The Nipple: A Simple Intersection of Mammary Gland and Integument ...

J Mammary Gland Biol Neoplasia (2013) 18:121–131 DOI 10.1007/s10911-013-9289-1

The Nipple: A Simple Intersection of Mammary Gland and Integument, but Focal Point of Organ Function Sachiko Koyama & Hsin-Jung Wu & Teresa Easwaran & Sunil Thopady & John Foley

Received: 9 March 2013 / Accepted: 29 April 2013 / Published online: 15 May 2013 # Springer Science+Business Media New York 2013

Abstract Having glands that secrete milk to nourish neonatal offspring characterizes all mammals. We provide a brief overview of the development and anatomy of nipples and mammary glands in monotremes, marsupials, and marine mammals, and focus on the nipples and mammary glands in terrestrial eutherian species. We first classify eutherians into three groups: the altricial, precocial, and arboreal types based on their rearing system. We then summarize the physiology of lactation and the cell biology of nipples with specific focus on comparing these in the mouse, cow, and human, which represent the three different groups. Finally we propose that the nipple is an example of specialized epidermis. As specialized epidermis, it is dependent the underlying stroma for development and maintenance in adult life. The development of the nipple and signaling pathways that regulate its formation are described.

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Keywords Nipple . Areola . Keratins . Lactation . Breast cancer

The possession of glands that secrete milk to nourish neonatal offspring characterizes all mammals. This makes the offspring completely dependent on their parents for nutrition and hydration, which facilitates immunological protection [1, 2], odor identification of the mother [3–5], and strong maternal attachment [3, 6, 7]. This strong bond between mother and offspring mediates the second stage of learning, which includes food identification [3] and acquisition of social behaviors [6, 8]. At the center of this process is the nipple, which discharges odorants and pheromones, provides nutrition to the young in a manner that prevents damage to maternal tissues, activates the neuroendocrine system to sustain lactation and in humans is a key component of female identity.

Abbreviations BMP Bone morphogenic protein BMPR1A BMP receptor 1A S. Koyama : H.10) and has a correspondingly increased number of nipples (6 to 9 pairs). This characteristic was likely obtained through the process of selective breeding associated with domestication, as wild boars produce small litters (3~5) and only have five pairs of nipples [17]. Domestication often affects both litter size and body size, but in some domesticated species, even the number of pairs of nipples can be changed which suggests the genetic mechanisms controlling mammary gland number are somewhat plastic [17]. Primates are uniquely evolved to utilize arboreal habitat [18]. Arboreal type animals in general don’t make elaborate nests and offspring are usually carried by the mothers until

Fig. 2 Location of Nipples and Glandular Arrangement of mammary Units in the Mouse, Cow and Human. a Mouse b Cow) Human

they obtain the locomotive ability to follow the troop or parents. Primate species that do not live in trees such as baboons and humans, still possess many morphological and behavioral characteristics common to other primate species. On the other hand, altricial-types that have adapted to arboreal niches, such as squirrels, retain the distribution of mammary glands found in these species. Most of the

