Precocious Eyelid Opening Research Articles

Malformation of Tear Ducts Underlies the Epiphora and Precocious Eyelid Opening in Prickle 1 Mutant Mice: Genetic Implications for Tear Duct Genesis.

PurposeObstruction of the tear drainage causes a range of ocular surface disorders. Hitherto, the genetics of tear duct development and obstruction has been scarcely explored, and related animal models are lacking. This study aims to study the potential role of the Wnt/PCP pathway mediated by Prickle 1 in tear duct development and diseases.MethodsA severe hypomorphic Prickle 1 mutant was generated. Histology and immunohistochemistry were performed to compare wild type, Prickle 1 hypomorphic, and null mutant tear ducts. In situ hybridization was conducted to identify the signaling components in the developing tear ducts. Three-dimensional (3D) reconstruction was used to detect the human embryonic tear duct.ResultsHere, we report that a severe Prickle 1 hypomorph mouse line exhibited epiphora. This phenotype was due to the blockage of the tear drainage by incompletely formed nasolacrimal duct (NLD) and lacrimal canaliculi (LC), which also causes precocious eyelid opening. We observed a dose-dependent requirement of Prickle 1 for tear duct outgrowth. An investigation of the expression of Wnt/PCP core genes demonstrated a subset of PCP signaling components expressed in the developing tear duct. Furthermore, Prickle 1 is not required for the expression of Fgfr2/Fgf10 and p63 genes, which are associated with the NLD and LC hypoplasia in humans. Last, we showed that Prickle 1 expression in the developing tear drainage system is conserved between mice and humans.ConclusionsThe study suggests that malformed tear ducts caused by disruption of Prickle 1 underlies the epiphora and precocious eyelid opening.

Ocular surface pathogenesis associated with precocious eyelid opening and necrotic autologous tissue in mouse with disruption of Prickle 1 gene

Ocular surface disease is one major type of eye diseases. Different etiologies trigger distinct pathological responses of the ocular surface. We previously reported that genetically engineered mice with ablation of Prickle 1 manifested precocious eyelid opening with ensuing cornea dysplasia. The current study aimed to characterize the molecular traits and the direct cause of ocular pathology associated with precocious eyelid opening in the Prickle 1 mutant mouse. Prickle 1 mutant mice exhibited a slew of ocular surface pathology including cell proliferation, cell fate transformation and inflammatory infiltration coinciding with the timing of the precocious eyelid opening. Forced eyelid opening in wild type mice did not induce cornea pathology comparable to that of the Prickle 1 mutants. Necrotic tissue debris was found associated with the lesioned cornea. RNAseq analysis of the mutant cornea revealed an expression profile shared by a range of dermatological diseases involving immune responses and cancer. Taken together, the data suggest that the necrotic eyelid debris plays an important role in ocular pathogenesis of the Prickle 1 mutant mouse, which may represent a type of non-infectious keratoconjunctivitis caused by damaged autologous tissues. Additionally, Prickle 1 mutant cornea pathogenesis may offer molecular insights into other types of epithelial pathogenesis.

