Histology of ferret skin: preweaning to adulthood.

Ferrets are important companion animals that incur a multitude of cutaneous diseases requiring diagnostic dermatohistopathology. This study provides a description of the histology of normal ferret skin, emphasizing changes in the interval from preweaning to adulthood, an essential basis for identification of pathological situations. Skin samples obtained post-mortem from 29 topographical sites on 41 ferrets, revealed in the haired, general body surface skin an epidermis consisting of strata basale, spinosum, granulosum, and corneum and a dermis consisting of strata papillare and reticulare. Adult skin contained compound hair follicles composed of one primary hair and a collection of secondary hairs with a primary to secondary ratio of 1/5-1/15. All hairs emerged through the same follicle outlet of the skin surface. There was associated with each primary follicle, an arrector pili muscle, a multilobular sebaceous gland, and a coiled tubular sweat gland, but secondary hairs lacked these features. Compound follicles, grouped mainly as triads across the body surface, were already fully formed in the youngest group studied (3 to 6 weeks). The secondary hairs all developed from one specific region of the primary follicles and smaller ones were formed with increasing age. The differences found in specialized body regions are described. Demodex sp. mites were found in follicles and sebaceous glands in nine of 25 individuals in the perianal, vulvar, preputial, facial, and caudal abdominal skin.

BMP-2 stimulates tyrosinase gene expression and melanogenesis in differentiated melanocytes.

Cells of the vertebrate neural crest (crest cells) differentiate in vitro to melanocytes and sympathoadrenal (SA) progenitor cells. We have shown previously, using primary J. quail neural crest cultures, the combinatorial effect of bone morphogenetic protein-2 (BMP-2) and cAMP signaling on SA cell development. Herein, we report that in primary J. quail neural crest cultures, BMP-2 and cAMP signaling similarly exert a combinatorial effect on melanocyte development. We demonstrate that BMP-2 treatment of neural crest cells increases melanogenesis by promoting the synthesis of melanin. This increased melanin synthesis by BMP-2 is effected by the selective increase in the transcription of the tyrosinase gene, encoding the rate-limiting enzyme of the melanin biosynthetic pathway. By contrast, BMP-2 exerts no effect on the expression of the tyrosine-related proteins 1 and 2 (Tyrpl and Dct), also involved in the melanin biosynthetic process, or on the expression of microphalmia (Mitf) gene, supporting the fact that BMP-2 does not affect melanocyte differentiation. Employing transient transfection analysis of tyrosinase-reporter constructs in B16 melanoma cells, we demonstrate that the BMP-2 response-element is localized between 900 and 1,100 bp upstream from the tyrosinase transcriptional start site. These studies support a role for BMP-2 in melanogenesis by selectively targeting the expression of the tyrosinase gene involved in melanin biosynthesis.

Differential expression of sympathoadrenal lineage-determining genes and phenotypic markers in cultured primary neural crest cells.

Bone morphogenetic protein-2 (BMP-2) promotes the development of primary neural crest cells grown in tissue culture to the sympathoadrenal (SA) lineage. Independent studies have characterized the expression patterns of SA-lineage genes in developing chicken embryo; however, studies using cultured primary neural crest cells have characterized only the expression patterns of the catecholaminergic markers, tyrosine hydroxylase (TH) and catecholamines (CAs). To further explore the molecular mechanisms that control SA-cell development using the in vitro model system, it is crucial to define the expression patterns of both the catecholaminergic markers and the genes regulating SA-lineage determination. Accordingly, we defined, in the absence and presence of BMP-2, the temporal expression patterns of TH and CA, the SA lineage-determining genes ASH-1, Phox2a, and Phox2b, the GATA-2 gene, and the pan-neuronal SCG10 gene. Comparison of these data with the reported temporal and spatial patterns of expression in vivo demonstrate that the inductive steps of SA-lineage determination, including the specification of neurotransmitter identity and neuronal fate, are recapitulated in the neural-crest culture system.

Cyclic AMP signaling functions as a bimodal switch in sympathoadrenal cell development in cultured primary neural crest cells.

