Lmx1a-Dependent Activation of miR-204/211 Controls the Timing of Nurr1-Mediated Dopaminergic Differentiation

The development of midbrain dopaminergic (DA) neurons requires a fine temporal and spatial regulation of a very specific gene expression program. Here, we report that during mouse brain development, the microRNA (miR-) 204/211 is present at a high level in a subset of DA precursors expressing the transcription factor Lmx1a, an early determinant for DA-commitment, but not in more mature neurons expressing Th or Pitx3. By combining different in vitro model systems of DA differentiation, we show that the levels of Lmx1a influence the expression of miR-204/211. Using published transcriptomic data, we found a significant enrichment of miR-204/211 target genes in midbrain dopaminergic neurons where Lmx1a was selectively deleted at embryonic stages. We further demonstrated that miR-204/211 controls the timing of the DA differentiation by directly downregulating the expression of Nurr1, a late DA differentiation master gene. Thus, our data indicate the Lmx1a-miR-204/211-Nurr1 axis as a key component in the cascade of events that ultimately lead to mature midbrain dopaminergic neurons differentiation and point to miR-204/211 as the molecular switch regulating the timing of Nurr1 expression.

MiR-204, together with miR-93 and miR-302d, has been shown to regulate the expression of Nurr1 in rat DA neurons [48]. In the same way, miR-204 seems to also regulate the expression of Nur77 in human brain microvascular endothelial cells [49].
Herein we investigate the link between miR-204/211 and the DA TFs Lmx1a and Nurr1. We demonstrate that Lmx1a induces the activation of miR-204/211 and, in turn, the latter regulates Nurr1 expression, influencing mDAn development. Thus, we believe that the Lmx1a/miR-204/211 axis controls the timing of differentiation of midbrain precursor cells, and its modulation could also be used to improve in vitro generation of mDAn, useful for the development of a model system or cell therapy.

miR-204/211 Is Expressed in Mesencephalic DA Progenitor Cells
To evaluate miR-204/211 expression in mDAn, we compared its expression in the cortex and midbrain of E14.5 and adult mice using qPCR. miR-204/211 shows enrichment in the midbrain compared to the cortex both in E14. 5  seed sequence and have only a single nucleotide difference in their mature forms. Because of this, they are functionally and structurally identical and are hereafter referred to as miR-204/211. miR-204/211 is widely expressed in neuronal tissues, including the cerebral cortex, hippocampus, eye, and choroid plexus [35,[42][43][44][45][46][47]. MiR-204, together with miR-93 and miR-302d, has been shown to regulate the expression of Nurr1 in rat DA neurons [48]. In the same way, miR-204 seems to also regulate the expression of Nur77 in human brain microvascular endothelial cells [49].
Herein we investigate the link between miR-204/211 and the DA TFs Lmx1a and Nurr1. We demonstrate that Lmx1a induces the activation of miR-204/211 and, in turn, the latter regulates Nurr1 expression, influencing mDAn development. Thus, we believe that the Lmx1a/miR-204/211 axis controls the timing of differentiation of midbrain precursor cells, and its modulation could also be used to improve in vitro generation of mDAn, useful for the development of a model system or cell therapy.

