Ectodermal Organs Research Articles

Reptile scale paradigm: Evo-Devo, pattern formation and regeneration.

The purpose of this perspective is to highlight the merit of the reptile integument as an experimental model. Reptiles represent the first amniotes. From stem reptiles, extant reptiles, birds and mammals have evolved. Mammal hairs and feathers evolved from Therapsid and Sauropsid reptiles, respectively. The early reptilian integument had to adapt to the challenges of terrestrial life, developing a multi-layered stratum corneum capable of barrier function and ultraviolet protection. For better mechanical protection, diverse reptilian scale types have evolved. The evolution of endothermy has driven the convergent evolution of hair and feather follicles: both form multiple localized growth units with stem cells and transient amplifying cells protected in the proximal follicle. This topological arrangement allows them to elongate, molt and regenerate without structural constraints. Another unique feature of reptile skin is the exquisite arrangement of scales and pigment patterns, making them testable models for mechanisms of pattern formation. Since they face the constant threat of damage on land, different strategies were developed to accommodate skin homeostasis and regeneration. Temporally, they can be under continuous renewal or sloughing cycles. Spatially, they can be diffuse or form discrete localized growth units (follicles). To understand how gene regulatory networks evolved to produce increasingly complex ectodermal organs, we have to study how prototypic scale-forming pathways in reptiles are modulated to produce appendage novelties. Despite the fact that there are numerous studies of reptile scales, molecular analyses have lagged behind. Here, we underscore how further development of this novel experimental model will be valuable in filling the gaps of our understanding of the Evo-Devo of amniote integuments.

Edar and Troy signalling pathways act redundantly to regulate initiation of hair follicle development

The development of ectodermal organs requires signalling by ectodysplasin (Eda), a tumor necrosis factor (TNF) family member, its receptor Edar and downstream activation of the nuclear factor kappaB (NF-kappaB) transcription factor. In humans, mutations in the Eda pathway components cause hypohidrotic ectodermal dysplasia, a syndrome characterized by missing teeth, sparse hair and defects in sweat glands. It has been postulated that Eda acts redundantly with another TNF pathway to regulate ectodermal organogenesis. A potential candidate is Troy (or TNFRSF19 or Taj), a TNF receptor which is homologous with Edar in its ligand-binding domain, and is expressed in an overlapping pattern. We have characterized Troy null mice and crossed them with Eda-deficient mice. Single Troy mutants had no defects in ectodermal organs. Analysis of the double mutants revealed an essential role for Troy in hair follicle development. In mice, hair follicles develop in three different waves. Only primary hair follicles are missing in Eda single mutants, whereas the compound mutants lacked also the follicles of the second wave, as well as all hair follicles in the middle of crown leading to focal alopecia. Assessment of NF-kappaB activity with a transgenic reporter construct indicated that Eda is the main activator of NF-kappaB signalling in developing skin appendages and surprisingly that the functional overlap of Troy and Eda signalling pathways is mediated by NF-kappaB independent pathways.

Identification of dkk4 as a target of Eda-A1/Edar pathway reveals an unexpected role of ectodysplasin as inhibitor of Wnt signalling in ectodermal placodes

The development of epithelial appendages, including hairs, glands and teeth starts from ectodermal placodes, and is regulated by interplay of stimulatory and inhibitory signals. Ectodysplasin-A1 (Eda-A1) and Wnts are high in hierarchy of placode activators. To identify direct targets of ectodysplasin pathway, we performed microarray profiling of genes differentially regulated by short exposure to recombinant Eda-A1 in embryonic eda −/− skin explants. Surprisingly, there were only two genes with obvious involvement in Wnt pathway: dkk4 (most highly induced gene in the screen), and lrp4. Both genes colocalized with Eda-A1 receptor Edar in placodes of ectodermal organs. They were upregulated upon Edar activation while several other Wnt associated genes previously suggested as Edar targets were unaffected. However, low dkk4 and lrp4 expression was retained in the absence of NF-κB signalling in eda −/− hair placodes. We provide evidence that this expression was dependent on Wnt activity present prior to Eda-A1/Edar signalling. Dkk4 was recently suggested as a key Wnt antagonist regulating lateral inhibition essential for correct patterning of hair follicles. Several pieces of evidence suggest Lrp4 as a Wnt inhibitor, as well. The finding that Eda-A1 induces placode inhibitors was unexpected, and underlines the importance of delicate fine-tuning of signalling during placode formation.

