Embryonic Chick Intestine Research Articles

Hox gene activity directs physical forces to differentially shape chick small and large intestinal epithelia.

Hox transcription factors play crucial roles in organizing developmental patterning across metazoa, but how these factors trigger regional morphogenesis has largely remained a mystery. In the developing gut, Hox genes help demarcate identities of intestinal subregions early in embryogenesis, which ultimately leads to their specialization in both form and function. Although the midgut forms villi, the hindgut develops sulci that resolve into heterogeneous outgrowths. Combining mechanical measurements of the embryonic chick intestine and mathematical modeling, we demonstrate that the posterior Hox gene HOXD13 regulates biophysical phenomena that shape the hindgut lumen. We further show that HOXD13 acts through the transforming growth factor β (TGF-β) pathway to thicken, stiffen, and promote isotropic growth of the subepithelial mesenchyme-together, these features lead to hindgut-specific surface buckling. TGF-β, in turn, promotes collagen deposition to affect mesenchymal geometry and growth. We thus identify a cascade of events downstream of positional identity that direct posterior intestinal morphogenesis.

Developmental Profile of Claudin‐3, ‐5, and ‐16 Proteins in the Epithelium of Chick Intestine

Proteins in the claudin family are a main component of tight junctions and form a seal that modulates paracellular transport in intestinal epithelium. This research tests the hypothesis that claudins 3, 5, and 16 will appear in the epithelium of embryonic intestine during functional differentiation. Immunohistochemistry is utilized to explore the developmental patterns of claudin-3, -5, and -16 proteins in the epithelium of embryonic chick intestine from 9 days prior to hatching through the early post-hatch period. These claudin proteins either changed their cellular localization or first appeared around the time of hatching. After hatching, claudin-3 expression was prominent in basal-lateral regions of the epithelium along the entire villus, but was absent from crypts. Claudin-5 was expressed most strongly in the crypt and lower villus epithelium within junctional complexes, whereas immunostaining of claudin-16 was localized within goblet cells of the upper villus region. The relative mRNA levels of claudin-3, -5, and -16 showed similar patterns; transcript levels rose between 18 and 20 days of development, then dropped by 2 days post-hatch. Results of this work indicate that the claudin proteins assume their final locations within the epithelium around the time of hatching, suggesting that in addition to their known barrier and fence functions within tight junctions, these claudins may have additional roles in the differentiation and/or physiological function of chick intestine. The localization of claudin-16 to goblet cells and its distribution in the more mature cells of the upper villus region suggest an unexpected role in goblet cell maturation and mucus secretion.

Regulation of goblet cell differentiation by calcium in embryonic chick intestine

The potential role of calcium as a regulator of goblet cell differentiation was tested by culturing duodenal explants from 14-day chicken embryos in media containing a range of calcium concentrations. Extracellular calcium in vitro approximated the range of plasma calcium concentration that was found to rise by 16% during the third week of embryonic development. As ionic calcium was increased from 0.9 to 2.0 mM in 48-h cultures, the number of goblet cells per 100 ridges increased progressively from 29 +/- 2.2 to 158 +/- 6.9 while attaining a distribution on the previllous ridges similar to that in ovo. The rate of goblet cell differentiation in vitro exceeded that in ovo when extracellular calcium was increased to 1.1 mM and was maximal at 1.6-2.0 mM calcium. Neither the growth of previllous ridges nor gross morphology of the tissue was altered as extracellular calcium was increased. Goblet cell differentiation in 1.3 mM calcium was stimulated by 0.1 microM calcium ionophore and inhibited by the calmodulin antagonist trifluoperazine, indicating an intracellular site for calcium action. These results indicate that calcium plays a primary role in regulating goblet cell differentiation in embryonic intestine and suggest that rising levels of plasma calcium influence the rate of differentiation in ovo.

