Characterization of mouse Frizzled-3 expression in hair follicle development and identification of the human homolog in keratinocytes.

Frizzled genes encode a family of Wnt ligand receptors, which have a conserved cysteine-rich Wnt binding domain and include both transmembrane and secreted forms. Work by others has shown that experimental perturbation of Wnt signaling results in aberrant hair formation, hair growth, and hair structure. To date, however, there is no information on the contribution of individual Frizzled proteins to hair development. We now report that Frizzled-3 expression in skin is restricted to the epidermis and to the developing hair follicle. Northern analysis on total mouse skin mRNA revealed a single Frizzled-3 transcript of 3.7 kb. Reverse transcription-polymerase chain reaction and in situ hybridization analysis revealed Frizzled-3 expression in epidermal and hair follicle keratinocytes. Frizzled-3 transcripts are first detected in discrete foci in the developing epidermis of 13 d embryos and later in the hair follicle placodes of 15 d embryos, suggesting a role for this Frizzled isoform in follicle development. In 17 d embryos and 1 d old newborn mice Frizzled-3 expression is limited to suprabasal keratinocytes and is not seen in pelage follicles until 3 d postpartum. In 7 d old neonatal skin, Frizzled-3 is expressed throughout the epidermis and in the outer cell layers of hair follicles. We have also identified the mRNA encoding human Frizzled-3 in epidermal keratinocytes and in the HaCaT keratinocyte cell line. Human Frizzled-3 mRNA encodes a 666 amino acid protein with 97.8% identity to the mouse protein. The human Frizzled-3 gene was mapped using a radiation-hybrid cell line panel to the short arm of chromosome 8 between the markers WI-1172 and WI-8496 near the loci for the Hypotrichosis of Marie Unna and Hairless genes.

Release of corticosterone from the rat adrenal cortex in response to administration of (1-24)adrenocorticotrophin.

The intraperitoneal injection of (1-24)ACTH (34-170 nmol/kg) in rats pretreated with betamethasone resulted in a biphasic rise in plasma corticosterone; only a single phase-response was observed after lower doses (3.4-17.0 nmol/kg). There was an initial rapid rise to a level of approximately 0.72 mumol corticosterone/l plasma over the first 10 min after injection, followed by a plateau phase in which there was no further significant rise in steroid levels up to 25 min. Between 25 and 30 min after injection there was a second significant rise in plasma corticosterone concentration. The amplitude of the first phase of steroid increase was essentially the same for all doses of (1-24)ACTH administered whereas the amplitude of the second phase was dose-dependent. The administration of the microtubule inhibitor, colchicine, greatly reduced the amplitude of the second phase of the response to (1-24)ACTH while not affecting the first phase. However, a second stimulus with (1-24)ACTH in colchicine-treated animals was without effect on plasma corticosterone concentrations; both the first and second phases of corticosteroid increase being abolished. It is proposed that the first phase of plasma corticosterone increase resulted from the release of corticosteroid from a storage form close to the cell membrane and not requiring microtubular transport, whereas the second phase was the result of transport from more distant storage sites or de-novo synthesis.

myc protooncogenes of wool and hair growth.

The growth of hard keratin fibers such as wool and hair is dependent on the proliferation of cells in the follicle bulb. If the cells leaving the bulb could be induced to undergo an extra division, then fiber growth should increase. The cellular division within the follicle is complex and probably involves one or more growth factors, which act by altering the expression of transcription factors and other nuclear proteins. We propose that the expression of the myc protooncogenes is a central part of this mechanism. In support of this hypothesis we have detected the mRNAs for TGF-beta 1, basic FGF, TGF-alpha, and c-myc in plucked wool follicles using PCR amplification. We have also shown that the TGF-beta 1, TGF-beta 2, TGF-beta 3, EGF, TGF-alpha, basic FGF, N-myc, and c-myc genes are expressed in mouse skin, and we looked for changes during the hair cycle. The PCR data suggest that in whole skin the levels of mRNA for TGF-beta 1, TGF-beta 2, TGF-alpha, and c-myc do not change. In Quackenbush mice the levels for N-myc, TGF-beta 3, and basic FGF mRNA appear to be lower at the end of the hair cycle. We have confirmed in CBA/C57 black mice that lower levels of N-myc mRNA are detected when hair growth ceases in catagen and telogen. To test our hypothesis further and to assess its practical application, we are making transgenic mice in which the N-myc gene is overexpressed in the hair follicle by way of a wool keratin promoter. The transgene consists of 3.3 kb of 5' sequence from an ovine type 1 IF gene, the murine N-myc genomic coding sequence, and an SV40 polyadenylation signal. The native keratin type 1 IF gene is expressed exclusively in the wool follicle, as shown by in situ hybridization. However, in mice the injection of the transgene has resulted in high embryonic mortality and some embryos with large body size and head malformations. Since these mice were not transgenic, this is likely to be an effect of transient expression of the transgene during embryogenesis. The two transgenic mice produced so far have a normal phenotype.

Effect of (1-24)adrenocorticotrophin stimulation on the rate of corticosterone synthesis by the rat adrenal cortex.