Transcription Factor Epiprofin Is Essential for Tooth Morphogenesis by Regulating Epithelial Cell Fate and Tooth Number

In tooth morphogenesis, the dental epithelium and mesenchyme interact reciprocally for growth and differentiation to form the proper number and shapes of teeth. We previously identified epiprofin (Epfn), a gene preferentially expressed in dental epithelia, differentiated ameloblasts, and certain ectodermal organs. To identify the role of Epfn in tooth development, we created Epfn-deficient mice (Epfn-/-). Epfn-/- mice developed an excess number of teeth, enamel deficiency, defects in cusp and root formation, and abnormal dentin structure. Mutant tooth germs formed multiple dental epithelial buds into the mesenchyme. In Epfn-/- molars, rapid proliferation and differentiation of the inner dental epithelium were inhibited, and the dental epithelium retained the progenitor phenotype. Formation of the enamel knot, a signaling center for cusps, whose cells differentiate from the dental epithelium, was also inhibited. However, multiple premature nonproliferating enamel knot-like structures were formed ectopically. These dental epithelial abnormalities were accompanied by dysregulation of Lef-1, which is required for the normal transition from the bud to cap stage. Transfection of an Epfn vector promoted dental epithelial cell differentiation into ameloblasts and activated promoter activity of the enamel matrix ameloblastin gene. Our results suggest that in Epfn-deficient teeth, ectopic nonproliferating regions likely bud off from the self-renewable dental epithelium, form multiple branches, and eventually develop into supernumerary teeth. Thus, Epfn has multiple functions for cell fate determination of the dental epithelium by regulating both proliferation and differentiation, preventing continuous tooth budding and generation.

Mammary glands and feathers: Comparing two skin appendages which help define novel classes during vertebrate evolution

It may appear counter-intuitive to compare feathers and mammary glands. However, through this Evo-Devo analysis, we appreciate how species interact with the environment, requiring different ectodermal organs. Novel ectodermal organs help define evolutionary directions, leading to new organism classes as exemplified by feathers for Aves and mammary glands for Mammals. Here, we review their structure, function, morphogenesis and regenerative cycling. Interestingly, both organs undergo extensive branching for different reasons; feather branching is driven by mechanical advantage while mammary glands nourish young. Besides natural selection, both are regulated by sex hormones and acquired a secondary function for attracting mates, contributing to sexual selection.

The development of a bioengineered organ germ method

To bioengineer ectodermal organs such as teeth and whisker follicles, we developed a three-dimensional organ-germ culture method. The bioengineered tooth germ generated a structurally correct tooth, after both in vitro organ culture as well as transplantation under a tooth cavity in vivo, showing penetration of blood vessels and nerve fibers. Our method provides a substantial advance in the development of bioengineered organ replacement strategies and regenerative therapies.

Ectodysplasin has a dual role in ectodermal organogenesis: inhibition of Bmp activity and induction of Shh expression

Ectodermal organogenesis is regulated by inductive and reciprocal signalling cascades that involve multiple signal molecules in several conserved families. Ectodysplasin-A (Eda), a tumour necrosis factor-like signalling molecule, and its receptor Edar are required for the development of a number of ectodermal organs in vertebrates. In mice, lack of Eda leads to failure in primary hair placode formation and missing or abnormally shaped teeth, whereas mice overexpressing Eda are characterized by enlarged hair placodes and supernumerary teeth and mammary glands. Here, we report two signalling outcomes of the Eda pathway: suppression of bone morphogenetic protein (Bmp) activity and upregulation of sonic hedgehog (Shh) signalling. Recombinant Eda counteracted Bmp4 activity in developing teeth and, importantly, inhibition of BMP activity by exogenous noggin partially restored primary hair placode formation in Eda-deficient skin in vitro, indicating that suppression of Bmp activity was compromised in the absence of Eda. The downstream effects of the Eda pathway are likely to be mediated by transcription factor nuclear factor-kappaB (NF-kappaB), but the transcriptional targets of Edar have remained unknown. Using a quantitative approach, we show in cultured embryonic skin that Eda induced the expression of two Bmp inhibitors, Ccn2/Ctgf (CCN family protein 2/connective tissue growth factor) and follistatin. Moreover, our data indicate that Shh is a likely transcriptional target of Edar, but, unlike noggin, recombinant Shh was unable to rescue primary hair placode formation in Eda-deficient skin explants.

