Master
Reference
A common genomic region regulates HoxD dynamic expression in the forming somites and in the vibrissae follicles
HINTERMANN, Aurélie
Abstract
During bilaterian embryonic development, Hox genes achieve critical functions in body patterning. The vertebrate trunk elongates by cyclic addition of blocks of mesoderm called somites. During this process, Hox genes instruct the forming segments with their positional identity. Hox genes have also been co-opted several times with the emergence of secondary structures. They have been described in axial tissues like digits, genitalia or hairs. In 2001, Zakany et al. showed that some Hox genes adopt a dynamic expression in the forming somites. In this study, we demonstrate that anterior Hoxd genes adopt dynamic transcription patterns in the vibrissae follicles, reminiscent to that found in the forming somites. Our results indicate that genomic features established in the somites have probably impacted the implementation of a new regulatory mechanisms acting in the whisker pad. Hence, our report supports the idea that higher order chromatin structure promotes the appearance of new enhancers.
HINTERMANN, Aurélie. A common genomic region regulates HoxD dynamic
expression in the forming somites and in the vibrissae follicles. Master : Univ. Genève, 2016
Available at:
http://archive-ouverte.unige.ch/unige:85255
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Université de Genève Faculté des Sciences
Département de
Génétique et Evolution Professeur Denis Duboule
A common genomic region regulates
HoxD dynamic expression in the forming somites and in the vibrissae follicles
Thèse
présentée à la Faculté des sciences de l'Université de Genève pour obtenir le grade de master ès sciences, mention biologie
par Aurélie HINTERMANN de Genève (GE)
27 juin 2016
Directeurs du travail Professeur Denis Duboule Dr Isabel Guerreiro
Dr Leonardo Beccari
Table of content
Table of content 3
Table of figure 5
Summary 7
Résumé 9
1 Introduction 11
1.1 Hox genes 11
1.1.1 Hox clustering and collinearity 12
1.1.2 Hox gene regulation 14
1.2 Somitogenesis -‐ building the main body axis of vertebrates 18
1.2.1 Segmentation 18
1.2.2 The segmentation clock 18
1.2.3 The determination front 19
1.2.4 Boundary specification 20
1.2.5 Somite identity specification 21
1.2.6 A molecular link between main body axis patterning and the segmentation clock 22
1.3 Hox gene function during hair follicle morphogenesis 23
1.3.1 Mammalian innovations 23
1.3.2 Architecture and function of the whisker pad 23 1.3.3 Molecular signalling during hair follicle morphogenesis 24
1.3.4 Adult hair cycling 28
1.3.5 Hox in hairs 29
1.4 Scope of the thesis 31
2 Results 32
2.1 Anterior Hoxd genes are transiently expressed in the developing whisker pad 32 2.2 Hoxd gene expression in vibrissae follicles is collinear 33 2.3 Hoxd1 expression is reported in pelage hairs 34 2.4 BAC(Hoxd1-‐Mtx2) drives a reporter gene expression in the whisker pad and in
pelage hairs 35
2.5 The HoxD cluster is not sufficient to activate the reporter gene in the whisker pad 37
2.6 A 140kb long region of the telomeric desert is sufficient to drive Hoxd1 expression
in the whisker pad 37
2.7 Screening for enhancer candidates driving Hoxd1 expression 40 2.8 Genomic contacts between Hoxd1 and the telomeric gene desert 43 2.9 Two enhancer candidate separately drive lacZ expression in the whisker pad and
in the somites 44
3 Discussion 46
3.1 Hoxd1 dynamic expression 46
3.2 Dynamism in the neighbouring genes 46
3.3 Hox collinearity in hair follicles 46
3.4 Hoxd1 is expressed in the follicles of pelage hairs 47
3.5 A common regulatory region 47
3.6 Identification of putative enhancers driving dynamic expression of anterior Hoxd gene 48
3.7 Interactions between Hoxd1 and the 140 kb region 48
3.8 Regulatory potential of enhancer candidates 49
3.9 Proposition of two evolutionary scenarios 49
3.10 Concluding remarks 50
4 Materials and methods 52
4.1 Mouse stocks 52
4.2 In situ hybridization 53
4.3 DAPI staining and imaging 53
4.4 ß-‐galactosidase staining 53
4.5 RT-‐qPCR 53
4.6 ChIP-‐sequencing 54
4.7 4C-‐ sequencing 54
5 Acknowledgments 55
6 References 56
Table of figure
Figure 1 - Representation of the HoxD cluster in different species ... 13
Figure 2 - Bimodal chromatin marks on the HoxD cluster ... 15
Figure 3 - Scheme of HoxD regulation in novel structures ... 16
Figure 4 - Regulatory deserts correspond to topological domains ... 16
Figure 5 - Shared and specific enhancers in digits and genitals ... 17
Figure 6 - Resegmentation of the sclerotome ... 21
Figure 7 - Spatial organization of the whisker pad ... 24
Figure 8 - Major steps of hair follicle morphogenesis ... 26
Figure 9 - Different cell lineages populate the hair bulb ... 28
Figure 10 - Hair cycle ... 29
Figure 11 - Hoxc13 expression in the whisker pad ... 30
Figure 12 - Hox gene expression during the hair cycle ... 30
Figure 13 - Characterization of Hoxd gene expression in the whisker pad ... 33
Figure 14 - Timing of activation of Hoxd1, Hoxd3 and Shh ... 34
Figure 15 - Hoxd1 expression on a larger time scale ... 36
Figure 16 - Regulatory potential of the telomeric gene desert in the whisker pad ... 39
Figure 17 - Hoxd1 expression is absent in the somites in Del(AttP-SB2) context. ... 40
Figure 18 - Promoter and enhancer activities of the close telomeric desert ... 42
Figure 19 - BAC recombination ... 45
Summary
During bilaterian embryonic development, Hox genes achieve critical functions in patterning the main body axis and secondary structures. In vertebrates, they are arranged in highly organized clusters. The relative positions of the genes within Hox clusters correspond to their sequential activation in time and space, a phenomenon termed collinearity.
The vertebrate trunk elongates by cyclic addition of blocks of mesoderm called somites. During this process, Hox genes instruct the forming segments with their positional identity. In addition to their roles in the trunk, Hox genes have been co- opted several times with the emergence of secondary structures. For example, they have been described in axial tissues like digits, limbs or genitalia. Hox gene expression was also reported in hairs, one of the most recent mammalian innovations.
In this context, they are thought to mainly function for the regionalization of the skin.
In 2001, Zakany et al. showed that some Hox genes adopt a dynamic expression in the forming somites, where they display rhythmic bursts of transcription (Zákány et al.
2001). In this study, we demonstrate that anterior Hoxd genes, and more specifically Hoxd1, adopt a dynamic transcription pattern in the vibrissae follicles, reminiscent to that found in the forming somites. We performed series of in situ hybridizations on whole-mount embryos to visualize the propagating wave of transcription in the vibrissae follicles. Staining embryos with DAPI allowed us to observe the developing structure of the whisker pad and to precisely stage our embryos.