Cells of the vertebrate neural crest (crest cells) are an invaluable model system to address cell fate specification. Crest cells are amenable to tissue culture, and they differentiate to a variety of neuronal and nonneuronal cell types. Earlier studies have determined that bone morphogenetic proteins (BMP-2, -4, and -7) and agents that elevate intracellular cyclic AMP (cAMP) stimulate the development of the sympathoadrenal (SA, adrenergic) lineage in neural crest cultures. To investigate whether interactive mechanisms between signaling pathways influence crest cell differentiation, we characterized the combinatorial effects of BMP-2 and cAMP-elevating agents on the development of quail trunk neural crest cells in primary culture. We report that the cAMP signaling pathway modulates both positive and negative signals influencing the development of SA cells. Specifically, we show that moderate activation of cAMP signaling promotes, in synergy with BMP-2, SA cell development and the expression of the SA lineage-determining gene Phox2a. By contrast, robust activation of cAMP signaling opposes, even in the presence of BMP-2, SA cell development and the expression of the SA lineage-determining ASH-1 and Phox2 genes. We conclude that cAMP signaling acts as a bimodal regulator of SA cell development in neural crest cultures.

Differential immediate early gene expression in conditional hepatitis B virus pX-transforming versus nontransforming hepatocyte cell lines.

We report construction and characterization of tetracycline-controlled hepatitis B virus pX-expressing hepatocyte (AML12) cell lines. These cell lines were constructed in AML12 clonal isolates (clones 3 and 4), which express constitutively the tetracycline-controlled transactivator. Since pX is implicated in HCC, this immortalized hepatocyte model system was used to investigate the mechanism of pX in transformation. Clonal isolates of 3pX and 4pX lineages display conditional synthesis of pX mRNA and protein and a 2-fold increase in growth saturation density following tetracycline removal, implicating pX in monolayer overgrowth. Interestingly, only 3pX clones display pX-dependent anchorage independence. Clone 3 lineages express hepatocyte nuclear factor-1alpha and hepatocyte-specific marker genes; clone 4 lineages express hepatocyte nuclear factor-1beta and reduced levels of hepatocyte-specific marker genes, suggesting the importance of the differentiated hepatocyte in pX-mediated oncogenic transformation. Importantly, 3pX and 4pX lineages display differential expression of immediate early genes c-fos and ATF3. The pX-transforming 3pX lineage displays early, pX-dependent induction of ATF3 and prolonged induction of c-fos. The nontransforming 4pX cells display an absence of pX-dependent ATF3 induction and transient induction of c-fos. Our results support the direct link of pX expression to oncogenic transformation in 3pX lineage clones and underscore the advantage of this conditional cellular model system for studying mechanisms of pX-mediated oncogenesis.

Effects of cholecystokinin on liquid diet intake of early weaned piglets.

The effect of cholecystokinin octapeptide (CCK-8) on the consumption of a liquid diet was investigated in 10 piglets, weaned on Day 1, housed individually in cages, and offered a commercial diet for suckling from feeding bottles nine times a day at 2-h intervals with an 8-h break during the night. CCK-8 was administered to piglets (n = 5) on Days 2, 4, 6, 8, 10, 12, 20, and 23 in single intraperitoneal doses of 4 microg x kg(-1) in saline, 5-7 min before the first morning feeding at 0600 h. The relative diet intake in CCK-treated animals was reduced significantly (p < 0.05) on Days 2, 4, 6, 8, 10, 12, and 23 compared to saline-treated controls, and on Days 2, 4, 6, 8, 10, 12, and 23 when the consumption at 0600 and 0800 h in CCK-treated piglets was compared. Our data support the notion that the short-term mechanisms involved in food intake control of piglets are relatively complex and operative at the time of birth, and that CCK plays a role in their food intake regulatory cascade.

The avian embryo responding to microgravity of space flight.

