Progress 10/01/03 to 09/30/09
Outputs
OUTPUTS: The epithelium of the vertebrate intestine is a dynamic structure, under constant renewal. Epithelial cells migrate up the villus while differentiating into enterocytes and goblet cells. Epithelial tight junctions form a barrier to the diffusion of molecules through the paracellular pathway. This project has utilized Western blotting, immunohistochemistry, microarrays, and real-time PCR to study the expression of several tight-junction proteins during the late embryonic and early post-hatch period of developing chick intestine. Claudin-3 and -5 proteins were detected within the epithelial cytoplasm of duodenum, jejunum, and ileum during the 12-18 day period of embryonic development. By 1-day post-hatch, these two proteins were concentrated in the epithelial basal-lateral and apical junction regions, respectively. The relative mRNA expression of both claudins increased between 18 and 20 days, then decreased. Thus, the increase in transcript expression is correlated with localization of these claudins within the epithelial plasma membranes. Claudin-16 protein was not detected until 19 days of development. At 20 days, it was present in goblet cells at the tips of long villi. At 1-day post-hatch, claudin-16 was found in goblet cells of duodenum, jejunum, and ileum and immunostaining was darker. A dramatic increase in claudin-16 transcripts occurred between 18 and 20 days of development, consistent with the emergence of claudin-16 protein at 20 days. Western blotting studies also revealed that claudin-2 increases between 18 and 20 days of embryonic development and continues to increase through 2 days posthatch. Like claudin-5, it is most strongly expressed within crypt epithelium. Conversely, claudin-4 was not detected in embryonic or early post-hatch intestine, but was present within the intestinal epithelium of 3-week old chicks. Addition of the synthetic glucocorticoid dexamethasone to an organ culture system increased the expression of several claudins in 18-day embryonic intestine within 12 hours. Taken together, these studies reveal a complex pattern of development and regulation of tight junctional components of chick intestinal epithelium and suggest important correlations with intestinal function after hatching. The results of the project have been disseminated at the annual meetings of the American Society of Cell Biology, the Developmental Biology Society, and the North Carolina Academy of Science. Two graduate theses have been published, and a manuscript titled "Developmental Profile of Claudin-3, -5, and -16 Proteins in the Epithelium of Chick Intestine" has been submitted to the Journal of Morphology. The findings have also been shared with faculty in the Department of Poultry Science at NCSU. In conjunction with this project, two graduate students have been trained and received degrees in the field of Zoology. Faculty in the Department of Poultry Science formed part of the graduate advisory committees and thus collaborated in the project. Additionally, Dr. Black served as an adviser on two committees for Poultry Science graduate students. PARTICIPANTS: Betty L. Black was the principal investigator/project director who designed experiments and supervised two graduate students working on the project. W. Jason Wingate and Ozkan Ozden received graduate degrees based on their work in the project. Christopher Ashwell (Department of Poultry Science) and Russell Borski (Department of Zoology) were collaborators at NCSU. They provided equipment and advice on molecular aspects of the project. TARGET AUDIENCES: Members of the Poultry Science community and scientists interested in basic research on intestinal development and function were the target audiences for the project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Based on findings from other vertebrate species, it is likely that claudins have the following functions in the epithelium of chick intestine: 1) Claudin-3 probably "tightens" the tight junctions, explaining why it was absent or weakly expressed in the crypts of 1-day old intestine (where the epithelium is "leaky" and secretion occurs), but strongly expressed on the villus (where a tight epithelial barrier is crucial for active transport of glucose and other essential nutrients); 2) Claudin-5 and claudin 2 may be associated with cell proliferation and/or regulation of secretion through paracellular channels, since they they found primarily in the crypt region of hatched chicks; 3) Claudin-16 may be required for secretion of mucous as the post-hatch intestine becomes functionally mature, since it was localized within the goblet cells in the upper (most mature) villus epithelium. 4) Since claudin-4 was not detected in embryonic or early post-hatch intestine, but was present within the intestinal epithelium of 3-week old chicks; its expression may be dependent on feeding or other physiological factors that occur after the second day post-hatch. The findings of this research have generated new knowledge by elucidating the developmental patterns and potential hormonal control of tight junctions in chicken intestine. This knowledge may be of use in understanding nutritional problems during the early post-hatch stage of both chickens and turkey poults. Since malnutrition is a contributing factor in early poult mortality, the increased understanding of epithelium maturation should be value in solving this problem.
