J Neural Transm (2006) [Suppl] 70: 47–55 # Springer-Verlag 2006

The engrailed transcription factors and the mesencephalic dopaminergic neurons

D. Gherbassi and H. H. Simon Department of Neuroanatomy, Interdisciplinary Center for Neuroscience, University of Heidelberg, Heidelberg, Germany

Summary. The engrailed genes belong to a 1999), echinoderms (Lowe and Wray, 1997) large family of homeobox transcription fac- and chordates, such as amphioxus (Holland tors. They are found throughout the animal et al., 1997) and multiple vertebrates (Joyner kingdom, are highly conserved in the DNA et al., 1985; Ekker et al., 1992; Logan et al., binding domain and have been investigated 1992; Koster et al., 1996; Lopez-Corrales for more than half a century. In the murine et al., 1998) including man (Zec et al., 1997; genome, two engrailed genes exist, called Barak et al., 2003). They belong to a large Engrailed-1 and Engrailed-2. Here, we sum- class of transcription factors with a con- marize the properties of the engrailed genes served DNA binding domain, the homeobox. and their functions, such as conserved struc- This group of genes is characterized by tures, cellular localisation, secretion and homeotic tissue transformation when their internalisation, transcription factor activity, expression is altered during early embryo- potential target genes and review their role in genesis. Some of them also take part in the the development of mesencephalic dopami- specification of neuronal properties, such as nergic neurons. During early development, directed axonal outgrowth, target recognition they take part in the regionalization event, and synaptogenesis. which specifies the neuroepithelium that pro- vides the precursor cells of the mesenceph- Engrailed genes across evolution alic dopaminergic neurons with the necessary and their molecular properties signals for their induction. Later in the post- mitotic neurons, the two transcription factors The first mentioning of the engrailed genes participate in their specification and are cell- was a spontaneously occurring mutation in autonomously required for their survival. Drosophila melanogaster characterized by the malformation of thoracic segments, scutel- Introduction lar modifications and distorted wings (Eker, 1929). The cloning of the underlying genes The engrailed genes have been investigated was achieved more than 50 years later (Fjose in species throughout the animal kingdom, in- et al., 1985; Kuner et al., 1985), its DNA cluding annelids (Wedeen and Weisblat, 1991), binding activity revealed (Desplan et al., molluscs (Wanninger and Haszprunar, 2001), 1985) and its function, as a transcriptional reg- insects and crustaceans (Patel et al., 1989; ulator, established (Han et al., 1989; Ohkuma Scholtz et al., 1994; Duman-Scheel and Patel, et al., 1990). In vertebrates, two engrailed 48 D. Gherbassi and H. H. Simon homologues exist (Ekker et al., 1992; Force regulatory target by affinity adjustment, but et al., 1999) with one exception, zebrafish, can also switch the transcriptional activity where genome duplication led to four para- from repressor to activator (Serrano and logues, each with distinct functional proper- Maschat, 1998; Gemel et al., 1999; Kobayashi ties (Ekker et al., 1992; Force et al., 1999). et al., 2003). Examples for engrailed asso- On the protein level, sequence differences ciated molecules are the hepatocyte nuclear between homologues (orthologues and para- factor 3 beta (HNF3b=Foxa2) (Foucher et al., logues) are significant, but the homeobox 2003) and the pre B-cell leukemia transcrip- domain is always highly conserved. Despite tion factor 1 (Pbx1) (Gemel et al., 1999). the large sequence differences outside the homeobox, the biochemical conservation of Cell localization and the engrailed genes is impressive. They show secretion=internalisation of engrailed functional homology over more than 100 proteins million years of evolution and the paralogues are in most aspects functionally redundant. As transcriptional regulators, the engrailed When mouse Engrailed-1 (En1) is replaced proteins are localized in the nucleus, how- by homologous recombination with its paralo- ever, a small proportion (5%) of the intra- gue Engrailed-2 (En2) or with its drosophila cellular protein is found in the cytoplasm homologue, the otherwise lethal phenotype, associated to membrane vesicles, that can be with significant defects in mid- and hind- transported anterogradely in the axon (Joliot brain, is rescued. The resulting animals are et al., 1997). Surprisingly, this cytoplasmic viable and fertile with normal brain morphol- proportion of the proteins can be secreted ogy (Hanks et al., 1995, 1998), suggesting and internalised by other cells. Export from that a common conserved genetic pathway the nucleus, secretion and internalisation exists in protostomia and deuterostomia. depends on the third helix of the homeobox The protein sequence analysis revealed and the first few aminoacids N-terminal to it five distinct subdomains in the engrailed pro- (Joliot et al., 1998; Maizel et al., 1999). This teins, designated as engrailed homology (EH) seemed to happen via an energy- and receptor- regions (EH1-5) (Logan et al., 1992). The protein-independent mechanism without in- EH4 region represents the homeodomain and volving classical exo=endocytosis (Han et al., includes approximately 60 amino acids, that 2000; Cosgaya