[The morphogenesis of mammalian cutaneous glands in evolutionary perspective].

The morphogenesis of mammalian cutaneous glands is considered based on the analysis of the literature and our own original data with the focus on the issues of gland polymorphism and specific features in postnatal development (from the case study of circumanal hepatoid glands of newborn domestic dogs), including the features reflecting the evolutionary relationships of various types of cutaneous glands. The hepatoid glands are a component of the glandular complex ofthe hair follicle, which also includes sebaceous and sweat glands; have a specific structure; and produce protein secretion by a merocrine pathway. Characteristic of these glands are wide polymorphism, sex- and age-related differences in the degree of development, occurrence in only a few phylogenetically related mammalian taxa (even-toed ungulates and carnivores); and a signaling type of their secretion. The data support the "generative concept," relying on the idea of a separate and independent origination of diverse derivatives of the external integuments.

Requirement for Shh and Fox family genes at different stages in sweat gland development.

Sweat glands play a fundamental role in thermal regulation in man, but the molecular mechanism of their development remains unknown. To initiate analyses, we compared the model of Eda mutant Tabby mice, in which sweat glands were not formed, with wild-type (WT) mice. We inferred developmental stages and critical genes based on observations at seven time points spanning embryonic, postnatal and adult life. In WT footpads, sweat gland germs were detected at E17.5. The coiling of secretory portions started at postnatal day 1 (P1), and sweat gland formation was essentially completed by P5. Consistent with a controlled morphological progression, expression profiling revealed stage-specific gene expression changes. Similar to the development of hair follicles-the other major skin appendage controlled by EDA-sweat gland induction and initial progression were accompanied by Eda-dependent up-regulation of the Shh pathway. During the further development of sweat gland secretory portions, Foxa1 and Foxi1, not at all expressed in hair follicles, were progressively up-regulated in WT but not in Tabby footpads. Upon completion of WT development, Shh declined to Tabby levels, but Fox family genes remained at elevated levels in mature sweat glands. The results provide a framework for the further analysis of phased down-stream regulation of gene action, possibly by a signaling cascade, in response to Eda.

A de Novo EDA-Variant in a Litter of Shorthaired Standard Dachshunds with X-Linked Hypohidrotic Ectodermal Dysplasia.

In this study, we present a detailed phenotype description and genetic elucidation of the first case of X-linked hypohidrotic ectodermal dysplasia in the shorthaired standard Dachshund. This condition is characterized by partial congenital hypotrichosis, missing and malformed teeth and a lack of eccrine sweat glands. Clinical signs including dental radiographs and histopathological findings were consistent with ectodermal dysplasia. Pedigree analysis supported an X-recessive mode of inheritance. Whole-genome sequencing of one affected puppy and his dam identified a 1-basepair deletion within the ectodysplasin-A (EDA) gene (CM000039.3:g.54509504delT, c.458delT). Sanger sequencing of further family members confirmed the EDA:c.458delT-variant. Validation in all available family members, 37 unrelated shorthaired standard Dachshunds, 128 further Dachshunds from all other coat and size varieties and samples from 34 dog breeds revealed the EDA:c.458delT-variant to be private for this family. Two heterozygous females showed very mild congenital hypotrichosis but normal dentition. Since the dam is demonstrably the only heterozygous animal in the ancestry of the affected animals, we assume that the EDA:c.458delT-variant arose in the germline of the granddam or in an early embryonic stage of the dam. In conclusion, we detected a very recent de-novo EDA mutation causing X-linked hypohidrotic ectodermal dysplasia in the shorthaired standard Dachshund.

Characterization of a target-derived neuronal cholinergic differentiation factor.