miR-204/211 Is Expressed in Mesencephalic DA Progenitor Cells
To evaluate miR-204/211 expression in mDAn, we compared its expression in the cortex and midbrain of E14.5 and adult mice using qPCR. miR-204/211 shows enrichment in the midbrain compared to the cortex both in E14. 5   (a-f) TaqMan assay for miR-204/211 (a,d) and qPCR for Trpm3 (b,e) and Trpm1 (c,f) on microdissected midbrain (Mid) and cortex (Ctx) at E14.5 (a-c) or 3 months (d-f). miR-204/211 values are normalized to sno-202 expression, while mRNA levels are normalized on the reference mRNA hypoxanthine phosphoribosyl transferase (Hprt). Bars represent mean ± SD of 2 −ΔCt values from four animals. * p < 0.05 (unpaired t-test with Welch's correction). (g-i) TaqMan assay for the expression of miR-204/211 in FACS-purified GFP + and GFPcells from Lmx1a-GFP (g), Th-GFP (h), and Pitx3-GFP (i) reporter mice at different developmental stages. Data are normalized to the average of the (a-f) TaqMan assay for miR-204/211 (a,d) and qPCR for Trpm3 (b,e) and Trpm1 (c,f) on microdissected midbrain (Mid) and cortex (Ctx) at E14.5 (a-c) or 3 months (d-f). miR-204/211 values are normalized to sno-202 expression, while mRNA levels are normalized on the reference mRNA hypoxanthine phosphoribosyl transferase (Hprt). Bars represent mean ± SD of 2 −∆Ct values from four animals. * p < 0.05 (unpaired t-test with Welch's correction). (g-i) TaqMan assay for the expression of miR-204/211 in FACS-purified GFP + and GFP − cells from Lmx1a-GFP (g), Th-GFP (h), and Pitx3-GFP (i) reporter mice at different developmental stages. Data are normalized to the average of the reference sno-202 and represent the mean ± SD of 2 −∆Ct values from three independent experiments. * p < 0.05 of GFP + with respect to GFP − (two-way ANOVA followed by Sidak).
To characterize whether miR-204/211 is ubiquitously expressed in all DA cells during development, we analyzed its expression in isolated mDAn, expressing Lmx1a, a marker of DA progenitors or early neurons, and Th or Pitx3, markers of differentiated mDAn. For this purpose, we purified GFP + neurons from the Lmx1a-GFP and Pitx3-GFP knock-in mouse lines, where the mDA neurons Lmx1a + or Pitx3 + were exclusively labelled by the GFP reporter, and from the Th-GFP transgenic mouse line, where the GFP was located downstream of the Th promoter [8,50,51].
We observed that miR-204/211 is enriched in GFP + cells derived from Lmx1a-GFP mice at E12.5 to E14.5 (Figure 1g) but is significantly reduced in GFP + (vs. GFP − ) cells purified from Th-GFP and Pitx3-GFP mice at E12.5 to E15.5 (Figure 1h,i). During development, in the ventral midbrain, Th and Pitx3 domains appear as subdomains of Lmx1a + cells from which they differentiate. Thus, the enrichment of miR-204/211 in Lmx1a + DA progenitorsbut not in differentiating (Pitx3 + ) or differentiated (Th + ) mDAn, suggesting miR-204/211 downregulation in postmitotic mDAn precursors-could identify the specific Lmx1a + sub-population involved in temporal and/or spatial regulation of the DA domain.

The Levels of Lmx1a Influence the Expression of miR-204/211 and Its Predicted Target Genes
Since only Lmx1a + cells are enriched for miR-204/211, we hypothesized that miR-204/211 plays a role in the Lmx1a-regulated commitment of mDAn and, therefore, that Lmx1a could influence its expression. To confirm this hypothesis, we used GFP + and GFP − cells isolated from E12.5 Lmx1a +/GFP and Lmx1a GFP/GFP mouse embryos corresponding to Lmx1a heterozygous (Lmx1a +/− ) and knock-out (Lmx1a −/− ) animals, respectively. Interestingly, in such a context, miR-204/211 was significantly enriched in GFP + cells derived from Lmx1a +/GFP embryos compared to those derived from Lmx1a GFP/GFP indicating that, in the absence of Lmx1a, the expression of the miR-204/211 is also reduced (Figure 2a,b). As a control, we analyzed the expression of two other miRNAs, miR-218 and miR-9. The first is known to be enriched in mDAn [52] while the second is broadly expressed in the brain [53,54]. Differently from miR-204/211, the expression of both miR-218 and miR-9 was not affected by the deletion of Lmx1a (Figure 2a,b), thus suggesting that miR-204/211 expression may be directly affected by Lmx1a.
To validate this hypothesis, we transfected the mes-cmyc-A1 cell line, an in vitro model of mDAn [55], with an inducible Lmx1a-Ires-GFP vector or with an empty Ires-GFP vector as a control. GFP + cells, overexpressing Lmx1a or not, were FACS-purified and analyzed for the expression of miR-204/211. As expected, we found an enrichment of miR-204/211 in the Lmx1a-Ires-GFP + cells but not in the control GFP + cells (Figure 2c), supporting our hypothesis that miR-204/211 expression is affected by Lmx1a activity.
To corroborate this idea, we analyzed previously published data obtained from E15.5 DA neurons derived from Lmx1a/b double KO (Lmx1a/b-KO) [56]. Thus, we scanned the entire list of differentially expressed genes (DEG) between KO vs. WT for the presence of putative binding sites for miR-204/211 by using the DIANA-microT-CDS prediction tool [57]. Out of 224 DEG, 27 (12%) were predicted to be targets for the miR-204/211 ( Figure 2d). Interestingly, 66.7% of these (18 genes) are upregulated in the Lmx1a/b-KO compared to the WT cells, while when looking at the unpredicted not targeted genes, they are equally distributed between down and upregulated (48.2% positive fold change; 51.8% negative fold change) (Figure 2e).

