2 - 4 - DEVELOPMENTAL BIOLOGY OF THE SKIN Flashcards
central cells of the epidermis and form the intermediate filament keratins that, among other roles, provide structural resiliency to cells.
Keratinocytes
central cells of the dermis that, among other roles, secrete collagen, which provides the substance for the dermis.
Fibroblasts
organized structures of keratinocytes and fibroblasts that act together to make hair follicles, eccrine sweat glands, apocrine glands, and the nail unit.
Appendages
cells that reside predominantly in the epidermis and synthesize melanin whose primary function is to absorb and block the sun’s damaging ultraviolet light.
Melanocytes
immune cells that reside predominantly in the epidermis, and internalize and present potentially harmful antigens encountered in the environment to initiate an immune response
Langerhans cells
monitor touch, pressure, temperature, and hair follicle movement
Sensory neurons
live in the epidermis and sense touch
Merkel cells
extracellular secreted proteins that likely have important posttranslational modifications such as palmitoylation and activate the frizzled receptors to eventually stabilize b-catenin, causing its nuclear translocation from cytoplasmicassociated or cytoskeletal-associated structures. B-Catenin controls epithelial differentiation, stem
cell function, appendage function
Wnt ligands
transcription factor with multiple isoforms that is perhaps the central regulator of epithelial identity. Without it, epidermis fails to stratify or fully form, which leads to failure of appendage formation.
p63
extracellular secreted proteins that bind the smoothened receptor and eventually activate the Gli family of transcription factors. It is very important to hair follicle formation and function
Shh ligands
receptor for the EDA (ectodysplasin A) ligand and part of the tumor necrosis factor (TNF) receptor family. It is critically important for the development of appendages
EDAR (ectodysplasin A receptor)
bind the receptor Notch which initiates transcription and epidermal differentiation
The ligands Delta or Jagged
family of ligands that bind BMP receptors and, among other functions, regulate hair follicle cycling and growth
BMP (bone morphogenic protein) ligands
The cell that composes the majority
of the epidermis is the ______
keratinocyte, so named because the structural protein keratin is abundant in that cell.
Keratinocytes are stacked in layers of increasingly more mature states until they are effectively ghosts of cells that serve only as a barrier and have little to no energy consumption. In aggregate, the epidermal layers are thicker in areas of the palms and soles, which are subjected to more pressure, and thinner in areas without such pressure, such as the eyelids. Each layer is defined by different keratins, and keratins also differ by body site. In fact, the same general arrangement of keratinocytes that mature from normal epidermis toward dead cells in the epidermis also is how teeth and hair are formed. Although in the general epidermis the dead keratinocytes are regularly released to allow for the next layer to mature and replace it, in hair and teeth those cells are retained to form the final “keratinized” structure. It is important to emphasize that many cells besides keratinocytes reside in the epidermis, including nerve cells that pierce the basal lamina, a vital scaffolding structure that separates the epidermis from the dermis, the next most internal layer of skin.
protein most abundant in the epidermis
keratin
most abundant protein in the dermis
Collagen
GENERAL SUMMARY OF EMBRYONIC DEVELOPMENT
Human development occurs through 40 weeks of gestation, and is commonly subdivided into trimesters (Table 4-1). The week of development is typically counted beginning from the last menstrual period. The developing human is called a fetus at around 8 weeks aafter arms and legs have developed and show motion. This occurs during the first trimester, from weeks 0 to 12 of estimated gestational age, when organogenesis has mostly completed. The second trimester occurs from weeks 12 to 26 and is marked by continuous development, for example, the appearance of downy hair in the infant. The third trimester runs from weeks 26 to 40 and is when most development completes, including the formation of the vernix caseosa from the skin whose function is thought to lubricate for the passage through the birth canal. Interestingly, skin function is not complete even at birth as final full-barrier formation occurs afterward.