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primates have one pair of nipples in their pectoral region (Fig. 2c) and usually give birth to one or two relatively mature offspring compared to altricial animal species. The locations of the nipples of old world monkeys and apes are similar to human, but those of the new world monkeys have their single pair of nipples located closer to the axilla, which enables nursing when the babies are riding on their backs [18]. Nipples/teats are found at the point where the mammary gland development initiates. Since the mammary gland forms from the mammary ridge, there is potential that nipples could be found at any point along the structure. However, species that produce precocial young or live in arboreal habitats have a restricted number of nipples located in very specific regions. The following factors are speculated to influence the selection of the location of the nipples/mammary glands. The sizes of mammary glands of animals with multiple pairs of nipples are not homogenous. The mammary glands in the inguinal region are generally larger (for example, the #4 and 5 mammary gland pairs in mice) (Fig. 2a). The precocial type species have nipples developed in the inguinal region (Fig. 2b). The limited number of offspring produced by these animals means there is not a need for a great number of nipples which essentially serve as points of milk outlet. The location where nipples are developed in the precocial type occurs in the region where the mammary gland appears to have the capacity to develop to a maximum size. This extensive glandular tissue in ungulates provides the rather large and active offspring of these animals with abundant milk, and this capacity has been further expanded in domesticated species. Most of the arboreal type species also produce only one or two offspring at a time, but have one pair of nipples developed in the pectoral region (Fig. 2c). So, why did arboreal type animals evolve such that the nipples are located in the pectoral region? Intriguingly, some of the primitive primate species have multiple sets of nipples that are located both in the inguinal region and pectoral regions. For example, some of the prosimians (lemurs, lorises, and tarsiers) have two pair of nipples located in both regions. Surprisingly the aye-aye has a single pair of nipples in the inguinal region [18]. It is possible to speculate that the typical location of nipples and mammary glands in the pectoral region in primates has evolved through the adaptation to the arboreal habitat. This has contributed to the development of various morphological characteristics in higher primates; for example, the positioning of the eyes permitting stereoscopic vision, flat nails and fingers of the hand that can oppose the thumb, enabling grasping branches of trees [18]. These grasping hands facilitate mothers holding their offspring in a manner that allows them to be face to face. Additionally the offspring can also grasp their mothers’ lateral sides while the mothers hold them stabilizing their location in the pectoral region of the mother. The secure grip of the young is crucial when the mothers walk and run or jump from branches to branches in the trees, enabling the

J Mammary Gland Biol Neoplasia (2013) 18:121–131

mothers to follow the foraging troop soon after the parturition. The presence of nipples and the mammary gland in the pectoral region would permit nursing during times not only when the mother is holding the infant while sitting on a branch, but during travel through the trees. Thus the pectoral mammary gland in primates can be a morphological adaption consistent with arboreal habitat. This review has centered on land mammals; however, marine mammals of the family Cetacea also produce milk and nurse their young. For purposes of streamlining required for efficient movement through the water, various dolphins and whales have nipples within folds located on either side of the female genital slit [19]. The nipple protrudes from the fold whenever the young are being suckled, and this always occurs underwater. Cetacean young lack lips and the mouth cannot grip the nipple, which occurs in most terrestrial mammals, so it appears that they cannot suck effectively [19]. As a result, muscular contractions occur along the mammary gland, and milk squirts into the mouth of the young [19]. It has been frequently observed that milk continues to squirt out of the nipple after the calves have disengaged [19], suggesting that milk release is independent of the direct interaction between the young and the Cetacean nipple.

Physiology and Cell Biology of the Eutherian Nipple The Nipple and the Physiology of Lactation The neural and endocrine circuitry that delivers milk and maintains its production provides one of the best-understood examples of how senses control physiology. Productive lactation requires the extensive ductal network and secretory alveoli that develop in the mammary gland under the influence of high levels of estrogen, progesterone and prolactin present during pregnancy. However, the production of large volumes of milk requires the fall of progesterone levels that occurs with the removal of the placenta after parturition [20]. This is the stage where suckling by the offspring is required to continue the endocrine and local mammary gland conditions required for sustained production of milk. Productive suckling of the offspring is detected by touch receptors within the teat or nipple. In the human these touch receptors are afferents that are part of the 3, 4 and 5th intercostal nerves that innervate the breast [21, 22]. Impulses from these touch receptors are transduced to the spinal cord and ultimately the information is relayed to projections in the median eminence of the hypothalamus [23]. This input results in four distinct impacts in pituitary function: 1) The most understood is the decrease of dopamine release from the arcuate nucleus neurons and its reduced diffusion through the portal vessels, lifting its inhibitory