The rate of corticosterone synthesis by the rat adrenal gland was measured in vitro, using a cell-free system, following the in vivo administration of (1-24)ACTH. The doses of ACTH used were 50, 100 and 250 micrograms ACTH/kg body weight. With all 3 doses of ACTH there was a significant increase in the rate of corticosterone synthesis within the first 5 min; this initial increase in rate did not vary with the dose of ACTH given. With both the 50 and 100 micrograms/kg doses the new rate of synthesis was maintained without further change, up to 30 min post-injection. In the case of the 250 micrograms/kg dose there was a second significant increase in the rate of corticosterone synthesis observed after 20 min. The results are discussed in the light of the hypothesis that the differential response of the adrenal gland represents the binding of ACTH to two receptors; a high affinity receptor of low abundance and a low affinity receptor of greater abundance. The results are consistent with the initial steroidogenic response resulting from binding to the high affinity receptors. Because of their low abundance these receptors may be completely occupied even at low doses of ACTH, thus explaining the dose-independent nature of the initial response to ACTH. Binding to the more abundant low affinity receptors may be associated with the secondary dose-dependent enhanced synthesis rate, a response which may be mediated via c-AMP.

The effect of acute nicotine administration on plasma levels of the thyroid hormones and corticosterone in the rat.

The effects of a single intraperitoneal injection of nicotine hydrogen tartrate (200 micrograms/kg) on the plasma levels of thyroxine, triiodothyronine and corticosterone were monitored over a 24 hour period. Nicotine did not alter the plasma levels of either of the thyroid hormones but did produce a significant increase in plasma corticosterone, an effect which peaked at 20 min post-injection and lasted for 45 min.

Effect of prolonged exposure to nicotine and stress on the pituitary-adrenocortical response; the possibility of cross-adaptation.

Daily IP injections of nicotine (200 micrograms/kg body weight) resulted in an adaptation of the nicotine induced rise in plasma corticosterone. By 30 days the plasma corticosterone rise was not significantly different from that seen in control animals receiving an injection of saline. A similar adaptation to the plasma corticosterone response to the stress of signalled, irregular footshock was also observed. However, in the case of the exposure to stress, while the corticosterone response at day 40 was significantly less than the response seen on day 1, it was still significantly greater than the plasma corticosterone level from unstressed control animals. Cross-adaptation experiments were conducted in which animals were adapted to the steroidogenic action of nicotine and then subjected to a novel exposure to footshock stress, and vice versa. In both situations the animals responded to the novel stimulus, either stress or nicotine, with a significant rise in plasma corticosterone. It was postulated that nicotine and psychological stress act upon the hypothalamo-pituitary-adrenal axis via functionally separate pathways at the level of the corticotrophin releasing factor neuron. The separate pathways appear to differ in their ability to be inhibited by corticosterone feedback.

Evidence for developmentally regulated transcriptional, translational and post-translational control of metallothionein gene expression in hair follicles.

The distribution of metallothionein (MT) and MT mRNAs was examined in hair (wool) follicles, where high levels of cell proliferation are found and where the resulting cells provide a temporal record of differentiation events. MT was found in the cytoplasm and some nuclei of follicle bulb cells of the proliferative zone, outer root sheath cells and in basal layer cells of sebaceous glands and sweat glands. The population of 5-bromo-2'-deoxyuridine (BrdU)+ cells in these tissues overlapped, but were not completely coincident with the distribution of MT staining. MT mRNA expression in hair (wool) follicles was assessed by in situ hybridization with four gene-specific sheep MT (sMT) isoforms. Intense signals were obtained with the sMT-Ib probe in follicle bulb cells from the proliferative zone to the keratogenous zone. Signals from the sMT-Ia probe were present in the same cells, but were much weaker. No signals were detected using the sMT-Ic and sMT-II gene-specific probes. The findings suggest that: (1) MT is important in cell proliferation and/or cell differentiation in the hair follicle bulb; (2) MT translation is inhibited during cell differentiation and migration; and (3) tissue-specific expression of uncharacterized sMT isoforms is likely.

Growth factor expression in skin during wool follicle development.

A variety of growth factors are likely to be involved in initiation and morphogenesis of wool follicles. To enable direct comparisons of the expression of different growth factors, reverse transcriptase-polymerase chain reactions (RT-PCR) were developed for ovine and murine TGF alpha, TGF beta 1, TGF beta 2, TGF beta 3, IGF1, IGF2, and FGF-2, which could all be carried out on a single cDNA sample. These RT-PCR were used with 16 sheep RNA samples from different foetal stages, neonatal sheep and mouse skin. The mRNAs for these growth factors were detected throughout gestation in sheep skin, except for TGF beta 1 mRNA which was not expressed in 51-day-old skin, but was expressed in 54-day and older samples. Since the first microscopically visible changes of follicle initiation occur around 62 days gestation, these results suggest that TGF beta 1 expression may be a signal for follicle initiation.

The plasma levels of ACTH following exposure to stress or nicotine.