Central questions and paradigms of developmental biology have in recent decades evolved to stress gene regulation. (See Moore, 1987 for an excellent review). However, during fertilization and the pre-embryonic cleavage stages, cells of identical genome are responding to multiple microenvironmental factors, e.g., pH, spatial position, temperature, osmotic and barometric pressure, light, electrical field charge, and the extracellular concentration of substrates, cellular byproducts, gases, and electrolytes. These cells commonly retain their full potential until chance of spatial positioning alters their access to the internal or external environment. (The commercial exploitation of these techniques at the 8-cell stage is now well-developed in beef and dairy breeding.) Importantly, these factors are exerting their influence prior to the genome repression/activation, all of which leads to germ layer definition and basic tissue differentiation. Of all the many potential and real microenvironmental influences, only gravity would appear to have remained relatively constant and ubiquitous for developing organisms. Histo-and organogenesis as well as differential growth of the embryo and fetus may have evolved with a constant environmental factor of gravity (G=6.7 x 10(-8) c.g.s. units). Embryos of 2-d and 9-d stage of incubation were flown in an incubator on the Space Shuttle during a 5-d mission. Significant differences in embryo response to this microgravity environment were observed. This paper offers an analysis and suggests mechanisms which may contribute to these results.

Dermal extracellular lipid in birds.

A light and electron microscopic study of the skin of domestic chickens, seagulls, and antarctic penguins revealed abundant extracellular dermal lipid and intracellular epidermal lipid. Dermal lipid appeared ultrastructurally as extracellular droplets varying from less than 1 micron to more than 25 microns in diameter. The droplets were often irregularly contoured, sometimes round, and of relatively low electron density. Processes of fibrocytes were often seen in contact with extracellular lipid droplets. Sometimes a portion of such a droplet was missing, and this missing part appeared to have been "digested away" by the cell process. In places where cells or cell processes are in contact with fact droplets, there are sometimes extracellular membranous whorls or fragments which have been associated with the presence of fatty acids. Occasionally (in the comb) free fat particles were seen in intimate contact with extravasated erythrocytes. Fat droplets were seen in the lumen of small dermal blood and lymph vessels. We suggest that the dermal extracellular lipid originates in the adipocyte layer and following hydrolysis the free fatty acids diffuse into the epidermis. Here they become the raw material for forming the abundant neutral lipid contained in many of the epidermal cells of both birds and dolphins. The heretofore unreported presence and apparently normal utilization of abundant extracellular lipid in birds, as well as the presence of relatively large droplets of neutral lipid in dermal vessels, pose questions which require a thorough reappraisal of present concepts of the ways in which fat is distributed and utilized in the body.

Teratogenicity of 3,3-dimethyl-1-phenyltriazene in the rat: histopathologic and ultrastructural alterations.

3,3-Dimethyl-1-phenyltriazene (DMPT) is a methylating agent which is teratogenic, carcinogenic, and mutagenic. A single intraperitoneal injection of 30 mg DMPT/kg given to pregnant rats on day 12 of gestation produces malformations with minimal maternal toxicity. Malformations include skeletal deformities such as micrognathism, cleft palate, and digital malformations, as well as central nervous system hypoplasia. The purpose of the present study was to characterize the light and electron microscopic alterations produced by DMPT. Electron microscopy (EM) revealed that at 4 hr postinjection of DMPT, rare cells of the neural tube contained few membrane-bound aggregations of organelles and condensed chromatin; this change was consistent with apoptosis, a type of cell death characterized by morphologic and biochemical alterations distinct from necrosis. At 8 hr postinjection, apoptosis was more prominent in the neural tube and also observed in the mandibular process. At 16 hr postinjection, numerous apoptotic cells were interspersed with unaffected cells that contained phagocytized apoptotic bodies. Light microscopic examination of DMPT-exposed conceptuses showed apoptosis in the neural tube at 24 hr postinjection. Forty-eight hours postinjection, apoptosis, in decreasing order of severity, was observed in the neural tube, craniofacial processes, limb buds, and somites and liver. Apoptosis was absent in all tissues by 72 hr postinjection. Nervous tissues failed to achieve proper histologic organization, but all other tissues appeared microscopically normal from 72 hr postinjection until the end of gestation. There appeared to be some degree of tissue specificity to the effects of DMPT.

Light and electron microscopy of keratinization in the laminar epidermis of the equine hoof with reference to laminitis.