LDL-receptor-related protein 4 is crucial for formation of the neuromuscular junction

Low-density lipoprotein receptor-related protein 4 (Lrp4) is a member of a family of structurally related, single-pass transmembrane proteins that carry out a variety of functions in development and physiology, including signal transduction and receptor-mediated endocytosis. Lrp4 is expressed in multiple tissues in the mouse, and is important for the proper development and morphogenesis of limbs, ectodermal organs, lungs and kidneys. We show that Lrp4 is also expressed in the post-synaptic endplate region of muscles and is required to form neuromuscular synapses. Lrp4-mutant mice die at birth with defects in both presynaptic and postsynaptic differentiation, including aberrant motor axon growth and branching, a lack of acetylcholine receptor and postsynaptic protein clustering, and a failure to express postsynaptic genes selectively by myofiber synaptic nuclei. Our data show that Lrp4 is required during the earliest events in postsynaptic neuromuscular junction (NMJ) formation and suggest that it acts in the early, nerveindependent steps of NMJ assembly. The identification of Lrp4 as a crucial factor for NMJ formation may have implications for human neuromuscular diseases such as myasthenia syndromes.

Hepatic Stem Cells: In Search of

The field of stem cell biology has exploded with the study of a wide range of cellular populations involving endodermal, mesenchymal, and ectodermal organs. One area of extensive study has included the identification of hepatic stem and progenitor cell subpopulations. Liver stem cells provide insights into the potential pathways involving liver regeneration that are independent of mature hepatocytes. Hepatic progenitor cells are either bipotent or multipotent and capable of multiple rounds of replication. They have been identified in fetal as well as adult liver. Various injury models have been used to expand this cellular compartment. The nomenclature, origin, and function of the hepatic progenitor cell populations are areas of ongoing debate. In this review, we will discuss the different definitions and functions of hepatic progenitor cells as well as the current research efforts examining their therapeutic potential.

Signs and symptoms from ectodermal organs in young Swedish individuals with oligodontia

The aim was to assess signs and symptoms from other ectodermal organs in addition to teeth in young individuals with oligodontia and to establish the prevalence of oligodontia. Children born 1981-94 reported by dental teams in the Public Dental Service to have oligodontia were asked to participate in a clinical study. The examinations comprised a structured interview on symptoms from ectodermal organs, and testing of salivary secretion. One hundred and sixty-two individuals met the inclusion criteria, and 123 individuals (75.9%) participated in the clinical study. Half of the individuals had one to four signs or symptoms from ectodermal organs beside oligodontia. The most common sign was low salivary secretion. Twelve individuals (9.6%) with isolated oligodontia reported impaired function of the sweat glands, hair, or nails. The prevalence of oligodontia was 0.090%. An early identification of individuals with oligodontia can be made in a majority of cases by checking that all permanent incisors have erupted at the age of 8 years. The validity in asking individuals about normal and abnormal function of ectodermal organs was found to be low. This indicates that there is a strong need to establish routine clinical criteria for dysplasia of ectodermal organs.