Publications
- No publications reported this period
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The epithelium of the vertebrate intestine is a dynamic structure, under constant renewal. The intestinal crypts represent a proliferative compartment that is monoclonal and is maintained by multiple stem cells. Epithelial cells migrate up the villus in sheets where they differentiate into enterocytes and goblet cells. A defining characteristic of the epithelial sheets are tight junctions that behave as a primary barrier to the diffusion of molecules through the paracellular pathway. This research has utilized Western blotting, immunohistochemistry, microarrays, and real-time PCR to study the expression of several tight-junction proteins during the late embryonic and early post-hatch period of developing chick intestine. The claudin protein family was of special interest, since claudins form the major part of tight junctions. Some claudins form a seal to prevent molecules from passing through the epithelial barrier via paracellular pathways, whereas other claudins may create ion-specific pores within the tight junctional complex. Several hormones, including glucocorticoids are known to promote functional maturation of the intestinal epithelium in chickens and turkeys. The ability of a synthetic hormone, dexamethasome, to alter gene expression of several tight junction proteins was also studied. The findings of this research elucidate the developmental patterns and potential hormonal control of tight junctions in chicken intestine and may be of use in understanding nutritional problems during the early post-hatch stage. A manuscript titled "Developmental Profile of Claudin-3, -5, and -16 Proteins in the Epithelium of Chick Intestine" has been submitted to the journal Developmental Dynamics to disseminate this information. The findings have also been shared with faculty in the Department of Poultry Science at North Carolina State University. PARTICIPANTS: Individuals who worked on the project are: Jason Wingate, MS candidate and Ozkan Ozden, Ph.D. candidate. TARGET AUDIENCES: Target audiences are basic researchers in the fields of Cell Biology, Developmental Biology, Intestinal Physiology, and Poultry Science. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Claudin-3 and -5 proteins were detected within the epithelial cytoplasm of duodenum, jejunum, and ileum during the 12-18 day period of embryonic development. By 1-day post-hatch, these two proteins were concentrated in the epithelial basal-lateral and apical junction regions, respectively. The relative mRNA expression of both claudins increased between 18 and 20 days, then dropped. Thus, the increase in transcript expression is correlated with localization of these claudins within the epithelial plasma membranes. Claudin-16 protein was not detected between 12 and 18 days of embryonic development, but was present in goblet cells at the tips of long villi by 20 days. At 1-day post-hatch, claudin-16 was found in goblet cells within the upper region of villus epithelium of duodenum, jejunum, and ileum and immunostaining was darker. A dramatic increase in claudin-16 transcipts occurred between 18 and 20 days of development, consistent with the emergence of claudin-16 protein at 20 days. Conversely, transcript levels of ZO-2 (another tight junction protein) decreased from 18 to 20 days and reached an even lower level by two days post-hatch. This indicates that the expression patterns of intestinal tight junctional proteins are not all alike. Based on findings from other vertebrate species, it is likely that claudins have the following functions in the epithelium of chick intestine: 1) Claudin-3 probably "tightens" the tight junctions, explaining why it was absent or weakly expressed in the crypts of 1-day old intestine (where the epithelium is "leaky" and secretion occurs), but strongly expressed on the villus (where a tight epithelial barrier is crucial for active transport of glucose and other essential nutrients); 2) Claudin-5 may be associated with cell proliferation and regulation of secretion through paracellular channels, since it is found primarily in the crypt region of hatched chicks; 3) Claudin-16 may be required for secretion of mucous as the post-hatch intestine becomes functionally mature, since it was localized within the goblet cells in the upper (most mature) villus epithelium. Additional studies on gene expression revealed that dexamethasone increased the expression of several claudins in 18-day embryonic intestine after 12 hours of exposure in an organ culture system. Taken together, these studies reveal a complex pattern of development and regulation of tight junctional components of chick intestinal epithelium and suggest important correlations with intestinal function after hatching.
Publications
- Wingate, Jason. 2008. Development of Claudin 2 and Claudin 4 in Embryonic and Hatched Chick Intestine. Thesis, North Carolina State University, (Under the direction of B.L. Black).
- Ozden, Ozkan. 2008. Expression of Claudin Tight Junction Proteins in Response to Varying Environmental and Physiological Conditions. Ph.D. dissertation, North Carolina State University, (Under the direction of B.L. Black and B.J. Grubb).