Figure 2.
Lmx1a levels influence miR-204/211 expression and its repressive activity. (a,b) TaqMan assay for miR-204/211, miR-218, and miR-9 on FACS-purified GFP + and GFP − cells isolated from microdissected midbrains of Lmx1a +/GFP and Lmx1a GFP/GFP E12.5 embryos. miRNA values are normalized on the reference sno-202 and represent relative to control as mean ± SD of 2 −ΔΔCt values from three embryos. * p < 0.05 (unpaired t-test with Welch's correction), (c) TaqMan assay for miR-204/211 on FACS sorted GFP + and GFPmes-c-myc-A1 infected with LV-Lmx1a-Ires-GFP or LV-Ires-GFP. Values are normalized on the reference sno-202 and represent the mean ± SD of 2 −ΔCt values from three independent experiments. * p < 0.05 (one-way ANOVA + Tukey post hoc test). (d) Representation of the frequency of miR-204/211 target genes among the 224 differentially expressed genes (DEG) in mDAn knocked out for Lmx1a/b [56]. The percentage and number of genes are reported. DIANA microT-CDS was used to identify targeted genes (miTG score > 0.5). (e) Fold change distribution for predicted and unpredicted target genes of the miR-204/211 among the 224 DEG derived from Chabrat's publication [56]. The percentage and number of genes are reported. Boxes in the violin plot represent the median, the 25th to 75th percentiles, and single values of log2 (Foldchange).

miR-204/211 Expression Is Regulated by Lmx1a
To confirm the role of Lmx1a in the regulation of miR-204/211 expression during mDAn development, we overexpressed the TF Lmx1a and Nurr1 alone or in combination, in four different in vitro models of mDAn differentiation. These are mes-c-myc-A1, E12.  TaqMan assay for miR-204/211, miR-218, and miR-9 on FACS-purified GFP + and GFP − cells isolated from microdissected midbrains of Lmx1a +/GFP and Lmx1a GFP/GFP E12.5 embryos. miRNA values are normalized on the reference sno-202 and represent relative to control as mean ± SD of 2 −∆∆Ct values from three embryos. * p < 0.05 (unpaired t-test with Welch's correction), (c) TaqMan assay for miR-204/211 on FACS sorted GFP + and GFP − mes-c-myc-A1 infected with LV-Lmx1a-Ires-GFP or LV-Ires-GFP. Values are normalized on the reference sno-202 and represent the mean ± SD of 2 −∆Ct values from three independent experiments. * p < 0.05 (one-way ANOVA + Tukey post hoc test). (d) Representation of the frequency of miR-204/211 target genes among the 224 differentially expressed genes (DEG) in mDAn knocked out for Lmx1a/b [56]. The percentage and number of genes are reported. DIANA microT-CDS was used to identify targeted genes (miTG score > 0.5). (e) Fold change distribution for predicted and unpredicted target genes of the miR-204/211 among the 224 DEG derived from Chabrat's publication [56]. The percentage and number of genes are reported. Boxes in the violin plot represent the median, the 25th to 75th percentiles, and single values of log 2 (Foldchange).

miR-204/211 Expression Is Regulated by Lmx1a
To confirm the role of Lmx1a in the regulation of miR-204/211 expression during mDAn development, we overexpressed the TF Lmx1a and Nurr1 alone or in combination, in four different in vitro models of mDAn differentiation. These are mes-c-myc-A1, E12.  These results confirm that miR-204/211 is regulated, directly or indirectly, by Lmx1a and indicate that miR-204/211 is not required for Th expression and is not influenced by Nurr1-mediated DA differentiation. Similar results were observed by using the human DA cellular system SHSY-5Y and the non-neuronal HeLa cells. The expression of Lmx1a in both cell lines promotes the upregulation of miR-204/211 suggesting that the same Lmx1a/miR-204/211 regulation occurs in humans as well ( Figure S1).