effects on prolactin release from the lactotrophs; 2) release of thyroid hormone stimulating hormone from the hypothalamus serves as a releasing factor for prolactin; 3) stimulation of paraventricular and supraoptic nuclei triggers oxytocin release from the posterior pituitary; 4) inhibition of arcuate nucleus neurons reduces gonadotropin-releasing-hormone and subsequently prevents the release of follicle-stimulating-hormone from the anterior pituitary [22, 23]. Prolactin signaling in the breast epithelial cells stimulates milk synthesis for the next round of nursing [20, 23]. Increased levels of circulating oxytocin leads to the processes of milk secretion (milk letdown) mediated by contractions of the myoepthelial cells in the alveoli, as well as the ducts that line the mammary gland [20, 23]. Suckling is not the only process that leads to oxytocin release and milk let down. Milk secretion can be stimulated by diverse stimuli including visual and auditory events [20, 23, 24]. Ultimately, the milk collects in large ducts or cisterni at the base of the nipple/teat. It is removed from these collection points by a combination of forces including contractions of myoepithelial cells, suckling and external pressure [20, 22]. Thus, the nipple serves to initiate the hormonal cascades that permit nursing and ensures milk production for future feedings, as well as regulating the reproductive status of the mother by preventing ovarian cycling in some species. Cell Biology of the Nipple Although differing greatly in size, the nipples/teats of the three above-mentioned terrestrial eutherian mammalian groups share one thing in common in that they are covered by a hairless epidermis. In the altricial type of nipple/teat, the nipples are quite small and tightly surrounded by hair that often obscures the appendage in the virgin animal. The nipple/teat lengthens dramatically during pregnancy and lactation. The teat of precocial mammals projects from an udder that is often a region with a sparse covering of hair. This teat also lengthens during pregnancy and lactation. Arboreal species have two types of nipples/teats. Many species of monkeys have nipples that in gross appearance are similar to the altricial type with the nipple that is tightly surrounded by abundant hair shafts. In contrast, some members of the Hominidae family including bonobos, orangutans and humans, have nipples surrounded by a hairless areola. The comparative histology of the nipple/teat has not been systematically studied in the modern era. To provide a general comparison of the cellular make up of nipples from the altricial, precocial or arboreal types, the mouse, cow and human are compared.

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two-layered stratified cuboidal epithelium, but in some regions may take on the appearance of transitional epithelia [25–28]. In the mouse these cells expresses high levels of keratin (K) 8 and 18 similar to luminal cells of the distal mammary tree, but both layers also express K14 and K5 [25]. In the mouse and the cow, there is a single lactiferous duct that intersects the epidermis at the apex of the teat, whereas the human has 15–20 ducts [25–28]. In the cow, the lactiferous duct expands distally to a great diameter composing the teat cistern [28]. The most superficial portion of the bovine lactiferous duct is a narrower teat canal and much of this is lined with epidermis [28, 29]. Fibroblasts and Extracellular Matrix In mouse, cow and human,, the nipple has an extensive papillary dermis with characteristic fine collagen bundles due in part to the deep infolding of the epidermis. Larger tightly packed collagen bundles can be found around the lactiferous ducts and between segments of smooth muscle. In cow and human, nipple/teat fibroblasts are inactive with heterochromatic nuclei and virtually no discernable cytoplasmic staining [28, 29]. In the areola, fibroblasts and matrix are similar to that of the nipple. In the mouse nipple, the fibroblasts have rounded euchromatic nuclei and more apparent stained cytoplasm than those of the surrounding intrafollicular dermis. It is presumed that the fibroblasts in the region are derived from the dense mammary mesenchyme [30]. Smooth muscle In mouse, cow and human, there are abundant smooth muscle cells within the connective tissue of the nipple/teat, and are located close to both lactiferous duct/s and the epidermis. The smooth muscle is orientated in both longitudinal and circular orientations relative to the lactiferous duct and this has been reported to be consistent with that of a sphincter [26–28]. In all three species, elastic fiber staining often labels the connective tissue immediately around the smooth muscle grouping suggesting that this is the cellular source of this in the nipple stroma [27, 31]. The human areola has abundant groupings of smooth muscle in the regions corresponding to the reticular dermis. In the human, contraction of smooth muscle is proposed to wrinkle the areola enhancing nipple projection[27]. The function of nipple smooth muscle in the other two species has not been determined. The developmental origin of the smooth muscle in the nipple remains undetermined [31].

