Research Report
Identification of differentially expressed genes in the developing mouse inferior colliculus

https://doi.org/10.1016/j.devbrainres.2005.06.010Get rights and content

Abstract

Although injured neurons of inferior colliculus (IC) display a robust axonal outgrowth through a lesion site at postnatal day six (P6) in vitro, and are capable to re-innervate their target cells, injured neurons from P10 IC are unable to regenerate their axons across the lesion site. This axonal regenerative failure has been attributed to an increase of expression of inhibitory molecules in endogenous tissue, during development. As a first step to identify such inhibitory molecules, the present study reports the isolation of molecules differentially expressed in the IC during development. A two-directional (forward and backward) suppression subtractive hybridization (SSH) was performed on IC tissue between P6 and P10 stages. One hundred cDNAs from P6 (P6−P10) and 200 cDNAs from P10 (P10−P6)-subtracted libraries were randomly sequenced. A dot-blot screening of sequenced cDNAs revealed the differential expression for the majority of these cDNAs at their respective developmental stage. Then, the analysis of sequenced clones showed that P6 library was highly enriched in molecules expressed early in the development, such as GAP43 or vimentin proteins. By contrast, the P10 library contained mostly molecules expressed at later stages of development in the central nervous system, such as myelin-related proteins. Our results show that SSH is a suitable method for identifying differentially expressed genes in the developing IC. In addition, these results provide a foundation for further studies dealing with molecules involved in the IC development before and at the onset of hearing, some of which being probably involved in the axonal outgrowth mechanism.

Introduction

Adult mammalian central nervous system (CNS) neurons are unable to regenerate axons following a trauma in vivo. In contrast, sectioned neonatal axons are able to re-grow, and to re-innervate initial target tissues. Then, the CNS regenerative capability disappears during early postnatal development [9], [10], [15], [17], [24], [27].

The inferior colliculus (IC) commissure has been previously used as an in vitro model to study axonal regeneration [9]. In organotypic cultures, severed IC commissural axons from postnatal day 6 (P6) display a robust regeneration after 6 days of recovery. Lesioned axons re-grew through the lesion site, and made synaptic contacts within the IC contralateral lobe [9]. This regenerative capability across the lesion site is completely lost by P10–12 [10], [11]. However, P10–12 neurons still retained an axonal outgrowth capability when provided with a favorable substrate in vitro (for example, C6 glia or polylysine/laminin).

The maturation of glia has been implicated in age-dependent axonal regeneration failure observed in the IC [10], [11]. In the latter study, the loss of axonal regeneration in the developing IC was shown to involve expression of endogenous tissue inhibitory molecules that interacted with injured axons. Indeed, blockage of protein synthesis from glial cells localized at the vicinity of the wounded zone, using cycloheximide (a protein synthesis inhibitor), improved P10–12 axonal regeneration through the lesion site [11].

It would be of interest to identify which molecules are involved in the axonal regeneration failure observed in the IC by P10. However, no data are available regarding the molecular expression in the central region of the IC at P10. In order to identify these molecules, the present study aimed to search for messengers differentially expressed in the IC during development. A suppression subtractive hybridization (SSH) was carried out between IC tissues from P10 and P6 aged mice. cDNA libraries were constructed, partly sequenced, and the differential expression of sequenced genes was analyzed by dot-blots. Finally, the subtractive protocol allowed for isolation and identification of specific endogenous molecules which are highly expressed in the central region of the IC at P6 and at P10.

Section snippets

Materials and methods

Mice of the C57BL/6 strain were used for this study. The care and use of animals was approved by the “Direction departementale des services veterinaires de la Gironde (authorization # 3303303)”.

Differential expression of cDNAs in the inferior colliculus at P6 and P10

In order to isolate cDNAs highly expressed in mouse inferior colliculus (IC) at P6 and P10, a suppression subtractive hybridization (SSH) procedure was performed using the intercollicular region (which contains the IC commissure) from P6 and P10 tissues. Forward and reverse hybridizations were carried out. Forward subtraction corresponded to P10 minus P6 (P10−P6), revealing molecules that are differentially expressed at P10. Reverse subtraction corresponded to P6 minus P10 (P6−P10), revealing

Discussion

In the present study, we used SSH in order to isolate genes that are differentially expressed in the mouse IC at developmental stages P6 and P10. Systematic sequencing of randomly chosen 300 clones from P6−P10 (n = 100) and P10−P6 (n = 200) libraries was performed. For most of the identified genes, differential expression was further confirmed by dot-blot screening. Sequence analysis revealed the presence of genes expressed early during CNS development within P6 library. In contrast, genes

Acknowledgment

This study was supported by Association Libre pour la Recherche sur la Moelle Epiniere (ALARME, France).

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