We next investigated the regulatory potential of the genomic landscape surrounding the HoxD cluster. We aimed to describe the region controlling Hoxd1 expression in the whisker pad and in the forming somites to see whether it implies common or distinct sets of enhancers between the two tissues. To do so, we used a combined approach involving genetic experiments and analyses of ChIP-seq and 4C-seq data.
To see which genomic domain is necessary for Hoxd1 cyclic expression in the whisker pad, we performed whole-mount in situ hybridizations on mouse embryos carrying different deletions of the telomeric gene desert. We highlighted a 140-kb large region, which is essential for Hoxd1 activation in the somites and in the whisker pad. ß-galactosidase experiments on BACs lines revealed that this region was also sufficient to activate gene expression in the whisker pad.
We then compared H3K27ac ChIP-seq profiles of embryonic trunks and whisker pads to evaluate the regulatory activity of the 140kb region in those tissues. We specifically looked for sequences that would be acetylated in both structures, reflecting potential common regulatory elements. We observed interesting acetylation on sequences that were comprised in the 140kb region, specifically enriched in the trunk and in the whisker pad.
Interestingly, the acetylated sequences matched specific peaks obtained by 4C-seq, using Hoxd1 as viewpoint. Hence, we selected two promising enhancer candidates located within the 140kb. We also noted numerous constitutive contacts between Hoxd1 and the telomeric gene desert in tissues where this gene is not expressed, reflecting that Hoxd1 is generally placed in close proximity from this genomic region.
We finally estimated the regulatory potential of the two candidates separately by reporter gene assays. We found that one of them could activate gene expression in the
somites but not in the whisker pad. The other sequence could activate transcription in the whisker pad. The expression in the somites for this construct remains to be determined.
Altogether, our study shows that regulatory activities sufficient to activate a dynamic gene expression in the forming somites and in the whisker pad are located within a 140kb of telomeric gene desert, directly adjacent to the HoxD cluster. We do not report specific enhancers that could drive expression in both tissues, but we do not rule out that they may exist.
Our results indicate that genomic features established in the somites have probably impacted, or participated to, the implementation of a new regulatory mechanisms acting in the whisker pad. Hence, our report provides new insights to the view of regulatory mechanisms recruitment from one developmental context to another. It also supports the idea that higher order chromatin structure promotes the appearance of new enhancers.
Résumé
Lors du développement embryonnaire des bilateriens, les gènes Hox remplissent des fonctions critiques pour le modelage de l'axe corporel et des structures secondaires.
Chez les vertébrés, ils sont arrangés au sein de complexes génomiques très organisés.
La position relative des gènes au sein de ces complexes correspond à leur activation séquentielle dans le temps et dans l'espace, un phénomène appelé collinéarité.
Le tronc des vertébrés s'allonge par ajout cyclique de blocs de mésoderme, les somites. Au cours de ce processus, les gènes Hox informent les segments en développement de leur position. En plus de leur rôle dans le tronc, les gènes Hox ont été co-optés plusieurs fois avec l'apparition de nouvelles structures. Ils ont par exemple été décrits dans des tissus axiaux tels que les doigts, les membres ou encore les organes génitaux. L'expression de gènes Hox a aussi été démontrée dans les poils, une des innovations morphologiques les plus récentes chez les mammifères. Dans ce contexte, ils fonctionnent probablement dans la régionalisation de la peau.
En 2001, Zakany et al. ont montré que certains gènes Hox adoptent une expression dynamique dans les somites en développement (Zákány et al. 2001). Dans ce travail, nous démontrons que les gènes Hoxd antérieurs, et plus spécifiquement Hoxd1, adoptent un pattern de transcription dynamique, évoquant celui ayant lieu dans les somites. Nous avons effectué des séries d'hybridations in situ sur des embryons entiers afin de visualiser la vague de transcription qui se propage à travers la zone des vibrisses. La coloration au DAPI nous a permis d' évaluer précisément le stade de développement des embryons.
Nous avons ensuite investigué le potentiel régulateur de l'environnement génomique du complexe HoxD. Nous voulions décrire la région qui contrôle l'expression de Hoxd1 dans les vibrisses et les somites, afin de savoir si les mêmes éléments régulateurs sont impliqués ou non. Pour accomplir cela, nous avons eu recourt à une combinaison d'approches, comprenant des expériences génétiques ainsi que des analyses de données de ChIP-seq et 4C-seq.
Afin de déterminer quel domaine génomique est nécessaire pour l'expression cyclique de Hoxd1 dans les vibrisses, nous avons effectué des d'hybridations in situ sur des embryons entiers portant différentes délétions du désert télomérique. Nous avons mis en évidence une région de 140kb, essentielle pour l'activation de Hoxd1 dans les somites et les vibrisses. Des expériences de coloration au X-gal sur des lignées contenant des BACs ont révélés que cette région était également suffisante pour activer l'expression de ce gènes dans les vibrisses.
Nous avons ensuite comparé des profiles de ChIP-seq de H3K27ac, effectués sur des troncs et des zones des vibrisses, dans le but d'évaluer l'activité régulatrice de la région de 140kb dans ces tissus. Nous avons spécifiquement recherché des séquences acétylées dans les deux structures, reflétant potentiellement des éléments régulateurs communs. Ces analyses ont révélé d'intéressantes marques d'acétylation spécifiquement enrichies dans le tronc et la zone des vibrisses.
De façon intéressante, les séquences acétylées correspondaient à certains pics spécifiques du profile de 4C-seq utilisant Hoxd1 comme point de vue. Par conséquent, nous avons sélectionné deux candidats enhancers au sein de la région de 140kb. Nous avons aussi remarqué de nombreux contacts constitutifs entre Hoxd1 et le désert
télomérique dans des tissus où ce gène n'est pas exprimé, reflétant la générale proximité de Hoxd1 avec cette région.
Finalement, nous avons évalué le potentiel régulateur des deux candidats séparément, par une méthode de gène rapporteur. Nous avons trouvé qu'un des deux candidats était capable d'activer l'expression du rapporteur dans les somites mais pas dans la zone des vibrisses. L'autre séquence pouvait quant à elle générer de la transcription dans la zone des vibrisses. L'expression dans les somites pour ce transgène reste à déterminer.
L'ensemble de notre étude démontre que des activités régulatrices, suffisantes pour activer l'expression dynamique de gènes dans les somites et la zone des vibrisses, sont situées au sein d'une région de désert télomérique. Cette région est large de 140 kb et se trouve dans le voisinage direct du complexe HoxD. Nous n'avons pas identifié d'éléments régulateurs qui pourraient activer l'expression de gènes dans les deux tissus, mais nous ne pouvons pas exclure leur existence.