Lactiferous Duct Nerve Endings In mouse, cow and human, the lactiferous duct, provides the core around which the rest of the tissue of the nipple/teat is built. In all three species, these ducts are lined mainly by a

The presence and function of nerve endings in the nipple have primarily been studied in the human. As compared to

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the skin of the breast, the nipple is about 2-fold more sensitive to all sensory modalities (light touch, temperature and vibration) except pain [21] [32]. The nipple does not have Meisner or Pancean corpuscles [26, 27], so it appears that free nerve endings mediate touch sensation in the nipple. The nerve endings are found close to the epidermis at the tip of the nipple, but not at its sides or in the areola. The lactiferous ducts and smooth muscle of the nipple and areola are richly supplied with nerve endings, and these are speculated to transduce the stimuli of suckling to the spinal cord [27].


unpigmented epidermis similar to the broken color patterns of the coat of most dairy breeds. This suggests that melanocytes are present in the teat epidermis of cows as well, but this had not been investigated at the cellular level. In pigmented strains of mice, the nipples are frequently transiently pigmented during hair shaft emergence [31]. In the adult mouse, a few melanocytes containing pigment granules can be observed in the connective tissue between the lactiferous duct and the epidermis [31]. Together these observations suggest that the growth factor environment of the nipple may promote the survival of melanocytes within tissue types where they are not typically found.

Sebaceous-like Glands Epidermis Large sebaceous glands are found at the tip of the human nipple but not the sides. These are somewhat unique in that hair follicles are not associated with these glands. As a result, these glands empty directly on the surface of the epidermis or into the distal ends of the lactiferous ducts [27]. It is speculated that the secretory products of these sebaceous glands help to prevent sore or chapped nipples [27]. Mice do not have sebaceous glands within the nipple [25, 33–35]. There have been reports that small clusters of pale staining cells were sebocytes, present within the epidermis of the bovine teat canal [28]. However, this has not been substantiated in more recent reports [29]. The human areola contains a few (~9 per side) large sebaceous glands called the tubercles of Montgomery [36]. There has been some controversy as to the precise nature of these glands with some groups identifying them as accessory mammary glands [27]. Also the tubercles of Montgomery appear to secrete pheromone substances that impact nursing efficacy in humans [36, 37]. In other species (for example, rabbits) pheromones appear to be present in the milk [5, 38, 39]. From the stand-point of understanding the signaling pathways that drive nipple morphogenesis, it would be interesting to determine the extent which other species have sebaceous glands associated with the nipple. Melanocytes The human nipple and areola can be found in a range of colors ranging from pink through yellows and orange to dark browns, indicating that its epidermis contains melanocytes. However melanocytes are not restricted to the epidermis, but are also very abundant in the basal layer of sebaceous glands and lactiferous ducts [26, 27]. The distinct coloration of the human nipple relative to the rest of the skin has been asserted to be an epigamic mark (attractive to the opposite sex) by the great dermatological-anatomist William Montagna [26, 27]. Of note is that nipples of other primates are not distinctively colored. Teats can be pigmented in cows and this is often manifest as patches of dark and