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Progress 10/01/06 to 09/30/07
Outputs
We have been studying the development of claudins in the intestinal epithelium of chick intestine. Recent work using Western blotting technique has confirmed that claudin 5 is present in the epithelial tight junctions of hatched chicks. Western blotting was performed on epithelial cell suspensions obtained from duodenum, jejunum, and ileum at 1-day post-hatch. Cell suspensions were homogenized and proteins separated by gel electrophoresis using 4-12% bis-tris gels and subsequent blots were incubated with rabbit polyclonal claudin 5 primary antibody (Zymed). Claudin 5 was present in cells from all intestinal regions, but was lower in ileum than in duodenum are jejunum. Immunohistochemistry using the same antibody revealed that this claudin is present as early as 12 days of embryonic development, but only within the apical cytoplasm. By day 18, claudin 5 staining was restricted to near the epithelial surface, and by 20 days it was clearly present between epithelial
cells. Thus claudin 5 appears to become localized within the epithelial tight junctions on the day before hatching. This is consistent with the need for a functional epithelial barrier when the chick begins to feed shortly after hatching. In mammalian skin, intestine, and kidney tubules there is good evidence that MAP kinase are involved in epithelial differentiation. One of these extracellular-signal-regulated kinases (ERK) is of special interest, since it controls the expression of a claudin in cultured kidney cells and is strongly expressed in developing kidney. Hence, we have begun a study of ERK expression during development of embryonic chick intestine. Our hypothesis is that ERK expression decreases as epithelial differentiation progresses, as occurs in adult mammals as intestinal cells emerge from the intestinal crypts and differentiate while migrating up the villi. Preliminary results utilizing Western blots indicate that ERK expression (in terms of amount of protein) is
inhibited by high calcium in cultures of 18-day embryonic intestine. This is consistent with our previous findings that intestinal development (and epithelial differentiation) is accelerated by high extracellular calcium. Future work will seek to determine the relationship between ERK expression and localization of claudin 5 within the tight junctional regions of intestinal epithelium.
Impacts
We anticipate that ERK expression and claudin 5 localization within epithelial tight junctions will be found to play an important role in development of the epithelial barrier during the final stage of embryonic chick development. Since an intact barrier in crucial in nutrient absorption and protection from intestinal pathogens, these findings could be important in the health and consequent growth rate of young chicks.
Publications
- No publications reported this period
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Progress 10/01/05 to 09/30/06
Outputs
We previously reported the unusual finding that claudin-16 is present in goblet cells of chick intestine. Additional study of intestinal claudins by immunostaining technique has revealed that the tight junction proteins claudin-3, and claudin-5 are also present, but have a distribution that differs from that of claudin-16. Claudin-3 was observed mainly in the epithelial cytoplasm and connective tissues of the intestine, whereas claudin-5 was seen mainly in the epithelial tight junctional areas and within the endothelial tight junctions of blood vessels. Claudin-16 was present only within the goblet cells and smooth muscle layers of the intestine. An analysis of developmental patterns also revealed differences in expression of the three claudins. Claudin-16 appeared within the intestinal muscle layers at 14 days of embryonic development, and became detectable in goblet cells between 19-20 days. Claudin-3 staining was observed as early as 12 days as was claudin-5 within
the epithelial cells. However, claudin-5 was not found in the tight junctional regions until day 20. We have also utilized Western blots to compare the three claudins. Homogenates of the three regions of the intestinal tract confirmed that claudins-3, -5, and -16 were present in 1-day old hatched chicks. Claudin-3 and claudin-5 were most abundant in the duodenum and least in the ileum whereas Claudin-16 had the opposite distribution. Claudin-16 was also detected in homogenates of epithelial cells isolated from the small intestine. These results support our hypothesis that all three claudins are involved the epithelial differentiation of embryonic and post-hatch chick intestine.
Impacts
This work has revealed the unexpected presence claudin-16 in intestinal goblet cells. We anticipate that claudin-16 and the tight junctional proteins, claudin-3 and claudin-5 will be found to play a role in epithelial cell differentiation and/or mucus secretion. Since an intact epithelium and mucus secretion are crucial to maintaining the intestinal barrier against pathogens, the role of claudins could be important in the health and consequent growth rate of young chicks.