P63 regulates multiple signalling pathways required for ectodermal organogenesis and differentiation

Heterozygous germline mutations in p63, a transcription factor of the p53 family, result in abnormal morphogenesis of the skin and its associated structures, including hair follicles and teeth. In mice lacking p63, all ectodermal organs fail to develop, and stratification of the epidermis is absent. We show that the ectodermal placodes that mark early tooth and hair follicle morphogenesis do not form in p63-deficient embryos, although the multilayered dental lamina that precedes tooth placode formation develops normally. The N-terminally truncated isoform of p63 (DeltaNp63) was expressed at high levels in embryonic ectoderm at all stages of tooth and hair development, and it was already dominant over the transactivating TAp63 isoform prior to epidermal stratification. Bmp7, Fgfr2b, Jag1 and Notch1 transcripts were co-expressed with DeltaNp63 in wild-type embryos, but were not detectable in the ectoderm of p63 mutants. In addition, beta-catenin and Edar transcripts were significantly reduced in skin ectoderm. We also demonstrate that BMP2, BMP7 and FGF10 are potent inducers of p63 in cultured tissue explants. Hence, we suggest that p63 regulates the morphogenesis of surface ectoderm and its derivatives via multiple signalling pathways.

Morphoregulation of teeth: modulating the number, size, shape and differentiation by tuning Bmp activity

During development and evolution, the morphology of ectodermal organs can be modulated so that an organism can adapt to different environments. We have proposed that morphoregulation can be achieved by simply tilting the balance of molecular activity. We test the principles by analyzing the effects of partial downregulation of Bmp signaling in oral and dental epithelia of the keratin 14-Noggin transgenic mouse. We observed a wide spectrum of tooth phenotypes. The dental formula changed from 1.0.0.3/1.0.0.3 to 1.0.0.2(1)/1.0.0.0. All mandibular and M3 maxillary molars were selectively lost because of the developmental block at the early bud stage. First and second maxillary molars were reduced in size, exhibited altered crown patterns, and failed to form multiple roots. In these mice, incisors were not transformed into molars. Histogenesis and differentiation of ameloblasts and odontoblasts in molars and incisors were abnormal. Lack of enamel caused misocclusion of incisors, leading to deformation and enlargement in size. Therefore, subtle differences in the level, distribution, and timing of signaling molecules can have major morphoregulatory consequences. Modulation of Bmp signaling exemplifies morphoregulation hypothesis: simple alteration of key signaling pathways can be used to transform a prototypical conical-shaped tooth into one with complex morphology. The involvement of related pathways and the implication of morphoregulation in tooth evolution are discussed.

Ectodysplasin A1 promotes placodal cell fate during early morphogenesis of ectodermal appendages.

Organs developing as appendages of the ectoderm are initiated from epithelial thickenings called placodes. Their formation is regulated by interactions between the ectoderm and underlying mesenchyme, and several signalling molecules have been implicated as activators or inhibitors of placode formation. Ectodysplasin (Eda) is a unique signalling molecule in the tumour necrosis factor family that, together with its receptor Edar, is necessary for normal development of ectodermal organs both in humans and mice. We have shown previously that overexpression of the Eda-A1 isoform in transgenic mice stimulates the formation of several ectodermal organs. In the present study, we have analysed the formation and morphology of placodes using in vivo and in vitro models in which both the timing and amount of Eda-A1 applied could be varied. The hair and tooth placodes of K14-Eda-A1 transgenic embryos were enlarged, and extra placodes developed from the dental lamina and mammary line. Exposure of embryonic skin to Eda-A1 recombinant protein in vitro stimulated the growth and fusion of placodes. However, it did not accelerate the initiation of the first wave of hair follicles giving rise to the guard hairs. Hence, the function of Eda-A1 appears to be downstream of the primary inductive signal required for placode initiation during skin patterning. Analysis of BrdU incorporation indicated that the formation of the epithelial thickening in early placodes does not involve increased cell proliferation and also that the positive effect of Eda-A1 on placode expansion is not a result of increased cell proliferation. Taken together, our results suggest that Eda-A1 signalling promotes placodal cell fate during early development of ectodermal organs.

Tooth patterning and enamel formation can be manipulated by misexpression of TNF receptor Edar.

