Shh promotes neuronal differentiation in P19 cells without treatment

Our previous study suggested that P19 cells, when replated as the aggregates, could differentiate into neural cells with RA (McBurney et al., 1987). However, P19/hShhN cells could differentiate into neurons without RA or aggregation culture.

To compare whether P19 cells were differentiated into neurons by Shh without treatment of RA or aggregation culture, P19 and P19/hShhN cells induced differentiation with and without treatment of RA or aggregation culture. Cells were differentiated in the absence or presence of 10µM forskolin. RT-PCR analysis of RNA isolated from differentiated P19 and P19/hShhN cells, which indicated that the expression of marker for neuron, NFM, for DAs, TH and Nurr1, and for MNs, Olig2 increased by Shh in current differentiation, but not in conventional differentiation (Figure 17). P19/hShhN cells could differentiate into neuronal cells without treatment of RA or aggregation culture. Compared to the conventional method, the current differentiation method without aggregation may provide a useful system for the study of the functions of Shh.

Nestin

Aggregate Agg. + Differentiation Nonagg. + Differentiation hShhN

Fig. 17. Shh promotes neuronal differentiation in P19 cells without treatment of RA or aggregation culture. P19 cells were differentiated into neurons by Shh without treatment of RA or aggregation culture, P19 and P19/hShhN cells induced differentiation with and without treatment of RA or aggregation culture. Cells were differentiated in the absence or presence of 10µM forskolin. RT-PCR analyzed expression of TH and Nurr1, as a Dopamine neurons marker and Olig2, as a Motor neurons transcription factor.

IV. DISCUSSION

In this study, we have further addressed the function of Shh in the proliferation and the motor neuron differentiation of P19 cells. Our studies reveal that overexpression of Shh can direct motor neuron differentiation of P19 cells in the absence of RA, when P19/hShhN cells were replated as the single sell suspension.

Furthermore, we show that Shh-overexpressing P19 cells differentiate exclusively into motor neuron.

Chemical inducers and aggregation are two key elements to impart fate choices of P19 cells. With aggregation, DMSO directs P19 cells to differentiate into mesoderm origin muscle cells. RA, however, induces P19 cells to differentiate into ectoderm-derived neural cells including neurons and astrocytes (E.M. Jones-Villeneuve et al., 1982, 1983; M.W. McBurney et al., 1982). According to the treatment of cells, the neural differentiation of RA-induced P19 cells can be divided into two sequential stages, a stage of induction and a stage of differentiation. During the first stage, P19 cells are allowed to aggregate in the Petri dish and induced with RA for four days. Based on cell morphology changes and gene expression profiles, it seems that pluripotent P19 embryonic carcinoma (EC) cells are determined into neural progenitor cells during the first RA-induction stage. In second stage, the induced P19 cells are replated into cell culture dish as the single cell suspension or aggregates and left to differentiate into mature neurons and astrocytes.

Shh could induce proliferation by a number of intracellular mechanisms.

Binding of Shh to Ptc releases this receptor’s repression of Smo, which then transduces the signal by acting on the transcription factor of Gli family. The transcriptionally activating Gli forms then upregulate Shh targets, including ptc, gli1, gli2 and Shh itself. Shh signaling may directly regulate the cell cycle, as it can

upregulate the expression of G1-phase cyclins of type D and E, and Pathed can act directly on the G2-phase cyclin B. Alternatively, Shh other activity may activate other signaling system that control cell proliferation. We observed that the Shh receptor Pathed and Smoothend and the target gene Gli is expressed in P19 cells. We have showed that Shh regulates the proliferation of P19 cells using growth kinetics and MTT assay. P19/hShhN cells increased the number of total cells in growth and differentiation condition and neutralized by 5E1. The biological activity of the Shh conditioned media was almost completely inhibited by 5E1, but unaffected by IgG.

The results suggest that the secreted hShhN was responsible for the stimulation of P19 cell proliferation .We conclude that Shh directly promotes P19 cells proliferation in vitro.

As the neurogenesis in vivo, neurons appear earler than glial cells during RA-induced P19 cell neural differentiation (E.M. Jones-Villeneuve et al., 1982).

Interestingly, our results show that P19/hShhN cells differentiate restrictively into neurons, but not glial cells. Our previous study suggested that P19/ShhN, when replated as the aggregates, could differentiate into neural cells without RA induction.

However, RA-inducted P19 cells were normally replated as the single cell suspension to enrich the cells of interest during their neural differentiation (M.W McBurney et

al., 1987, 1995). The wild-type P19 cells were used as the negative control. At day 3, P19/hShhN cells started to send out long neurite-like process. At day 4, P19/hShhN cells sent out many neurite-like process to form the networks. In parallel, the expression of Tuj1 and NF-M, as a neuronal specific marker, increased during the differentiation of P19/hShhN. This suggests that Shh may promote the neuronal differentiation in P19 cells without RA or aggregation culture.

Shh is necessary for the induction of both spinal motor neurons and midbrain dopaminergic neurons. In the developing midbrain, Shh was first characterized for its ability to induce the production of dopaminergic neurons. In the developing spinal cord, the induction of motor neuron progenitors depends on Shh activity. We have showed that Shh promotes motor neuron differentiation than dopaminergic neurons in current differentiation method. P19/hShhN cells increased expression of motor neurons transcription factor, homeodomain or specific marker gene, Nkx.1, Olig2, Ngn2, NeuroD, Isl -1, Hb9 and ChAT. But, the expression of TH decreased in P19/hShhN cells. Nkx6.1 is expressed in progenitors for both motor neurons and ventral interneurons. The expression of Olig2 and Ngn2 was further correlated with subtype-specific neuronal marker at E12.5, the stage when progenitors actively generate various neuronal subtype. The onset of Olig2 and Ngn2 expression precedes those of Hb9 and Isl -1, indicating that they are one of the earlist transcription factors expressed in the motor neuron lineage. Thus, among many members of bHLH factors, the coexpression of Olig2 and Ngn2 is specifically correlated to motor neuron generation. Isl-1 and Hb9 are defined markers for motor neurons and their

progenitors in the ventral neural tube (Tsuchida et al., 1994; Arber et al., 1999;

Thaler et al., 1999). These results suggest that Shh could be promoted into motor neuron progenitor cells and motor neuron differentiation and expressed transcription factors and HD.

The results here supported previous suggestions that Shh may be useful vehicles for a variety of neurological diseases, as Parkinson disease or Spinal cord injury.

V. CONCLUSIOS

Shh increases both proliferation and neuronal differentiation in P19 cells and promote the neuronal differentiation in P19 cells without RA or aggregation culture.

In the neuronal differentiation, Shh may promote proliferation of neuronal precursor cells and thereby increase expression of motor neuron specific proteins. Compared to the conventional method, the current differentiation method without aggregation may provide a useful system for the study of the functions of Shh.

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-국문 요약-

세포 증식과 분화에서 Shh 의 효과를 알아보았다. Shh 이 과 발현되고 있는

핵심어 : : : : Sonic hedgehog, Proliferation, Differentiation, Motor neurons, P19 cells