The Lmx1a-miR-204/211 Axis Controls the Timing of Midbrain Precursors Differentiation
Lmx1a controls the differentiation of DA progenitors from the FP, promoting the transition from the progenitors' domain to more ventral and differentiating domains. Its expression maintains progenitor proprieties and neurogenic potential, regulating proneural genes. Similarly, miR-204/211 has been reported to maintain adult neural stem cells (NSCs) in an undifferentiated state but primed for neurogenesis [45] in order to control the balance between self-renewal and differentiation and to modulate the gene These results confirm that miR-204/211 is regulated, directly or indirectly, by Lmx1a and indicate that miR-204/211 is not required for Th expression and is not influenced by Nurr1-mediated DA differentiation.
Similar results were observed by using the human DA cellular system SHSY-5Y and the non-neuronal HeLa cells. The expression of Lmx1a in both cell lines promotes the upregulation of miR-204/211 suggesting that the same Lmx1a/miR-204/211 regulation occurs in humans as well ( Figure S1).

The Lmx1a-miR-204/211 Axis Controls the Timing of Midbrain Precursors Differentiation
Lmx1a controls the differentiation of DA progenitors from the FP, promoting the transition from the progenitors' domain to more ventral and differentiating domains. Its expression maintains progenitor proprieties and neurogenic potential, regulating proneural genes. Similarly, miR-204/211 has been reported to maintain adult neural stem cells (NSCs) in an undifferentiated state but primed for neurogenesis [45] in order to control the balance between self-renewal and differentiation and to modulate the gene expression programs that mediate eye development [42,43]. Thus, we hypothesize that Lmx1a may control, at least in part, progenitor identity via miR-204/211.
Through further analysis, we observed the existence of a temporal correlation during epiSC DA differentiation between the expression of Lmx1a, miR-204/211, Nurr1, and Pitx3. Indeed, the initial phases of differentiation, between in vitro day 5 and day 9, were characterized by an increased expression of Lmx1a and miR-204/211 (Figures 4e and S3a). In this window, the expression of Nurr1 was maintained at a low level, as well as that of its known target Pitx3. Conversely, regarding the differentiation process, between days 9 and 14, we observed a further increase in Lmx1a expression, a decrease in miR-204/211, and a progressive upregulation of Nurr1 and Pitx3 mRNAs (Figure 4e). These data suggest that Lmx1a initially regulates the expression of miR-204/211 that, in turn, influences the timing of Nurr1 and Pitx3/Th expression and mDAn differentiation.
Interestingly, Nurr1 and Gdnf are the only genes with a clear opposite expression to that of miR-204/211, both at day 9 and day 14 of the epiSC DA differentiation. They are low during the initial phases of differentiation but increased later during differentiation (Figures 4d and S3). These trends were similar but less evident for a few other identified genes (Elavl2, Sox4, Plxna2, and Rasgef1b) (Figures 4d, S3 and S4).
These observations highlight the importance of finely regulated expression timing for miR-204/211, and in turn, its target genes, during early mDAn development, and suggest a role of this miRNA in defining specific mDAn subpopulations (Figure 4f). In this context, considering the screening results, Nurr1 came out as a strong potential candidate for further investigation.

