FOXP1 belongs to the forkhead box (Fox) family of transcription factors which all contain a ~100 amino acid winged helix/forkhead box DNA binding domain (Tang et al., 2012). The FOXP division of the transcription factor family also contain a zinc finger domain and a leucine zipper motif which is involved in transcriptional repression, these reside in the N-terminus of the protein and allow the transcription factors to form homo- and heterodimers with each other see Figure 1.9 (Tang et al., 2012, Li et al., 2004, Shu et al., 2001). Foxp1 is widely expressed in E12.5 mice with high levels of expression in the lung, and lower levels expressed in intestinal, neural and cardiovascular tissue (Shu et al., 2001). There are at least four splice isoforms of Foxp1 two of which have been detected in the brain which are; 3.2 kb
and 2.4 kb and are expressed at similar levels, the FOXP1 consensus binding site in promoter regions is TATTTG/AT (Wang et al., 2003).
Figure 1.9: Schematic highlighting the important features in human FOXP1. An N
terminal Glutamine rich domain and a centrally located C2H2 zinc finger and leucine zipper,
and the C terminal Forkhead domain and acidic sequence
An alternative splice form of FoxP1 has been detected in human and mouse, which is expressed in pluripotent ES cells (Gabut et al., 2011). In human, exon 18 is substituted for exon 18b and in mouse exon 16 is substituted for 16b in the ES form; the expression of the “b form” of the exon decreases on differentiation, the splice variant alters the amino acid sequence of the forkhead domain affecting DNA binding (Gabut et al., 2011). Foxp1 has been observed in the very early developing zebrafish from the one cell stage from the maternal transcript, decreasing in expression at the developmental stage of 30% epiboly and increasing again at 75% epiboly in the whole zebrafish embryo and at later stages localised to the developing midbrain, hindbrain and spinal cord (Cheng et al., 2007). There was a marked reduction of expression of Foxp1 in a Dlx1/2-/- mouse SVZ and MZ of the LGE and in maturing striatal neurones, suggesting that FoxP1 is an important transcription factor in later striatal development (Long et al., 2009). In the mouse, DLX5/6 were also expressed in the mantle zone and could induce the expression of
Foxp1 (Tamura et al., 2004).
At E12.5 in the mouse, Foxp1 was shown to be expressed in the developing spinal cord motor neurons and possesses a similar expression pattern to Islet1 (Shu et al., 2001, Ericson et al., 1992, Tsuchida et al., 1994). However, its expression was observed later in the developing basal ganglia at E16.5 (Shu et al., 2001). Mouse in
situ hybridisation and immunohistochemistry showed FoxP1 protein and mRNA to
be expressed in the SVZ of the ganglionic eminence at E14.5, therefore localised to the migratory/post-migratory neurons of the striatum (Ferland et al., 2003). Striatal expression of FOXP1 has been observed from later embryonic developmental stages right through to the adult mouse (Ferland et al., 2003). In the adult mouse, FOXP1 expression was observed elsewhere in the brain including the substantia nigra, the ventral striatum, anterior olfactory nucleus, olfactory tubercle, thalamus, pontine nucleus, inferior olive, deep layers of the superior colliculus and cortical layers III-V (Ferland et al., 2003). Tamura et al., (2003) performed in situ hybridisation in the mouse forebrain and detected Foxp1 expression earlier at E13.5 in the post-mitotic neurons of the caudatoputamen. At E15.5, expression of Foxp1 was also detected elsewhere including the paraventricular thalamic nucleus, lateral preoptic area and preoptic area (Tamura et al., 2003). At E17.5, the expression of
Foxp1 increased in the caudatoputamen and paraventricular thalamic nucleus, but
decreased in the lateral preoptic area and preoptic area but was also detected in the CA1 region of hippocampus (Tamura et al., 2003). Foxp1 mRNA was only expressed in MAP2+ neurons of the mouse striatum, with over two thirds of MSNs (DARPP32+/GluR2/3+) expressing Foxp1 mRNA, and no Foxp1 mRNA expressed in interneurons (Tamura et al., 2004). This expression pattern suggested Foxp1 is more likely to be a matrix marker than a striosomal patch marker in mouse (Tamura et al., 2004). More recent findings by Martín-Ibáñez et al., (2012) have localised FOXP1 expression to the matrix in the mouse as it co-localises with HELIOS expression. However, FOXP1 has been shown to be expressed in both the striosomal patches and matrix compartments in the P3-20 rat striatum (Takahashi et al., 2003), indicating possible species variations in the location of FOXP1 expression in the striatum. Foxp1 mRNA was first detected in the rat at E14 in the mantle zone of the LGE, at E16-20 Foxp1 expression in the SVZ was lower than the
MZ, with low level expression in the primordium of septum and amygdala unlike the expression of the closely related transcription factor which was also striatally enriched, Foxp2 (Takahashi et al., 2003). FOXP1 was detected in the 11 week old human fetus in the LGE in both precursors and differentiated medium spiny neurons (Delli Carri et al., 2013a).
