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. 2014 Nov 14;9(11):e111430.
doi: 10.1371/journal.pone.0111430. eCollection 2014.

Boolean modelling reveals new regulatory connections between transcription factors orchestrating the development of the ventral spinal cord

Anna Lovrics  1 , Yu Gao  1 , Bianka Juhász  1 , István Bock  2 , Helen M Byrne  3 , András Dinnyés  4 , Krisztián A Kovács  5
Affiliations

Boolean modelling reveals new regulatory connections between transcription factors orchestrating the development of the ventral spinal cord

Anna Lovrics et al. PLoS One. .

Abstract

We have assembled a network of cell-fate determining transcription factors that play a key role in the specification of the ventral neuronal subtypes of the spinal cord on the basis of published transcriptional interactions. Asynchronous Boolean modelling of the network was used to compare simulation results with reported experimental observations. Such comparison highlighted the need to include additional regulatory connections in order to obtain the fixed point attractors of the model associated with the five known progenitor cell types located in the ventral spinal cord. The revised gene regulatory network reproduced previously observed cell state switches between progenitor cells observed in knock-out animal models or in experiments where the transcription factors were overexpressed. Furthermore the network predicted the inhibition of Irx3 by Nkx2.2 and this prediction was tested experimentally. Our results provide evidence for the existence of an as yet undescribed inhibitory connection which could potentially have significance beyond the ventral spinal cord. The work presented in this paper demonstrates the strength of Boolean modelling for identifying gene regulatory networks.

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Conflict of interest statement

Competing Interests: Al, YG, BJ and KK were affiliated to Biotalentum Ltd. IB and AD are still affiliated to Biotalentum Ltd. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Transcription factor profiles in the ventral spinal cord.
(A) Neural progenitor cells along the dorsal-ventral axis of the ventral spinal cord and (B) the corresponding TFs. Figure 2A,B from modified, with permission. (C) Binary representation of TF expression (present or absent) in each progenitor cell type in the order (Nkx2.2,Nkx6.1,Olig2,Pax6,Irx3,Dbx2,Nkx6.2,Dbx1).
Figure 2
Figure 2. Gene regulatory networks.
(A) GRN of ventralization assembled from published experimental data. TFs are denoted by ellipses. Transcriptional inhibitions between TFs and target genes are denoted by blunted arrowheads. Associations depicted by continuous lines were demonstrated experimentally; associations denoted by dotted lines were tested with Boolean simulations. (B) Minimal GRNs obtained from Boolean simulations. Inhibition represented by continuous lines were experimentally verified, additional dashed lines represent connections present in the minimal matching GRNs. The line between Nkx2.2 and Dbx2 contains a circle at both ends representing that the direction of inhibition is not defined in the minimal GRNs.
Figure 3
Figure 3. Boolean simulation results of the minimal GRN where Nkx2.2 inhibits Dbx2.
In contrast to figure 2, the nodes in this figure represent the TF expression states as a vector of zeros and ones in the order (Nkx2.2, Nkx6.1, Olig2, Pax6, Irx3, Dbx2, Nkx6.2, Dbx1). Red nodes stand for attractor states and are denoted by the name of the corresponding neural progenitor (p0, p1, p2, pMN and p3 cells), other nodes are labelled by the expression level of each transcription factor. As a result of the asynchronous update in the Boolean model, one state can have more than one successor states, but the figure only shows the most probable update route(s) for each node.
Figure 4
Figure 4. Relative luciferase activity.
The light emission of luciferase was measured after addition of Luciferase Assay Reagent II, normalized against the protein content in samples transfected by pGL4-Irx3 (Irx), promoterless pGL4.12 (pB), pGL4-Irx3 plus increasing molar ratio of Nkx2.2 (N1, N2, N3) and promoterless pGL4.12 plus Nkx2.2. In the promoterless reporter vectors there is virtually no activity. The luciferase activity in samples N1, N2, N3 significantly differ from that of sample Irx3 (significance level formula image0.05). The reduction of the luciferase activity shows that Nkx2.2 negatively regulates Irx3 promoter activity, however no dose-dependent inhibition was observed.
Figure 5
Figure 5. Relative expression of Nkx2.2 in the transfected cells (cells with exogenous expression of Nkx2.2: Nkx2.2_a and Nkx2.2_b) and non-transfected cells (cells which have only endogenous expression of Nkx2.2: control_a and control_b).
Relative expression was calculated using the formula image method. Ct values were normalized to the reference gene GAPDH and the average expression level of the non-transfected cells (control_a, control_b) were normalized to 1. Expression level of Nkx2.2 in transfected cells significantly differ from expression level of Nkx2.2 in non-transfected cells (significance level formula image0.05).
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