Calcium and Inorganic Phosphate Transport in Embryonic Chick Intestine: Triiodothyronine Enhances the Genomic Action of 1,25-Dihydroxycholecalciferol

The influence of triiodothyronine (T3) on the induction of intestinal calcium and inorganic phosphate (Pi) transport by 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) was studied in 48 h cultures of embryonic chick jejunum. While T3 alone had no effect on calcium uptake by gut segments cultured on d 20 of embryonic development, the thyroid hormone amplified the effect of 1,25-(OH)2D3 on calcium transport and effectively shifted the dose-response curve to lower 1,25-(OH)2D3 concentrations. T3 had a dual effect on Pi uptake by cultured jejunum: It induced transport activity even in the absence of the steroid hormone, and, in addition, synergistically raised 1,25-(OH)2D3-related Pi uptake. In d 17 embryonic small intestine, which does not respond to 1,25-(OH)2D3 by a significant increase in Pi transport, T3 permitted the induction of Pi transport by the sterol. In general, the thyroid hormone enhanced the responsiveness of cultured embryonic intestine toward 1,25-(OH)2D3 by two orders of magnitude, resulting in facilitated induction of calcium and Pi transport by the sterol and, in particular, modulated the stage-specific expression of 1,25-(OH)2D3 action on intestinal Pi transport.

Purification and properties of an 18-kilodalton, 1,25-dihydroxyvitamin D3-modulated protein from embryonic chick intestine

An 18,000-dalton protein (pI = 5.1) shown previously to be modulated by 1,25-dihydroxyvitamin D3 was purified to allow its further characterization. This protein from embryonic chick intestine was shown to comigrate during two-dimensional electrophoresis with an abundant protein from the intestine of 4-week-old chickens. The protein was purified from 4-week chick intestine and analyzed for amino acid composition, and 28 amino acids of its N-terminal sequence were determined. The N-terminal amino acid sequence had significant homology to cellular retinol binding protein II, an intestinal protein that has been recently sequenced. The purified 18-kilodalton protein was shown to bind retinol by fluorescence spectrophotometry. This 18-kilodalton protein is dramatically changed by 1,25-dihydroxyvitamin D3 in the chick embryonic organ culture system. Therefore, further study of it may lead to a better understanding of vitamin A and D interaction and how 1,25-dihydroxyvitamin D3 acts through proteins to stimulate intestinal calcium and phosphate transport.

Development of glucose active transport in embryonic chick intestine. Influence of thyroxine and hydrocortisone

1. 1. Glucose active transport is detectable in 12-day-old embryonic chick duodenum and increases at least 11-fold after 4 days of postnatal life. 2. 2. Glucose active transport develops at the in vivo rate in 72-hr cultures of 14-day embryonic duodenum. 3. 3. In the presence of either 1 nM thyroxine or 1 μM hydrocortisone in vitro, glucose active transport reaches levels approximately 200% of control values (equivalent to 18–19 day levels in vivo). 4. 4. Thyroxine and hydrocortisone act by different mechanisms based on their antagonistic interaction and differences in time course of action, requirement for protein synthesis and modulation by extracellular calcium.

1 alpha,25-Dihydroxyvitamin D-induced modification of a cytosolic protein in embryonic chick intestine.

Two-dimensional electrophoresis together with radiolabeling experiments was used to examine cytosolic proteins of embryonic chick duodenum for responses to 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 caused a striking decrease in [3H]leucine content of an 18,000-dalton protein (approximate pI, 5.1) after a 10-min pulse with radioisotope followed by a 4-h chase. Decreased [14C]leucine content of the same protein was also observed at various times following 1,25-dihydroxyvitamin D3 addition to culture media; a significant decrease in radiolabel incorporation occurred within 30 min after addition of the hormone. The results argue that 1,25-dihydroxyvitamin D3 causes either a decreased synthesis rate or a post-translational modification of this protein. This change joins the biosynthesis of calcium-binding protein as an early event in the response of chick embryonic intestine to 1,25-dihydroxyvitamin D3.

Cells isolated from embryonic intestine synthesize 1,25-dihydroxyvitamin-D3 and 24,25-dihydroxyvitamin-D3 in culture.

Cells isolated from embryonic chick intestine in culture convert 25-hydroxyvitamin-D3 to a number of more polar metabolites. Two of these metabolites have been identified with chemical and chromatographic methods as 24,25-dihydroxyvitamin-D3 (24,25(OH)2D3) and 1,25-dihydroxyvitamin-D3 (1,25(OH)2D3). The enzymes conform to substrate saturation kinetics. The apparent Km for substrate for the synthesis of 1,25(OH)2D3 and 24,25(OH)2D3 is 70 nM and 29 nM, respectively.

Competence of 1 alpha, 25-dihydroxyvitamin D3 receptor system in embryonic chick intestine.