Nos résultats indiquent que des caractéristiques génomiques établies dans les somites ont probablement impacté, ou participé à l'implémentation de nouveaux mécanismes de régulation actifs au sein de la zone des vibrisses. Ainis, notre rapport amène de nouvelles connaissances dans les mécanismes de régulation et leur éventuel recrutement d'un contexte développemental à un autre. Il supporte notamment l'idée que l'organisation structurelle de la chromatine puisse éventuellement promouvoir l'apparence de nouveaux éléments régulateurs.
1 Introduction
1.1 Hox genes
A simple glance is enough to notice the astonishing morphological diversity of animals, where sea urchins and snakes are one example among many. For over 30 years, the study of Hox function and regulation has provided important contributions to this field of research (Carroll 2008; Pearson et al. 2005; Moczek et al. 2015;
Guerreiro and Duboule 2014). Indeed, Hox genes are thought to have largely contributed to morphological diversification (Hrycaj and Wellik 2016). For example, pioneer studies revealed that Hox gene misexpression could result in homeotic mutations whereby the identity of a body segment transforms into another. For example, in Drosophila embryos, ectopic Antennapedia expression results in the conversion of the antennas into an additional pair of legs (Lewis 1978).
Following their identification in the fruit fly, Hox genes were found in numerous species, including mouse and human (McGinnis et al. 1984; Graham et al. 1989;
Duboule and Dollé 1989). Their ancient origin is currently dated at the divergence between diplo- and triploblasts, as they were discovered in bilaterians and cnidarians, but not in sponges (Ferrier and Holland 2001), or non-metazoans (Holland 2013;
Hrycaj and Wellik 2016). Hox genes contain a highly conserved sequence called the homeobox, which encodes the homeodomain, a 61 amino acid-long helix-turn-helix DNA-binding motif (Gehring 1987; Gehring et al. 1990). The large homoeobox superfamily is thought to arise from extensive duplication events of common ancestral sequences (Holland 2013). In addition to Hox genes, it comprises many genes with regulatory roles in development, like ParaHox or NK members (Pearson et al. 2005;
Holland 2013; Hrycaj and Wellik 2016).
In many cases, HOX transcription factors operate either alone or in cooperation with other transcription factors, including the member of the PBC and MEIS families.
They form heterotrimers that bind DNA regulatory regions to control the expression of downstream targets (Mann and Affolter 1998; Pearson et al. 2005). Some of these targets have been shown to regulate signalling molecules such as ephrins, BMP (Stadler et al. 2001) and Shh (Zákány et al. 2004; Kmita et al. 2005) or transcription factors like Hox themselves (Svingen and Tonissen 2006; Ernesto Sánchez-‐
Herrero et al. 2013). In other cases, they activate effector genes, which encode proteins that participate in a large range of cell behaviours like division, migration, adhesion or death, thereby driving the formation of body structures (Stadler et al.
2001; Chow and Emmons 1994; Pearson et al. 2005; Kalyani et al. 2016).
The most ancestral function of Hox genes is the control of axial morphology in the main body plan (Lewis 1978; Burke et al. 1995; Mallo et al. 2010; Hrycaj and Wellik 2016). Besides their role in the trunk, Hox genes diversified over million years of evolution, becoming highly pleiotropic. They acquired novel functions in the patterning of secondary structures, including limbs (Davis and Capecchi 1996), digits (Zákány et al. 1997a; Montavon et al. 2011), genitals (Spitz et al. 2001), caecum (Delpretti et al. 2013), kidneys (Spitz et al. 2001, 2003; Dollé et al. 1989; Di-Poï et al.
2007) or hair follicles (Godwin and Capecchi 1998).
1.1.1 Hox clustering and collinearity
Clustering
In some species, Hox genes tend to be arranged in clusters. This peculiar topography is not limited to the Hox family and was discovered by pioneer studies in bacteria, where genes that regulate related functions are often found in close genomic proximity. These "linked genes" mostly mediate growth and developmental pathways, for example in the control of histidine biosynthesis in Salmonella (Demerec 1964) or lactose metabolism in E.coli (Jacob and Monod 1961). In the case of Hox genes, the eight loci of the fly genome appeared to be arranged in two complexes, Antennapedia and Bithorax (Lewis 1963, 1978; McGinnis and Krumlauf 1992). An analogous functional organization was found in higher vertebrates, where several clusters were discovered (Duboule and Dollé 1989; Graham et al. 1989; Holland 2013). The clustered organization is thus conserved among species, which suggests functional importance in developmental strategies (Darbellay and Duboule 2016).
The organization of Hox clusters varies considerably in the different animal clades (Duboule 2007)(Figure 1). The ancestral cluster emerged probably from cis- duplications of an original protoHox gene, generating a series of paralogous loci in close physical proximity. It is thought that the ancestral protoHox gene appeared in cnidarians and that the number of Hox genes per cluster has been increasing during early bilaterian evolution. Deuterostomian genomes were reported to comprise up to fifteen Hox genes, as exemplified by the prototypical Amphioxus cluster (Lemons and McGinnis 2006; Holland et al. 2008; Amemiya et al. 2008).
Two successive genome duplications at the basis of vertebrate phylogeny (Ohno,S 1970), in addition to secondary losses of individual genes, are thought to have resulted in a minimum of four Hox clusters found in vertebrate species. These clusters display some remarkable properties: they are polarized, i.e.: with all their genes being transcribed from the same DNA strand, particularly compact and organized, with sizes spanning around 100 kb, they contain no unrelated genes and very few repeated sequences (Fried et al. 2004; Amemiya et al. 2008). Vertebrate clusters are composed of 9 to 11 Hox genes, classified depending on their sequences into 13 groups of paralogy (Krumlauf 1994; Garcia-‐Fernàndez 2005). Mammalian genomes account for 39 Hox genes distributed on four clusters, HoxA to HoxD, located on different chromosomes (Garcia-Fernàndez 2005).
Figure 1 - Representation of the HoxD cluster in different species
Modified from (Duboule 2007). Hox gene arrangements within their cluster are represented.
Drosophila counts two sub-clusters, Amphioxus and Mouse one cluster.
Collinearity
In bilaterians, the order of Hox genes within their cluster reflects the anterior position of the corresponding transcripts along the main body axis. Lewis enunciated this phenomenon called collinearity in Drosophila melanogaster (Lewis 1978). This characteristic was subsequently uncovered in mice (Gaunt, Duboule 1988) and it finally appeared to be a conserved feature among most species (Krumlauf 1994;
Duboule 2007). In vertebrates, gene activation times follow their genomic arrangement, reflecting a temporal component of collinearity (Izpisúa-‐Belmonte et al. 1991; Kmita and Duboule 2003). The early expression of murine Hoxd genes in the primitive streak remarkably illustrates this intrinsic property: 3' genes like Hoxd1, also called "anterior genes", start to be expressed at embryonic day 7.75, while Hoxd13, a 5' posterior gene, is only activated at E9 (Jacqueline Deschamps and Nes 2005; Tschopp et al. 2009).