The epidermis of the nipple/teat is histologically distinct from that of the surrounding breast or abdominal skin of all three species. The key distinguishing feature is the epidermal ridges that deeply penetrate the papillary dermis [25–28, 34, 36] (Fig. 3). This is speculated to be a morphological adaption to the attrition to which the nipple/teat is subjected to during suckling [27]. The mouse nipple epidermis is composed of ~6 layers which makes it much thicker than surrounding 3-cell trunk epidermis [34] (Fig. 3a,b). In the human, the areola has similar invaginated folds but these are not as impressive or extensive as the nipple (Fig. 3c-e). However, in the mouse, human and cow there is only a modest change in the keratinized material overlying the nipple relative to that of surrounding skin [27, 34, 40] (Fig. 3) In humans, a unique “clear” epithelial cell that expresses the luminal marker K7 is frequently located in the basal or suprabasal layers at the apex of the nipple in the proximity of lactiferous ducts [41–43]. These were first described by Cyril Toker, the renowned surgeon, pathologist and author, as a larger non-squamous cells, with poor staining cytoplasm, and nucleus with scanty stained chromatin [41]. These features were consistent with morphology of luminal cells from small mammary ducts [41]. Superficially, these resemble the malignant cells found in the nipple of Paget’s disease patients, in which ductal carcinoma in situ, or invasive breast cancer has migrated to the nipple causing eczema-like lesions [44]. However extensive studies of nipples from patients without apparent breast malignancies, or who have had prophylactic mastectomies suggest that the “Toker” cells as detected by K7 expression are present in the majority of human breasts from both males and females [42]. The source of these cells remains obscure, but their presence in supernumerary nipples [45] leads to speculation that these clear cells may reflect glandular differentiation of the epidermal cells, reflecting plasticity of the ectodermal components associated with the mammary gland. It is possible that the pale-staining cells of the epidermis of the teat


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In the human, the nipple and areola have not been considered an example of specialized epidermis. In general,

canal in the cow may be similar to the human Toker cell, and the identification of this cell type in other eutherians may provide insight into the origin of these unique cells. Nipple as a Site of Specialized Epidermis Virtually all mammals, birds, reptiles and amphibians have regions of their skin where the epidermis is specialized as compared to that which covers the trunk. These regions are crucial for interaction with manipulation of elements in the environment. The epidermis of these regions is characterized by the following: 1) reduced numbers of predominant appendage (hair, scales, feathers) and this is often coupled with the presence of unique appendages such as glands or specialized sensory hairs; 2) distinct patterns of epidermal stratification; 3) the expression of unique differentiation markers (often keratins) that are not present in trunk epidermis [46]. The mouse has several regions of specialized epidermis including the vibrissae containing muzzle, ear, tail, footpad and the genital regions [47]. In the human, the lips, palms, soles, and anal genital region are considered specialized regions. So, is the nipple an example of specialized epidermis? First it is glabrous and also associated with a unique gland. Second, in the mouse, the increased numbers of suprabasal cells is distinctly different from the stratification pattern of murine trunk skin. Third, the mouse nipple expresses a number of keratins that are absent in the intrafollicular epidermis of the trunk. These include keratin 2e, keratin 6 and the keratin associated protein epiplakin [34, 35]. Interestingly, the expression of one of these differentiation markers, K2e is down regulated during nursing [35]. Thus, it does appear that the murine nipple is actually specialized epidermis. During lactation, nipple epidermis is subject to a unique combination of insults including sucking pressure, mechanical strain associated with pulling and stretching, as well as prolonged exposure to the high moisture and the digestive enzymes in saliva. To maintain integrity in the face of these diverse insults, it is likely that the nipple epidermal cells would have a very unique cytoskeleton, consistent with the structure being an example of specialized epidermis. Fig. 3 Masson’s Trichrome Stain of Skin and Nipples from Mouse and Human a Murine ventral skin. b Murine nipple. c Human breast skin (note the red stain of the collagen bundles in the reticular dermis as indicated by the black & symbol is an artifact of poor fixation). d Apex of human nipple. e Human areola. f The entire tissue section showing the human nipple and areola. The boxes indicate are where micrographs in D and E are from. Scale bar 650 μm in A-E and 4 mm in F. The epidermis is the red tissue layer at the top of each section and the blue stains the underlying extracellular matrix of the connective tissue. Arrows indicate red stained smooth muscle. The white # symbol indicates lactiferous ducts. The * indicates sebaceous glands associated with hair follicles in A and B, whereas these glands are associated with the lactiferous duct in C