Plasma ACTH and corticosterone profiles were measured following the intraperitoneal injection of nicotine or ACTH, or following exposure of rats to unpredictable stress. The elevation in plasma corticosterone was biphasic in nature in all cases, whereas plasma ACTH levels demonstrated only a single peak which rapidly declined to a sustained plateau level. The evidence supports the hypothesis that the adrenal cortex may exert a differential response to a single plasma peak in ACTH. The level of ACTH during its plateau phase may effect the duration rather than the magnitude of the corticosterone elevation. The differences between the ACTH and corticosterone profiles, resulting from the three different stimuli, are discussed.

The action of nicotine on the pituitary-adrenal cortical axis.

Nicotine was found to raise dramatically plasma corticosterone levels in a dose-dependent manner. Plasma corticosterone time course for the 100 microgram/kg dose of nicotine produced an initial increase in corticosterone elevation which did not return towards control levels until after 30 min. The 200 and 500 microgram/kg doses, however, indicated a biphasic response for nicotine-induced steroid secretion. The first steroid peak occurred 1-15 min after nicotine administration and was followed by a greater elevation at 20 min. Plasma nicotine levels did not show the same biphasic pattern. The nicotine-induced plasma steroid elevation was completely abolished by hypophysectomy, indicating that the response to nicotine must be mediated through ACTH release from the anterior pituitary. The administration of exogenous ACTH to rats pretreated with betamethasone was found to result in a biphasic plasma steroid elevation similar to that seen after injection of 200 microgram/kg nicotine. It was postulated that this may reflect a differential response of the adrenal cortex to a single ACTH outflow.

Renin synthesis and gene cloning.

1. The present study describes the biosynthesis and gene cloning of mouse submandibular gland renin. 2. After translation of mRNA coding for renin a Mr 46 000 protein is produced (preprorenin). However, rapid removal of the 'pre' segment from the nascent chain would account for the fact that in pulse-chase and continuous labelling experiments the largest renin-immunoreactive protein seen was of Mr 44 500, pI 6.4 (determined by 2-dimensional gel electrophoresis). This 'prorenin' was rapidly converted to a Mr 40 000, pI 6.2 species and then more slowly to forms of Mr 35 500, pI 5.6 and Mr 34 000, pI 5.4, which correspond in Mr and pI to renin isolated in pure form from mouse submandibular glands. 3. Colonies of the bacterium Escherichia coli strain RRI, containing plasmid pBR322 into which mouse submandibular gland cDNA had been inserted at the PstI site, were screened with affinity-purified anti-renin. Of 500 colonies, two were found that reacted positively with the antibody. These contained protein that could be immunoprecipitated with anti-renin and cDNA which was capable of inter-colony hybridization and which had a HinfI restriction site in each case.

Celite multi-column chromatography for the simultaneous separation of progesterone, deoxycorticosterone and 17 alpha-hydroxyprogesterone from small plasma or tissue samples.

A rapid, inexpensive and reliable chromatographic method has been developed for the simultaneous separation of progesterone, deoxycorticosterone and 17 alpha-hydroxyprogesterone on 15-cm Celite columns using n-heptane-benzene-methanol-water (100:35:80:20) as mobile phase. Six columns can be run in parallel within an hour with the assistance of nitrogen pressure. There is negligible column-to-column or batch-to-batch variation with consistent high recoveries. Columns are easily and quickly prepared and being inexpensive are disposable which is important when working with radioactivity. Using this method three important corticosteroids can be rapidly and reliably isolated from 20-30 small plasma or tissue samples per day.

Clustered arrangement of keratin intermediate filament genes.

We report here that component members of the keratin intermediate filament (IF) type I and type II gene families of sheep are closely linked but apparently the two families are not. Nine genes, accounting for up to half of the keratin IF gene repertoire, were mapped in four cosmid clones and the linkage between the genes ranged from several kilobases to 20 kilobases. In one cosmid, three tandem type I genes had the same transcriptional arrangement and were regularly spaced. In another cosmid, tandem genes encoding type II keratins were identified and, surprisingly, a solitary exon was discovered in the intergene region between the two type II genes. In a normal gene this exon encodes one of the most conserved amino acid regions of IF proteins, the C-terminal end of the alpha-helical core. Homologous C-terminal protein subdomains were encoded by two wool keratin type II genes and we suggest that this arrangement may also exist in the other wool keratin type II genes.

Gene expression in sheep skin and wool (hair).

We sequenced 2939 ESTs from fetal and adult sheep skin. Stages of gestation were picked to coincide with the major events in skin appendage (wool follicle) formation. Clustering analysis generated a nonredundant set of ESTs 2435 strong (83% nonredundant). Approximately 24% of these gave no hit to NCBI build 29 of the human genome, while 35% were tentatively classified by putative function based on BLASTX hits with a p(N) of <10(-4). In addition to bioinformatics analysis of our ESTs and gene mapping, we have generated a large EST spatial expression data set using in situ hybridization. One thousand one hundred forty-two ESTs have been used for in situ localization; about 31% are from adult sheep skin, 39% from late gestation fetal sheep skin, and 30% from midgestation fetal sheep skin. These probes have been used in over 3000 hybridization experiments. In this report, we summarize the results of in situs on adult sheep skin.