The most complicated steps of development occur at the earliest times when the skin begins to form within the first 2 weeks of development. Several canonical structures characterize early development: morula, blastula, gastrula, and then somatogenesis, and organogenesis. After fertilization, the initial unstructured multiplication of cells leads to the formulation of a morula. The morula divides to form a more complicated structure called a blastula, which has 2 main parts. The first is the trophoblast, which is destined to become the parts of the placenta of fetal (as opposed to maternal) origin, namely the chorion, amnion, allantois. The second is the inner cell mass (ICM), which is destined to become the actual embryo. As the ICM differentiates into 3 germ layers it becomes the gastrula, which is the first stage where skin development separates from the development of other organs.
The 3 germ layers that form during gastrulation include the ectoderm, the mesoderm, and the endoderm. The ectoderm forms eventually the epidermis and melanocytes, but also the nervous system. The mesoderm forms fibroblasts, blood vessels, muscles, and bone. The endoderm does not contribute to skin development. There are some exceptions to these generalities, such as some fibroblast subpopulations that actually originate from the ectoderm, as they are thought to be derived from the neural crest.
NEURAL CREST AND SOMITE DEVELOPMENT
Neural crest development is important in the skin as it contributes to structures such as melanocytes and fibroblasts, and is therefore also involved in some clinical diseases where neural crest does not mature correctly.
Neural crest development initiates during the third week of fetal life when the ectoderm forms the neural plate within it. The ectoderm is the outermost layer of the ICM and sits upon the mesoderm. A plate of cells within the ectoderm differentiates into the future central nervous system. However, at the junction between the neural plate and the ectoderm, a distinct group of cells, known as the neural crest form. During gastrulation, the neural plate forms a valley in the ectoderm, and as it does, the edges of the valley actually rise up; this raised area is the boundary between the ectoderm and the neural plate and is termed the neural crest, as the crest of a hill. Eventually the valley becomes so deep and narrow that top parts of the ectoderm fuse and the neural plate detaches to become the neural tube. The neural crest cells, however, are not retained in either the ectoderm or the neural tube, but instead remain free in the mesoderm. Interestingly, there are some differences
in terms of when the neural tube closes and when the neural crest cells migrate away, probably as a clue to their function. In the head, the neural crest migrate even before neural tube closure, and contribute to dermal fibroblasts in the face and anterior scalp. In the trunk, it is the last event. Neural crest cells are thought to specifically secrete the Wnt1 ligand, an important signaling molecule that activates the transcription factor and cytoskeletal protein, β-catenin.
Neural crest cells continue to migrate after neural tube morphogenesis. After detaching from the ectoderm or the neural tube, neural crests migrate either dorsally or ventrally. Persistent neural crest cells that do not complete migrations and differentiate into melanocytes are hypothesized to contribute to common blue nevi.
By the third week of human development, the mesoderm condenses into regular-spaced cuboidal segments termed somites, which are lateral to the neural tube. Although they mostly contribute to the axial skeleton and muscles, early somite fibroblasts are also precursors for dermal fibroblasts. Many of these fibroblasts—especially from body locations such as the back—originate from the dorsolateral portions of the somite, which is also called the dermatomyotome. Many diseases are associated with defects in neural crest migration, some of which affect melanocytes (see section “Melanocyte and Differentiation”). Non–melanocyte-related diseases of neural crest migration include DiGeorge syndrome where 22q11.2 deletion results in defects in cardiac, craniofacial, and endocrine organs, among others.
EPIDERMIS DIFFERENTIATION
Figure 4-2 Epidermis development. Shown are the intermediate stages of skin development and acquisition of postnatal keratin expression.
The final epidermis is a strong barrier consisting of a carefully stratified sequential layer of keratinocytes, so named because of their abundant synthesis of keratins, which are intermediate filaments with broad roles in regulating cell function even outside of their central role of providing structural support. 2 The final mature layers of the epithelium are very well defined with characteristic keratins expressed at each stage, typically with unique pairs of both a type I keratin and a type II keratin. The lack of development of some these keratins through mutations causes some blistering epidermolysis bullosa diseases, and is just one example of a family of proteins necessary for epidermal function. Understanding epithelial development (Fig. 4-2) will guide understanding of these diseases.
The development of the epidermis is unique in that it is destined to function at the air interface, but develops in the liquid environment of the amnion, and thus undergoes some unique stages that are never repeated in life. These stages are named after the layers that form such that the first is periderm formation, followed by intermediate layer formation, and, finally, full maturation.