b

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these sites in the human are associated with increased layers of keratinocytes, a more prominent cornified layer, as well as more pronounced rete pegs [46]. Since the nipple and areola only clearly demonstrate one of these features (more pronounced rete pegs), it is plausible that this region has been simply overlooked in terms of being specialized epidermis. Evaluation of keratin expression in the human nipple and the nipples/teats of other species would help determine if the nipple is an example of specialized epidermis in all eutherian mammals. The development and maintenance of the characteristics of specialized epidermis is dependent on inductive signals from the underlying dermis [48]. In the classical mammalian sites of specialized epidermis, the underlying mesenchymes that gave rise to the dermis in these locations have unique developmental histories. The dermal fibroblasts in the trunk are derived from two sources: the mesenchyme of the dorsal surface is derived from the dermomyotome, whereas the ventral arises from the somatopleural mesoderm [49]. These provide the basic mesenchymal cells that participate in hair follicle development and support the intrafollicular epidermis [49]. The mesenchymes that underlie sites of specialized palmar, plantar, lip/muzzle and genital region have positionspecific Hox gene-driven expression signatures [50]. In the case of in the head, the source of the mesenchyme is derived from the neural crest [49]. Whereas the nipple, the stroma that underlies this epidermis has its origins in mammary gland development [21]. Development of the Murine Nipple The development of the nipple has been most extensively studied in the rodents and the following discussion will be limited to these species. The formation of the nipple is but one part of the multicellular events within the middle period of mammary gland development after the mammary bud forms. The formation of the nipple becomes apparent as the primary duct migrates from the mammary bud and penetrates the primary mammary mesenchyme and invades the mammary fat pad [51]. The first morphological evidence of nipple formation is the thickening and invagination of the edge of the nipple epidermis forming a bat-winged structure typically apparent after E-17 [25, 52–54]. From this point, until several days after birth, there are no apparent morphological changes in the nipple epidermis. At 9 days after birth macroscopically, the nipple is a slightly sunken region of epidermis with smooth texture due to the lack of erupting hair shafts [31]. It is not until after puberty that the nipple will begin to grow and eventually extend above the surrounding epidermis, suggesting that this results from the action of estrogen and/or progesterone [31, 55, 66]. During pregnancy the nipple triples in size and this expansion is mediated by the action of the hormones estrogen and relaxin


[55–57]. The precise location of the estrogen receptor within the mouse nipple has not been determined, but analysis of the relaxin receptor (leucine-rich repeat-containing G proteincoupled receptor 7/LGR7) beta-galactosidase-knockin mouse indicates that the connective tissue cells immediately under the nipple epidermis express high levels of the gene encoding the relaxin receptor [58]. The nipple does not lengthen during lactation but there is a continued high rate of epidermal proliferation [55, 56]. Thus, the nipple, like the rest of mammary gland itself, is formed in embryonic life but undergoes much of its development in the adult and appears to be extensively influenced by hormones associated with pregnancy and lactation. What is known about the molecular signaling that facilitates nipple development stems from a unique transgenic mouse. In this mouse, the human K-14 promoter was used to drive the peptide growth factor parthyroid hormone-related protein (PTHrP), which is ancestrally related to parathyroid hormone (PTH) [59, 60]. High levels of PTHrP expression is restricted to developing epidermal appendages such as the hair follicle and the mammary gland, hence the K14 promoter resulted in ectopic expression over the entire developing epidermis [25, 61]. The PTH/PTHrP receptor (PTH1R) is expressed in the developing dermis and the primary mammary mesenchyme [61–63]. Surprisingly the main skin phenotype of the K14-PTHrP mouse was a hairless but markedly thickened wrinkled ventral epidermis [59, 64]. In addition the developing ventral dermis of the mouse exhibited expression of markers of primary mammary mesenchyme including tenascin C, androgen receptor expression as well as evidence of canonical Wnt signaling [25]. Ultimately markers epiplakin, K2e and K6 were found to be expressed in the ventral skin of the K14-PTHrP mouse suggesting that this was in fact one large nipple. These findings integrated well with the phenotypes observed in PTHrP- and PTH1R-knockout mice in which mammary development was stalled at the bud stage and the primary duct failed to sprout, the nipple epidermis did not form and markers of primary mammary mesenchyme were not expressed around the bud [52, 54]. Together these observations suggest that PTHrP stimulation of the PTH1R is required to fully differentiate the mammary mesenchyme, which in turn produces signals that maintain mammary fate of the epithelium, induce sprouting of the primary duct and direct the formation of a patch of nipple epidermis [25, 51]. The signaling pathways active in the primary mammary mesenchyme downstream of PTHrP and PTH1R are beginning to be determined, and have produced insights into key aspects of nipple formation. In several organ systems, activation of the PTH1R on mesenchymal cells increases bone morphogenetic protein (BMP) and wingless/Wnt signaling. A recent report indicates that beta-catenin is required for the differentiation of the mammary mesenchyme [65]. A more complete story has emerged regarding the PTH1R regulation


