Ontogeny of enhancing factor in mouse intestines and skin

Summary Enhancing Factor (EF) is a 14 kDa protein isolated from mouse small intestines, which enhances the binding of ¹²⁵I-EGF to A431 cells. This observation as well as our earlier in vitro studies have indicated that EF is a modulator of EGF. In adult mice, localization of EF by immunohistochemistry shows it is present pre-dominantly in the Paneth cells of the small intestines and to a lesser extent in the stomach and colon. This study of the ontogeny of EF shows that the appearance of the protein coincides with the appearance of mature Paneth cells. In new born mouse skin EF is localized in the hair follicles in the first hair cycle from day 2 to day 8. It is however absent in the adult skin. Thus EF is associated with tissues which have a high growth rate.     

Expression of enhancing factor gene and its localization in mouse tissues.

Enhancing factor (EF), a 14 kDa protein, isolated from mouse small intestines, has been reported from this laboratory. Based on our earlier studies EF has been implicated in cell proliferation. Preliminary immunohistochemical studies have shown EF to be localized in the Paneth cells of small intestines. In this paper we report the tissue distribution of EF using conditions optimized for immunohistochemical staining. In addition, the data are supported by northern blot analysis using a nick translated cDNA probe specific for EF. The results indicate that EF gene is actively transcribed mainly in the intestines. The chief source of synthesis of EF appears to be the Paneth cells located at the base of the crypts of Lieberkühn.     

Expression of enhancing factor gene and its localization in mouse tissues

Summary Enhancing factor (EF), a 14 kDa protein, isolated from mouse small intestines, has been reported from this laboratory. Based on our earlier studies EF has been implicated in cell proliferation. Preliminary immunohistochemical studies have shown EF to be localized in the Paneth cells of small intestines. In this paper we report the tissue distribution of EF using conditions optimized for immunohistochemical staining. In addition, the data are supported by northern blot analysis using a nick translated cDNA probe specific for EF. The results indicate that EF gene is actively transcribed mainly in the intestines. The chief source of synthesis of EF appears to be the Paneth cells located at the base of the crypts of Lieberkühn.     

Trace amounts of enhancing factor/phospholipase A2 in mouse peritoneal exudate cells

Enhancing factor (EF), a mouse phospholipase A₂ (PLA₂), has been purified from the small intestines, based on its ability to increase the binding of epidermal growth factor in a radioreceptor assay. EF/PLA₂ was found to be localized predominantly in the Paneth cells in the small intestines. Whether mouse intestinal EF/PLA₂ is identical/similar to mouse secretory PLA₂ was to be determined. Phospholipases are known to play a crucial role in the process of inflammation. This paper reports the presence of trace amounts of EF/PLA₂ in the peritoneal exudate cells. Western blot analysis of the acid extracts showed the presence of a 14 kDa immunologically cross-reactive protein. RT-PCR analysis using EF specific primers amplified a ∼700 bp product which was further confirmed to be EF-specific by nested PCR analysis and sequencing. Presence of EF in the peritoneal exudate cells could be a unique mode of transport of growth factor modulator to the site of injury to aid in regeneration/cell proliferation of damaged tissue.     

Reprogramming adult dermis to a neonatal state through epidermal activation of β-catenin

Hair follicle formation depends on reciprocal epidermal-dermal interactions and occurs during skin development, but not in adult life. This suggests that the properties of dermal fibroblasts change during postnatal development. To examine this, we used a PdgfraEGFP mouse line to isolate GFP-positive fibroblasts from neonatal skin, adult telogen and anagen skin and adult skin in which ectopic hair follicles had been induced by transgenic epidermal activation of β-catenin (EF skin). We also isolated epidermal cells from each mouse. The gene expression profile of EF epidermis was most similar to that of anagen epidermis, consistent with activation of β-catenin signalling. By contrast, adult dermis with ectopic hair follicles more closely resembled neonatal dermis than adult telogen or anagen dermis. In particular, genes associated with mitosis were upregulated and extracellular matrix-associated genes were downregulated in neonatal and EF fibroblasts. We confirmed that sustained epidermal β-catenin activation stimulated fibroblasts to proliferate to reach the high cell density of neonatal skin. In addition, the extracellular matrix was comprehensively remodelled, with mature collagen being replaced by collagen subtypes normally present only in developing skin. The changes in proliferation and extracellular matrix composition originated from a specific subpopulation of fibroblasts located beneath the sebaceous gland. Our results show that adult dermis is an unexpectedly plastic tissue that can be reprogrammed to acquire the molecular, cellular and structural characteristics of neonatal dermis in response to cues from the overlying epidermis.     