Epidermal development begins soon after gastrulation. Although it is mostly complete in the first trimester, it is not fully complete until after birth. Skin forming begins when the ectoderm converts to a single layer
known as the germinativum—a cuboidal, mitotically active and undifferentiated layer. It expresses the gene p63, which is vital for epidermal differentiation and also corrupted in the EEC syndrome. Coincident with p63 expression is conversion from the more primitive cytokeratins KRT8 and KRT18 to the more mature basal layer keratins KRT5 and KRT14. 3 At 15 days the periderm layer forms above it, which appears as flattened cells with tight junctions and polarized cytoskeletal adhesions. The periderm initially expresses the cytokeratin KRT17 and then cytokeratin KRT6. Genes that control periderm formation include SFN, IRF6, and IKKα, likely partially through the nuclear factor kappa B (NF-κB) pathway. Hypothesized functions for the periderm include transport of and/or protection from material from the amnion, regulation of the dermis, and contribution to epithelial maturation. Recent evidence suggests it is also a protective layer that prevents pathologic adhesions to adjacent epithelium, so that lack of periderm formation leads to the human cocoon syndrome.4
At around 60 days of gestation, the intermediate layer is formed between the periderm on the outside and the germinativum layer. The intermediate layer probably forms through asymmetric cell divisions in basal layer keratinocytes, 5 much as is thought to occur for suprabasal layer formation in adults. In this case, 1 daughter cell continues to function as a stem cell in the basal layer, while its asymmetric sibling cell moves upward to begin differentiation. Following the formation of these 3 layers of embryonic skin, stratification of the epidermis begins coincident with the conversion to a fetus. Barrier formation is patterned, at least in mice, initiating at the dorsum and head. From there, it moves posteriorly and toward dorsal and ventral midlines. Characteristics of low transepidermal water loss and full exclusion of dyes as tests of mature epidermis are not reached until after birth.
Defects in the establishment of the skin barrier in humans are related to the clinical ichthyosis syndromes, also called defects in cornification. A wide variety of genes are important in the establishment of the skin barrier, and disruption of any of them can, in varying degrees, cause disease. Perhaps the most common is ichthyosis vulgaris with defects in the filaggrin protein, which is present in keratohyalin granules in the upper epidermis, from which the term granular layer derives. But others include defects in keratins of the suprabasal layer (KRT1 and KRT10), or in enzymes that crosslink proteins to make an impermeable layer (transglutaminases), or in lipid-metabolizing enzymes whose products are important for cornification (ALOX)
MESENCHYME/ FIBROBLAST AND ADIPOSE DEVELOPMENT
The dermis is formed from the mesodermal layer of the embryo; consequently, it is referred to as mesenchyme. Important structures, such as blood vessels
and nerves, are discussed in sections “Development of the Cutaneous Nerves and Vasculature”. Besides these, the primary cell type that supports the dermis is the fibroblast, which—despite a monomorphic appearance on histology—is likely a very heterogenous population. Although fibroblasts are most appreciated for their ability to abundantly secrete collagens and other extracellular matrix molecules, they are very critical even to epithelial cell function in many contexts, often depending on their location in the body and their subtype within that area of skin.7
The first type of fibroblast heterogeneity is the diversity of distinct lineages found at distinct body locations such as those at the distal fingertips compared to the proximal arm. Whereas fibroblasts from the ventral body are thought to derive from lateral plate mesoderm, fibroblasts from the head are thought to develop more from neural crest precursors, and those, for example, of back skin are more likely to develop from somites (and more specifically dermatomyotomes). This heterogeneity of fibroblast origin might partially explain the different features of skin in these areas, such as the high density of hair follicles in the head compared to the abdomen (see next paragraph). Even within a body part such as the limb, fibroblasts have distinct developmental origins whose signature is retained even in adulthood. 8 In particular, fibroblasts are programmed with a specific combination of HOX genes, which, even after culturing of fibroblasts, are retained to specify, for example, proximal to distal location with functional consequences such as the thick hairless skin on the palms and soles. This is consistent with the vital role of HOX in specifying body patterning, where mutations can lead to large-scale changes, such as legs appearing where antennae should be in Antennapedia Drosophila mutants.