of BMP receptor 1A (BMPR1A) levels, modulating the sensitivity of mesenchymal cells to BMP ligands [53, 66]. This also appears to occur in the mammary development where PTHrP-induced increased BMPR1A levels lead to greater responsiveness to BMP4 that is preferentially expressed in the mesenchyme that underlies the ventral ectoderm/epidermis of mouse embryos at E12.5 to 13.5 [53]. The combination of PTH1R and BMPR1A signaling differentiate the mesenchyme surrounding the mammary bud into primary mammary mesenchyme [53]. Repression of hair follicle formation is essential to the formation of specialized epidermis [46]. Among the genes co-upregulated by PTHrP and BMP signaling is msh homeobox 2 (Msx2), and crossing the K14-PTHrP transgene onto a Msx2-/- background restored hair follicle formation on the ventral epidermis; however, the nipple of the Msx2-/- mice does appear relatively normal [53, 67]. In contrast, overexpression of the BMP inhibitor, Noggin, with the K14 promoter cassette leads to nipples that have ectopic hair follicles [67]. These conflicting findings suggest that Msx2 may represent one of several signals downstream of BMP that blocks morphogenesis of the hair follicle and associated sebaceous gland in the region of the nipple. Overall, PTHrP appears to mediate nipple formation in part by enhancing sensitivity to ventrally restricted BMP signaling which is one major regulators of placement of hair follicles as well as the hair cycle [68–71], as would be expected in the eutherian mammary gland and nipple which develops independently of pilosebaceous units.

Conclusion The mammary gland defines mammals, but its components and development in monotremes and marsupials suggest an evolutionary relationship to pilosebaceous unit, which is the predominant epidermal appendage of this class. In many eutherian mammals the mammary gland develops independently of pilosebaceous units, and appears to coopt the BMP signaling pathway to recruit mesenchymal cells for organogenesis. The differentiated primary mesenchyme cells are incapable of responding to the subsequent signaling required for formation of pilosebaceous units. So despite differences in the location, numbers sizes and shapes among the eutherian groups, the nipple is first of all a hairless patch of epidermis. The similarity of the connective tissue of the nipple in the altricial, precocial and arboreal species in terms of extracellular matrix morphology, presence of smooth muscle suggest a common set of events occur as the primary mammary mesenchyme is replaced by dermis. Research leading to understanding the cellular and molecular events that lead to the production of this connective tissue capable of producing the unique epidermis of the nipple, will provide insights into basic questions surrounding how specialized

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epidermis developments and is maintained, and the mechanisms by which skin cells are responsive to hormones associated with pregnancy and lactation. In addition, such work should further provide approaches to correcting developmental defects such as inverted nipples, as well as developing improved strategies for the replacement of nipples in patients who have undergone mastectomies.

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