Principe de la reconstruction 3D de la forme de la molaire

Dans le document Évolution et Développement d'un organe sériel - la molaire : Transcriptomique comparée des bourgeons de molaire chez les rongeurs (Page 105-115)

A. La forme à l’interface entre l’épithélium et le mésenchyme va donner la forme de la molaire au stade adulte (représenté en jaune). B. Le marquage de la lame basale par la lamininα5 permet de délimiter l’interface entre épithélium et le mésenchyme. Ce marquage nous a permis de reconstruire la forme 3D de la partie épithéliale du bourgeon de molaire à l’aide du logiciel Amira.

bucco-linguale

Matériels et méthodes du chapitre 11.

6.1 Hybridation in situ : caractérisation du patron d’expression de

Bmper

J’ai commencé à caractériser l’expression de bmper chez la souris pas in situ in toto. Pour cela, j’ai cloné une sous-partie du transcrit Bmper chez la souris afin de réaliser un sonde pour les hybridations in situ. En parallèle, j’ai disséqué des germes de molaire à différents stades (ED15,ED15.5 et ED16) puis j’ai réalisé des hybridation in situ pour Bmper ainsi que pour Fgf4 afin d’avoir un contrôle positif de la manipulation et aussi pour pouvoir marquer la position des noeuds d’émail. J’ai observé le patron d’expression de bmper au stade ED15.5 sur germe entier. J’ai ensuite inclus ces germes de molaires ayant subi l’hybridation in situ en paraffine afin de réaliser des coupes pour effectuer une une meilleure caractérisation du patron spatial d’expression. Pour contrôler l’orientation des germes, j’ai pré-inclus en agarose, puis j’ai déshydraté le tout avant de l’inclure en paraffine et d’effectuer les coupes au microtome. Cependant, le processus de déshydratation (en vu de l’inclusion en paraffine) lavait trop le signal de l’hybridation in situ in toto. Pour pallier à ce problème il faudrait mettre au point une in situ sur coupe cryostat.

6.2 Mutants bmper et coupes histologiques

Nous nous sommes demandé si bmper jouait un rôle dans le développement de la molaire chez la souris. Comme il existait déjà des mutants de Bmper chez la souris, nous avons collaboré avec Pamela Lockyer du laboratoire de Cam Patterson qui nous a fourni le matériel (adultes et embryons) pour caractériser la morphologie dentaire des souris mutantes Bmper, dont la

Chapitre 6. Bmper : gène candidat de l’asymétrie bucco-linguale

mutation a été obtenu par délétion, créant l’allèle Bmpert m1C p at[Kelley et al. , 2009]. Les mutants bmper homozygotes meurent à la naissance car ils ne peuvent pas respirer (malformation de la trachée). Je n’ai donc pas pu travailler directement avec la molaire minéralisée des homozygotes. J’ai cependant pu photographier et comparer la dentition des mutants bmper hétérozygotes avec celle de la souche sauvage, mais je n’ai pas observé de différences notoires avec la den-tition de la souche sauvage. En parallèle j’ai travaillé sur des embryons au stade ED18.5 pour la souche sauvage, hétérozygote et homozygote mutante. Nous avons choisi le stade ED18.5 car il correspond à la fin de la mise en place des cuspides. De plus, à ce stade le processus de minéralisation n’a pas encore commencé ce qui permet l’analyse en coupes histologiques. J’ai donc reçu les échantillons en Bouin (traitement qui permet de déminéraliser et de fixer les tissus), que j’ai déshydraté et inclus en paraffine. À l’aide d’un microtome, j’ai ensuite réalisé des coupes histologiques (de 6µm d’épaisseur) que j’ai coloré au trichrome de Masson afin de mettre en évidence les différents tissus de la dent, puis j’ai analysé ces coupes au microscope. J’ai ensuite photographié des séries complètes (1 section toutes les 3 sections) pour observer et caractériser la morphologie embryonnaire (pour 5 homozygotes mutés / 1 hétérozygote / 3 sauvages). Dans le but de reconstruire la morphologie 3D du bourgeon de molaire chez des embryons homozygotes bmper, j’ai aussi testé, sur des échantillons de souris sauvages, un traitement PTA (Phospho Tungstic Acid) afin de contraster les tissus mous pour permettre d’imager ces tissus mous par micro-tomographie à rayons X (microCT scan) [Metscher, 2009; Hoorebeke, 2013]. Cependant, cette approche n’a pas été concluante due à la difficulté de la reconstruction du bourgeon de molaire qui est encapsidé dans le cartilage de la mâchoire et qui ne permet pas une reconstruction simple de la morphologie du bourgeon et un travail plus important de mise au point serait nécessaire.