The competence of the duodenal 1α, 25-(OH)2-D3 receptor system in embryonic chicks was studied in the reconstituted cytosol-chromatin system. The sucrose density gradient centrifugation following incubation of duodenal cytosol with 1α, 25-dihydroxy-[3H]-vitamin D3 revealed that a cytosol 1α, 25-dihydroxyvitamin D3 receptor existed in the duodenum of 18-day-old embryos. In the duodenum of 14-day-old embryos, however, the cytosol 1α, 25-dihydroxyvitamin D3 receptor was not detected. In the reconstituted cytosol-chromatin system, duodenal cytosol of 18day-old embryos stimulated the association of 1α, 25-dihydroxy-[3H] vitamin D3 with chromatin of the vitamin D-deficient chick duodenum. The association of 1α, 25-dihydroxy-[3H]-vitamin D3 was shown to be saturable and temperature dependent. In a system where duodenal chromation of 18-day-old embryos was reconstituted with cytosols, 1α, 25-dihydroxy-[3H]-vitamin D3 was revealed to associate specifically with the chromatin in the presence of duodenal cytosol obtained from 18-day-old embryos or vitamin Ddeficient chicks. No significant association of 1α, 25-dihydroxy-[3H]vitamin D3 with duodenal chromatin of 18-day-old embryos occurred in the absence of duodenal cytosol. From these results, it was strongly suggested that the intestinal 1α, 25dihydroxyvitamin D3 receptor system in embryonic chicks has become competent for the intracellular transfer of 1α, 25-(OH)2-D3 before hatching.

Embryonic chick intestine in organ culture: Hydrocortisone and vitamin D-mediated processes

The effects of the glucocorticoid, hydrocortisone (HC), on vitamin D-mediated responses were examined in the organ-cultured, embryonic chick duodenum. In this system tissue responses to vitamin D-steroids in the culture medium include increased cAMP concentration, de novo synthesis of a specific calcium-binding protein (CaBP), enhanced uptake and transmucosal transport of calcium, and increased alkaline phosphatase activity. HC at levels ≥ 27.5 n m increased vitamin D-induced CaBP concentration: This apparently represents the first report of an interaction of HC with another steroid, vitamin D, in the regulation of the concentration of a specific protein. High levels of HC (≥27.5 μ m) in the culture medium reduced duodenal calcium uptake and transmucosal transport regardless of the presence of vitamin D. However, at lower concentrations of HC (≤2.75 μ m), only vitamin D-independent calcium uptake (basal calcium uptake) was reduced. Actinomycin D had no effect on HC reduction of basal calcium uptake suggesting that new protein synthesis is not involved in this action. In other experiments either HC or vitamin D stimulated phosphate and glucose uptake, and this uptake was potentiated by the presence of both steroids. HC also stimulated alanine uptake. Either HC or vitamin D increased both alkaline phosphatase activity (APA) and cAMP concentration, but together their activities were only additive. The data accumulated thus far indicate that HC directly influences calcium (and other nutrient) uptake by the duodenum and increases the concentration of the vitamin D-induced CaBP. Other vitamin D-mediated responses (APA and cAMP) were influenced by HC but there was no readily discernible relationship to nutrient uptake.

Morphological development of the epithelium of the embryonic chick intestine in culture: influence of thyroxine and hydrocortisone.

A culture system with defined medium has been utilized to elucidate the role of thyroxine (T4) and hydrocortisone (HC) in growth and differentiation of the epithelium of embryonic chick duodenum during the third week of development. Duodenal explants maintained a basically normal morphology for 48 to 72 hours, but during subsequent culture, muscle and mesenchyme deteriorated, previllous ridges shrank, the epithelial surface became pitted, and the tissue lost weight. These degenerative processes were retarded by addition of HC to the culture medium. Previllous ridges of cultured duodenum failed to develop into true villi and ridge growth was subnormal. Epithelial mitotic counts were increased by T4 or HC, as compared to cultured control tissue, but dropped below values in vivo, by 72 hours in culture. Epithelial differentiation proceeded more rapidly in duodenum cultured without hormones than in vivo. Increase in cell height, flattening of the epithelial surface, attainment of uniform staining of the brush border with periodic acid-Schiff, increase in microvillar density, and formation of a terminal web were accelerated by 24 to 48 hours in culture. In the presence of T4, cell height and microvillar growth were stimulated further, reaching the state found at 19 to 21 days in vivo.