Clustering was initially believed to be necessary for collinearity, but this notion was nuanced. Some modifications of the HoxD cluster provoked a delayed gene activation, without affecting the final spatial distribution of the transcripts (Zákány et al. 1997b; Tschopp et al. 2009). Findings in urochordates reinforced this idea by revealing a collinear distribution of Hox transcripts along the main body axis of the Oikopleura larvae, although the genes do not physically group into a cluster (Seo et al. 2004; Duboule 2007). The contribution of the HoxD cluster in implementing both types of collinearity was further examined (Tschopp and Duboule 2011; Tschopp et al. 2009; Monteiro and Ferrier 2006). It resulted that local regulatory regions seem to control a great part of spatial collinearity, while temporal collinearity relies on regulatory elements surrounding the cluster. Therefore, genomic clustering appears as an essential component of the precise activation timing of Hoxd genes, while it is not systematically required for the correct establishment of expression boundaries (Tschopp et al. 2009).
Comparison studies between numerous organisms, concerning Hox genes collinearity and their genomic organization in clusters, contributed to major conceptual understandings concerning global regulation of animal genomes. Thus, Hox regulation is a central theme both in the molecular theory of anatomical evolution
(Hrycaj and Wellik 2016) and in paradigmatic explanation of general genomic mechanisms (Tschopp and Duboule 2011, 2011).
1.1.2 Hox gene regulation
The accurate and intricate regulation of developmental genes is essential for proper growth and morphogenesis. Remarkably, body patterning in species that display different forms and shapes rely on functionally conserved proteins, suggesting that the wide diversity of animal anatomies arises, at least in part, from differences in the regulation of developmental genes (Wittkopp and Kalay 2012; Guerreiro et al. 2013).
In this regard, proper Hox gene regulation, which is greatly implemented by transcription factors binding genomic sequences called "regulatory elements", is central in phenotypic evolution (Carroll 2008). These elements are classified depending on their relative position to the protein coding sequence. A first distinction is made between promoters and enhancers: they are respectively located in close proximity of the transcription start site, or at distances varying from a few to hundreds of kb (Ohler and Wassarman 2010). Enhancers are remarkably modular regulatory elements with established properties: they can generally act upstream or downstream of the promoter and in both backwards and forwards directions. Moreover, they often display weak promoter specificity, implying the possibility to regulate several genes at once (Pennacchio et al. 2013). Thus, the precise, refine transcription profile of a gene at a given time and place results from the complex combination of regulatory inputs provided by promoters and short- to long-range enhancer activities.
Hox landscape - gene regulatory deserts
The HoxD cluster relies on the activity of numerous enhancers and stands as paradigm for more general gene regulation. It spans around 110 kb and comprises nine Hoxd genes. Short-range enhancers located within the cluster are in charge of the expression occurring in the main body axis (Duboule and Dollé 1989, 89). In contrast, Hoxd gene regulation in secondary structures mainly relies on long-range enhancers located in two wide genomic domains flanking the cluster. These regions are called 5' (centromeric) and 3' (telomeric) "regulatory gene deserts" because they contain almost no genes but hold important regulatory potential (Andrey et al. 2013;
Montavon et al. 2011).
Gene differential coverage by histone modifications
The sequential activation of Hoxd genes in the trunk was shown to correspond to a progressive change in chromatin marks. As transcription starts, the permissive H3K4me3 gradually replaces the repressive H3K27me3 histone modification (Soshnikova and Duboule 2009)(Figure 2). In addition to these biochemical characteristics, investigating the 3D organization of Hox clusters revealed a physical separation of inactive and active genes in distinct spatial compartments (Noordermeer et al. 2011). Thus, temporal collinearity is apparently supported by dynamic histone modifications and spatial compartmentalization of Hox clusters.
Figure 2 - Bimodal chromatin marks on the HoxD cluster
Adapted from (Soshnikova and Duboule 2009). A scheme of the HoxD cluster is depicted above the profiles. Transcription levels are in green, H3K27me3 repressive histone mark in blue and H3K4me3 permissive mark in red. Embryos were dissected at E8.5 and E9.5 and profiles are compared with ESC stage, in order to observe the evolution of bimodal chromatin marks following temporal collinear activation of Hoxd genes. H3K27me3 covers inactive genes and H3K4me3 active loci.
Secondary structure regulation
The biochemical and three-dimensional separation of active and inactive Hoxd genes was also observed in secondary structures, for example in the digits (Montavon et al.
2011) or the caecum (Delpretti et al. 2013). In these tissues, loci belonging to the transcribed compartment of the cluster contact several long-range enhancers located in either one or the other of the surrounding gene deserts. The development of the limb is a good illustration of this bimodal regulation: a first series of contiguous Hoxd genes is involved in the patterning of the proximal limb and activated by the telomeric domain. On the opposite, a second subset of Hoxd genes is necessary for the patterning of the digits and relies on the centromeric gene desert (Andrey et al.
2013)(Figure 3).
Figure 3 - Scheme of HoxD regulation in novel structures
Modified from (Andrey et al. 2013). Representation of the HoxD locus and its surrounding regulatory landscape, illustrating that long-range regulatory elements located in the gene deserts influence the expression of Hoxd gene subsets in recently evolved structures like digits or limbs. This case exemplifies how enhancers active in digits are located in a separate region and affect distinct genes than elements involved in forearms.
Chromatin conformation
Remote regulatory elements populating the deserts are often examined through sequence conservation and combined analyses of H3K27ac histone mark, which generally covers active enhancers and promoters, as well as 4C and Hi-C interaction profiles. In particular, Hi-C analyses of the HoxD regulatory landscape revealed the correspondence between regulatory gene deserts and topological associated domains (TAD)(Figure 4). The higher order organization of chromatin is thought to potentially facilitate the appearance of new enhancer sequences or the recruitment of regulatory modalities from one developmental context to another (Darbellay and Duboule 2016).
Figure 4 - Regulatory deserts correspond to topological domains
Adapted from (Beccari et al. 2016) Each desert matches to a distinct topological domain and the border between these two TADs is located within the HoxD cluster. Each TAD contains regulatory elements that modulate overlapping but distinct subsets of Hoxd genes, as indicated by the blue and the green rectangles.
For instance, common genomic regions were shown to regulate Hoxd9 to Hoxd11 in both the caecum (Delpretti et al. 2013) and the budding limbs (Andrey et al. 2013).
Besides, overlapping sets of enhancers located in the centromeric gene desert were shown to activate the expression of Hoxd genes in the digits and the genitals (Lonfat
et al. 2014a)(Figure 5).
Figure 5 - Shared and specific enhancers in digits and genitals
Adapted from (Lonfat et al. 2014a) Il-1 is specific to the limb and GT2 to the genital bud. On contrary, Prox is able to drive reporter expression in both structures.