The sympathetic innervation of rat sweat glands undergoes a target-induced switch from a noradrenergic to a cholinergic and peptidergic phenotype during development. Treatment of cultured sympathetic neurons with sweat gland extracts mimics many of the changes seen in vivo. Extracts induce choline acetyltransferase activity and vasoactive intestinal peptide expression in the neurons in a dose-dependent fashion while reducing catecholaminergic properties and neuropeptide Y. The cholinergic differentiation activity appears in developing glands of postnatal day 5 rats and is maintained in adult glands. It is a heat-labile, trypsin-sensitive, acidic protein that does not bind to heparin-agarose. Immunoprecipitation experiments with an antiserum directed against an N-terminal peptide of a cholinergic differentiation factor (CDF/LIF) from heart cells suggest that the sweat gland differentiation factor is not CDF/LIF. The sweat gland activity is a likely candidate for mediating the target-directed change in sympathetic neurotransmitter function observed in vivo.

Gene-activated matrix/bone marrow-derived mesenchymal stem cells constructs regenerate sweat glands-like structure in vivo.

It is a significant challenge to regenerate full-thickness skin defects with sweat glands. Various skin substitutes have been developed to resolve this issue with minimal success. In this study, to yield a novel construct for in situ regeneration of sweat glands, the collagen-chitosan porous scaffold was combined with Lipofectamine 2000/pDNA-EGF complexes to obtain the gene-activated scaffold (GAS), which was then seeded with bone marrow-derived mesenchymal stem cells (BM-MSCs). The porous scaffold functionalized as a reservoir for the incorporated gene complexes which were released in a sustained manner. The seeded BM-MSCs were transfected in situ by the released complexes and specially differentiated into sweat gland cells in vitro under the induction of the expressed epidermal growth factor (EGF). Application in vivo of the GAS/BM-MSCs constructs on the full-thickness skin defects of SD rats confirmed that GAS/BM-MSCs could accelerate the wound healing process and induce the in situ regeneration of the full-thickness skin with sweat gland-like structures. Analyzed by immunohistochemical staining, RT-qPCR and Western-blotting, the levels of the major sweat gland markers such as carcino-embryonic antigen (CEA), cytokeratin 8 (CK8) and cytokeratin 14 (CK14) were all up-regulated, indicating that GAS/BM-MSCs can facilitate the regeneration of sweat glands-like structure in vivo.

Target regulation of neurotransmitter phenotype.

Studies of sympathetic neurons developing in cell culture revealed a surprising degree of transmitter plasticity and established the role of environmental factors in determining transmitter choice. The sympathetic neurons that innervate sweat glands undergo a change in neurotransmitter phenotype from noradrenergic to cholinergic during normal development similar to that observed in culture. Cross-innervation experiments indicate that the target sweat glands induce the switch and thereby specify the phenotype of the neurons that innervate them. Thus, both the transmitter plasticity and the role of environmental influences initially elucidated in culture are part of the developmental repertoire of sympathetic neurons in vivo. Further, these findings extend considerably our understanding of the role that targets may play during development; targets may not only determine how many neurons survive but also what their properties will be.

Catecholamines are required for the acquisition of secretory responsiveness by sweat glands.

The sympathetic innervation of sweat glands undergoes a developmental change in transmitter phenotype from catecholaminergic to cholinergic. Acetylcholine elicits sweating and is necessary for development and maintenance of secretory responsiveness, the ability of glands to produce sweat after nerve stimulation or agonist administration. To determine whether catecholamines play a role in the development or function of this system, we examined the onset of secretory responsiveness in two transgenic mouse lines, one albino and the other pigmented, that lack tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. Although both lines lack TH, their catecholamine levels differ because tyrosinase in pigmented mice serves as an alternative source for catecholamine synthesis (Rios et al., 1999). At postnatal day 21 (P21), 28 glands on average are active in interdigital hind footpads of albino TH wild-type mice. In contrast, fewer than one gland is active in albino TH null mice, which lack catecholamines in gland innervation. Treatment of albino TH null mice with DOPA, a catecholamine precursor, from P11 to P21 increases the number of active glands to 14. Pigmented TH null mice, which have faint catecholamine fluorescence in the developing gland innervation, possess 12 active glands at P21, indicating that catecholamines made via tyrosinase, albeit reduced from wild-type levels, support development of responsiveness. Gland formation and the appearance of cholinergic markers occur normally in albino TH null mice, suggesting that catecholamines act directly on gland cells to trigger their final differentiation and to induce responsiveness. Thus, catecholamines, like acetylcholine, are essential for the development of secretory responsiveness.