Alkaline phosphatase and maltase activity in the embryonic chick intestine in culture: Influence of thyroxine and hydrocortisone

In strips of duodenum from 14-day chick embryos explanted into defined medium, alkaline phosphatase (ALP) activity accumulated in the tissue at a faster rate than in vivo for about 48 hr, but failed to increase thereafter. The addition of thyroxine (T4) to the medium at 10 −8 M or less both enhanced the early accumulation and elicited a very large increase in ALP activity comparable to that normally occurring during the last 2 days in ovo. Activity with phenylphosphate (PhP) was more strongly affected than that with β-glycerophosphate (βGP), so that the high PhP β GP ratio attained in vivo at 18 days was reached after 24 hr in T4-supplemented medium. Hydrocortisone (HC) evoked APL activity only slightly above that in unsupplemented medium and only during the first 48 hr in culture, but it precociously elevated PhP β GP ratio to the normal maximum. In the presence of T4 or HC, maltase activity rose in explanted strips at the same rate as in the intact duodenum, but it lagged in unsupplemented medium. Assay of the medium revealed, however, that under all conditions of culture a large amount of both maltase and ALP activity had been released from the tissue. This effect was especially pronounced in the presence of T4, so that explants and medium together accumulated ALP and maltase equivalent to the high peaks of activity found in the intact duodenum at hatching. With 10 −8 M T4, ALP activity began to rise above that of control explants after 8 hr, with accumulation in the medium beginning about 4 hr later. Combining 2 × 10 −6 M HC with a range of T4 concentrations produced greater than additive effects, particularly with ALP, but did not lead to enhanced retention of either enzyme in the tissue strips. Prolactin, pentagastrin, and insulin were without effect alone, but the latter inhibited the effects of both T4 and HC.

Discrimination between nicotinic receptors in vertebrate ganglia and skeletal muscle by alpha-bungarotoxin and cobra venoms.

1. We have used snake neurotoxins, alpha-bungarotoxin and venoms from Naja naja siamensis and Naja nivea, to distinguish the nicotinic receptors of ganglia from those of skeletal neuromuscular junctions. 2. These neurotoxins failed to block responses of isolated guinea-pig longitudinal muscle with adherent myenteric plexus to the nicotinic agonists, nicotine or dimethylphenylpiperazinium, to acetylcholine (ACh), or to electrical field stimulation. 3. The toxins failed to affect responses of the isolated guinea-pig stomach to pregnaglionic stimulation by way of the vagus nerves or of the vas deferens to preganglionic stimulation via the hypogastric nerves. 4. Snake neurotoxins did not block non-adrenergic inhibitory responses of the rabbit small intestine to nicotine or electrical field stimulation. 5. Neurotoxins were ineffective blockers against nicotinic agonists in new-born rabbit or embryonic chick intestine. 6. Attempts to increase the penetration of the toxins into tissues with dimethylsulphoxide, exposure to hypertonic solutions, or to ethylene-diaminetetracetic acid did not enable the toxins to act as nicotinic antagonists. 7. In contrast to diaphragmatic or oesophageal skeletal neuromuscular junctions no binding of rhodamine or tritium labelled toxins to structures in ganglia could be detected. 8. No potential permeability barriers were found by electron microscopy of the ganglia of the guinea-pig myenteric plexus. 9. The tracers, lanthanum ion and ruthenium red, readily penetrated into all regions of the myenteric plexus including synaptic gaps. 10. It is concluded that the failure of snake neurotoxins to act as nicotinic antagonists or to bind to ganglia is not due to their inability to reach ganglionic nicotinic receptors. Therefore, it is likely that ganglionic nicotinic receptors are different from those of the skeletal neuromuscular junction.

Embryonic chick intestine in organ culture: Stimulation of calcium transport by exogenous vitamin D-induced calcium-binding protein

Vitamin D 3 or a potent metabolite, 1,25-dihydroxycholecalciferol, induces calcium-binding protein (CaBP) synthesis and stimulates transmucosal calcium transport in embryonic chick duodena maintained in novel organ culture apparatus. When added to the sterol-free culture medium, highly purified chick intestinal CaBP, similarly and specifically, stimulates calcium transport in the cultured duodena. These results clearly demonstrate the involvement of CaBP in intestinal calcium transport.