1.2 Somitogenesis -‐ building the main body axis of vertebrates
In vertebrates, Hox gene mediated patterning of the anteroposterior (AP) axis is associated with somitogenesis and the correct coordination of both mechanisms is essential for harmonious morphogenesis (Burke et al. 1995; Mallo et al. 2010; Hrycaj and Wellik 2016; Lewis 1978; Duboule and Noordermeer 2013). The main body plan is built by the addition of repetitive metameric units called somites, which are the embryonic precursors of the vertebrae and their associated skeletal muscles, peripheral nerves, blood vessels, part of the dermis and tissues of the limbs (Hubaud and Pourquié 2014a; Takahashi 2001; Gilbert 2000; Hirsinger et al. 2000; Christ et al.
2007). Concurrently with their production, somites acquire a molecular identity based on the combinatorial expression of different molecular markers. In this process, Hox genes are critical as they confer positional information to the tissues (Krumlauf 1994;
Deschamps and Nes 2005; Iimura and Pourquié 2006).
1.2.1 Segmentation
Somite formation, a process termed segmentation, occurs during embryo elongation (Saga and Takeda 2001; Dequéant and Pourquié 2008; Hubaud and Pourquié 2014a).
A metameric body plan is not an exclusivity of vertebrates but is also observed in large groups of invertebrates such as annelids or arthropods. However, distinct modes of segmentation are operating in those species. In long-germ-band insects like Drosophila melanogaster, the metamerization of the body occurs at once, while vertebrates and short-germ-band insects undergo sequential segmentation (Nagy 1994; Sarrazin et al. 2012). Segmentation is thus an interesting case of convergent evolution, suggesting that periodic body patterning appeared independently several times over the course of evolution (Bénazéraf and Pourquié 2013; Peel et al. 2005).
In vertebrates, somites sequentially form from the rostral end of the presomitic mesoderm (PSM). This tissue is part of the paraxial mesoderm, a transient structure that consists of two longitudinal stripes of mesenchymal cells bordering both sides of the neural tube. During gastrulation, the cells that contribute to the PSM ingress caudally through the primitive streak. When gastrulation terminates, the primitive streak originates the tailbud, which continues to provide mesenchymal cells to the PSM further being segmented. Therefore, during most of the segmentation process, the PSM length remains approximately constant (Bénazéraf and Pourquié 2013).
Segmentation is an intrinsic property of the PSM and it proceeds until all somites are shaped. The total number of somites and the segmentation rate are species-specific characteristics that may vary greatly from one lineage to another. For instance, mice have 65 pairs of somites and each set needs about 120 minutes to form, while there are 50 pairs in the chicken with a period of 90 minutes (Gomez et al. 2008). The range of body segment numbers is as wide as from 26 in the platyfish to 344 somites in some snake species. Accordingly, segmentation participates greatly in body shape diversification of vertebrates.
1.2.2 The segmentation clock
The molecular mechanisms controlling the segmental patterning of embryos have long remained mysterious. Nevertheless, in 1976, the clock-and-wavefront model was proposed as a theoretical attempt to explain the periodicity of segmentation (Cooke
and Zeeman 1976). It predicted the coexistence of a molecular oscillator, called segmentation clock, and a deterministic level of morphogenetic gradients, the determination front. Both components would work together to cyclically determine the presumptive somite boundaries.
The first molecular evidence of the segmentation clock was provided by the characterization of c-hairy1 expression. c-hairy1 is the avian homolog of the hairy segmentation gene in Drosophila. It is periodically transcribed in the chicken PSM, generating waves of mRNA that propagate anteriorly. This first observation suggested that cyclic activities occurring in the PSM are required to establish the rhythmicity of new segments (Hubaud and Pourquié 2014a).
Numerous additional genes were shown to adopt rhythmic behaviours in the PSM of different species including chicken, mice and zebrafishes. Most of them are involved in cyclic activities of the Wnt, FGF or Notch pathways, such as Lfng or Hes-related gene activation, ERK phosphorylation or Axin2 transcription (Goldbeter and Pourquié 2008; Li et al. 2007; Aulehla et al. 2003; Dequéant and Pourquié 2008).
A comparative study of the chicken, zebrafish and murine transcriptomes revealed that if Wnt, FGF and Notch pathways are conserved features of the vertebrate segmentation, the individual identity of the cyclic genes can greatly differ from one species to another (Krol et al. 2011).
Molecular oscillations often arise from negative regulatory feedback loops. For example, the cleavage of NOTCH activates the pathway by liberation of the intracellular domain NICD, which enters the nucleus where it regulates downstream targets. It namely triggers Lfng expression, which in turn represses Notch signalling.
As a consequence, the level of Lfng decreases, allowing the re-activation of the Notch pathway. Similarly, Wnt signalling, mediated by the ß-catenin pathway, promotes Axin2 accumulation and Axin2 inhibits the Wnt pathway by contributing to ß-catenin degradation. Finally, FGF signalling enhances Dusp6 activity, a phosphatase that reduces FGF signalling (Aulehla et al. 2008; Dequéant and Pourquié 2008).
In short, periodic activations of signalling inhibitors produce autonomous and continuous oscillations of the three main signalling pathways involved in the segmentation clock: Notch, Wnt and FGF.
1.2.3 The determination front
The determination front (also called wavefront) is a region along the AP axis set by antagonist Wnt/FGF and RA gradients (Dubrulle and Pourquié 2004). During embryo elongation, these gradients are displaced and the position of the wave front shifts posteriorly, so that its relative distance to the anterior PSM remains constant (Dubrulle et al. 2001). Only cells belonging to the wavefront are competent to respond to the molecular oscillators of the clock, which prevents posterior PSM cells to respond to the segmentation machinery. Hence, the periodicity of cyclic gene expression is converted into rhythmic segmentation (Hubaud and Pourquié 2014a).
The FGF gradient arises from an mRNA gradient and by diffusion. Fgf gene transcription is restricted to the primitive streak and tailbud. Its mRNA level decays progressively while the cells ingress and travel along the PSM. The Wnt gradient is presumably established by similar mechanisms. On the other hand, the Retinoic Acid (RA) gradient results from joined activities of RA metabolic enzymes in a source-sink mechanism: RA is synthesized from retinaldehyde by RALDH2 anteriorly to the PSM
whilst it is degraded by CYP26 in the tail bud (Aulehla and Pourquié 2010). Of note, the RA gradient appears to be necessary for the formation of bilateral, symmetric somites in addition to its role in determining the position of the wave front (Vermot et al. 2005).
In brief, two antagonistic dynamic gradients, a caudorostral Wnt/FGF and a rostrocaudal RA, control the level of the determination front, which is critical to translate the periodic instructions of the segmentation clock into the initiation of a somitic boundary.
