Bhlhb5, Soxe With Necl-1 Role In Neural Development And Differentiation

The vertebrate central nervous system (CNS) is a very complex and highlyorganized system that is comprised of thousands of distinct neuronal and glial cell types.Cellular diversity is a remarkable feature of the nervous system structure. Although neuronand glia play important function, the detailed mechanism regulating their developmentremains unclear. The combinatorial action of numerous transcription factors and celladhesion molecules is required for the development and function of the nervous system.In this study, I focus on the development of oligodendrocytes and neurons throughexamining the function of transcription factors Bhlhb5, SoxE and cell adhesion moleculesNecl-1 by gain-of-function and loss-of-function assays.Accumulating evidence has suggested that the basic helix-loop-helix (bHLH)transcription factors play important roles in controlling neuronal fate specification anddifferentiation in the developing CNS. Bhlhb5 is one of the bHLH transcription factorsand belong to Olig family with Olig1, Olig2, Olig3 and Bhlhb4. In this study, weperformed a detailed immunological study on the temporal and spatial expression ofBhlhb5 in embryonic mouse spinal cord with a newly-developed antibody and BrdUlabeling experiment. At the early stage of neural development (E10.5), Bhlhb5 isspecifically expressed in dI6 dorsal interneurons, and in V1 and V2 ventral interneurons.At late stages of development (E17.5) , Bhlhb5 expression is detected in a subset oflate-born dorsal association interneurons that migrate into the uppermost layer of the dorsalhorn. BrdU birthdating and double immunostaining experiments suggested that Bhlhb5+dIL neurons were predominantly generated from the dorsal neural progenitor cells at E12.5and later. Take together; these results suggest that Bhlhb5+ neurons progressively migrateinto the superficial laminae of the dorsal horn between E14.5 and E17.5. Bhlhb5 maybehave relationship with the generation and switch of neurotransmitter in interneurons.SoxE subfamily (Sox8, Sox9 and Sox 10) belongs to a large family of transcriptionfactors Sox (SRY related HMG-box gene, Sox). The sox proteins can implicate in a varietyof developmental processes, including roles in neural crest, gonad, skeletal and central nervous system development. Previous evidence comes from gene loss of functionexperiment appear the function redundancy on oligodendrocyte development. We use inovo electroporation technique to reveal the effect of SoxE proteins on development ofneuron and oligodendrocyte. The in situ hybridization experiment showed the expressionpattern of SoxE in the embryonic chick spinal cord. The results indicated that Sox9 isrelated to the generation of neuron and oligodendrocyte precursor cells, whereas Sox8 andSox10 are involve in specification of oligodendrocytes. Over expression of Sox8 and Sox9in chick spinal cord could directly induce oligodendrocytes generation at early stage ofdevelopment and also can promote oligodendrocyte differentiation. On the contrary, forcedexpression of Sox9 can inhibit neuronal differentiation. In order to elucidate therelationship of the SoxE transcription factor, we check the expression level of the othermembers of SoxE protein when overexpress one member of SoxE. There are reciprocalinteractions among SoxE members and other pivotal transcription factors in thedevelopment of oligodendrocytes. To analyze the detail molecular mechanism of SoxEproteins, the DNA binding domain HMG of SoxE was fused with VP 16 activation domainand EnR repressor domain. Misexpression of HMG-VP16 in chick spinal cord cansuppress the generation of interneuron and induce oligodendrocyte differentiation. Theseresults mimic the function of full length SoxE protein. Electroporation of HMG-EnRshowed no obvious effect on neural cell development. The soxE transcription factor canregulate the spinal cord development as an activator. These findings support importantroles for SoxE proteins controlling both oligodendrocyte and neuronal progenitor cellgeneration and specification as a complicated network.Cell adhesion molecules can play an important role in various aspects ofdevelopment of central nervous system which including morphogenesis of brain,differentiation and proliferation of neural stem cells, synaptic plasticity and the function oflearning and memory. Necl-1 (TSLL1/SynCAM3) belongs to the Ca²⁺-independentimmunoglobulin superfamily cell adhesion molecules. Necl-1 specifically expresses in thenervous system and localizes at the contact sites among neuron/neuron axon terminals andneuron/glia interactions. It is speculated that Necl-1 may regulate neuronal migration,axonal outgrowth, synapse assembly and myelin formation during neural development. In this study, we mainly use conventional gene knockout and in ovo electroporationtechniques to investigate the function of Necl-1 in neuronal development. The in situhybridization and immunostaining showed that the Necl-1 was broadly expressed in thedeveloping and postnatal spinal cord gray matter and brain including cortex, hippocampus,cerebellum and hindbrain. In order to detect the detail function of Necl-1 in developingneural tube, we overexpressed Necl-1 molecule in chicken embryonic spinal cord by in ovoelectroporation. The results indicated that over-expression Necl-1 does not affect thegeneration and localization of motoneurons and interneurons at early stage of spinal corddevelopment. Necl-1 mutants were generated by the conventional gene targetingapproach. A Necl-1 targeting vector was constructed by replace exon 2-5, that is the firstIg-like domain and the partial second Ig-like domain withβ-galactosidase gene and thefunction of Necl-1 will be disrupted. The targeting vector then was introduced intoembryonic stem (ES) cells for homologous recombination. Mutant ES cells were screeningby genome Southern blot and PCR. The positive clone has been used to generate mutantmouse lines. Now we have got heterozygote mice. The future work will focus onhomozygous mutant animal analyses. Neuronal migration, axonal outgrowth, synapseformation will be examined by immunological studies, retrograde labeling andelectrophysiological measurements. Also myelination will be investigated by electronicmicroscope.The study of these genes provides important clues about understanding how thenervous system develops and how the brain works.