| Summary: | 碩士 === 國立陽明大學 === 神經科學研究所 === 100 === Foxp1, Foxp2, and Foxp4 belong to the subgroup of the FOX family of winged-helix/ forkhead transcription factors and are expressed as early as E12.5. In the forebrain, they share high homology in DNA sequence of the crucial DNA-binding domain. They form homodimers and heterodimers, which are thought to be necessary for their efficient binding to target DNA. Foxp2 gene is well-known for its involvement in language disorders. The KE family with spoken language disorders carries a heterozygous missense mutation (R553H) in FoxP2 gene. Foxp2 regulates neurite outgrowth. Foxp1 mutation has recently been shown to be associated with autism and language disorders. Similar expression patterns in the embryonic brain and high sequence homology suggest that Foxp2 and its closest relative, Foxp1, may regulate some common processes in developing forebrain.
The first part of my thesis study is to investigate the biological function of Foxp1 in the developing forebrain. By crossing Dlx5/6-Cre mice with conditional FOXP1 (R542H) mutant mice, we generated conditional R542H Foxp1 mutant mice. The myc-tagged Foxp1 mutant protein was expressed in the striatum. We examined the expression of Lhx8 and Fezf2 by immunohistochemistry and in situ hybridization with 35S-labeled probe (35S-ISH), which are involved in neuronal differentiation. Lhx8, a cholinergic interneuron marker, was increased in rostral and middle mutant striatum. Next, we focused on the regulation neuronal morphology by Foxp1 R542H. We cultured E15.5 striatal cells and confirmed myc-tagged Foxp1 mutant protein expression by immunostaining. We quantified the total neurite lengths, the longest neurite length, and primary neurite branch. These three parameters had a trend of decrease in Dlx5/6-Cre; mtFoxp1 group compared to that in Dlx5/6-Cre group. These results suggest that Foxp1 R542H mutant protein might interfere with neuronal differentiation and neurite outgrowth of striatal neurons.
The second part of my study focused on Mef2C, a potential target gene of Foxp2. Mef2C is known to inhibit formation of glutamatergic synapses. In cerebral cortex, Mef2C and Foxp2 are both expressed in deep layers. We used immunohistochemistry (IHC) to analyze the expression profile. In striatum, the signal was too weak to be detected but abundant expression was observed in cortex. The knockout of Foxp2 gene did affect the number of Mef2C-positive cells in motor and somatosensory cortex, but increased Mef2C expression in the layerⅥ of motor cortex of postnatal day 14 Foxp2 knockout mice. These results suggest that Foxp2 may function as an inhibitory regulator of Mef2C.
The third part of my thesis was to characterize Foxp1 and Foxp4 conditional knockout targeting constructs. I confirmed the clones of gene targeting plasmid by PCR. I further designed 5’ and 3’ probes for screening of homologous recombination ES cells by Southern blotting. I also confirmed the correct presence of the third loxP site in the targeted ES cells.
In summary, although Foxp1, Foxp2 and Foxp4 belong to the same gene subgroup, further study is needed to clarify the functions of Foxp family in striatal development.
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