Although the evidence is somewhat mixed, the mesenchymal cells are considered to be regulators of keratinocyte function. 9 In tissue-swapping studies where positionally mismatched epidermis and dermis layers are juxtaposed (eg, palmo-plantar dermis with haired epidermis), fibroblasts can “reprogram” keratinocyte function in some cases. Although contamination remains a theoretical concern, 10-14 this is one example suggesting that fibroblasts are not merely important for synthesis of extracellular proteins, but actually help dictate epidermal identity and function.
There is also heterogeneity of fibroblasts within a given location of skin. A common fibroblast progenitor (in mice characterized by the markers PDGFRa, Dlk1, and Irig1) gives rise to two general linages of fibroblasts, one destined for the upper dermis and one for the lower dermis. The upper dermal fibroblast progenitor (PDGFRa+, Blimp1+, Dlk−, Irig1+) become dermal papillae (a ball of fibroblasts that control hair keratinocytes), arrector pili muscle (muscle attached to hair that causes goosebumps), and the fibroblast of the upper dermis termed papillary fibroblasts. These fibroblasts are unique in their greater density and biased synthesis of collagens such as collagen III over collagen I. Interestingly, more detail is emerging on how these precursors differentiate and localize, such
as the role of nephronectin as a maturation signal in the hair follicle stem cell compartment to induce arrector pili muscle differentiation. 15 The lower dermal fibroblast progenitor (PDGFRa+, Blimp1−, Dlk1+) give rise to not only adipocytes but to the reticular fibroblasts (lower density and biased collagen I over collagen III production). These reticular fibroblasts also differentiate to myofibroblasts during wounding, which promotes wound closure and likely also scarring. A separate group has defined another population of fibroblasts that promotes scarring in wounds and is classified by high engrailed (eng1) expression during development. 16 Although more work is needed to define the intersections of these populations and what differentially controls them, the startling heterogeneity of fibroblasts is quite clear.
One interesting developmental feature of fibroblasts is that they maintain their mesodermal capacity to transdifferentiate, for example, into bone and fat in culture, especially those from the dermal papillae, or fibroblasts surrounding the hair follicle in the hair sheath. These are classic defining features of mesenchymal stem cells derived from the bone marrow, but mesenchymal stem cells are thought to be distinct from most resident dermal fibroblasts, and not a normal component of the dermis, and do not contribute to dermis formation. 7,17 Similarly, pericytes, which are fibroblasts closely associating with blood vessels, also likely do not contribute to dermis formation.
Adipose cells have long been appreciated to have important roles in energy balance. Recently, however, a wider role has been appreciated, such as in immune surveillance 18 and coordinating hair cycling. 19 Adipocytes develop from lower dermal fibroblast progenitors. Similar to the diversity of fibroblasts, there also is an important diversity of adipocytes. Perhaps the central distinction is between white adipose cells, which function to store energy as lipid, and brown adipose, which function to burn energy through the uncoupling of oxidative phosphorylation to generate heat. In humans, brown adipose cells are thought to be located in the subcutaneous tissue at the paracervical/interscapular and supraclavicular areas. 20 Even though peroxisomeproliferator-activated receptor γ (PPARγ) is an important transcription factor for both white and brown adipose development, Prdm16 appears to be uniquely important for brown adipose development. 21 How brown adipocytes might modulate skin function in health and disease is an important area of future study.
Hallmark clinical syndromes of defects in mesenchymal development include focal dermal hypoplasia or Goltz syndrome caused by mutations in the PORC gene, which is important for Wnt molecule secretion. In Goltz syndrome, dermal atrophy manifests as fat herniations appearing as soft yellow to red nodules appearing in Blaschko lines and ulcers at sites of absent skin, among other symptoms. Analogous clinical syndromes for adipose tissue include the lipodystrophy syndromes such as Berardinelli-Seip where a lack of adipose development occurs from defects in the lipid synthesizing AGPAT2 gene.