Developmental expression of the high affinity choline transporter in cholinergic sympathetic neurons.

Choline uptake by the high affinity choline transporter (CHT) is the rate-limiting step in acetylcholine synthesis. Induction of CHT is therefore a critical step in cholinergic differentiation, and we examined the developmental expression of CHT in cholinergic sympathetic neurons that innervate rodent sweat glands. During postnatal development the earliest sympathetic axons in the rear footpads are noradrenergic, containing intense tyrosine hydroxylase immunoreactivity and lacking CHT-immunoreactivity (CHT-IR). By postnatal day 7 (P7) in mouse, and P10 in rat, weak CHT-IR appeared in axons associated with the sweat gland anlagen. CHT staining intensity increased during the following weeks in conjunction with plexus arborization and gland maturation. The pattern of CHT-immunoreactivity (CHT-IR) in the sweat gland innervation was similar to staining for the vesicular acetylcholine transporter and vasoactive intestinal peptide. Immunoblots of tissue from sympathectomized rats confirmed that most of the CHT in footpad was contained in sympathetic neurons. Although CHT expression has been reported in noradrenergic sympathetic neurons of the superior cervical ganglion, these data indicate that in the sympathetic neurons projecting to sweat glands CHT is present at detectable levels only after association with the glands.

Changes of expression of intermediate filament proteins during ontogenesis of eccrine sweat glands.

The intermediate filament expression in fetal and adult human eccrine sweat glands was studied by immunoperoxidase microscopy performed on cryostat sections using monoclonal antibodies against various cytokeratins (CK), vimentin, and actin. In palmar skin of 14-week-old fetuses, the early dermal cords showed a primitive CK pattern similar to that of epidermal basal cells. From week 15 on (distal finger skin), inner cells of the proximal (ductal) portion of the glandular anlagen expressed CK 1/10/11 and 19 (markers of adult eccrine ductal luminal cells). In addition, CK 4 was expressed in ductal luminal cells mainly in the fetal period. In the distal portion of the sweat gland anlagen the increased or new expression of the simple-epithelium-type CK 7, 8, 18, and 19 was detected at week 15, indicating the onset of the secretory differentiation pathway. Two subsegments of the prospective secretory portion could be distinguished (elongated part and end bud). Interestingly, in fetuses, most secretory portion cells co-expressed vimentin in addition to CK. From week 22 on, peripheral cells of the secretory portion were stained for CK 17 and smooth-muscle-type actin, suggesting myoepithelial differentiation. In newborn and adult eccrine glands, secretory cells expressed mainly CK 7, 8, 18, and 19, whereas myoepithelial cells were conspicuous by their co-expression of certain CK (including CK 5 and 17), vimentin, and smooth-muscle-type actin and sometimes even glial filament protein (GFP), similar to myoepithelial cells of other glands. These results throw further light onto the complex processes of fetal development of eccrine sweat glands and their cellular diversification. The possible biologic significance of the differential CK expression in the various glandular cell types is discussed.

Matrilysin (PUMP) correlates with dermal invasion during appendageal development and cutaneous neoplasia.