Studies on calciferol metabolism: Metabolism of 25-hydroxy-Vitamin D 3 by the chicken embryo

It is known that after birth of a vertebrate there is a requirement for the metabolism of Vitamin D 3 (cholecalciferol) to 1,25-(OH) 2-Vitamin D 3 to produce the hormonally active form essential for calcium homeostasis. However it is not known whether the enzymatic capability to produce 1,25-(OH) 2-D 3 only appears after birth or whether it is generated in the embryo. Presented in this paper are results of studies designed to measure the production and localization of 1,25-(OH) 2-D 3 in the embryo. It was found that the renal enzyme, 25-OH-cholecalciferol-1-hydroxylase, which is capable of producing 1,25-(OH) 2-D 3, is present as early as day 9 of incubation (12 days before hatch) in White Leghorn chicks. Further, the enzyme activity increases 6-fold to a maximal level which occurs on the day of hatching. 1,25-(OH) 2-D 3 was shown to be produced in vivo at day 17 and was found then in low levels in the embryonic intestine and kidney. Thus we have shown that 1,25-(OH) 2-D 3 is made by embryonic chick kidneys and is found in low levels in embryonic chick intestine and kidney significantly before hatch.

Embryonic chick intestine in organ culture: response to vitamin D 3 and its metabolites.

Embryonic chick intestine maintained in organ culture responded to vitamin D(3) and its metabolites 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol by synthesis of calcium-binding protein and enchanced calcium-45 uptake. The dihydroxy metabolite was by far the most potent inducer of the protein and also acted more rapidly than vitamin D(3) to stimulate isotope uptake. Despite its lower potency, vitamin D(3) itself was effective.

Vitamin D3: induction of calcium-binding protein in embryonic chick intestine in vitro.

Induction of the synthesis of calcium-binding protein in chick embryonic intestine maintained in vitro was accomplished by simply adding vitamin D(3) to the culture medium. Accompanying the induction of this protein, there was enhanced radiocalcium uptake by the intestine. These observations represent the first demonstration of an in vitro physiological effect of vitamin D(3) on the calcium absorptive mechanism of the intestine.

EFFECT OF PUROMYCIN ON CHOLINESTERASE ACTIVITY OF EMBRYONIC CHICK INTESTINE IN ORGAN CULTURE.

THE acetylcholinesterase (AChE) activity of a number of chick embryo tissues cultured in vitro can be influenced by the presence of acetylcholine (ACh) or other substrates in the nutrient media1–4. For example, in the presence of 0.02 M ACh, the AChE activity of primary explants of chick embryo lung increased 3–7-fold over a period of four days3. It would seem that a case of enzyme induction had been demonstrated. However, no direct evidence has been provided which would support this hypothesis. This report presents evidence that certain changes in AChE and cholinesterase (ChE) activity of chick embryo intestine maintained in vitro are due to changes in protein synthesis, a finding that is compatible with the hypothesis that the synthesis of AChE can be induced.

Growth of Entamoeba invadens in organotypic cultures of embryonic chick intestine.

Entamoeba invadens from axenic and monoxenic cultures was inoculated into expiants of embryonic chick intestine, which were then cultured in perfusion chambers at 30 °C. The growth and metabolic activity of the explants in cultures were evaluated in terms of fibroblastic outgrowth and extent of liquefaction of the plasma clots in which they were embedded. The effects of several media used to fill the perfusion chambers on the survival of the explants were studied. It was found that amoebae developed best in those explants which themselves showed most vitality; this was in turn related to the kind of fluid medium used in the culture. Amoebae in the explants fed on mucous secretion and on dead cells and penetrated into intact tissue without apparent histolytic activity. It is suggested that the living explants provided the amoebae with certain enzymes which the latter were unable to produce at the temperature of incubation. Approximately 40% of all cultures made became positive for amoebae. This is attributed to the fact that not all explants retained the amoebae injected into them, before they were placed in culture.

Cell growth of hydrophobic substrates.

1. Embryonic chick liver and intestine expiants give rise to monolayer epithelial sheets when planted on a water-repellent silicone substratum or on glass in the presence of a serum exsudate. 2. Mitotic activity of fibroblasts from embryonic chick heart is lower on silicone than on glass while the areas of outgrowth differ lesser. 3. The presence of serum is necessary for the attachment and proliferation of cells in primocultures on plane hydrophobic substratum. 4. The possible mechanism of this phenomenon is briefly discussed.