are largely unknown. The engrailed (En) homeobox transcription factors are candidate regulators of this process in the dorsal midbrain (tectum) and anterior hindbrain (cerebellum). En1 mutants lack most of the tectum and cerebellum and die at birth, whereas En2 mutants are viable with a smaller cerebellum and foliation defects. Our previous studies indicated that the difference in phenotypes is due to the earlier expression of En1 as compared with En2, rather than differences in protein function, since knock-in mice expressing En2 in place of En1 have a normal brain. Here, we uncovered a wider spectrum of functions for the En genes by generating a series of En mutant mice. First, using a conditional allele we demonstrate that En1 is required for cerebellum development only before embryonic day 9, but plays a sustained role in forming the tectum. Second, by removing the endogenous En2 gene in the background of En1 knock-in alleles, we show that Drosophila en is not sufficient to sustain midbrain and cerebellum development in the absence of En2, whereas En2 is more potent than En1 in cerebellum development. Third, based on a differential sensitivity to the dose of En1/2, our studies reveal a genetic subdivision of the tectum into its two functional systems and the medial cerebellum into four regions that have distinct circuitry and molecular coding. Our study suggests that an 'engrailed code' is integral to partitioning the tectum and cerebellum into functional domains.