Expression of enhancing factor/phospholipase A2 in ICRC mice

Enhancing factor (EF), a growth factor modulator, recently identified as the mouse secretory phospholipase A₂ (PLA₂), has been isolated in our laboratory from the intestines of mice. EF modulates the action of epidermal growth factor (EGF) by mediating an almost 2-fold increase in EGF binding in a radioreceptor assay. EF has been localized immunohistochemically to the Paneth cells of the intestine, adjacent to the proliferating stem cell population. Although very weak staining was observed in the intestines of ICRC mice (ICRC is an inbred strain of mouse developed at this Institute) as compared to Balb/c mice, the enhancing activity was not detected in the partially purified, acid soluble intestinal proteins of the ICRC strain. However, studies using polyclonal antibodies against purified EF demonstrated that EF from Balb/c and ICRC intestines are either immunologically identical or closely related to each other although, quantitatively, EF was very low in ICRC mice. RFLP studies indicated that ICRC mice carry a mutation in the coding region of the EF gene resulting in loss of the BamHI. restriction site. On sequencing, a T insertion was found at position 166 from the ATG site thereby causing a disruption in the ORF. This probably results in undetectable levels of enhancing activity. In this paper we report the molecular characterization of the ICRC mouse with respect to theenhancing factor gene     

The Distribution of Polycomb-Group Proteins During Cell Division and Development in Drosophila Embryos: Impact on Models for Silencing

The type I keratin 17 (K17) shows a peculiar localization in human epithelial appendages including hair follicles, which undergo a growth cycle throughout adult life. Additionally K17 is induced, along with K6 and K16, early after acute injury to human skin. To gain further insights into its potential function(s), we cloned the mouse K17 gene and investigated its expression during skin development. Synthesis of K17 protein first occurs in a subset of epithelial cells within the single-layered, undifferentiated ectoderm of embryonic day 10.5 mouse fetuses. In the ensuing 48 h, K17-expressing cells give rise to placodes, the precursors of ectoderm-derived appendages (hair, glands, and tooth), and to periderm. During early development, there is a spatial correspondence in the distribution of K17 and that of lymphoid-enhancer factor (lef-1), a DNA-bending protein involved in inductive epithelial–mesenchymal interactions. We demonstrate that ectopic lef-1 expression induces K17 protein in the skin of adult transgenic mice. The pattern of K17 gene expression during development has direct implications for the morphogenesis of skin epithelia, and points to the existence of a molecular relationship between development and wound repair.     

Slow cycling intestinal stem cell and Paneth cell responses to Trichinella spiralis infection

There is limited information regarding responses by slow cycling stem cells during T. spiralis-induced T-cell mediated intestinal inflammation and how such responses may relate to those of Paneth cells. Transgenic mice, in which doxycycline induces expression of histone 2B (H2B)-green fluorescent protein (GFP), were used. Following discontinuation of doxycycline (“chase” period), retention of H2B-GFP enabled the identification of slow cycling stem cells and long-lived Paneth cells. Inflammation in the small intestine (SI) was induced by oral administration of T. spiralis muscle larvae. Epithelial retention of H2B-GFP per crypt cell position (cp) was studied following immunohistochemistry and using the Score and Wincrypts program. Compared to non-infected controls, there was significant reduction in the number of H2B-GFP-retaining stem cells in T. spiralis-infected small intestines. H2B-GFP-retaining stem cells peaked at around cp 4 in control sections, but smaller peaks at higher cell positions (>10) were seen in sections of inflamed small intestines. In the latter, there was a significant increase in the total number of Paneth cells, with significant reduction in H2B-GFP-retaining Paneth cells, but a marked increase in unlabelled (H2B-GFP-negative) Paneth cells. In conclusion, following T. spiralis-infection, putative slow cycling stem cell numbers were reduced. A marked increase in newly generated Paneth cells at the crypt base led to higher cell positions of the remaining slow cycling stem cells.     

In situ localization of agouti signal protein in murine skin using immunohistochemistry with an ASP-specific antibody

Switching between production of eumelanin or pheomelanin in follicular melanocytes is responsible for hair color in mammals; in mice, this switch is controlled by the agouti locus, which encodes agouti signal protein (ASP) through the action of melanocortin receptor 1. To study expression and processing patterns of ASP in the skin and its regulation of pigment production in hair follicles, we have generated a rabbit antibody (termed alphaPEP16) against a synthetic peptide that corresponds to the carboxyl terminus of ASP. The specificity of that antibody was measured by ELISA and was confirmed by Western blot analysis. Using immunohistochemistry, we characterized the expression of ASP in the skin of newborn mice at 3, 6, and 9 days postnatally. Expression in nonagouti (a/a) black mouse skin was negative at all times examined, as expected, and high expression of ASP was observed in 6 day newborn agouti (A/+) and in 6 and 9 day newborn lethal yellow (A(y)/a) mouse skin. In lethal yellow (pheomelanogenic) mice, ASP expression increased day by day as the hair color became more yellow. These expression patterns suggest that ASP is delivered quickly and efficiently to melanocytes and to hair matrix cells in the hair bulbs where it regulates melanin production.     