1.2.4 Boundary specification
The combined actions of the wave front and the segmentation clock initiate somite boundary formation through periodic transcription of segmentation genes. Namely, Mesp2, a basis helix-loop-helix (bHLH) transcription factor activated by Notch, is first expressed as a large stripe in the anterior PSM, where it defines the size of the presumptive somite. Mesp2 expression is later restricted to the rostral half of the nascent segment, where it establishes its anteroposterior polarity (Takahashi et al.
2000; Saga and Takeda 2001). At this stage of somite maturation, oscillating gene expression stops and Mesp2 influences Eph/ephrin signalling. This pathway in turn regulates adhesion proteins and cellular reorganization,leading to the physical separation of the somite from the unsegmented PSM.
Early somites are epithelial spheres containing a core of mesenchymal cells that form via mesenchymal-to-epithelial transition (MET). This morphogenetic process regulated byParaxis, another bHLH transcription factor (Burgess et al. 1995;
Dahmann et al. 2011). It was described in chicken that once newly formed somites separated from the PSM, their cells undergo additional subdivisions into different lineages under the influence of signals arising from neighbouring tissues. Namely, cells of the ventral somitic wall undergo massive proliferation in response to a Shh signal secreted by the notochord. Shh activates the expression of Pax1, which commits the ventral part of each segment to become the sclerotome, the actual precursor of vertebrae. On the other hand, the dorsal portion of the somite is instructed by BMP-4, secreted by the dorsal part of the neural tube, as well as Noggin and MyoD. The dorsal somite contributes to both dermatome and myotome, originating respectively a portion of the dermis and the skeletal muscles of the trunk (Christ et al. 1997; Takahashi et al. 2000; Gilbert 2000).
After its separation from the dorsal part of the somite, the sclerotome undergoes a rostrocaudal reorganization called re-segmentation (Figure 6). It was stated very early on that somitic borders do not match vertebrae limits (Remak R. 1855) and this process has been more recently described in both chick (Stern and Keynes 1987) and zebrafish (Morin-Kensicki et al. 2002). First, the sclerotome segments separate into rostral and caudal halves. Then, the posterior half of the rostral segment and the anterior half of the caudal somite join together. Thus, one somite generates the neighbouring sclerotome halves of two adjacent vertebrae, and one vertebra is composed of two halves from adjacent somites (Saga and Takeda 2001; Senthinathan et al. 2012).
Figure 6 - Resegmentation of the sclerotome
Adapted from (Saga and Takeda 2001). The scelrotome undergoes a second segmentation, in which the the fusion of two adjacent somite halves gives rise to a vertebra.
1.2.5 Somite identity specification
The vertebrate spine is subdivided into distinct anatomical regions classified in cervical, thoracic, lumbar, sacral and caudal domains. This subdivision is largely controlled by the precise distribution of HOX transcription factors along the anteroposterior axis (Krumlauf 1994). The collinear Hox gene distribution along the main body axis results from their sequential onset of expression in the epiblast, early in development and is refined by molecular regulatory influences as they travel the PSM (Forlani et al. 2003; Deschamps et al. 2004). As described in the previous section about collinearity, Hox genes located in 3' of the cluster are activated earlier than the 5' Hox genes (Duboule and Morata 1994; Kmita and Duboule 2003). This temporal Hox gene activation is converted into anteroposterior coordinates for HOX proteins: the first cells produced by the mesodermal precursors are going to form anterior structures and express 3' Hox genes exclusively, while posterior structures arise from later ingression of cells that contain both 3' and 5' Hox gene transcripts.
More than the combination of HOX proteins, the most posterior Hox gene expressed within a given region is the main determinant of segmental identity. Indeed, it has been described that 5' Hox genes have a dominant function over 3' Hox genes. This hierarchy between HOX protein functions is termed as posterior prevalence (Duboule 1991; Duboule and Morata 1994; Duboule 2007).
In summary, vertebral identity is largely established by differential Hox expressions along the anteroposterior axis, which is determined both by their early colinear activation in the epiblast and the subsequent refining of their expression domains in the PSM.
1.2.6 A molecular link between main body axis patterning and the segmentation clock
For a harmonious development of the main body axis, Hox gene expression needs to be precisely coordinated with somitogenesis. Although the Hox code is initially determined by the progressive transcriptional activation of these genes, it is not fixed and will be refined after the emergence of the cells from the primitive streak. As they transit in the PSM, the cells receive patterning and positioning instructions by exposure to the Wnt/FGF and RA morphogenetic gradients, which help them to acquire their definitive Hox expression (Deschamps and Nes 2005). In addition, genes that function in the segmentation program have been described to influence also Hox expression in the anterior PSM (Zákány et al. 2001; Cordes et al. 2004). Indeed, the discovery that Hoxd1 is transcribed in stripes of expression that correspond to the phase of Mesp2 and Lnfg cyclic transcription, two effectors of the Notch pathway, revealed for the first time a molecular link that is likely to coordinate vertebral patterning with the segmentation clock. Furthermore, comparable patterns of expression were discovered for Hoxd3, Hoxa1 and Hoxb1. Confirming the previous indication for a connection between somitogenesis and Hox expression, the knockout of RBPJk, one of the major Notch modulators, results in severely reduced Hoxd1 and Hoxd3 expression levels in emerging somites (Zákány et al. 2001).
Interestingly, it was observed that the bursts of Hoxd1-Hoxd3 transcription are likely due to a unique shared regulatory sequence located outside of the HoxD cluster.
Indeed, transgenic experiments demonstrated that the striped pattern of expression is not inherent to an individual Hox gene as it cannot be recapitulated by randomly integrated Hox transgenes. Moreover, the replacement of the whole HoxD cluster by a lacZ reporter could perfectly produce the dynamic transcription patterns, strongly supporting the idea that that the putative enhancer is located outside of the cluster (Zákány et al. 2001).
In summary, the regulatory impact of segmentation signaling on Hox gene dynamic transcription, via the Notch pathway, suggests a molecular link between segmentation and Hox gene regulation, which could be relevant for the coordination of body elongation and determination of vertebrae morphological fates.
1.3 Hox gene function during hair follicle morphogenesis 1.3.1 Mammalian innovations
We have seen that in addition to a universal role in patterning the main body axis, Hox genes were co-opted along with the emergence of novel structures.
Following this observation, extensive Hox gene expressions were reported in hairs, a defining feature of the mammalian lineage. Hairs are keratinized skin appendages produced by very small organs, the hair follicles, during the whole life span of the animal (Awgulewitsch 2003). Sensation and thermoregulation were proposed as ancestral hair functions, in addition to which they acquired numerous novel tasks such as communication, protection from direct sunlight, sensory perception, camouflage or sexual attraction (Dhouailly 2009; Maderson 2003) as they differentiated into several types of hairs (Sennett and Rendl 2012). One of the most divergent categories of hairs are the long and stiff vibrissae, which are specialized in tactile sensing and provide highly accurate perception of the environment, namely object recognition and spatial localization. Of note, vibrissae are thought to be one of the most recently evolved mammalian sensory systems, as they are found in both eutherians and marsupials, but not in monotremata (Pocock 1914; Brecht et al. 1997).