gans : the mouse first upper and lower

molar

Introduction

How to link differences of the developmental program to changes in morphology ? We tackle this question at the whole transcriptomic level in the mouse first upper and lower molars.

The molars are serial organs, meaning that they are the same module patterned in different location of the body. In the case of upper and lower molar they deployed in the upper and the lower jaws. As already discussed in the introduction of this thesis (see chapter 3, p. 41), the upper and the lower jaw identities are defined around ED8.25-ED9 by homeobox genes. For example Dlx5/6 are essential in the first pharyngeal arch to determine the identity of the lower jaw [Depew, 2002; Beverdam et al. , 2002]. Afterwards, the odontogenic homeobox code is known to regulate the type of the tooth [Cobourne & Sharpe, 2003; Catón & Tucker, 2009]. For example, regions of the jaw expressing Dlx1/2 [Qiu et al. , 1997; Thomas et al. , 1997] and Barx1 [Tucker, 1998; Miletich et al. , 2005] are the presumptive regions where molars will develop.

An interesting feature about mouse first molars is that they present extreme divergent morpho-logies between the upper and the lower jaw. Thus the mouse first upper molar develop two supplementary cusps. How to increase the number of cusps patterned during development ? Experiments of tissue recombination show that cusp patterning is tightly linked to mesenchyme identity and proportion [Schmitt et al. , 1999; Hu et al. , 2006; Cai et al. , 2007; Ishida et al. , 2011]. Gene-centered studies have already identified some genes and pathways responsible for the patterning of supplementary cusps [Tucker et al. , 2004; Kassai et al. , 2005; Charles et al. , 2009a; Harjunmaa et al. , 2012] (see chapter 3, p. 52 ). However, most of these gene-centered studies were done in mouse lower molars only and were not interested in identifying the developmental bases responsible for the increase number of cusps in mouse upper molar.

Chapitre 7. Transcriptomic signature of serial organs : the mouse first upper and lower molar

In the lab we are interested in how the mouse upper molar is able to develop these two supple-mentary cusps. Answering this question implies comparing developmental programs of upper and lower molars. Historically this was done at a gene by gene basis. However, the rise of mi-croarrays and RNA-seq techniques enable to address the developmental program at the scale of the whole transcriptome. The transcriptomics studies already performed in mouse were either focusing on lower molars development [Landin et al. , 2015] or in a single developmental stage [Laugel-Haushalter et al. , 2013], or were focusing on earlier stages [O’Connell et al. , 2012; Jia et al. , 2012]. The novelty of our study is to survey both the development of mouse first upper and lower molars at the transcriptomic level during crown morphogenesis, when cusps are patterned sequentially from ED14.5 to ED18 (sampled every 0.5 days). We proposed to study cusps patter-ning in both upper and lower molars at the whole transcriptomic level first to gain insight into the transcriptomics signatures that characterize crown morphogenesis and second to compare the development programs between upper and lower molars to identify the modifications of the developmental program that enable the patterning of two supplementary cusps in mouse upper molar. Most transcriptomics studies comparing developing embryos or organs looked for the main pattern of variation in transcriptomes, which are typically extracted by multivariate analysis and identified different species or organs through developmental time [Yanai et al. , 2004; Liao & Zhang, 2005; Brawand et al. , 2011; Lin et al. , 2014; Gilad & Mizrahi-Man, 2015]. As discussed by Breschi et al. [2016] the transcriptome is made of different signatures. To go beyond the identification of which transcriptomics signature prevails, it is of interest to identify the patterns of expression responsible for each signature and to quantify the relative contribution of those signatures to the final transcriptome. Such a characterization would enable to efficiently compare expression profiles between homologous organs or between species and tackle ques-tions such as : how differences in transcriptomics signatures enlighten us of the developmental basis of morphological disparities between homologous organs ?