FOXP1 overexpression in mouse striatal cells induced an increase in expression of
Follistatin, Fos and activating transcription factor 3 (Tang et al., 2012). siRNA
mediated knockdown of Foxp1 decreased their expression, suggesting these are targets of FoxP1 (Tang et al., 2012). FOXP1 overexpression also up-regulated genes involved in glutamate receptor function, GABA receptor signalling, G protein coupled receptor signalling and cAMP-mediated signal transduction (Tang et al., 2012). In total, there are currently 40 FoxP1 targets associated with the Huntington’s disease signalling pathway, mainly involved in BDNF signalling, caspase activity and glutamate and calcium signalling (Tang et al., 2012). FOXP1 regulates its own expression as well as increasing expression of Kcnip2 (Tang et al., 2012), KCNIP2 was significantly decreased in expression in human HD samples (Desplats et al., 2006). Interestingly, both FOXP1 and CTIP2 (another transcription factor involved in striatal development) showed a significant decrease in their expression in the striatum of an R6/1 HD transgenic mouse model compared to that of wild type littermates at 6 months of age (Desplats et al., 2006). The Foxp1 promoter does not contain a CTIP2 binding site but Foxp2 does (Desplats et al., 2008). However, Ctip2 does possess many FOXP1 binding sites in its promoter region (Tang et al., 2012). It may become apparent on transfection that Foxp1 transfection up-regulates Ctip2 expression through direct activation of the promoter. FOXP1 may be involved in immunosuppression in the CNS and is predominantly expressed in the neurons as shown by co-labelling with NeuN, but has been detected in qRT-PCR analysis of activated microglia cultures (Tang et al., 2012). In
vivo, increased FOXP1 expression induced an increased expression of suppressor
of cytokine signalling 5 (SOCS5), and so FOXP1 could be seen as being a neuro- protective transcription factor by counteracting glia activation (Tang et al., 2012). FOXP1 has been shown to regulate retinoic acid signalling in the developing lateral motor column of the spinal cord at brachial and lumbar levels (Rousso et al., 2008), and could be important in the developing striatum since retinoic acid signalling has been shown to induce expression of DARPP-32 (Liao et al., 2005, Liao & Liu 2005). In the mouse Foxp1 knockout, there was a loss of the lateral motor column and an increased size of the medial motor column more laterally (Rousso et al., 2008). FOXP1 and ISLET1 are co-expressed in the lateral motor column (Rousso et al., 2008) and in the more caudal regions of the brachial and lumbar divisions of the lateral motor column, expression of OCT6, another transcription factor important for striatal development, has been observed (Dasen et al., 2005, Luria & Laufer 2007), and so there could be activation of Foxp1 by OCT6 or vice versa and the same with
Foxp1 and ISLET1. The Pitx3 gene promoter contains seven FOXP1 binding sites
and FOXP1 is co-expressed in the Pitx3+ post mitotic midbrain dopamine neurons in the E12.5 mouse, FoxP1 forced expression in mES cells led to expression of Pitx3+/TH+ dopamine neurons when differentiated in monolayer culture (Konstantoulas et al., 2010).
FoxP1 will be transfected into neural stem cells as it is important for MSN development as it is highly expressed in post-mitotic neurons of the developing striatum and co-localises in the developing and adult striatum with the MSN marker DARPP32. FOXP1 also co-localises with other transcription factors important for MSN development. The phenotype of FOXP1 transfected cells will need to be determined as there is disparity in the literature as to whether FOXP1+ cells correspond to matrix or a patch phenotype.