In this chapter, we are going to focus on this last type of hairs, the vibrissae. We will try to describe their function and the arrangement of their follicles on the skin.
Then, established molecular pathways of hair follicle morphogenesis will be presented, followed by basic explanations about cyclic growth. Finally, we will try to summarize what we know about Hox expression in pelage hairs and vibrissae, which functions in skin appendages remain to be characterized today.
1.3.2 Architecture and function of the whisker pad
Vibrissae are grouped on each side of the face in a grid-like structure commonly called "whisker pad", where they arrange in a highly ordered manner according to their lengths and orientations. The whisker pad comprises macro- and micro- vibrissae. Micro vibrissae are arranged in dense arrays and principally function for close exploration and object recognition (Brecht et al. 1997). Macro vibrissae, on the other hand, cooperate to generate information about object detection and spatial orientation. Since the scope of this work mainly involves gene expression and regulation in macro vibrissae, I will from now on use the term "vibrissae” to refer to this type of hair.
The architecture of the whisker pad is central to encode the sensorial information, which is transduced to the brain by sensory neurons (Jadhav and Feldman 2010). The functional importance of the mystacial apparatus is reflected by its wide representation in the primary somatosensory cortex, the part of the brain that computes environmental stimuli. Remarkably, the topographic map of the neuron projections in the cortex corresponds to the anatomical organization of the vibrissae within the pad (Jadhav and Feldman 2010; Deschênes et al. 2012).
The whisker pad is a remarkably ordered structure, which is determined by the distribution of vibrissae follicles in regular rows and columns (Figure 7).
Interestingly, the exact distribution and number of follicles are not variable among individuals of the same species. Mice and rats typically have around thirty mystacial vibrissae arranged in five rows and up to seven columns. The importance of a
conserved whisker pad organization for environmental sensing has been established, as it allows coordinating the inputs of individual vibrissae. For example, the differences of length between vibrissae that belong to a same row are constant and enable distance detection (Brecht et al. 1997). Many mammalian species rapidly move the vibrissae back and forth to methodically scan the environment. This active sensing behavior has been well characterized in rodents and marsupials (Wineski 1985;
Mitchinson et al. 2011; Deschênes et al. 2012). The sophisticated, controlled movements of the vibrissae rely on two types of musculatures. While back and forth movements are controlled by intrinsic muscles that link the hairs of a same row together, an extrinsic musculature originating from the skull allows the whole pad to move in different directions (Dörfl 1982; Haidarliu et al. 2010; Grant et al. 2013).
Figure 7 - Spatial organization of the whisker pad
from (Haidarliu et al. 2010). Mystacial pad of the rat, xylène staining of the blood sinuses that surround whisker follicles. α-‐δ signify the four most caudal vibrissae. A to E represent the rows, 1 to 7 the columns of smaller vibrissae.
Spatial organization of the whisker pad
Thus, vibrissae functions rely in part on the very ordered manner the individual hairs arrange on the whisker pad (Figure 7). This distinctive anatomy appears early during embryogenesis, as hair follicles display this ordered organization already during the very first stages of morphogenesis. The vibrissa follicle is an elaborate structure that anatomically differs from pelage follicles in that it is surrounded by large blood vessels and is enclosed in a thick collagen capsule (Haidarliu et al. 2010;
Grant et al. 2013). Although the development of vibrissae follicles starts earlier and proceeds faster, respectively at embryonic stages E11.5 and E14.5, it seems mostly similar to that of pelage follicles (Duboule 1998; Kanzler et al. 2004). Because little is known about the molecular development of the vibrissae follicles, some general molecular pathways involved in hair formation are presented below to illustrate their morphogenesis.
1.3.3 Molecular signalling during hair follicle morphogenesis
The regular distribution of emerging structures on the epidermis is a recurrent theme in skin development. Placodal fate determination through local competition is thought to involve reaction-diffusion systems proposed by Alan Turing in 1952. In these models, the system requires at least two chemical substances, an activator and an
inhibitor, that need to diffuse, regulate each other and decay, in order to organize an initially homogeneous tissue in regular arrays of, for example, hair placodes (Turing 1952; Pispa and Thesleff 2003; Jiang et al. 2004; Sick et al. 2006). According to this view, patterns could arise from the molecular interactions of the diffusible activators and repressors found early in the presumptive skin. Indeed, the biochemical characteristics of the main factors involved in early follicle morphogenesis tend to meet the reaction-diffusion system requirements (Awgulewitsch 2003).
The embryonic epidermis comprises a unique layer of undifferentiated epithelial keratinocytes, while the embryonic dermis is composed of mesenchymal cells (Fuchs 2008). The vertebrate skin generates an impressive number of different appendages, which implicates major cellular rearrangements of the surface ectoderm and involves numerous molecular factors necessary for cell growth, migration, adhesion or compaction (Hardy 1992). Remarkably, the first steps of these reorganizations are very similar among hairs, mammary glands, feathers or teeth, as they all imply the reorganization of epithelial cells into a placode, accompanied by the condensation of underlying mesenchymal cells in a precursor dermal papilla. The placode appears as a local thickening of the skin and is induced by series of interactions between the mesenchymal and epithelial cells. The appendages acquire their specific identity later, at the time of layer folding. In hairs, the placode invaginates into the skin and the follicle develops into a peg that finally differentiates into several epithelial components of the mature hair shaft (Hardy 1992; Mikkola 2007; Sennett and Rendl 2012; Pispa and Thesleff 2003; Millar 2002). Numerous signalling events take place at each of these follicle morphogensis stages. Below, we will try to present some of the basic, established molecular communication pathways occurring from placode induction to hair shaft differentiation.
Early stages: placode induction and dermal condensation
At the cellular level, placode induction represents the switch from epidermal to follicular fate and is linked with major, controlled changes in cell behaviour, including compaction or migration (Hardy 1992; Sennett and Rendl 2012; Ahtiainen et al. 2014). It is regulated by sequential mesenchymal-epithelial interactions, mediated by signalling pathways such as Wnt, Eda, FGF and BMP (Millar 2002;
Sennett and Rendl 2012)(Figure 8). In vivo, pioneer tissue recombination experiments between mouse and chicken, involving heterospecific grafting of epithelium and mesoderm into ectopic locations, has shown the inductive effect of the dermis for placode initiation (Dhouailly 1973). This dermal signal was then shown to belong to the Wnt family and long considered as the first step of placode initiation. However, recent studies show that epithelial Wnt signaling would even be responsible for the activation of the dermal Wnt expression (Chen et al. 2012).
