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

Abstract

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.

Distinct cerebellar foliation anomalies in a CHD7 haploinsufficient mouse model of CHARGE syndrome

“…The foliation defects identified in Chd7 gt/+ mice share some features with En1/En2 deficient mice (Cheng et al, 2010; Joyner, Herrup, Auerbach, Davis, & Rossant, 1991; Millen, Wurst, Herrup, & Joyner, 1994; Orvis et al, 2012; Sgaier et al, 2007). The Engrailed genes encode homeodomain containing transcription factors that are master regulators of cerebellar patterning (Desplan, Theis, & O'Farrell, 1985; Joyner, Kornberg, Coleman, Cox, & Martin, 1985; Joyner & Martin, 1987; Sillitoe, Stephen, Lao, & Joyner, 2008; Sillitoe, Vogel, & Joyner, 2010).…”

“…These genes are critical for normal cerebellar development, even after their early role in maintaining Fgf8 expression in the isthmic organizer is complete. In fact, in later stages of cerebellar development, En1/2 are essential for anterior‐posterior and medial‐lateral patterning of the cerebellum (Cheng et al, 2010; Joyner et al, 1991; Sgaier et al, 2007; Sillitoe et al, 2008; Sillitoe et al, 2010). Interestingly, our recent RNA‐seq analysis of Chd7 ‐deficient cerebellar GCps showed that En1 expression was upregulated in these cells, implicating CHD7 as a potential repressor of En1 expression (Whittaker et al, 2017).…”

Gli3 coordinates three-dimensional patterning and growth of the tectum and cerebellum by integrating Shh and Fgf8 signaling

“…Deletion of exon 8 and splicing from exon 7 to 9 result in a frameshift mutation upstream of the DNA-binding domain (Gli3 rec ). Since we previously showed that a short period of gene function can be sufficient to rescue the most severe defects observed in null mutants Sgaier et al, 2007), En1-Cre was used to remove Gli3 specifically in the mes/r1 by E9.0, about 36 hours after the onset of Gli3 expression (Hui et al, 1994;Kimmel et al, 2000;Li et al, 2002). To assess whether Gli3 is required for mes/r1 development after midgestation, Nestin-Cre was used to remove Gli3 from the entire neural tube around E11.5 Graus-Porta et al, 2001;Tronche et al, 1999).…”

Clonal analysis reveals granule cell behaviors and compartmentalization that determine the folded morphology of the cerebellum

“…Moreover, the pattern of cerebellar folds is conserved between species, suggesting developmental regulation of the allocation of cells to each fold. Indeed, in mouse mutants lacking alleles of engrailed 1 and 2 (En1/2) homeobox genes, the timing and placement of particular anchoring centers in the Cb is altered and is accompanied by changes in the shape of the intervening folia (Cheng et al, 2010;Millen et al, 1994;Orvis et al, 2012;Sgaier et al, 2007). It is therefore crucial to understand how the correct number of cells is allocated to each folium to produce the foliation pattern essential for normal cerebellar circuit formation and function.…”