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

Lovrics A, Gao Y, Juhász B, Bock I, Byrne HM, Dinnyés A, Kovács KA - PLoS ONE (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Biotalentum Ltd., Gödöllö, Hungary.

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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Related in: MedlinePlus

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  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 0.05).
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pone-0111430-g005: 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 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 0.05).

Mentions: Calcium-phosphate mediated transient co-transfection experiments were performed in HEK293T [45] cells. The pGL4-Irx3 construct or a promoterless pGL4.12 was introduced into the cells with or without different amounts of the pcDNA3-Nkx2.2 plasmid (see figure 4(a)). Green fluorescent protein (GFP) expressing control plasmid, that does not bind to or regulate the expression of Irx3, was spiked onto every condition to verify the transfection efficiency (see a representative photo of transfected cells in figure S1) while the total amount of DNA and the amount of pGL4-Irx3 or promoterless pGL4.12 reporter plasmid were kept constant. Additionally, qPCR was used to confirm that the transfected cells expressed exogenous Nkx2.2 (see figure 5). The amount of cotransfected plasmids in each condition for qPCR and luciferase measurements are listed in table S6.


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

Lovrics A, Gao Y, Juhász B, Bock I, Byrne HM, Dinnyés A, Kovács KA - PLoS ONE (2014)

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  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 0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232242&req=5

pone-0111430-g005: 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 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 0.05).
Mentions: Calcium-phosphate mediated transient co-transfection experiments were performed in HEK293T [45] cells. The pGL4-Irx3 construct or a promoterless pGL4.12 was introduced into the cells with or without different amounts of the pcDNA3-Nkx2.2 plasmid (see figure 4(a)). Green fluorescent protein (GFP) expressing control plasmid, that does not bind to or regulate the expression of Irx3, was spiked onto every condition to verify the transfection efficiency (see a representative photo of transfected cells in figure S1) while the total amount of DNA and the amount of pGL4-Irx3 or promoterless pGL4.12 reporter plasmid were kept constant. Additionally, qPCR was used to confirm that the transfected cells expressed exogenous Nkx2.2 (see figure 5). The amount of cotransfected plasmids in each condition for qPCR and luciferase measurements are listed in table S6.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Biotalentum Ltd., Gödöllö, Hungary.

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.

Show MeSH
Related in: MedlinePlus