et al., 1998; Joliot et al., 1998), form three alpha helices, common with all but it is nevertheless a highly regulated pro- classes of homeodomain proteins (Manak and cess, since phosphorylation of the serine-rich Scott, 1994). The third helix is responsible domain inhibits secretion and nuclear export, for the binding to specific sequences in the and simultaneously increases DNA binding large groove of double stranded DNA by several fold (Maizel et al., 1999, 2002). (Desplan et al., 1988; Kissinger et al., 1990; The nuclear export opens the possibility, that Ades and Sauer, 1994). Besides the DNA engrailed can function outside the nucleus and binding activity, the engraileds can also act as intercellular polypeptidic messengers establish proteic interactions through the (Joliot et al., 1998; Maizel et al., 1999) and EH1, 2, 3 and 5 domains. The proteins act might for example locate mRNAs into axonal mostly as transcriptional repressors via the terminals (Joliot et al., 1997). Most of these EH1 and EH5 domains (Han et al., 1989; studies have been performed in cell culture Ohkuma et al., 1990; Smith and Jaynes, and they still need confirmation in vivo, how- 1996), however this inhibitory function is ever they suggest that homeoprotein transcrip- not a fixed property. Protein interactions me- tion factors may have non-cell-autonomous diated by EH2 and EH3 can determine the signaling properties and act in a paracrine The engrailed transcription factors and the mesDA neurons 49 manner (Prochiantz, 1999; Prochiantz and pression in the midbrain, however it is essen- Joliot, 2003). tial for the maintenance of this expression (Danielian and McMahon, 1996). Expression of engrailed in the murine brain, pattern formation Differentiation of mesDA neurons of mid=hindbrain and neurogenesis and survival of mesDA neurons Briefly after the rostral to caudal neuroaxis has The most common function of the engrailed been laid down, mesDA neurons are induced genes in all investigated phyla is an in- in the ventral midbrain by the combined in- volvement in pattern formation during early teraction of two diffusible molecules, sonic embryogenesis (Hidalgo, 1998; Wurst and hedgehog (SHH) and the fibroblast growth Bally-Cuif, 2001). Later in development, they factor 8 (FGF8), released by the floor plate take part in neurogenesis (Condron et al., and isthmus, respectively (Ye et al., 1998). 1994; Harzsch et al., 1998) and neuronal Thereafter, cells become postmitotic (Altman differentiation (Saueressig et al., 1999; Marie and Bayer, 1981) and begin to express the first et al., 2000). In vertebrates, the early devel- markers specific for their neurotransmitter opmental role of En1 and En2 arises from phenotype (Perrone-Capano and Di Porzio, their expression in a broad band rostral and 2000). Three regulatory pathways involved in caudal to the isthmus, the border between the differentiation of the mesDA neurons mid- and hindbrain (Fig. 1C, D). Targeted have been identified so far. The key transcrip- inactivation of the genes results in a gene- tion factors for these pathways are Nurr1, dose dependent deletion of mid- and hind- Lmx1b and the engrailed genes (reviewed in brain structure mostly affecting the colliculi, Simon et al., 2003). In contrast to Nurr1, the the cerebellum (Fig. 1G, H) and ventrally engrailed expression in the postmitotic neu- located neuronal populations like the seroto- rons begins relatively late and is initially only nergic neurons of dorsal raphe nucleus, the detectable in the surrounding midbrain tissue. noradrenergic cells of the locus coeruleus and The onset of expression in the postmitotic the motor nuclei of the III and IVth nerve neurons occurs gradually over 72 hours, with (Wurst et al., 1994; Simon et al., 2005). Their the first TH positive cells in the ventral mid- regional expression is probably determined by brain expressing the two genes at E11, the other, earlier expressed transcription factors, latest start to express them at E14 (Alberi like Otx2, Pax2, Pax5 and Gbx2 (Wurst and et al., 2004). This expression persists from Bally-Cuif, 2001), since their mutant pheno- then on throughout the life of the animal type in respect to the midbrain is rather simi- (Fig. 1A, B). lar (McMahon and Bradley, 1990; Acampora It seems that the neuroepithelial precursor et al., 1995; Ang et al., 1996; Schwarz et al., cells never express the engrailed genes. The 1997) and their ectopic expressions by domain role of the two transcription factors is in- shifts alter the engrailed expression domains direct, arising from the fact that they regulate and mid=hindbrain structures in manner con- the FGF8 expression, which is essential for the cordant with this notion (Hidalgo-Sanchez induction of these precursor cells. In embryos et al., 1999; Brodski et al., 2003; Puelles et al., homozygous null for En1 and En2, the initial 2003, 2004). Another molecule that plays a FGF8 expression at E8 is wild type-like, key role in the regulation of engrailed ex- however no FGF8 is detectable at the mid= pression during early embryogenesis is Wnt1, hindbrain border at E9 (Liu and Joyner, 2001). a diffusible signaling protein. Its expression The lower number of postmitotic mesDA neu- has no influence on the initial engrailed ex- rons in E12 engrailed double mutant embryos 50 D. Gherbassi and H. H. Simon The engrailed transcription factors and the mesDA neurons 51

