Profiling, Bioinformatic, and Functional Data on the Developing Olfactory/GnRH System Reveal Cellular and Molecular Pathways Essential for This Process and Potentially Relevant for the Kallmann Syndrome

Abstract

During embryonic development, immature neurons in the olfactory epithelium (OE) extend axons through the nasal mesenchyme, to contact projection neurons in the olfactory bulb. Axon navigation is accompanied by migration of the GnRH+ neurons, which enter the anterior forebrain and home in the septo-hypothalamic area. This process can be interrupted at various points and lead to the onset of the Kallmann syndrome (KS), a disorder characterized by anosmia and central hypogonadotropic hypogonadism. Several genes has been identified in human and mice that cause KS or a KS-like phenotype. In mice a set of transcription factors appears to be required for olfactory connectivity and GnRH neuron migration; thus we explored the transcriptional network underlying this developmental process by profiling the OE and the adjacent mesenchyme at three embryonic ages. We also profiled the OE from embryos null for Dlx5, a homeogene that causes a KS-like phenotype when deleted. We identified 20 interesting genes belonging to the following categories: (1) transmembrane adhesion/receptor, (2) axon-glia interaction, (3) scaffold/adapter for signaling, (4) synaptic proteins. We tested some of them in zebrafish embryos: the depletion of five (of six) Dlx5 targets affected axonal extension and targeting, while three (of three) affected GnRH neuron position and neurite organization. Thus, we confirmed the importance of cell–cell and cell-matrix interactions and identified new molecules needed for olfactory connection and GnRH neuron migration. Using available and newly generated data, we predicted/prioritized putative KS-disease genes, by building conserved co-expression networks with all known disease genes in human and mouse. The results show the overall validity of approaches based on high-throughput data and predictive bioinformatics to identify genes potentially relevant for the molecular pathogenesis of KS. A number of candidate will be discussed, that should be tested in future mutation screens.

Systems biology of facial development: contributions of ectoderm and mesenchyme

“…In temporal terms, this study bridges the gap between an early gene expression atlas (up to E10.5) based on laser-capture samples (Brunskill et al, 2014) and several studies of later structures undergoing terminal differentiation including teeth, tongue, salivary glands and olfactory epithelium (Garaffo et al, 2013; Musselmann et al, 2011; O’Connell et al, 2012). In terms of resolution, this study delineates the ectoderm and mesenchyme components that are not resolved in whole prominence studies (Feng et al, 2009), while retaining the global profile of prominence expression that is not possible with the restricted sampling of laser capture (Brunskill et al, 2014).…”

“…Although it lacks the spatial resolution to examine the dynamics of gene expression at the site of fusion of the lip and primary palate, its statistical robustness makes it well suited to layer with other more spatially refined datasets (e.g. Brunskill et al, 2014; Garaffo et al, 2013; O’Connell et al, 2012; A. S. Potter and S. S. Potter, 2015) to address roles of specific tissues and pathways in facial development and palate fusion.…”

“…To understand the ectodermal genetic programs and how they coordinate and integrate with the mesenchymal programs to drive facial development, it is important to obtain a comprehensive understanding of the cellular and molecular changes that occur in each of these tissues during the early stages of facial development. Previous transcriptome studies of human, mouse or chick facial development have provided considerable information concerning gene expression in discrete tissues or regions at specific developmental stages or have generated gene expression profiles of whole prominences over a defined time course (Bhattacherjee et al, 2007; Brinkley et al, 2016; Brugmann et al, 2010; Brunskill et al, 2014; Buchtová et al, 2010; Cai et al, 2005; Ding et al, 2016; Feng et al, 2009; Garaffo et al, 2013; Han et al, 2014; Iwata et al, 2012; Mima et al, 2013; Musselmann et al, 2011; O’Connell et al, 2012; A. S. Potter and S. S. Potter, 2015; Warner et al, 2014).…”

The Dlx5 and Foxg1 transcription factors, linked via miRNA-9 and -200, are required for the development of the olfactory and GnRH system

“…Tables VIIA, B, C and VIIIA, B). Amongst these we noted Foxp1 and Foxg1 , which however were not differentially expressed in the Dlx5 −/− OE with the adopted cut-off value and statistical parameters ( Garaffo et al, 2013 ). Two possible explanations: either changes in the abundance of miR-9 and miR-200 -class cause changes in the abundance of target RNAs that are too modest to pass the imposed cut-off value, or these miRs preferentially affect translation and not stability of the target mRNAs.…”

“…Next we intersected the predicted miR-9 and miR-200 -class targets with the coding mRNAs found to be differentially expressed in the Dlx5 −/− OE compared to the WT ( Garaffo et al, 2013 ). A significant enrichment of miR-9 and miR-200 -class target sequences was detected in the 3′ UTR of genes up-regulated in the Dlx5 −/− OE ( Table 1 A, B).…”