stereotaxic-surgery-for-genetic-manipulation-striatal-cells-neonatal

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

National Yang-Ming University

Proper development of vertebrate embryos depends not only on the crucial funtions of key evolutionarily conserved transcriptional regulators, but also on the precisely spatiotemporal expression of these transcriptional regulators. The mouse Nolz-1/Znf503/Zfp503 gene is a mammalian member of the conserved zinc-finger containing NET family. The expression pattern of Nolz-1 in mouse embryos is highly correlated with that of its homologues in different species. To study the spatiotemporal regulation of Nolz-1, we first identified two evolutionarily conserved cis-elements, UREA and UREB, in 5' upstream regions of mouse Nolz-1 locus. We then generated UREA-LacZ and UREB-LacZ transgenic reporter mice to characterize the putative enhancer activity of UREA and UREB. The results indicated that both UREA and UREB contained tissue-specific enhancer activity for directing LacZ expression in selective tissue organs during mouse embryogensis. UREA directed LacZ expression preferentially in selective regions of developing central nervous system, including the forebrain, hindbrain and spinal cord, whereas UREB directed LacZ expression mainly in other developing tissue organs such as the Nolz-1 expressing branchial arches and its derivatives, the apical ectodermal ridge of limb buds and the urogenital tissues. Both UREA and UREB directed strong LacZ expression in the lateral plate mesoderm where endogenous Nolz-1 was also expressed. Despite that the LacZ expression pattern did not full recapitulated the endogenous Nolz-1 expression and some mismatched expression patterns were observed, co-expression of LacZ and Nolz-1 did occur in many cells of selective tissue organs, such as in the ventrolateral cortex and ventral spinal cord of UREA-LacZ embryos, and the urogenital tubes of UREB-LacZ embryos. Taken together, our study suggests that UREA and UREB may function as evolutionarily conserved cis-regulatory elements that coordinate with other cis-elements to regulate spatiotemporal expression of Nolz-1 in different tissue organs during mouse embryogenesis.

Identification of two evolutionarily conserved 5' cis-elements involved in regulating spatiotemporal expression of Nolz-1 during mouse embryogenesis.

The zinc finger-containing gene Nolz-1/Zfp503 is a developmentally regulated striatum-enriched gene. In the present study, we characterized the cell type-selective expression pattern of Nolz-1 protein in the developing mouse striatum. Nolz-1 immunoreactivity was present in Isl-1-positive ventral LGE (vLGE, striatal primordia), but absent in Pax6-positive dorsal LGE (dLGE, non-striatal primordia). In the vLGE, Nolz-1 immunoreactivity was detected in early differentiating TuJ1-positive neurons, but not in Ki67-positive proliferating progenitor cells. Moreover, many Nolz-1-immunoreactive cells co-expressed Foxp1 or Foxp2, markers for striatal projection neurons. To further characterize Nolz-1 expression with respect to D1R-containing striatonigral and D2R-containing striatopallidal projection neurons, we used the Drd1-EGFP and Drd2-EGFP transgenic mice. Nolz-1 and EGFP double labeled neurons were found in the developing striatum of Drd1-EGFP and Drd2-EGFP mice, indicating Nolz-1 expression in both populations of striatal projection neurons. Notably, Nolz-1 protein was not expressed in Nkx2.1-positive interneuron progenitors, Lhx8-positive cholinergic interneuron progenitors, nNOS and calretinin-positive interneurons in E18.5 striatum. In the developing nucleus accumbens and olfactory tubercles of ventral striatum, many Nolz-1-positive cells co-expressed Sox1, an important transcriptional regulator for ventral striatum, suggesting a role of Nolz-1 in regulating development of the ventral striatum. Finally, in contrast to postnatal down-regulation of Nolz-1 in the dorsal striatum, Nolz-1 protein was persistently expressed in the olfactory tubercle from E15.5 to adulthood. Taken together, our study suggests that Nolz-1 serves as a marker for early differentiating striatal projection neurons and that Nolz-1 may regulate development of striatal projection neurons. 

Cell type-selective expression of the zinc finger-containing gene Nolz-1/Zfp503 in the developing mouse striatum.

Nolz-1, as a murine member of the NET zinc-finger protein family, is expressed in post-mitotic differentiating neurons of striatum during development. To explore the function of Nolz-1 in regulating the neurogenesis of forebrain, we studied the effects of ectopic expression of Nolz-1 in neural progenitors. We generated the Cre-loxP dependent conditional transgenic mice in which Nolz-1 was ectopically expressed in proliferative neural progenitors. Ectopic expression of Nolz-1 in neural progenitors by intercrossing the Nolz-1 conditional transgenic mice with the nestin-Cre mice resulted in hypoplasia of telencephalon in double transgenic mice. Decreased proliferation of neural progenitor cells were found in the telencephalon, as evidenced by the reduction of BrdU-, Ki67- and phospho-histone 3-positive cells in E11.5-12.5 germinal zone of telencephalon. Transgenic Nolz-1 also promoted cell cycle exit and as a consequence might facilitate premature differentiation of progenitors, because TuJ1-positive neurons were ectopically found in the ventricular zone and there was a general increase of TuJ1 immunoreactivity in the telencephalon. Moreover, clusters of strong TuJ1-expressing neurons were present in E12.5 germinal zone. Some of these strong TuJ1-positive clusters, however, contained apoptotic condensed DNA, suggesting that inappropriate premature differentiation may lead to abnormal apoptosis in some progenitor cells. Consistent with the transgenic mouse analysis in vivo, similar effects of Nozl-1 over-expression in induction of apoptosis, inhibition of cell proliferation and promotion of neuronal differentiation were also observed in three different N18, ST14A and N2A neural cell lines in vitro. Taken together, our study indicates that ectopic expression of Nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation and induces abnormal apoptosis in the developing telencephalon. 

Ectopic expression of nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation accompanying with abnormal apoptosis in the developing mouse telencephalon.

Cortico-basal ganglia circuits are critical for speech and language and are implicated in autism spectrum disorder, in which language function can be severely affected. We demonstrate that in the mouse striatum, the gene Foxp2 negatively interacts with the synapse suppressor gene Mef2c. We present causal evidence that Mef2c inhibition by Foxp2 in neonatal mouse striatum controls synaptogenesis of corticostriatal inputs and vocalization in neonates. Mef2c suppresses corticostriatal synapse formation and striatal spinogenesis, but can itself be repressed by Foxp2 through direct DNA binding. Foxp2 deletion de-represses Mef2c, and both intrastriatal and global decrease of Mef2c rescue vocalization and striatal spinogenesis defects of Foxp2-deletion mutants. These findings suggest that Foxp2-Mef2C signaling is critical to corticostriatal circuit formation. If found in humans, such signaling defects could contribute to a range of neurologic and neuropsychiatric disorders.

Article	Year
Identification of two evolutionarily conserved 5' cis-elements involved in regulating spatiotemporal expression of Nolz-1 during mouse embryogenesis. PloS one\| PubMed ID: 23349903	2013
Cell type-selective expression of the zinc finger-containing gene Nolz-1/Zfp503 in the developing mouse striatum. Neuroscience letters\| PubMed ID: 23684982	2013
Ectopic expression of nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation accompanying with abnormal apoptosis in the developing mouse telencephalon. PloS one\| PubMed ID: 24073229	2013
Foxp2 controls synaptic wiring of corticostriatal circuits and vocal communication by opposing Mef2c. Nature neuroscience\| PubMed ID: 27595386	2016

Shih-Yun Chen

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