Sox 14 is essential for initiation of interneuron differentiation 6 in the chick spinal cord


 The neural tube comprises several different types of progenitors and postmitotic neurons that coordinately act with each other to play integrated functions. Its development consists of two phases: proliferation of progenitor cells and differentiation into postmitotic neurons. How progenitor cells differentiate into each corresponding neuron is an important question for understanding the mechanisms of neuronal development. Here we introduce one of the Sox transcription factors, Sox14, which plays an essential role in the promotion of neuronal differentiation. Sox14 belongs to the SoxB subclass and its expression starts in the progenitor regions before neuronal differentiation is initiated at the trunk level of the neural tube. After neuronal differentiation is initiated, Sox14 expression gradually becomes confined to the V2a region of the neural tube, where Chx10 is co-expressed. Overexpression of Sox14 restricts progenitor cell proliferation. Conversely, the blockade of Sox14 expression by the RNAi strategy inhibits V2a neuron differentiation and causes expansion of the progenitor domain. We further found that Sox14 acted as a transcriptional activator. Taken together, Sox14 acts as a modulator of cell proliferation and an initiator protein for neuronal differentiation in the intermediate region of the neural tube.


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The neural tube is the embryonic organ of the central nervous system, and several distinct types

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As most transcription factor expression starts after proneural gene expression is initiated, we 94 attempted to identify the onset of Sox14 expression. We performed in situ hybridisation analysis of 95 sections of the neural tube at various embryo stages. As a result, we found that Sox14 is already 96 expressed in the neural tube at HH stage 14 (Fig. 1a) before neuronal differentiation starts. Moreover,

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Next, we investigated when Chx10 expression began. Chx10 expression was not observed 101 before the initiation of neuronal differentiation (Fig. 1d). The initial expression was found at the V2a 102 region after neuronalisation started (Fig. 1e), and a stronger expression was found at HH stage 22 (Fig.   103 1f). These findings suggest that Sox14 has upstream factors other than Chx10 for its initial expression

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The same concentration of Shh was used to induce Chx10; however, expression began 24 h 115 after the start of culture, suggesting that the onset of Chx10 expression was later than that of Sox14 (

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Results indicated that expression of Sox2, a neural progenitor marker, was reduced in the 128 electroporated cells, while no change was found by electroporation of the control vector ( Fig. 2a- 129 n=5 for control and n=8 for Sox14). We reasoned that the cells might have precociously differentiated 130 into neurons; however, Sox14 overexpression also reduced p27 KIP1 expression, suggesting that neuronal 131 maturation was also perturbed by Sox14 overexpression (Fig. 2c-d'). Therefore, we hypothesised that 132 the initial stage of the neurons was induced by Sox14, and investigated the expression of p57 KIP2 , which

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Sox14. In addition, the IdU injection did not change the rate of incorporation as in the control (Fig.   193 5g,g'; n=8), which differs from the phenotype observed in wild-type Sox14 (Fig. 3b,c).

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In this study, we analysed the function of one of the Sox transcription factors, Sox14, and 200 demonstrated that Sox14 is required for the progression of neuronal differentiation (Fig. 4). In addition,

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Sox14 was initially identified as a SoxB subclass gene, which encompasses Sox1-3, Sox14, 204 and Sox21 [13]. A subsequent study demonstrated that Sox14 expression was found in the V2a neuronal 205 region, and its expression was overlapped, at least in part, with that of Chx10 and Lhx3/Lim3 [20].

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Moreover, a recent study showed that electroporation of Chx10 induces ectopic expression of Sox14,

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indicating that Chx10 is a sufficient upstream factor for Sox14 [11]. In contrast, our analysis revealed 208 that Sox14 expression onset occurred earlier than that of Chx10 (Fig. 1), suggesting that Chx10 is not

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In contrast, our analyses revealed that Sox14 is required for the promotion of neuronal 228 differentiation. Knockdown of the Sox14 gene caused the abolishment of neuronal differentiation, rather 229 than producing different types of neurons (Fig. 4). Therefore, it can be said that Chx10 and Sox14 play

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In our analyses, Sox14 acted as a transcriptional activator (Fig. 5). We recognise that Sox14 239 has been suggested as a transcriptional repressor, which is controversial for its mode of action. The

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repressor assumption was based on its amino acid sequence [13], and we predicted that the mode of 241 action would change in a context-dependent manner.

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In this study we demonstrated that Sox14 induces the p57 KIP2 expression; however, whether this 243 induction is direct is still elusive. Thus future studies will focus on searching for the direct target genes 244 of Sox14 using chromatin immunoprecipitation. Moreover, it is highly possible that the target genes of 245 Sox14 in neural progenitor cells and those in V2a neurons are distinct. Therefore, in addition to 246 searching for different target genes depending on the neural differentiation steps, it would be useful to 247 identify cofactors that bind to Sox14. These cofactors would include the general proneural genes, such 248 as Neurogenin1/2 or NeuroD2/4/6, and would modulate the functions of Sox14.

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Understanding the function of each transcription factor is useful for generating specific 250 functional neurons from stem cells [33]. We envision that the findings of this study will partly contribute 251 to regenerative medicine in the future.

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Quantitative data are presented as means ± SEM, and differences were evaluated using the two-tailed

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Ectopic (e,f) and reduced (b,c) expression is indicated by filled and outlined arrowheads, respectively.

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Note that the GFP expression in (c',f') was taken from the adjacent sections. Scale bars in (b) for (b-  Table   386 Supplementary Tab. S1 The RT-qPCR primers used in this study.