miR-204/211 Regulates Nurr1 Expression and Influences DA Differentiation
To investigate whether the Lmx1a-miR-204/211 axis could have a role in controlling Nurr1 expression and timely regulating the mDAn differentiating program, we used the computational prediction tools TargetScan 7.2 (http://www.targetscan.org/vert_72/ accessed on 20 June 2022) and DIANA-microT-CDS (http://diana.imis.athena-innovation. gr/DianaTools/ accessed on 20 June 2022). We found that miR-204/211 has a conserved 7mer-m8 binding site on the 3 UTR of both mouse Nurr1 transcripts (Figure 5a). To validate this prediction and investigate whether miR-204/211 could really act as a posttranscriptional regulator of Nurr1, we performed a luciferase reporter assay in HeLa cells by cloning 1233 bp of Nurr1 3 UTR (corresponding to mouse Ch2: 56,997,526 to 56,998,759), containing the predicted miR-204/211 binding site, downstream of the luciferase reporter gene stop codon in the pMIR-Report. The Nurr1-3 UTR (WT) or the same sequence deleted in the binding site for miR-204/211 (MUT) were co-transfected in combination with a miR-204/211 expressing vector. As shown in Figure 5b, 48 h after transfection, miR-204/211 was able to significantly reduce luciferase activity by approximately 33%. This effect was absent when the miR-204/211 binding site on the Nurr1-3 UTR was deleted and specific to the miR-204/211, since miR-218, which is not predicted as a potential regulator of Nurr1 expression, did not affect the luciferase activity.
of miR-204/211 (FDR = 7.2 × 10 −8 Figure S2a), while a reduced number of miR-204/211 targets was identified in ventral lateral genes ( Figure S2b). Following the same approach, we also observed that miR-204/211 is enriched in NSCs and might target 29,7% of NSC genes (FDR = 7.1 × 10 −7 ; Figure S2C) [45]. Altogether, these results point to the potential role of miR-204/211 as a regulator of the DA differentiation process. To select key miR-204/211 targets in the early phases of DA differentiation, we combined these data with our previously published array data obtained from epiSCs differentiated towards the DA phenotype [36]. Here, by using DIANA-microT-CDS and TargetScan algorithms, we selected a set of FP and NSC genes being predicted as a miR-204/211 targets and showing an opposite expression profile to that of miR-204/211 during the in vitro epiSC to mDAn differentiation (Figure 4a; see section 4 for details). Following this approach, we identified 14 FP genes (Nurr1, Gdnf, Tcf12, Rasgef1b, Itpr1, Samd5, Wnt4, Arx, Nr3c1, Glis3, Tmem64, Adamts9, Gcnt2, and Fam43a) and 8 NSC genes (Nurr1, Elavl2, Elavl4, Plxna2, Sox4, Sox11, Khdrbs1, and Sox4) (Figures 4b-d and S3 and S4). Interestingly, Nurr1 was identified as the only common miR-204/211 target between the FP and NSC genes.  predicted as a potential regulator of Nurr1 expression, did not affect the luciferase activity. These data show that miR-204/211 is able to bind the 3′UTR of Nurr1 and downregulate its expression and are in accordance with previous reports [48,49].
Interestingly, the miR-204/211-mediated downregulation of Nurr1 affects the expression of specific Nurr-1 downstream targets such as Th, Vamt2, and DAT that were significantly reduced upon miR-204/211 overexpression in differentiated mes-c-myc-A1 cells (Figure 5c-f). This observation suggests that miR-204/211 overexpression keeps the cells in a more undifferentiated stage.

Discussion
During midbrain development, changes in gene expression result in the transition from the progenitor to the differentiative phase that anticipates the generation of distinct neuronal populations. The process is finely regulated in space and time since inappropriate timing could result in premature differentiation with consequently reduced numbers of mature neurons.
A specific set of transcription factors acts in a concerted temporal fashion to promote cell cycle exit and initiate DA differentiation. In this context, the TF Lmx1a regulates, among other functions, the neurogenic potential of DA precursors, controlling the expression of floor plate progenitor genes. Thus, by regulating the proneural factor Ngn2 and inhibiting alternative factors specific to other neuronal types, Lmx1a determines the These data show that miR-204/211 is able to bind the 3 UTR of Nurr1 and downregulate its expression and are in accordance with previous reports [48,49].
Interestingly, the miR-204/211-mediated downregulation of Nurr1 affects the expression of specific Nurr-1 downstream targets such as Th, Vamt2, and DAT that were significantly reduced upon miR-204/211 overexpression in differentiated mes-c-myc-A1 cells (Figure 5c-f). This observation suggests that miR-204/211 overexpression keeps the cells in a more undifferentiated stage.