Publications
- No publications reported this period
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Progress 10/01/04 to 09/30/05
Outputs
Additional work has been completed concerning the presence of claudin-16 in goblet cells of chick intestine. We have confirmed that claudin-16 is present in intestinal goblet cells by 19-20 days of embryonic development. By one day post-hatch, the goblet cells are distributed in a gradient along the villus, with more goblet cells in the distal regions. Goblet cells are also present in the intestinal crypts, as confirmed by alcian blue staining of goblet cell mucin. Claudin-16 is more prominent in goblet cells located distally on the villus and is completely absent from goblet cells within the crypts, suggesting that claudin plays a role in functional differentiation as villus epithelial cells migrate from villus crypt to tip. We have also detected large amounts of claudin-16 in intestinal goblet cells of 6-week old chickens and in adult mice. Hence this claudin is not species specific and is not found only at embryonic and neonatal stages. We have initiated a new line
of investigation to determine whether goblet cells contain claudin-16 when embryonic intestine is maintained in an organ culture system. Preliminary results indicate that claudin appears sooner in organ-cultured chick intestine than in vivo. We have detected claudin-16 in goblet cells of 14-day embryonic intestine cultured for 3 days and in 18-day intestine cultured for 24 hours. This finding parallels the acceleration of goblet cell differentiation in culture previously reported by our laboratory and lends support to the hypothesis that claudin-16 is involved in cell differentiation.
Impacts
This work has revealed the unexpected presence claudin-16 in intestinal goblet cells. We anticipate that claudin-16 will be found to play a role in goblet cell differentiation and/or mucus secretion. Since mucus secretion is crucial to maintaining the intestinal barrier against pathogens, the role of claudin-16 could be important in the health and consequent growth rate of young chicks.
Publications
- O. Ozden, B.L. Black, and B. Brizuela. 2004. Novel role of claudin 16 tight junctional protein in the goblet cells of chick intestine. Molecular Biology of the Cell 15: 68a.
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Progress 10/01/03 to 09/30/04
Outputs
Tight junction (TJ) proteins form major barriers in epithelial and endothelial tissues and are responsible for regulating paracellular transport. Claudin protein family members are important structural and functional components of the tight junction complex. Mutations in Cla16 have been linked to impaired reabsorption of magnesium and calcium leading to kidney disease, and Cla16 is reported to play a role in forming aqueous pores in the paracellular pathway within Henle's loop. This work investigates, for the first time, the localization of Cla16 in the intestine of chick embryos during the week before hatching and in one-day old chicks. The localization of Cla16 was determined in sections of frozen and paraffin-embedded tissue from duodenum, jejunum and ileum using an immunostaining protocol. Interestingly, the expression of Cla16 was primarily detected in two regions of chick intestine: 1) goblet cells within the intestinal epithelium and 2) smooth muscle layers. A
gradient in Cla16-staining was present along the crypt-villus axis in duodenum, jejunum and ileum with more claudin-containing goblet cells in the upper half of villi and none in the crypts. This differed from the pattern of mucus-containing goblet cells (ascertained by staining with alcian blue) in that goblet cells were present in similar numbers from crypt to villus tip. Cla16 was not detectable in embryonic epithelium until day 19 (2 days before hatching) even though goblet cells containing mucus appeared at 14 days and were abundant by day 18. Cla16 was present in embryonic circular muscle from 14 days (the earliest stage examined) through posthatch. Light microscopy of the goblet cell associated Cla16 suggests that it resides within the membranes of mucus granules. Thus it may play a role in the mucus secretion process of mature goblet cells, possibly by creating calcium channels or junctional complexes during exocytosis. Cla16 in the smooth muscle fibers might function in
calcium movement between cells or across organelle membranes. Preliminary work with other claudins has revealed the presence of Cla3 and Cla5 within the epithelial tight junctions of chick epithelium. Further studies are underway to elucidate the novel role of TJ proteins in goblet cells and smooth muscle and further explore the developmental pattern and functional relevance of claudins 3 and 5.
Impacts
Elucidation of the roles of claudins in intestinal function can shed light on the mechanism of mucus secretion (Cla16) and the integrity of the epithelial barrier (Cla3 and Cla5). Mucus secretion and the epithelial barrier are both crucial in thwarting infectious disease, diarrheal diseases are often associated with inappropriate ion flux across epithelial tight junctions, and an intact epithelial barrier is necessary for optimal function of nutrient transport systems. Thus an understanding of the role that claudins play in epithelial function could lead to better prevention and/or treatment of intestinal diseases in poultry as well as to improvement of nutrient absorption and consequent growth rates in young chicks.
Publications
- Ozden, Ozkan. 2004. Developmental profile of claudin-3, claudin-5 and claudin-16 tight junction proteins in the chick intestine. Thesis, North Carolina State University.
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