Development of the Mouse Dermal Adipose Layer Occurs Independently of Subcutaneous Adipose Tissue and Is Marked by Restricted Early Expression of FABP4

The laboratory mouse is a key animal model for studies of adipose biology, metabolism and disease, yet the developmental changes that occur in tissues and cells that become the adipose layer in mouse skin have received little attention. Moreover, the terminology around this adipose body is often confusing, as frequently no distinction is made between adipose tissue within the skin, and so called subcutaneous fat. Here adipocyte development in mouse dorsal skin was investigated from before birth to the end of the first hair follicle growth cycle. Using Oil Red O staining, immunohistochemistry, quantitative RT-PCR and TUNEL staining we confirmed previous observations of a close spatio-temporal link between hair follicle development and the process of adipogenesis. However, unlike previous studies, we observed that the skin adipose layer was created from cells within the lower dermis. By day 16 of embryonic development (e16) the lower dermis was demarcated from the upper dermal layer, and commitment to adipogenesis in the lower dermis was signalled by expression of FABP4, a marker of adipocyte differentiation. In mature mice the skin adipose layer is separated from underlying subcutaneous adipose tissue by the panniculus carnosus. We observed that the skin adipose tissue did not combine or intermix with subcutaneous adipose tissue at any developmental time point. By transplanting skin isolated from e14.5 mice (prior to the start of adipogenesis), under the kidney capsule of adult mice, we showed that skin adipose tissue develops independently and without influence from subcutaneous depots. This study has reinforced the developmental link between hair follicles and skin adipocyte biology. We argue that because skin adipocytes develop from cells within the dermis and independently from subcutaneous adipose tissue, that it is accurately termed dermal adipose tissue and that, in laboratory mice at least, it represents a separate adipose depot.     

Immunohistochemical characterization of a crypt cell-specific plasma membrane protein in rat small intestine epithelium using a monoclonal antibody

Proteins of the basolateral membrane (BLM) of small intestine epithelial cells of adult rats, in the MW ranges of 50-65 KD, 85-100 KD, and over 100 KD, were obtained as follows. After isolation of the BLM and subsequent SDS-PAGE and transblotting of the proteins on nitrocellulose sheets, the bands in these MW ranges were cut out of the nitrocellulose sheet and extracted. Balb/C mice were immunized with these protein fractions and a monoclonal antibody (MAb) was then produced. MAb SI/CC1 obtained via immunization with the 50-65 KD protein fraction shows specificity for the crypt epithelium of the small intestine. It can be used to characterize, by light and electron microscopic immunohistochemical methods, a crypt cell protein (SI/CC1-Ag) with a very specific localization. Fluorescence labeling shows that the SI/CC1-Ag can be found only in the epithelium of small intestine crypts (except for the granules in eosinophilic granulocytes). The epithelium of the colon, as well as the epithelia of other organs, could not be labeled. In the small intestine crypts, SI/CC1-Ag is found only in the Paneth cells located in the basal crypt section, and in the undifferentiated cells in the middle crypt section; it is lacking in the cells of the upper crypt section. Gold labeling shows that SI/CC1-Ag in the undifferentiated cells is localized exclusively in the basolateral PM domain. On the Paneth cells, the content of the secretory granules is labeled, along with the basolateral PM domain; the labeling sometimes present on their luminal part is probably due to passively absorbed secretion from these cells. The SI/CC1-Ag in the BLM of undifferentiated and Paneth cells is found only on Days 21-23 post partum, whereas the Paneth cell granules could be labeled as early as the Day 16 post partum. With immunodetection with SI/CC1, one band at about 55 KD is specifically labeled in the protein pattern of the isolated small intestine cell BLM. In the protein pattern of the isolated crypt cells two bands were labeled, again one at 55 KD and one at about 120 KD. These findings indicate that SI/CC1-Ag is a 55 KD protein that appears on Days 21-23 post partum in the BLM of undifferentiated cells and of Paneth cells.     

Tissue and cell distribution of the multidrug resistance-associated protein (MRP) in mouse intestine and kidney.