Signaling by Edar, a tumor necrosis factor receptor, is required for the development of ectodermal organs. Mutations in Edar or other molecules of the same signaling pathway cause ectodermal dysplasias in humans and mice. In these diseases, teeth are missing or malformed, and the development of hairs and several glands is hypoplastic. During tooth and hair development, Edar expression becomes patterned to ectodermal placodes and signaling centers. This localization has been suggested to be required for organogenesis. We have expressed Edar throughout the ectoderm using the keratin 14 promoter and show that this misexpression disrupts tooth patterning and differentiation. Tooth shape and cusp number are differentially affected, depending on the amount of transgene expression. In addition, tooth enamel formation is defective in a dose-dependent manner. We speculate that the tooth patterning defects are caused by ectopic Edar activity outside the signaling centers.

Morpho-Regulation of Ectodermal Organs: Integument Pathology and Phenotypic Variations in K14-Noggin Engineered Mice through Modulation of Bone Morphogenic Protein Pathway

Ectodermal organs are composed of keratinocytes organized in different ways during induction, morphogenesis, differentiation, and regenerative stages. We hypothesize that an imbalance of fundamental signaling pathways should affect multiple ectodermal organs in a spatio-temporal-dependent manner. We produced a K14-Noggin transgenic mouse to modulate bone morphogenic protein (BMP) activity and test the extent of this hypothesis. We observed thickened skin epidermis, increased hair density, altered hair types, faster anagen re-entry, and formation of compound vibrissa follicles. The eyelid opening was smaller and ectopic cilia formed at the expense of Meibomian glands. In the distal limb, there were agenesis and hyperpigmentation of claws, interdigital webbing, reduced footpads, and trans-differentiation of sweat glands into hairs. The size of external genitalia increased in both sexes, but they remained fertile. We conclude that modulation of BMP activity can affect the number of ectodermal organs by acting during induction stages, influence the size and shape by acting during morphogenesis stages, change phenotypes by acting during differentiation stages, and facilitate new growth by acting during regeneration stages. Therefore during organogenesis, BMP antagonists can produce a spectrum of phenotypes in a stage-dependent manner by adjusting the level of BMP activity. The distinction between phenotypic variations and pathological changes is discussed.

Lineage of non-cranial neural crest cell in the dental mesenchyme: using a lacZ reporter gene during early tooth development.

The tooth is one of the ectodermal organs controlled by reciprocal interactions between the epithelium and the mesenchyme. Mesenchymal cells in the developing tooth, so-called dental mesenchymal cells, are derived from two different origins: the cranial neural crest (CNC) and the non-CNC. These CNC-derived cells migrate, proliferate and differentiate into odontoblasts, cementoblasts, fibroblasts, osteoblasts and chondroblasts. Tooth germs of wild-type mice were transplanted into the kidney of adult lacZ-transgenic mice. After 1 week of transplantation, a few lacZ-expressing cells and many red blood cells were found near or inside the blood vessels in the pulp of wild-type tooth germs. This result shows that circulating cells of the adult host could invade the dental pulp during tooth development, through the blood vessels, and be a part of dental pulp tissue. Therefore, it can be suggested that these circulating progenitor cells could be the origin of non-CNC-derived cells in tooth germ and their migration pathways would be the blood vessels invading the dental pulp during tooth development. If variations of this experiment were suitably adjusted, such as the embryonic stage of the tooth germ, duration of transplantation, etc., this transplantation experiment using adult lacZ-transgenic mice could be a good system to reveal the origin and migration pathway of cells in developing organs as well as in dental mesenchymal cells.

Msx homeobox gene family and craniofacial development.

Vertebrate Msx genes are unlinked, homeobox-containing genes that bear homology to the Drosophila muscle segment homeobox gene. These genes are expressed at multiple sites of tissue-tissue interactions during vertebrate embryonic development. Inductive interactions mediated by the Msx genes are essential for normal craniofacial, limb and ectodermal organ morphogenesis, and are also essential to survival in mice, as manifested by the phenotypic abnormalities shown in knockout mice and in humans. This review summarizes studies on the expression, regulation, and functional analysis of Msx genes that bear relevance to craniofacial development in humans and mice. Key words: Msx genes, craniofacial, tooth, cleft palate, suture, development, transcription factor, signaling molecule.