MELANOCYTE DIFFERENTIATION
The dynamics of melanocyte development are clues to multiple human pigmentation inherited diseases, and perhaps even melanoma. For example, multiple hypotheses exist to explain the treatment-resistance and metastatic potential of melanomas such as their intrinsic ability to resist oxidative damage. Another posits that their considerable migration during development might be recapitulated in metastatic potential. Indeed, the great distances melanoblasts migrate beg the question on how the behavior might be different among less versus more migratory populations once the melanoblasts reach their destination. Melanocytes can be detected by the eighth week estimated gestational age (EGA) 23 in human epidermis.
Melanocytes derive from the neural crest as described in section “Neural Crest and Somite Development” migrating from the closing neural tube. Their precursor is a SOX10-positive progenitor, which also can differentiate into glial cells in addition to melanocytes. When differentiating into melanocytes, they begin to express the critical transcription factor MITF (microphthalmia-associated transcription factor), and also DCT and KIT proteins. These precursor cells actually can lose expression of MITF and KIT to revert to a melanoblast—the adult melanocyte stem cells—that live in the hair follicle bulge stem cell compartment. These melanoblasts differentiate through asymmetric divisions to mature melanocytes (expressing TYR [Tyrosinase]) that populate the hair follicle to give pigment to hair. 24 In addition to populating hair follicles, melanoblasts are thought to home to sweat glands where they might contribute to acral melanomas. These melanocyte stem cells supply interfollicular melanocytes as well, most dramatically during ultraviolet light therapy for patients with vitiligo.
There are a number of clinical correlates to the development of melanocytes. Melanocytes are present in the dermis more in development and less in adults. However, dermal melanocytes are thought to persist after birth in several locations, including the dorsa of the hands and feet, the sacrum/buttocks, and the scalp. These areas are clinically important because they are also common sites for blue nevi. Another example of defective melanocyte development is piebaldism where disrupted melanocyte migration secondary to defects in, for example, c-kit growth factor signaling lead to areas of albinism.
ADNEXAL DEVELOPMENT
Figure 4-3 Hair follicle development. Shown are the stages of hair follicle during development, with a focus on the overlapping signaling pathways between initial hair follicle morphogenesis and postnatal hair cycling. (Adapted from Lee J, Tumbar T. Hairy tale of signaling in hair follicle development and cycling. Semin Cell Dev Biol. 2012;23(8):906-916, with permission. Copyright © Elsevier.)
Adnexal structures refer to all the skin appendages, or specialized arrangements of keratinocytes for unique functional purposes such as hair, nail, or sweat production. These appendages actually have similarities in development to actual limb appendages, as seen in mice where deletions in β-catenin or p63 lead to embryos with limb deformations as well as skin appendage formation.
The best studied of these appendages is the hair follicle. The exciting analogy to development of the hair follicle is that even in the adult, the hair goes through repeating cycles of apoptosis followed by regeneration, which partially recapitulates development. During normal hair cycling, however, the bulge stem cell compartment is maintained, unlike during development where it is formed de novo. Nevertheless, it has been rediscovered that—at least in mice—adults may fully recapitulate the organogenesis of hair follicles, with even the reformation of the stem cell compartment.
How does embryogenesis of a hair occur (Fig. 4-3)? One consensus is that the earliest step in hair development is initiated by the activity of β-catenin in keratinocytes rather than fibroblasts, but this opinion has shifted and it is now thought that initiation of development by β-catenin activity in keratinocytes and fibroblasts might be effectively simultaneous. These epidermal and dermal signals determine how the regular spacing of hair follicles is generated. Mathematical models of reaction–diffusion involving both activators
and inhibitors can effectively generate regular repeating nodes of activity, which, given the similarity to hair follicle regular arrangement, suggests that this occurs in vivo. 32 Signals involved in patterning are thought to include Wnt, but also BMP and FGF (fibroblast growth factor) signaling, among others. In this complete process, signals emanate at first broadly, and then become more localized and patterned to reflect the final tissue architecture. The broad outline of hair development shares similarities with the development of both teeth and mammary glands, among other structures.