The multidrug resistance-associated protein (MRP) that is involved in drug resistance and the export of glutathione-conjugated substrates may not have the same epithelial cell membrane distribution as the P-glycoprotein encoded by the MDR gene. Because intestinal and kidney epithelial cells are polarized cells endowed distinct secreting and absorptive ion and protein transport capacities, we investigated the tissue and cell distribution of MRP in adult mouse small intestine, colon, and kidney by immunohistochemistry. Western blot analyses revealed the 190-kD MRP protein in these tissues. MRP was found in the basolateral membranes of intestinal crypt cells, mainly Paneth cells, but not in differentiated enterocytes. All the cells lining the crypt-villous axis of the colon wall contained MRP. MRP was found in the glomeruli, ascending limb cells, and basolateral membranes of the distal and collecting tubule cells of the kidney but not in proximal tubule cells. Cultured mouse intestinal m-ICcl2 cells and renal distal mpkDCT cells that have retained the features typical of intestinal crypt and renal distal epithelial cells, respectively, also possess MRP in their basolateral membranes. The patterns of subcellular and cellular distribution indicate that MRP may have a specific role in the basolateral transport of endogenous compounds in Paneth, renal distal, and collecting tubule cells.     

新生BALB/c小鼠肠道潘氏细胞的分布规律

观察BALB/c小鼠小肠潘氏细胞的分布规律。应用石蜡切片、H&E染色技术和LeicaQwin显微图像处理系统,分别对2 d、4 d、6 d、8 d、10 d BALB/c小鼠小肠内潘氏细胞的形态发育、分布规律进行观察和分析。结果发现,4日龄前的BALB/c小鼠各段小肠中肠腺发育尚不完整,未见有潘氏细胞。6日龄后,潘氏细胞开始出现,数量随日龄增加呈递增趋势增长,各日龄间差异显著(P<0.05)。BALB/c小鼠肠道潘氏细胞存在于小肠,十二指肠和空肠较少,回肠较多,各组(不同日龄/肠段)之间差异显著(P<0.05)。     

Purification of the enhancing factor from mouse intestines

A unique polypeptide, called enhancing factor (EF), which enhances the binding of labeled epidermal growth factor (EGF) to cells, has been isolated. It has been purified to homogeneity from the acid-soluble proteins of mouse intestines. Earlier, EF was partially purified by two cycles of gel-permeation chromatography on Bio-Gel columns. We now report the final purification of EF on high-performance liquid chromatography (HPLC), using a reverse-phase column (mu Bondapak C18). The purity of the protein was confirmed when a single peak was obtained in HPLC. Also, a single protein band was obtained in SDS-PAGE. Purified EF has the same properties in vitro as those reported earlier for partially purified EF.     

Hes1-deficient mice show precocious differentiation of Paneth cells in the small intestine

We have previously shown that Hes1 is expressed both in putative epithelial stem cells just above Paneth cells and in the crypt base columnar cells between Paneth cells, while Hes1 is completely absent in Paneth cells. This study was undertaken to clarify the role of Hes1 in Paneth cell differentiation, using Hes1-knockout (KO) newborn (P0) mice. Electron microscopy revealed premature appearance of distinct cells containing cytoplasmic granules in the intervillous region in Hes1-KO P0 mice, whereas those cells were absent in wild-type (WT) P0 mice. In Hes1-KO P0 mice, the gene expressions of cryptdins, exclusively present in Paneth cells, were all enhanced compared with WT P0 mice. Immunohistochemistry demonstrated increased number of both lysozyme-positive and cryptdin-4-positive cells in the small intestinal epithelium of Hes1-KO P0 mice as compared to WT P0 mice. Thus, Hes1 appears to have an inhibitory role in Paneth cell differentiation in the small intestine.     

Identification of a novel fibroblast growth factor, FGF-22, preferentially expressed in the inner root sheath of the hair follicle

We isolated cDNA encoding a novel fibroblast growth factor (FGF-22) (170 amino acids) from human placenta. Of the FGF family members, FGF-22, which appears to be a secreted protein, is most similar to FGF-10 and FGF-7 (approximately 46% and approximately 40% amino acid identities, respectively). The human FGF-22 gene was localized on chromosome 19p13.3. We also isolated mouse cDNA encoding FGF-22 (162 amino acids) from the skin. Mouse FGF-22 shows high homology (87% amino acid identity) to human FGF-22. Mouse FGF-22 mRNA was found to be preferentially expressed in the skin among the mouse adult tissues examined by Northern blotting analysis. By in situ hybridization, FGF-22 mRNA in the skin was found to be preferentially expressed in the inner root sheath of the hair follicle. Therefore, FGF-22 is expected to be a unique FGF that plays a role in hair development.