Matrix-degrading metalloproteinases play a major role in tissue remodeling. Recent studies have shown that enzymes of this class are constitutively expressed primarily by stromal cells and not by epithelium. Here we present immunohistochemical evidence that matrilysin is localized within epidermal cells in developing skin and in tumor cells of cutaneous malignancies. The expression of matrilysin protein in developing fetal skin (6-15 weeks) is localized primarily to the germinative basal cell layer of fetal epidermis and early appendageal buds. The buds continue to express matrilysin during mesenchymal invasion. As development progresses (15-19 weeks) matrilysin is concentrated only in cells at the distal portion of the invading follicular and sweat gland appendageal cords. In adult skin, matrilysin was localized specifically to the outer root sheath of the hair follicles and the secretory cells of the eccrine glands but was absent in the epidermis. Nodulocystic, keratotic, adenoid basal cell carcinomas (BCCs) did not express matrilysin. In contrast, in the more aggressive morpheaform (infiltrative) BCCs and recurrent BCCs, matrilysin was localized at the tumor-stromal interface. In squamous cell carcinomas matrilysin was present in tumor cells at the stromal interface surrounding the tumor nests. The demonstration of matrilysin protein in germinal basal cells during fetal skin development and its presence in tumor cells at the stromal junction suggests that this enzyme may contribute to the proteolytic activity associated with cell-extracellular matrix interactions during appendageal development and tumor invasion.

Development of muscarinic receptors and regulation of secretory responsiveness in rodent sweat glands.

Sweat glands are innervated by sympathetic neurons which undergo a change in transmitter phenotype from noradrenergic to cholinergic during development. As soon as the glands begin to differentiate, M3 muscarinic receptor mRNA and binding sites are detectable. Receptor expression appears in the absence of innervation and is maintained after denervation. While receptor expression is not regulated by innervation, secretory responsiveness is. Muscarinic blockade during development or in adult animals results in the loss of responsiveness and its reappearance requires several days. Cholinergic muscarinic activation is most likely to regulate one or more steps in the signalling cascade that are downstream of calcium mobilization. The anterograde regulation of sweat gland responsiveness is one facet of the reciprocal interactions are required to establish a functional synapse in this system.

In vivo effects of epidermal growth factor on epidermal pattern formation and hair follicle initiation in the marsupial bandicoot Isoodon macrourus.

The extrauterine development of marsupial pouch young (northern brown bandicoot Isoodon macrourus) has facilitated the study of the effects of murine epidermal growth factor (mEGF) on pattern formation in skin. Hair follicle initiation and development, which in the mouse would occur from about Days 13-14 of gestation onward, occurs postnatally. In the present study the effect in vivo of mEGF on developing skin corresponding to mouse gestational ages from Day 13 onward was examined. Subcutaneous injections of mEGF (0.5, 1.0 and 2.0 microg g[-1] body weight) or equivalent volumes of saline (0.9% w/w) were administered daily, before and during hair follicle initiation and development. Murine EGF inhibited the formation of hair follicles, hair follicle sweat glands, sebaceous glands and dermal papillae. The pattern of follicle initiation was perturbed. The characteristic trio follicle grouping was absent, and follicle rudiment densities (no. per mm2 skin surface) were significantly lower in animals treated with mEGF, whereas follicle diameters were increased. These data may reflect a role for the epidermal growth factor (EGF) receptor in epidermal pattern formation. The EGF receptor and its potential ligands (such as EGF, transforming growth factor (TGF-alpha) or other yet-to-be-discovered ligands) perhaps act as parts of a pattern-forming system in vertebrate skin.

[The morphology and postnatal development of the skin in purebred and mixed-breed cattle (Bos taurus)]. / Morfologiia i postnatal'noe razvitie kozhnogo pokrova krupnogo rogatogo skota (Bos taurus) chistoporodnykh i pomesnykh zhivotnykh.

Growth and differentiation of skin layers, hair follicles, and skin glands were studied in cattle of two genotypes during the period from early postnatal development until the age of 30 months. Development of skin and its derivatives was shown to proceed irregularly and include alternating periods characterized by an increase, temporary arrest, or even decrease of corresponding morphometric indices. On the basis of these data a conclusion is drawn that periodic changes in thickness of skin and its individual layers during ontogenesis are a natural phenomenon. It is suggested that a complex of morphometric indices can reflect specific characteristics of individual breeds and their productivity.