Discussion
During midbrain development, changes in gene expression result in the transition from the progenitor to the differentiative phase that anticipates the generation of distinct neuronal populations. The process is finely regulated in space and time since inappropriate timing could result in premature differentiation with consequently reduced numbers of mature neurons.
A specific set of transcription factors acts in a concerted temporal fashion to promote cell cycle exit and initiate DA differentiation. In this context, the TF Lmx1a regulates, among other functions, the neurogenic potential of DA precursors, controlling the expression of floor plate progenitor genes. Thus, by regulating the proneural factor Ngn2 and inhibiting alternative factors specific to other neuronal types, Lmx1a determines the DA progenitor identity. Indeed, at E12.5, the apical portion of the Lmx1a domain maintains, up to birth, progenitor proprieties as demonstrated by the positive staining for Ngn2, Ascl1, Ki67, Sox2, and Nestin, while in the same domain, the expression of the differentiating factor Nurr1, which marks mainly post-mitotic mDAn [6,9,59], is kept repressed. Together with Pitx3 and before the Nurr1 expression, Lmx1a appears when the transition from proliferation to differentiation occurs, playing an essential role in this process.
Alongside transcription factors, microRNAs emerged as a fine-tuner of specific gene networks responsible for the differentiation process for most neuronal types. In oligodendrocytes, the homeobox gene Sox10 is essential for the differentiation and expression of miRNAs with relevance in oligodendrocytes development, including miR-338, miR-335, and miR-155 [60,61]. In DA neurons, miR-34b/c and miR-135 instead regulate the expression of key components of the Wnt pathway facilitating the expansion of the progenitor pool, just before the start of progenitor differentiation [36,38]. Similarly, in motor neurons, miR-218 is essential to repressing alternative differentiation programs during motor neuron differentiation, facilitating the proper establishment of the neuro-muscular junction [62].
In a similar way, miR-204/211 mediates retina development and lens formation by controlling the Meis2/Pax6, as well as sustaining the undifferentiated state of the neuronal stem cells [42,45].
Herein, we described a new regulatory axis that starts with Lmx1a and controls the timing for mDAn differentiation through the negative regulation of the post-mitotic transcription factor Nurr1, via miR-204/211. Thus, in the midbrain, Lmx1a mediated the expression of miR-204/211, preventing the start of terminal differentiation, driven by Nurr1, which in turn promotes the expression of terminal DA markers. In addition, the enriched expression of miR-204/211 in Lmx1a + cells, but not in Pitx3 + or Th + cells, reveals a subpopulation of primed DA neurons similar to that identified in choroid plexus cells, where miR-204/211 keeps primed but quiescent neuronal stem cells to timely regulate cellular differentiation [45]. We believe that this process occurs through the direct control of Lmx1a on miR-204/211, since the ectopic overexpression of Lmx1a in non-neuronal cells such as HeLa is sufficient to drive miR-204/211 expression. However, other factors may also intervene in this process, since it has been recently shown that miR-204/211 expression can be regulated through epigenetic processes [63]. In addition, since, in the midbrain, Lmx1a/b drives the transition from the proliferative to the DA progenitor phase and the loss of Lmx1a/b activity results in the reduction of Th + neurons during adulthood, we cannot rule out the possible involvement of Lmx1b in the process, although our data points to a clear role for Lmx1a.
Failure in miR-204/211 expression could result in an anticipated mDAn priming during development with a possible reduction of DA neurons in adult animals.
Indeed, several pieces of evidence point to the observation that, during development, neurogenic priming relies not only on the regulation of the translational repression complex elF4E1/4E-T that traps mRNA encoding for neurogenic transcription factors, but also on miRNA-mediated mechanisms [38,64].
Interestingly, most of the miR-204/211 targets we identified are directly linked to Nurr1. These include Gdnf [65], Elavl2 [66], Itpr1, Elavl4, Khdrbs1 [67], Rasgef1b [67], and Tcf12 [68]. Some of them are also involved in the differentiation of retinal progenitor cells and in the development of the eye (Nurr1, Elavl2, Plxna2, Rasgef1b, Wnt4, Sox11), tissues in which miR-204/211 shows a relevant role [42,44,66,[69][70][71][72][73] In a precise time window during embryonic development, miR-204/211 holds Nurr1 and its downstream targets repressed, preventing an early DA maturation. Later, during mDAn development, once the precursors are at the right differentiative stage, miR-204/211 expression becomes no longer necessary, Nurr1 expression is "released", and the DA maturation can progress. In the case of miR-204/211 downregulation, early activation of Nurr1 would occur, resulting in premature DA differentiation and a consequent permanent reduction in the overall number of DA neurons. Our findings highlight the importance of fine-tuning miR-204/211 expression during development as an additional mechanism to control the timing of DA neuron differentiation.
We finally believe that our data are of relevance in the optic of human disorders. Indeed, the miR-204 expression has been reported as dysregulated in PD patients [39][40][41], and recently identified as a potential tumor suppressor in glioblastoma [74]. Thus, modulation of its expression could be relevant as a potential therapeutic approach.

