Multiple Regionalized Genes and Their Putative Networks in the Interpeduncular Nucleus Suggest Complex Mechanisms of Neuron Development and Axon Guidance

Abstract

The interpeduncular nucleus (IPN) is a highly conserved limbic structure in the vertebrate brain, located in the isthmus and rhombomere 1. It is formed by various populations that migrate from different sites to the distinct domains within the IPN: the prodromal, rostral interpeduncular, and caudal interpeduncular nuclei. The aim here was to identify genes that are differentially expressed across these domains, characterizing their putative functional roles and interactions. To this end, we screened the 2,038 genes in the Allen Developing Mouse Brain Atlas database expressed at E18.5 and we identified 135 genes expressed within the IPN. The functional analysis of these genes highlighted an overrepresentation of gene families related to neuron development, cell morphogenesis and axon guidance. The interactome analysis within each IPN domain yielded specific networks that mainly involve members of the ephrin/Eph and Cadherin families, transcription factors and molecules related to synaptic neurotransmission. These results bring to light specific mechanisms that might participate in the formation, molecular regionalization, axon guidance and connectivity of the different IPN domains. This genoarchitectonic model of the IPN enables data on gene expression and interactions to be integrated and interpreted, providing a basis for the further study of the connectivity and function of this poorly understood nuclear complex under both normal and pathological conditions.

Keywords: axon guidance; cadherin; ephrin; interactome; interpeduncular nucleus; neuronal migration; rostral hindbrain; transcription factors.

FIGURE 1

Regional gene expression in the IPN at E18.5 (I). (A) Schemes representing the dorsal and lateral views of the IPN with its main sub-divisions. In the dorsal view, a, b represent the median and paramedian planes of the sections analyzed, respectively. The dashed areas correspond to the lateral sub-divisions of the IPN that were not included in this study. (B–O) and Figure 2 show representative parasagittal sections centered on the IPN in the rostral hindbrain, highlighting the regionalized expression of genes. The rostral end is situated to the left in all images. The different expression patterns, corresponding to combinations of the IPN sub-divisions, are defined in the vertical boxes and followed by images of representative genes fitting each pattern. The abbreviations used are specified in the main text. Scale bars = 200 μm.

FIGURE 2

Regional gene expression in the IPN at E18.5 (II). Additional expression patterns (vertical boxes) followed by images of their corresponding genes (A–O) as displayed in Figure 1. Scale bars = 200 μm.

FIGURE 3

Global distribution of the regionalized genes in relation to the IPN sub-divisions. (A) Scheme representing the number of genes expressed in either the Pro, IPR or IPC, or combinations of these domains. (B) Scheme representing the rostro-caudal and deep-to-superficial sub-divisions of the IPN in colors: Pro (yellow), IPRa (gray), IPRi (blue), IPRb (green), IPC (pink). The entire IPR is filled with a dotted background. (C–H) Lists of the genes expressed in the Pro (C), IPRa (D), IPRi (E), IPRb (F), entire IPR (G), and/or IPC (H).

FIGURE 4

Overrepresentation test and the interaction analysis of the regionalized genes within the IPN. (A) Graphic results of the PANTHER Statistical Overrepresentation Test (p < 0.00005). For each functional category, the orange bar represents the percentage of genes in the analyzed (test) list (135 genes: see “Materials and Methods” section) that match this category, while the blue bar corresponds to the percentage of genes in the reference list (2,038 genes: see “Materials and Methods” section) associated to the same category. (B–F) Respective STRING networks of the genes expressed in the Pro (E), IPRa (B), IPRi (D), IPRb (F), and IPC (C). Each node represents a gene and each line symbolizes a predicted interaction (with a minimum required confidence score of 0.7: see “Materials and Methods” section). The thickness of the line represents the strength of the data supporting the predicted interaction. The green nodes correspond to genes that belong to every GO family represented in (A), the red nodes symbolize genes included only in both the cell morphogenesis (GO: 0000902) and the cellular component morphogenesis (GO: 0032989) families, the blue nodes correspond to genes belonging exclusively to the neuron development family (GO: 0048666) and the light pink nodes represent genes not included in any of the GO categories listed in (A).