(Fig. 1E, F) (Simon et al., 2001; Alberi et al., RNA interference experiments on primary 2004) could be explained by this altered midbrain cell cultures showed that cell death FGF8 expression. A conserved region in is induced in less than 24 hours after trans- the large intron of the FGF8 gene with an fection, indicating that the engraileds may active En1 binding site exists in the genome regulate one or more genes in the apoptosis of higher vertebrates (Gemel et al., 1999 and pathway. our own unpublished database search; Simon et al., 2004) suggesting a direct regulation of Engrailed regulatory targets FGF8 by the engrailed genes. Ectopic FGF8 expression in chicken embryonic midbrain The close connection of mesDA neurons to one infected with retroviruses expressing En1 of the most prominent human neurodegen- supports this idea (Shamim et al., 1999). erative disorders makes it of high interest to The most noticeable function of the identify genes that are involved in the survival engrailed genes in respect to mesDA neurons of these cells. Since the role at the engrailed is their close connection to the survival of genes in the survival of mesDA neurons can- these cells. Despite the early morphological not be a direct consequence of their DNA deletion of mid=hindbrain structures, mesDA binding property or protein–protein interac- neurons are generated in the engrailed dou- tions with other transcriptional regulators, the ble mutant mice and start to express markers effector genes must be downstream to them. typical for their neurotransmitter phenotype. The most interesting candidate target gene to However, shortly thereafter the mesDA neu- our opinion is a-synuclein, a presynaptic pro- rons die with signs of apoptosis and at E14 tein, not expressed in mesDA neurons of all are lost (for P0 Fig. 1I–L), suggesting that the engrailed double mutants (Simon et al., in parallel to the onset of genes expression in 2001). a-Synuclein was the first gene asso- the wild type, a requirement for the engrailed ciated to familial form of Parkinson’s disease genes sets in which leads to death of the cells (Polymeropoulos et al., 1997; Kruger et al., if not met in the mutant (Simon et al., 2001; 1998), however its inactivation by homol- Alberi et al., 2004). By performing cell mix- ogous recombination in mice leads only to ing in vitro and in vivo (chimera), we demon- minor changes in the nigrostriatal system strated that the engrailed requirement for the (Abeliovich et al., 2000). survival of mesDA neurons is cell-autono- An approach to isolate genes downstream mous and not attributable to the deletion of of the engrailed was recently attempted in midbrain tissue in the mutant. Furthermore, our laboratory (Thuret et al., 2004a, b). We

1 Fig. 1. Engrailed expression in mouse brain and loss of mesDA neurons in absence of both engrailed genes. Sagittal sections of adult (A, B) and P0 brains (G–L) and two whole mount preparation (C–F), immunostained against the b-gal, to detect the En1=tauLacZ reporter (A, C, D) and against tyrosine hydroxylase (TH) (B, E, F, I–L), and Nissl stained (G, H). A, B The expression pattern of the tauLacZ reporter genes demonstrates that in adult animals En-1 is expressed in the inferior colliculus, the cerebellum and the mesDA neurons. The axonal projection to the basal ganglia revealed by the reporter is identical to TH. C, D At E9, En-1 is expressed in a broad band rostral and caudal to the isthmus (arrow) in the wild type, while in the engrailed double mutant embryos (En1-=-;En2-=-) part of mid=hindbrain is absent and the expression of the En1=tauLacZ reporter is reduced to a small domain in the ventral midbrain (E, F). At E12, TH positive cells are detectable in the ventral midbrain of wild type and double mutants. The mutant domain is smaller and no axonal outgrowth is detectable (G, H). The Nissl staining of the P0 brain section reveals loss of inferior (IC) and superior colliculus (SC), cerebellum (Cb) and large parts of the periaqueductal central grey (CG) in the mutant (I–L). The sagittal sections reveal that all mesDA neurons are lost in the engrailed double mutants at PO. Ac nucleus accumbens, OT olfactory tubercle, SN substantia nigra, St striatum, VT ventral tegmentum 52 D. Gherbassi and H. H. Simon compared the expression pattern of ventral gives rise to the neuroepithelium that provides midbrain between wild type and engrailed the precursors cells in the ventral midbrain double mutant embryos with a PCR based with the FGF8 signal necessary for their in- differential display technique at the age duction. In the postmitotic neurons, the two when the mesDA neurons are disappearing, transcription factors are essential for their and isolated synaptotagmin 1 (Syt1), v-erb-a survival by preventing the induction of apop- erythroblastic leukemia viral oncogene ho- tosis. Furthermore, the continuous expression molog 4 (ErbB4) and microtubule-associated of the engrailed genes from midgestation to protein 1 B (Mtap1b). Syt1 is an integral adulthood suggests that they may participate membrane protein of synaptic vesicles and in the long-term maintenance of this neuronal acts as Ca2þ sensor in vesicle exocytosis population. (Fernandez-Chacon et al., 2001). 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