Polipose palpebral por hidrocistoma écrino / Eyelid polyposis caused by eccrine hidrocystoma

Os hidrocistomas écrinos são lesões raras, císticas e benignas, que resultam em deformidades nas regiões palpebrais bilateralmente. Vários tratamentos são citados na literatura, porém nenhum deles é considerado padrão de referência. Deve-se somar as impressões obtidas à avaliação clínica e aos recursos disponíveis. Paciente de 73 anos, feminino, apresentando tumorações em regiões palpebrais bilaterais, de crescimento lento, com obstrução parcial do campo de visão e ectrópio. Foi submetida a dois procedimentos cirúrgicos para ressecção, em 2007 e em 2008. Apresentou melhora significativa do contorno palpebral bilateral, com boa simetria entre as regiões. O exame histopatológico concluiu: hidrocistoma écrino. O tratamento cirúrgico para a polipose palpebral bilateral relacionada ao hidrocistoma écrino mostrou-se uma modalidade que pode apresentar bons resultados estéticos e funcionais, sendo reprodutível, e sem causar maiores morbidades pós-operatórias ao paciente.

Eccrine hidrocystomas are rare lesions, cysts, or benign tumors, which lead to bilateral deformities in the eyelid areas. Several treatments are described in the literature. However, none of them has been established as the gold standard. Hence, it becomes necessary to consider the contribution of different opinions and available resources to clinical evaluation. A 73-year-old female patient presented with slow-growing tumors on both eyelids, which consequently led to partial visual field obstruction and ectropion. She underwent 2 surgical resections, one in 2007 and the other in 2008. As a result, she showed significant improvement of the bilateral eyelid contours and satisfactory symmetry between the areas. Histopathological diagnosis indicated eccrine hidrocystoma. The surgical treatment of bilateral eyelid polyposis associated with eccrine hidrocystoma proved to be a reproducible procedure that may ensure satisfactory aesthetic and functional results, without causing major postoperative morbidities to the patient.

Postnatal expression and denervation induced up-regulation of aquaporin-5 protein in rat sweat gland.

The evolution of aquaporin-5 (AQP5) expression during postnatal development has not been defined in the sweat gland. Previous studies have suggested that AQP isoforms in several peripheral targets are regulated by a neural mechanism. We have examined, in rat sweat glands, the expression of AQP5 during postnatal development and the effects of denervation on AQP5 expression. Both AQP5 mRNA and protein begin to be expressed at postnatal day 10, before sweat-secretory responsiveness first appears; this expression coincides with the occurrence of vasoactive intestinal peptide (VIP) immunoreactivity. Early noradrenergic and later cholinergic interaction between sweat glands and their innervation are disrupted by neonatal chemical sympathectomy or postnatal severance of the sciatic nerve. Examination of such denervated developing rats has shown that secretory responsiveness fails to arise later in the adults, and AQP5 immunostaining increases in the denervated glands, whereas gland morphogenesis and the occurrence of AQP5 expression proceed normally. Immunobloting has revealed an increase of AQP5 abundance after the denervated mature glands lose their secretory ability. These findings suggest that AQP5 protein is necessary for sweat secretion, and that the expression of AQP5 in rat sweat glands is independent of sympathetic innervation. Our data also indicate that factor(s) regulating the normal morphological development of sweat gland might be responsible for controlling AQP5 expression.

[Relationship of ectodysplasin gene signaling with development and regeneration of sweat glands].

OBJECTIVE: To investigate the expression of ectodysplasin (EDA) gene signaling and its relationship with the development and regeneration of sweat glands. METHODS: The articles concerned in the latest years were extensively reviewed. RESULTS: EDA gene is an important signaling pathway associated with the developmental procedure of sweat glands in early fetal stage. Abnormality or depletion of function in sweat glands partially owed to the defect of EDA gene. CONCLUSION: EDA signaling has its biological significance in inducing development and morphogenesis of sweat glands and in maintaining physiological function of skin. It could be a new approach to repair or regenerate the sweat glands for clinical therapy by regulating the expression of EDA gene.