Identification of a secreted BMP antagonist, ectodin, integrating BMP, FGF, and SHH signals from the tooth enamel knot

We have identified mouse and human cDNAs encoding a novel secreted BMP inhibitor, which we have named ectodin. It is most homologous (∼37% amino acid identity) to sclerostin that is a secreted BMP antagonist. Recombinant ectodin protein produced in cultured cells was efficiently secreted as a antagonist. Ectodin inhibited the activity of BMP2, BMP4, BMP6, and BMP7 for mouse preosteoblastic MC3T3-E1 cells, and bound to these BMPs with high affinity. Ectodin is intensely expressed in developing ectodermal organs, including teeth, vibrissae, and hair follicles. However, it is absent from the hair placodes and from the enamel knot signaling centers in teeth. In addition, several cell layers surrounding the enamel knots were completely devoid of ectodin transcripts. We analyzed the regulation and function of ectodin in tooth germs. Recombinant ectodin protein antagonized the BMP-mediated induction of Msx2 expression in cultured tooth explants, indicating that ectodin is a secreted BMP inhibitor. BMP2 and BMP7 stimulated ectodin expression in tooth explants, showing that it is part of a feedback mechanism controlling the activity of BMPs. The stimulation of ectodin expression by BMP was prevented by SHH and FGF4 but not by Wnt6. Hence, the feedback mechanism whereby BMPs upregulate their own inhibitor is counteracted by signals coexpressed with BMPs in the enamel knot. We conclude that ectodin is a novel BMP inhibitor which integrates BMP signaling with the SHH and FGF signal pathways and contributes in defining the exact spatiotemporal domain of BMP target field around the ectodermal signaling centers.

Ectodysplasin, Edar and TNFRSF19 are expressed in complementary and overlapping patterns during mouse embryogenesis

Ectodysplasin (Eda), a member of the tumor necrosis factor (TNF) superfamily, and its receptor Edar are necessary components of ectodermal organ development. Analysis of their expression patterns and mutant phenotypes has shown that during mouse hair and tooth development they may be involved in signalling between separate epithelial compartments. Here we have analysed ectodysplasin and Edar expression in other embryonic mouse tissues, and show that Edar mRNA is confined to the epithelium. Ectodysplasin and Edar are expressed in separate epithelial compartments in the developing brain and the lacrimal gland. In the salivary gland ectodysplasin is expressed in the mesenchyme and Edar in the epithelium. This is the first indication of ectodysplasin–Edar signalling between the epithelium and the mesenchyme. We also studied the expression pattern of a related TNF receptor, TNFRSF19, and show that it is expressed in an overlapping domain with Edar in the tooth, mammary gland, whiskers, and limb bud suggesting a potentially redundant role.

Stimulation of ectodermal organ development by Ectodysplasin-A1

Organs developing as ectodermal appendages share similar early morphogenesis and molecular mechanisms. Ectodysplasin, a signaling molecule belonging to the tumor necrosis factor family, and its receptor Edar are required for normal development of several ectodermal organs in humans and mice. We have overexpressed two splice forms of ectodysplasin, Eda-A1 and Eda-A2, binding to Edar and another TNF receptor, Xedar, respectively, under the keratin 14 (K14) promoter in the ectoderm of transgenic mice. Eda-A2 overexpression did not cause a detectable phenotype. On the contrary, overexpression of Eda-A1 resulted in alterations in a variety of ectodermal organs, most notably in extra organs. Hair development was initiated continuously from E14 until birth, and in addition, the transgenic mice had supernumerary teeth and mammary glands, phenotypes not reported previously in transgenic mice. Also, hair composition and structure was abnormal, and the cycling of hairs was altered so that the growth phase (anagen) was prolonged. Both hairs and nails grew longer than normal. Molar teeth were of abnormal shape, and enamel formation was severely disturbed in incisors. Furthermore, sweat gland function was stimulated and sebaceous glands were enlarged. We conclude that ectodysplasin–Edar signaling has several roles in ectodermal organ development controlling their initiation, as well as morphogenesis and differentiation.