Importance of cellular proportion in organ identity

In this article we aimed to identify the transcriptomic differences that underly the development of mouse first upper and lower molar during crown morphogenesis. We found that part of the upper/lower transcriptomic differences are explained by three transcriptomics signatures that are shaped by relative abundance of cellular type.

First, differences in epithelial/mesenchymal relative proportion participate to differences bet-ween upper/lower molars, together with differences of expression within tissue, at least in the mesenchyme. Second, a significant heterochrony explained part of the upper/lower differences. This heterochrony could be modeled by the increase in proportion of the tooth germ occupied by cusp tissue. Third, an early-mid (ED15.5-ED16) transient excess of upper/lower differences have been identified. Analysis of the genes responsible for this transient excess suggested that the upper molar experience exaggerated morphogenetic processes (with less mitotic cells and more migrating cells).

Our analysis suggested that gene expression in a complex tissue is not only the result of

however it provides valuable insight in understanding the cellular processes responsible for differences in organ development, as exemplified also during limb development (cf. chapter A figure 7).

Participation to this work

The article corresponding to this work has been accepted for publication in Genome Biology. Even though my participation (detailed below) is minor in this analysis, I present the full manuscript (in Appendix A), because it is not yet available online, and I figured that the results may be useful for the understanding of the other sections of the manuscript.

For this work, I developed a visualization tool for GO analysis to overcome one of the problem of GO analysis which is to compare results between two different GO analyses, for example one obtained for genes differentially expressed in lower molar versus one obtained for genes differentially expressed in upper molar (see later chapter 10). I made a visualization tool to look at specific subset of GOTerms (chosen by the user ). Each plot include information on the size on the GOTerm, its odds ratio in the dataset analyzed and its statistical significance. Size, or number of genes in the GOTerm, is proportional to the width of the bar, enrichment in the data set, the odd ratio, is proportional to the length of the bar. An bar are color coded to represent the significance of the GOTerm in the dataset. I coupled it with a pairwise comparison of GOTerms (based onχ2test) to find if one GOTerm is more enriched in one condition than in another one, and represent the statistical significance of an enrichment with red stars. I used this tool to perform the Supplementary figure 2 of the paper presented in appendix A. I re-use this visualization tool for my bucco-lingual analysis of upper and lower molars in chapter 10.

developing organs

Temporal signal in transcriptomes throughout development

Several studies analyzing transcriptomes during development found a temporal ordering of samples when performing multivariate analyses [O’Connell et al. , 2012; Levin et al. , 2012; Anavy et al. , 2014; Tschopp et al. , 2014]. For example, Principal Component Analysis (PCA) of Xenopus embryos transcriptomes revealed that samples ordered according to their stages of development, as illustrated in figure 8.1 [Anavy et al. , 2014]. Levin et al. [2012] note that adjacent stages have more similar expression patterns than temporally distant stages, thus reflecting the progression of development.

The difficulty when working with developmental samples is to be able to stage them correctly. Typically, stages are defined by morphological characters, such as the advancement of limb development in tetrapods. Finer staging in model organisms such as mice can also be inferred from weight. For example, for mice embryos within the same litter, which are supposed to be at the same development stages, exhibit differences in weight that are correlated with their stages of development [Peterka et al. , 2002]. There are a lot of cases when the morphological staging is not possible, for example between two closely sampled developmental stages, or at stages when there are no morphological milestones to be exploited. Even the weight staging in mice embryo is not satisfying between litters.

Anavy et al. [2014] used the transcriptomic signature of time present in timeseries transcriptomes of whole embryos to order transcriptomic samples according to their stages of development. They develop a method named BLIND, Basic Linear Index Determination of transcriptomes, to order transcriptomic samples of developing embryos. This method takes into account the distance between every two samples on the PCA axis corresponding to time ordering, together with the use of a salesman algorithm to find the shortest path between consecutive samples. However, it is poorly known what kind of expression differences are responsible for this strong temporal signal.

Chapitre 8. Temporal signal in transcriptome of developing organs

Dans le document Évolution et Développement d'un organe sériel - la molaire : Transcriptomique comparée des bourgeons de molaire chez les rongeurs (Page 105-115)