Origins of Growth Factors: NGF and EGF

Growing up on the streets of Brooklyn, I remember being interested in how things worked: taking apart an old telephone and the gears of my new 4-speed bicycle was most enjoyable. As a biology major at Brooklyn College in 1945, I was fascinated by embryology. How does an egg turn into a chicken or a frog or a person? My only insight into the problem was the thought that it was necessary to understand the chemical reactions inside the egg and embryo and not simply observe biological structures. I, therefore, became a double major in chemistry and biology (also with the hope of earning a living as a laboratory technician). My first job after graduating was working nights as a bacteriologist in a milk processing plant. One of my professors at Brooklyn College wrote asking whether I was interested in going to graduate school. The college had been sending one student a year to the Biology Department at Oberlin to work as a laboratory assistant while studying for a master's degree. Because Oberlin offered to pay my tuition plus some $300 a semester for living expenses, I jumped at the chance. School and science were both interesting and fun. Upon graduating, I continued my education toward a Ph.D. working as an assistant in the Biochemistry Department of the University of Michigan under Howard B. Lewis. My research involved studying the Krebs urea cycle in the common earthworms that I collected from the university campus. I had been told at Oberlin that the yellow cells surrounding the gut in the worm corresponded to the mammalian liver. I decided to check whether the enzymatic reactions in worms were similar to those in mammals. They were (1). My first “real job” was in the Pediatrics Department at the University of Colorado studying creatine metabolism in premature infants under Harry Gordon. I think he hired me because he was impressed by my ability to stomach tube earthworms. After several years, I decided I needed to learn the then new technique of using radioisotopes in metabolic studies, and I obtained an American Cancer Society fellowship to work in the Radiology Department of Washington University under Martin Kamen where I combined this new knowledge with my interest in embryology. Although fertilized frog eggs and early embryos are impermeable to small molecules (amino acids, phosphates, etc.), sufficient amounts of C14O2 were metabolized by intact eggs and embryos to permit the identification of the radioactive compounds present at various stages of development (2). Upon completion of my fellowship, Dr. Kamen recommended me for a position in the Zoology Department of Washington University in the laboratory of Viktor Hamburger and Rita Levi-Montalcini. This move was critical in determining the direction of my research for the next 40-some odd years: the isolation, structure, and function of the first of the “growth factors,” nerve growth factor (NGF) and epidermal growth factor (EGF).

Origins of Growth Factors: NGF and EGF

This presentation describes the events that led to the sequential discoveries of nerve growth factor (NGF) and epidermal growth factor (EGF). Each was isolated during attempts to understand the biochemical basis for unexpected biological observations. The in vivo stimulation of the growth of specific nerve fibers following the transplantation of a mouse sarcoma into a chick embryo eventually resulted in the finding of NGF, not only in the transplanted tumor but also in snake venom and in the mouse salivary gland. Unexpectedly, treatment of newborn animals with extracts of the mouse salivary gland resulted not only in enhancement of nerve growth but also in precocious eyelid opening and tooth eruption. The latter observations led to the isolation of both EGF and its tyrosine kinase-active receptor.

Metabolism and Effects of Epidermal Growth Factor and Related Growth Factors in Mammals*

The initial observations of Stanley Cohen in the 1960s established that EGF induced in vivo effects such as precocious eyelid opening and tooth eruption. Subsequently the actions of EGF have been extensively explored in cell culture systems. The receptor for EGF was characterized as a prototype model for other growth factors and the now extensive in vitro data indicate multiple functions for EGF. Moreover, EGF and EGF receptors have been characterized in many tissues, and EGF has been identified in most body fluids of several mammalian species. Interestingly, neither EGF antibody administration to newborn animals nor passive immunization of pregnant rodents against EGF has caused major deleterious effects (except the delay in epidermal maturation events), as might be expected from the in vitro studies. This is in contrast to the effects of nerve growth factor antiserum in developing rodents. Also, to date, no pathological EGF deficiency disorder has been characterized. However, the EGF family of growth factors appears to be important in mammalian development and function, although the precise roles and significance are not yet clear. Members of the family may have a role in embryogenesis and fetal growth since receptors have been identified in fetal tissues. Available evidence suggests that TGF alpha subserves the growth factor family roles in fetal development. In the developing postnatal animal pro-EGF mRNA, immunoreactive EGF, immunoreactive TGF alpha, and EGF receptors are present in many tissues. EGF also is produced and secreted by the maternal mammary gland, and mammary derived EGF appears to be important in gut development in the neonatal rodent. There is now extensive data to indicate important hormone-EGF interactions. In the postnatal period, thyroid and steroid hormones including retinoic acid have been shown to modulate EGF and/or EGF receptors in several tissues. GH increases EGF binding in liver and increases urine EGF concentrations. Moreover, EGF stimulates secretion of several hypothalamic and pituitary hormones, increases placental production of hCG and human chorionic somatomammotropin, increases adrenal cortisol production, and inhibits testicular, ovarian, and thyroid hormone secretions. As summarized in this review EGF has been implicated in a number of developmental events including palate and skin differentiation, growth of hair follicles, eye opening and tooth eruption, lung maturation, gut and liver growth, and differentiation of neurons. These EGF actions probably are mediated via autocrine, paracrine, and endocrine routes. A role for salivary and urine EGF in the maintenance of adult stomal, gut, and urinary epithelial surface integrity seems likely, although not yet proven.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization and localization of receptors for epidermal growth factor in ovine skin.