Ethics Statement
Mice were bred in-house at the Institute of Genetics and Biophysics "Adriano Buzzati Traverso", C.N.R., Naples, Italy. Selected animals were sacrificed in accordance with the recommendations of the national legislation as well as the European Commission (EU Directive 2010/63/EU for animal experiments). All the procedures involving mice were approved by the Ethic Scientific Committee for Animal Experiments (project id code: 491/2017-PR).

Tissues Collection
Mice brains were dissected under sterile conditions in PBS supplemented with glucose 33mM. Cortex and midbrain tissues at different ages were isolated under a stereomicroscope and processed for FACS sorting, RNA extraction, or primary cultures, as described below.

GFP + Cell Sorting
Freshly dissected ventral midbrains from Th-GFP mouse embryos were enzymatically dissociated by incubation for 3 min at 37 • C in a trypsin solution (0.25% trypsin in 33 mM glucose/PBS; Sigma-Aldrich, Milan, Italy) containing 0.01% pancreatic DNAse (Sigma), and cell suspensions were sorted by BD FACSAria III into GFP-positive and -negative fractions. Cells were stored in RNAlater (Thermo Fisher Scientific, Milan, Italy) for further investigations.

RNA Extraction and Real-Time qPCR
RNA was extracted from cells and tissues using the miRVana miRNA isolation kit (Ambion, Milan, Italy). The yield and the integrity of RNA were determined by spectrophotometric measurements, and agarose gel electrophoresis was used to determine the yield and quality of extracted RNA. One microgram of RNA was reverse-transcribed by 400 units of SuperScript III (Thermo Fisher Scientific, Milan, Italy) with 6 µM random hexamers (New England Biolabs) and RNAaseH (2U/µL; Thermo Fisher Scientific).
Real-time qPCR was performed on 2 µL of previously diluted cDNA (1:4) template, corresponding to 25 ng of original RNA amount, using the Power SYBR Green Master Mix (Thermo Fisher Scientific), in the presence of 0.5 µM of specific oligos. All analyzed genes shown in Table 1 were normalized vs. hypoxanthine phosphoribosyl transferase (Hprt). Analysis of relative expression of target genes was performed by the 2 −∆Ct or 2 −∆∆Ct methods.
A luciferase assay was performed by using the Luciferase Reporter Assay System (Promega, Milan, Italy), following the manufacturer's instructions. First, 400 ng of the pMIR-Report containing WT 3 UTR or mutated 3 UTR was co-transfected with 400 ng of the Tet-O-FUW-miR-204/211 and 400ng of the rtTA-expressing vector (400 ng) using Lipofectamine2000 (Invitrogen) in HeLa cells seeded the day before at a density of 40 × 10 3 in a 48-well culture plate in DMEM, supplied with 10% FBS, Pen/Strep, 2 mM glutamine, 1 mM sodium pyruvate, and 100× non-essential amino acids. An empty pMIR-Report vector or Tet-O-FUW-miR-218 was used as further controls. Transfection efficiency was evaluated by the pRL-SV40 Renilla luciferase reporter vector (Promega). The firefly luciferase luminescent signal was normalized on the Renilla luciferase signal.
Microarray data obtained for epiSC differentiated toward the DA phenotype and previously reported (GEO: GSE110270) [36] were analyzed to extract the fold-change values for Lmx1a, Nurr1, Th, miR-204/211, and selected genes specific for FP and NSC identity [45,58]. Venn diagrams were generated using InteractiVenn at http://www.interactivenn.net/ accessed on 1 December 2021 [83] while microarray values are represented by the ggplot2 library in R.