Engrailed1 and Engrailed2 (En1 and En2) are two developmental genes of the homeogene family expressed in the developing midbrain. En1 and, to a lesser degree, En2 also are expressed in the adult substantia nigra (SN) and ventral tegmental area (VTA), two dopaminergic (DA) nuclei of the ventral midbrain. In an effort to study En1/2 adult functions, we have analyzed the phenotype of mice lacking one En1 allele in an En2 wild-type context. We show that in this mutant the number of DA neurons decreases slowly between 8 and 24 weeks after birth to reach a stable 38 and 23% reduction in the SN and VTA, respectively, and that neuronal loss can be antagonized by En2 recombinant protein infusions in the midbrain. These loss and gain of function experiments firmly establish that En1/2 is a true survival factor for DA neurons in vivo. Neuronal death in the mutant is paralleled by a 37% decrease in striatal DA, with no change in serotonin content. Using established protocols, we show that, compared with their wild-type littermates, En1ϩ/Ϫ mice have impaired motor skills, an anhedonic-like behavior, and an enhanced resignation phenotype; they perform poorly in social interactions. However, these mice do not differ from their wild-type littermates in anxiety-measuring tests. Together, these results demonstrate that En1/2 genes have important adult physiological functions. They also suggest that mice lacking only one En1 allele could provide a novel model for the study of diseases associated with progressive DA cell death.
Engrailed transcription factors regulate the expression of guidance cues that pattern retinal axon terminals in the dorsal midbrain. They also act directly to guide axon growth in vitro. We show here that an extracellular En gradient exists in the tectum along the anterior-posterior axis. Neutralizing extracellular Engrailed in vivo with antibodies expressed in the tectum causes temporal axons to map aberrantly to the posterior tectum in chick and Xenopus. Furthermore, posterior membranes from wild-type tecta incubated with anti-Engrailed antibodies or posterior membranes from Engrailed-1 knockout mice exhibit diminished repulsive activity for temporal axons. Since EphrinAs play a major role in anterior-posterior mapping, we tested whether Engrailed cooperates with EphrinA5 in vitro. We find that Engrailed restores full repulsion to axons given subthreshold doses of EphrinA5. Collectively, our results indicate that extracellular Engrailed contributes to retinotectal mapping in vivo by modulating the sensitivity of growth cones to EphrinA.
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