Specific receptor sites for murine epidermal growth factor (EGF) have been characterized and their distribution determined in ovine skin. Binding of 125I-labelled EGF to skin membrane particles was temperature- and time-dependent, with equilibrium being reached within 1 h at 23 degrees C. Analysis of skin biopsies collected from ten castrated Merino sheep demonstrated the presence of a single class of saturable, high-affinity binding sites with a dissociation constant of 64 +/- 4 (S.E.M.) pmol/l and a binding capacity of 33.8 +/- 4.5 fmol/mg protein. Skin particle binding of 125I-labelled EGF was inhibited equipotently by mouse salivary gland EGF, EGF produced by recombinant DNA procedures and urogastrone. The EGF peptides 1-48, 6-53 and 7-53, derived from the native molecule by enzymatic cleavage, were much less potent. The relative binding potency of these molecules was correlated with their ability to induce precocious eyelid opening in mice and to inhibit wool follicle activity. Synthetic fragments representing the major structural domains of the EGF molecule (EGF(29-44), EGF(33-42) and EGF(3-31] were inactive in both the receptor and bioassays. Autoradiography of skin sections incubated with 125I-labelled EGF in vitro or of sections from skin which was perfused with 125I-labelled EGF in vivo demonstrated that EGF receptors were localized in undifferentiated cells of the epidermis and sebaceous glands, the inner and outer root sheath and bulb of wool follicles and in dermal arterioles. Differences in receptor concentration were observed between follicles following in-vivo perfusion of 125I-labelled EGF but not when the in-vitro labelling technique was used. The presence of receptors in these regions is consistent with the morphological changes in sheep skin in response to EGF administration which have been reported previously.

Epidermal growth factor has both growth-promoting and growth-inhibiting effects on physical and neurobehavioral development of neonatal mice

The protein molecule epidermal growth factor (EGF) exerts powerful effects on mouse physical development, since repeated subcutaneous administrations of murine EGF (3.5 mg/kg, from postnatal day 2 to postnatal day 10) cause precocious eyelid opening (as early as day 8 instead of day 13 in control littermates receiving 3.5 mg/kg cytochrome c) and precocious eruption of the lower incisors (day 6 instead of day 8). By contrast, the same EGF treatment retards both the rate of body growth and the full appearance of several neurobehavioral signs of maturation, such as righting and grasping responses. Neuronal mice receiving 5 mg/kg murine nerve growth factor (NGF) under the same treatment schedule, although showing a significant retardation in body weight gain, exhibited only limited changes in neurobehavioral maturation. Specifically, the appearance of slow and swift righting, response to strong tactile stimulation, hindlimb and forelimb grasping, pole grasping, and vertical screen and screen climbing were significantly retarded by EGF and slightly advanced by NGF (the only significant NGF effect was an acceleration of swift righting maturation). Polypeptide growth factors seem to play an important role in physical and neurobehavioral development of altricial rodents, orchestrating the relative maturation of different tissutal targets on different developmental stages.

An autoradiographic study on the effect of epidermal growth factor on cell proliferation in erupting mouse incisors

Epidermal growth factor (EGF) is a small polypeptide that induces precocious eyelid opening and incisor eruption in newborn mice; it stimulates cell proliferation in various cell types in vitro and in vivo. EGF was injected twice daily into newborn mice (0.4 μg/g) and tritiated thymidine was injected subcutaneously 6 h before killing the mice. Longitudinal sections of the lower incisors showed significantly more thymidine-labelled mitoses in the pre-odontoblast and pre-ameloblast layers at the basal ends of incisors in EGF-treated mice than in control mice. Although these results indicate that the EGF-induced precocious tooth emergence is associated with a stimulation of cell proliferation in the root sheath, this tissue may not be the target for the actions of EGF. Earlier studies have shown that root growth is not a factor in the generation of the eruptive force and, as neither the pre-ameloblasts nor pre-odontoblasts express EGF-receptors, the stimulation of cell proliferation in the root-sheath region appears to be a result and not a cause of accelerated tooth eruption, i.e. the increase in root growth may meet the need to maintain the tooth positionally during the accelerated eruptive process.