Figure 8 - Major steps of hair follicle morphogenesis
modified from (Millar 2002). Schematic representation of major steps of hair follicle morphogenesis, coloured arrows symbolize intercellular signalling.
Early Wnt dermal signalling subsequently regulates activators and inhibitors of placodal fate located in the overlying epithelial cells, which result in the appearance of orderly arranged hair placodes (Zhang et al. 2009). Several studies show the central role of the canonical Wnt/ß-catenin pathway, mediated by the transcription factors LEF1/TCF, in placode initiation. For example, in mice carrying a mutation in the LEF-1 gene, the development of organs requiring epithelial-mesenchymal interactions is impaired, including a reduced number of pelage hair follicle and a complete lack of vibrissae (Genderen et al. 1994; DasGupta and Fuchs 1999). Moreover, LEF-1 overexpression in tissue that normally do not wear hairs, like the lip epithelium, leads to the emergence of ectopic hairs and teeth (Zhou et al. 1995).. In vibrissae, a reporter gene mimicking the activity of LEF-1, presumably activated by Wnt, allowed to visualize that these pathways are signalling in both epithelium and mesenchyme (DasGupta and Fuchs 1999). These results support a collaboration between ß-catenin and LEF-1 as well as their deep implication in several steps of hair follicle morphogenesis (Kratochwil et al. 1996; DasGupta and Fuchs 1999).
Slightly after placode induction, Wnt and Eda are upregulated in the nascent follicles.
Eda is a ligand that belongs to the TNF family and presumably acts for placode stabilization by mediating downstream effectors via the binding to its receptor, Edar, and the subsequent activation of the NFκB transcription factor (Mikkola et al. 1999;
Schmidt-Ullrich et al. 2001; Mikkola 2007; Zhang et al. 2009). In addition to Wnt and Eda, Fgf10 and its receptor Fgfr2 have been shown to be necessary for placode growth (Celli et al. 1998; Petiot et al. 2003). TGF-ß2 is another necessary and sufficient positive regulator of placode induction, as supported by experiments involving TGF-ß2 knockout mice and treatments of embryonic skin explants with beads soaked with TGF-ß2 proteins (Foitzik et al. 1999; Jamora et al. 2004). On the contrary, members of BMP family prevent placodal fate in the interfollicular cells.
BMPs are expressed in the placodes themselves, but they are thought to act on surrounding interfollicular cells through lateral inhibition, while their effects in the placodes seem to be repressed locally by BMP inhibitors. Indeed, it has been proposed that Eda/Edar signalling indirectly promotes placodal fate through BMP repression, via follistatin activation in the placodes (Botchkarev et al. 1999;
Botchkarev and Paus 2003).
Once placode initiation has started on the epidermis, epithelial Wnt and Eda signallings activate Shh and Fgf20, which in turn trigger the dermal condensations of underlying mesenchymal cells (St-Jacques et al. 1998; Pummila et al. 2007; Huh et al. 2013; Rishikaysh et al. 2014). This cellular compaction under the placode is the precursor of a dermal papilla, a population of cells that is tightly associated with the mature follicle (Sennett and Rendl 2012).
In summary, at early stages of placode formation, Wnt, Eda, FGF and TGFß2 positively regulate placodal fate, while BMPs act as repressors on interfollicular cells.
Slightly later, dermal condensates form by compaction of underlying mesenchymal cells.
Invagination of the placode: late dermal to epithelial signalling. The peg stage
Following placode induction and dermal condensation, the development of the hair follicle continues with the downgrowth of the placode, a stage called hair peg, driven by the influence of Shh emitted by the placode (Pispa and Thesleff 2003; Sennett and Rendl 2012). After its early activation in the placode, Shh expression is localized in the tip of the bulb that invaginates in the dermis, where it regulates proliferation and growth (Iseki et al. 1996; Sennett and Rendl 2012). At this stage, anatomical differences between skin appendages arise as a consequence of dissimilar folding and rearrangements of the epithelium (Jamora et al. 2004). For example, hair follicules invaginate, while feather follicles are protruding (Pispa and Thesleff 2003). In addition to epidermal Shh, dermal TGFß2 is another key component that binds to epithelial receptors in order to promote follicle development beyond the bud stage (Foitzik et al. 1999; Jamora et al. 2004).
The bulbous peg stage
While invagination progresses, the lower part of the hair peg adopts a bulbous shape, epithelial cells of the placode divide to form the matrix. Most cells of the matrix are undifferentiated keratinocytes, they wrap around the dermal papilla, which will remain engulfed in the bulb during the whole life span of the animal, where it retains its important inductive properties for the control of hair cycle. The upper portion of the peg then differentiates into three epithelial cylinders: the hair shaft is produced in the centre by the hair follicle (Schneider et al. 2009) and is surrounded by two distinct layers of keratinocytes called root sheets, which provide physical support. The inner root sheet (IRS) guides the protruding hair fibre to the outside and the outer root sheet (ORS) separates the whole structure from the dermis (Stenn and Paus 2001). As development progresses, a companion layer appears between the ORS and IRS, and both IRS and the hair shaft originate three further layers, bringing to seven the number of concentric rings of differentiated tissue within the mature follicle. It is noteworthy that each of these layers is distinct by its structure and protein content, i.e.
differentially expressed keratins (Fuchs 2007)(Figure 9). How transcriptional regulation of keratins is achieved and the way it promotes hair layer specification will be presented below, with the data supporting a putative role for Hoxc13 (Godwin and Capecchi 1998; Tkatchenko et al. 2001).
Figure 9 - Different cell lineages populate the hair bulb
Modified from (Fuchs 2007). Schematic representation of a mature hair bulb. Different genetic markers of the seven differentiated lineages are indicated on the top and Ki67 indicate proliferative matrix cells.
Ch, hair shaft cuticle; Co, cortex; Cp, companion layer; DP, dermal papilla; Me, medulla
1.3.4 Adult hair cycling
During the hair cycling program, many steps of embryonic patterning are deployed, like induction, differentiation, epithelial-mesodermal interactions, or cell migration. It is important not to confuse follicle morphogenesis and hair cycle. While follicle morphogenesis happens only once, during embryonic and perinatal development, the mature follicle constitutes a sophisticated "mini-organ" that repetitively produces hair shafts throughout adulthood (Stenn and Paus 2001). This process involves sequential phases termed as anagen (growth), catagen (regression) and telogene (rest) (Hardy 1992; Müller-Röver et al. 2001)(Figure 10). Several studies highlighted that common signalling pathways and genetic programs are acting during both hair development and cycling (Oro and Scott 1998; Müller-‐Röver et al.
2001; Jamora et al. 2004; Awgulewitsch 2003). Shh, Wnt, Delta/Notch, BMPs and FGFs account for a great part of these shared factors, in addition to which Hox genes are promising patterning candidates, as suggested by the several expression patterns reported (Awgulewitsch 2003; Tkatchenko et al. 2001, 13; Kanzler et al. 2004).
