FoxP2 acts as a regulator in neuronal subtype specification

During development, neurons are instructed to migrate and send projections to specific regions in the brain. A fundamental question is how cells differentiate into specific neuronal subtypes. The subdivision of the GE can give rise to different subtypes of neurons by the expression of transcription factors. The LGE gives rise to not only cortical GABAergic interneurons but also striatal projection neurons and the MGE gives rise to many cortical GABAergic interneurons (Sussel et al., 1999). Several recent studies have shown that the GE regional identities are maintained through cross-regulatory interactions by transcription factors (Tucker et al., 2008) For example, homeobox transcription factors Dlx1 and Dlx2 are expressed by progenitors within the ventral telencephalon, regulate the fate and migration of ventral precursors, and promote GABAergic interneuron differentiation (Maqdelena et al., 2007). Another

transcription factor Mash1 has a role in the specification of neuronal

identity in different regions of the nervous system and can induce expression of markers of GABAergic neurons in the cerebral cortex (Fode et al. 2000). Moreover, Shh signaling is centrally involved in the patterning of the nervous system. It has been shown that expression of Shh is required for the specification of neuronal identity (Ericson et al., 1996) and GABAergic populations such as interneurons derived from the MGE also depend on Shh-signaling (Nery et al., 2001). Many

Shh-dependent homeodomain transcription factors are involved in ventral patterning in GE (Corbin et al., 2003). Previous study has demonstrated that Gli family of transcriptional regulators – Gli1, Gli2 are collectively necessary and sufficient for all aspects of intracellular Shh-responses involved in ventral forebrain patterning (Matise et al., 1998). Another study shows that the Shh-mediated induction of Nkx2-1 expression initiates MGE development (Xu et al., 2005). However, the mechanisms controlling the segregation of LGE and MGE neuronal populations

remain poorly understood. Previous reports have shown that transcription factors are thought to play a major role in regulating the specification of neuronal subtype. For example, one previous study showed that FoxP2 was expressed by LGE-derived neurons and absent from MGE-derived interneurons (Takahashi et al., 2009). Therefore, FoxP2 may be important to specify GE-derived neuronal subtypes. Using loss-of-function

approaches, my experiments demonstrate that loss of FoxP2 expression results in a reduction of LGE-derived medium spiny neurons. In contrast, MGE-derived interneurons are increased. These results suggest that in the absence of FoxP2, LGE progenitors are specified to a more MGE fate.

This transformation leads to a dramatic change in the number of GABAergic cortical interneurons, striatal interneurons and projection neurons derived from the MGE and LGE, respectively (Sussel et al., 1999). Furthermore, other factors may be involved in transcriptional regulation of neuronal subtype specification. I believe that evolution has selected distinct regulatory networks of gene expression for the cell types derived from each of the two GE. For example, Nkx2-1, unlike FoxP2, is expressed in the MGE and is critical for the development of the

mammalian forebrain (Gulacsi et al., 2006). Also, previous studies have revealed that Nkx2-1 regulates the specification of MGE-derived cells during early patterning of the telencephalon (Corbin et al., 2003).

Interestingly, FoxP2 inhibits Nkx2-1 transcription activity in alveolar cells (Zhou et al., 2008). It is likely that FoxP2 interacts with other transcription factors to regulate neuronal subtype specification. Taken together, it implies that these two transcription factors FoxP2and Nkx2-1 may regulate the specification of neuronal fates in the ventral forebrain.

Overall, in this study, I investigate the effects and mechanisms of FoxP2 in embryonic neurogenesis. Therefore, the next step is to investigate whether FoxP2 and Shh/Nkx2-1 together control the

specification of GABAergic neurons and potential interactions between FoxP2 and Shh/Nkx2-1 in the embryonic brain.

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Figure 1: Overexpression and knockdown of FoxP2 in primary cell culture by electroporation. To set up gain-of-function experiments, I overexpressed FoxP2 and GFP in E13.5 cortical progenitor cells and

cultured them for 2 days. Transfected cells were identified by GFP (green) staining and FoxP2 expressing cells were identified by FoxP2 (red)

staining. Arrows indicate GFP- and FoxP2-double positive cells;

arrowheads indicate GFP-positive but FoxP2-negative cells. The expression of FoxP2 in FoxP2 overexpression group was significantly increased (A, left two columns). For the loss-of-function experiments, I knocked down FoxP2 with shRNA against FoxP2 in GE progenitor cells and cultured them for 5 days. The expression of FoxP2 in the knockdown group was significantly decreased than in the control group (A, right two columns). For quantification anaylysis of US2 and FoxP2 groups,

numbers of FoxP2 and GFP double-positive cells were normalized to the numbers of total GFP-positive cells. n=3, ≧360 GFP-positive cells were counted in each group (B). For quantification anaylysis of UI4 and

shFoxP2 groups, n=3, ≧320 GFP-positive cells were counted in each group (C). Data are presented as means ± SEM. ** compared to the control group, p < 0.01 (t-test); scale bar: 50μm.

Figure 2: Test of knockdown efficiency of shFoxP2 constructs. To test the knockdown efficiency of three shFoxP2 constructs (shFoxP2-5, shFoxP2-6, shFoxP2-7), P19 cells were transfected with control vector, shFoxP2-5, shFoxP2-6, shFoxP2-7 along with GFP- and

FoxP2-expressing constructs and fixed 3 days after transfection (A).

Transfected cells were identified by GFP (green) staining and FoxP2 expressing cells were identified by FoxP2 (red) staining. Arrows indicate GFP- and FoxP2-double positive cells; arrowheads indicate GFP-positive but FoxP2-negative cells. More GFP-positive cells were FoxP2-negative in shFoxP2-6 group than in the control one (A). For quantification

anaylysis of (A), numbers of FoxP2 and GFP double-positive cells were normalized to the numbers of total GFP-positive cells. n=3, ≧197

GFP-positive cells were counted in each group (B). Western blot analysis

of shFoxP2 knockdown efficiency in P19 cells (C). Cells were lysed 3 days after transfection. Expression levels of β-tubulin (55 kDa) were used as loading controls. Quantification of FoxP2 protein levels was

determined as relative intensity of FoxP2 over β-tubulin. FoxP2

expression was reduced significantly in P19 cells transfected with two shRNA expression constructs (shFoxP2-6 or shFoxP2-7) (D). Data are presented as means ± SEM. * compared to the control group. **: p < 0.01;

*: p < 0.05 (t-test); scale bar: 50 μm.

Figure 3: FoxP2 is sufficient and necessary for neuronal differentiation in the embryonic telencephalon. To investigate whether FoxP2 regulats neuronal differentiation, I overexpressed FoxP2 and GFP in the cortical

在文檔中 FoxP2在胚胎時期的神經新生中所扮演的角色 (頁 35-82)