Epidermal growth factor (hEGF) has no effect on murine intestine epithelial damage and regeneration after melphalan.

The effect of epidermal growth factor (hEGF) on intestinal epithelial damage by melphalan was explored in CBA mice. Human EGF was administered in doses of 100 micrograms kg-1 or 1000 micrograms kg-1 using a variety of schedules. Mucosal damage was assessed 4, 8 and 13 days later, by [14C]-xylose uptake and by microcolony survival of jejunum, ileum and colon. The only regimen to show enhanced jejunal crypt survival was administration of hEGF, 100 micrograms kg-1, i.p., 8 hourly, beginning 24 h before melphalan treatment. Oral administration of hEGF had no effect on melphalan induced damage nor on subsequent recovery of intestinal mucosa. Activity of hEGF in mice was confirmed by demonstration of precocious eyelid opening in newborn mice. No consistent protective or restorative effect of hEGF on melphalan-induced intestinal epithelial damage could be demonstrated with the doses and schedules used.

Human transforming growth factor-alpha causes precocious eyelid opening in newborn mice.

Both murine and human epidermal growth factors (EGFs) are known to cause precocious opening of the eyelids in newborn mice. Another set of peptides that are structurally and functionally homologous to murine and human EGFs are the murine and human type-alpha transforming growth factors (TGF-alpha s), TGF-alpha s have been found in many cancer cells and it has been suggested that their autocrine action may play an important part in malignant transformation. In several in vitro systems murine and human TGF-alpha s are functionally interchangeable with murine and human EGFs. However, the in vivo activity of the TGF-alpha s has not been characterized, as only small amounts of these peptides were available until recently. The cloning of the gene for human TGF-alpha and its expression in Escherichia coli now allow us to demonstrate that human TGF-alpha is as active as murine EGF in promoting eyelid opening in newborn mice. Furthermore, we show in a dose-dependent eyelid opening assay that human EGF is as potent as its murine homologue with respect to this biological property.

Overproduction of human epidermal growth factor/urogastrone in Escherichia coli and demonstration of its full biological activities

A man-made gene coding for [21-Leu] human epidermal growth factor (hEGF)/β-urogastrone was constructed, inserted into a lacZ fusion-gene expression vector, and cloned into Escherichia coli. In the cloned cells the β-galactosidase/hEGF fusion gene was efficiently expressed and the yield of the hybrid protein reached 10% of the total cellular protein. The [21-Leu] hEGF synthesized in the bacterial cells was proved to be as functional as the natural hEGF or urogastrone and mouse EGF by the following criteria: (1) stimulation of cell proliferation, (2) stimulation of thymidine incorporation into cultured cells, (3) competition with mouse EGF for binding sites on the plasma membrane of human KB cells, and (4) stimulation of phosphorylation of a membrane-bound protein of human KB cell, which has an apparent molecular weight of 150 000 to 170 000 and is indistinguishable from the protein phosphorylated in the presence of mouse EGF in the sodium dodecyl sulfate—polyacrylamide electrophoretic gel. The hEGF produced in the bacterial cells also resulted in precocious eyelid-opening by the daily subcutaneous injection into newborn mice and in accelerated incisor eruption in the animals as mouse EGF did, indicating that the hEGF is also fully active in vivo or physiologically.

Human epidermal growth factor: isolation and chemical and biological properties.

A polypeptide hormone has been isolated from human urine, human epidermal growth factor. It was assayed by its ability to compete with 125I-labeled mouse-derived epidermal growth factor in binding to human foreskin fibroblasts. The biological effects of the human polypeptide are similar to those previously described for the mouse hormone. These include the stimulation of the growth in vitro of human foreskin fibroblasts and corneal epithelial cells in organ culture, and the in vivo induction of precocious eyelid opening in the newborn mouse. The amino acid compositions of the two polypeptides differ, although certain similarities are present. The estimated molecular weight of the human polypeptide, 5300-5500, is slightly lower than that of the mouse hormone. Both polypeptides apparently compete for the same site on the cell membrane; and antibodies to the mouse polypeptide crossreact to some extent with the human hormone.