The earliest morphologic changes are the formation of a hair placode in the epidermis, where the keratinocytes become thinner, columnar, and tightly packed. This occurs around 75 days EGA, and is accompanied by accumulations of congregated or condensed fibroblasts beneath the epidermis. The following stage is the hair germ phase (80 days EGA; sometimes referred to as hair bud), which is the more pronounced downward movement of epithelium forming a clear nubbin, followed by continued downward epithelial invasion through the peg stage (100 days EGA). During the peg stage, the epithelium begins to wrap around and encompass the associated inductive fibroblast population, which is now called the dermal papillae. Some authors include a final bulbous peg stage, by the end of which the sebaceous glands are formed. At the end of hair follicle development, the follicle is capable of creating a keratinized shaft, lanugo hair. Lanugo hair refers to the first wave of hair production and emerges around 130 days EGA. Interestingly, not all hairs develop simultaneously, and most lanugo hair is shed before birth.
The hair follicle stem cell compartment, termed the bulge, is so named because during development this area (located roughly between the arrector pili muscle and the sebaceous gland in the area termed the isthmus) is rounded out as a bulge in the otherwise straighter edge of the hair follicle. 33 In recent years, novel, distinct, stem cell populations of the hair follicle are being defined, some even outside of the bulge region, with many marked by the specific members of the LGR family of Wnt coreceptors.34
As mentioned above, an array of signals helps define the patterning of hair follicle development. Similarly, an orchestra of signals coordinate the final steps of morphogenesis. The activity of β-catenin is perhaps the most important for appendage development. Mice with activated versions of β-catenin in keratinocytes, for example, form supernumerary hair follicles. Downstream genes that are activated and necessary for morphogenesis include the ectodysplasin ligand (EDA) and receptor (EDAR) functions. For example, human mutations in EDAR cause syndromes where individuals are born with fewer hair follicles and sweat glands. Other genes important for hair development include the Shh, BMP, and FGF pathways. 35 Interestingly, different structures within the hair respond to these signals differently. For example, sebaceous gland differentiation is dependent on low activity of the β-catenin pathway, but also c-Myc, the androgen receptor, and p53, among others.
Sebaceous glands develop during the bulbous peg phase from SOX9+ LRIG1+ hair follicle keratinocyte stem cells during 13th to 14th weeks of fetal life. Interestingly, while sebaceous glands have a dip in activity during childhood, they are more active during embryogenesis (in the production of the vernix caseosa) and puberty.36 Classic sebaceous glands are always associated with hair follicles and do not exist alone, but variations of the sebaceous gland labeled as orphan sebaceous glands include the meibomian gland of the eye, the Fordyce spots of the mouth, the Tyson glands of the prepuce, and the Montgomery glands of the female areola.
Despite increased attention, less is known regarding the developmental dynamics of other appendage structures, such as the orphan sebaceous glands. Many overlaps exist, such as the primacy of epithelialmesenchymal interactions and the involvement of the β-catenin and EDAR pathways. For example, sweat gland and nail development require EDAR, as evidenced even in small natural variants of EDAR (V370A), which confer changes in humans such as thick hair, incisor tooth shoveling, and increased eccrine sweat gland density. 37 Other similarities are that just as lanugo hair is an initial wave of keratinization, nails also have a preliminary nail that is complete and then shed in utero during the second trimester. Nail development also requires HOX programming (see HOX discussion in section “Mesenchyme/Fibroblast and Adipose Development”) as is evident by patients with nail-patella with mutations in the HOX gene LMX1b. 21 Sweat glands in the volar skin develop around 12 to 13 weeks EGA, and the rest of the body at 20 weeks, following the same pattern of placode formation as a hair follicle.
Besides hair, nails, and sweat glands, the final appendage of the skin is the apocrine gland found in the hormonally responsive areas of the axillae and genitals. As apocrine glands are absent from most, if not all, regions of the mouse, little research has defined their developmental biology. Also, besides the above appendageal organs, there are other specialized keratinized structures, such as dermatoglyphs, the technical term for ridges responsible for characteristic fingerprints. Volar dermatoglyphics development begins around week 7 EGA when protuberances in palms and soles, called volar pads, appear. 38 The substance of volar pads are accumulations of grouped fibroblasts. As the volar pads involute, epidermal ridges appear as a result of local hyperproliferation of the basal layer of epidermis. There are several rounds of epidermal ridge development and also much more to learn about this process.