The laminar epidermis (epidermis parietis) of hooves from 14 clinically normal horses, 6 months to 15 years old, was examined by light and electron microscopy and immunofluorescence to measure the contributions of this region to the formation of the hoof wall. By their progressive keratinization to form primary epidermal laminae, the secondary epidermal laminae ultimately contributed about 20% of the thickness of the hoof wall (as revealed in the white line [zona alba]). The keratinized, primary epidermal laminae were developed to a height of 4 mm during their proximodistal-course, much of this obscured because of their basal portion being embedded in the cap horn epidermis. From evaluation of structural changes accompanying keratinogenesis in the cell and determination of the contribution of the laminar epidermis to the formation of laminar horn, cap horn, connecting horn, terminal horn, and the white line, we concluded that the sterile bed concept of a nongerminative role for the secondary epidermal laminae is no longer tenable.

Lymph heart musculature in birds.

Development and innervation of the lymph heart musculature of chicken, emu, rhea, and duck was studied by electron microscopy at post-hatch ages from 3 days to adulthood. Development of innervation was monitored by acetylcholinesterase staining. Horseradish peroxidase was used to determine the extent of the transverse tubule network. Chickens were unusual among these birds in that lymph heart myocytes had already undergone a definitive differentiation and degeneration by 3 days. In ducks and ratite birds, lymph heart myocytes more slowly but progressively differentiate a cytomorphology that does not conform in all characteristics to cardiac or skeletal muscle and even resembles in some aspects, smooth muscle. Myofibrils become the dominant cytoplasmic structure, transverse tubules form "internal couplings" with agranular reticulum cisternae, and "external couplings" are formed between myocytes at myomyal junctions. The myomyal junctions also contain AChE-positive reaction product and some subplasmalemmal vesicles that lack a dense core. The lymph heart myocardium of ducks of 2 weeks demonstrated mitotic figures. In adult ducks the myosatellite cell numbers diminish and a characteristic pattern of myocyte degeneration appears. In juvenile ducks and ratites some myocytes differentiate to conductile cells, much as the conductile myocytes and myofibers of the blood heart. The lymph heart innervation is described, and the role of nerve in differentiation and maintenance of myocyte morphology in the lymph heart is discussed.

Postpartum atony of the small and large intestine in a Holstein cow: a case of pseudo-obstruction.

A four-year-old Holstein, 23 days postpartum, was presented with abdominal distention. Gas filled viscera were detected in the right paralumbar fossa and pelvic canal. Surgery revealed cecal and colonic distention and generalized intestinal atony. There was no other evidence of gross pathology. The condition deteriorated during five post-operative days. Gross postmortem findings were not remarkable; histopathology revealed ganglionitis of the cranial mesenteric ganglion and myenteric plexus. A brief review of relevant literature for a differential diagnosis and an etiology is discussed.

Testicular organogenesis in the fetal calf: interstitial endocrine (Leydig) cell development.

The development of the interstitial endocrine (Leydig) cells of the fetal testis in the calf is described and correlated with a swelling reaction of the gubernaculum and normal, prenatal descent of the testis. An hydroxysteroid dehydrogenase (HSD) procedure is used to determine the onset of functional activity for the interstitial endocrine cells (IEC). The NADH control procedure was strongly positive for the IECs at all ages investigated, indicating that these cells utilize the pyridine nucleotide as a coenzyme for oxireduction conversions. The 3 alpha- and 3 beta-HSD reactions were strongly positive and lightly positive, respectively, demonstrating that these cells contain the HSDs commonly utilized in the early steroidogenesis. TEM revealed structural evidence of this differentiating steroidogenic capability within IECs. During the period of the swelling reaction there is a functional IEC population, but there is no evidence presented by this study for a causal relationship of the gubernacular swelling reaction and subsequent normal descent of the testis into the scrotum.

Descent of the testis in the fetal calf. A summary of the anatomy and process.

The descent of the testis in the fetal calf is reviewed, and the role in that process of the swelling reaction of the gubernaculum testis is discussed. The testes of 30 Dutch Friesian fetuses were examined by dissection and light microscopy of sections prepared from chemically and frozen-fixed specimens. The gubernaculum remains unattached to the scrotal fasciae until descent is completed. Shortening of the intra-abdominal gubernaculum and displacement of the testis begins at fetal week 11; the swelling reaction of the gubernaculum occurs between weeks 14 and 15. The testis is at the deep inguinal ring by week 15, and by week 20 it is in the scrotal position and the gubernaculum has regressed. It is proposed that the swelling of the gubernaculum dilates the vaginal ring and enlarges the inguinal canal. The clinical importance of these anatomical relationships and changes is discussed.