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The tadpole gut shortens dramatically during metamorphosis. The components of the gut depicted here are as follows: es, esophagus; st, stomach; du, duodenum; il, ileum; co, colon; re, rectum. The duodenum and part of the ileum comprise the intestine’s outer loops that coil in a counterclockwise direction (ventral perspective). The ileum reverses direction at the switchback point (arrowhead) and coils clockwise with the colon to form the intestine’s inner loops. The rectum is the final internal structure of the gut. ( A ) The gut of a premetamorphic NF 54 tadpole ( Inset ) is shown both in situ (upper) and excised and uncoiled (lower). ( B ) A prometamorphic NF 58 tadpole ( Inset ) has a much larger and longer gut than an NF 54 tadpole. ( C ) In a tadpole at metamorphic (NF 62) climax ( Inset ), the length of the gut has begun to shorten, and the number of outer and inner coils has decreased. ( D ) By the end of metamorphosis (NF 66) ( Inset ), the gut has shortened by Ϸ 75% of its original length. (Scale bars: 2 mm.)
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Thyroid hormone controls remodeling of the tadpole intestine during the climax of amphibian metamorphosis. In 8 days, the Xenopus laevis tadpole intestine shortens in length by 75%. Simultaneously, the longitudinal muscle fibers contract by about the same extent. The radial muscle fibers also shorten as the diameter narrows. Many radial fibers unde...
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... TH. A second class is the inverse of the first; gene expression is high before climax, then drops at climax (‘‘eclipses’’) and rises again at the end of metamorphosis. This eclipse has also been described for genes expressed specifically in the epithelium of the posterior intestine (6) and in the epithelium of the stomach (8). To date, no gene that is expressed exclusively in the larval intestine has been identified. This paper addresses two features of intestinal remodeling: the phenomenon of shortening and narrowing, and the remodeling of the intestinal epithelium. We describe the TH-induced contraction of longitudinal muscle fibers that accompanies intestinal shortening and the contraction and death of radial muscles as the diameter narrows. During this time, epithelial cells facing the lumen undergo cell death. A net effect of these size changes is transient ‘‘heaping’’ of the epithelium into many layers. In situ hybridization of genes that are expressed in the epithelium reveals a uniform change in all of the epithelial cells as the intestine transforms into its convoluted state of crypts and villi. Similarly, DNA replication occurs uniformly throughout the larval epithelium until days after metamorphosis is completed, when it localizes to the intestinal crypts. chemistry, and in Situ Hybridization. Tadpoles were staged by the tables of Nieuwkoop and Faber (NF) (9). Gastrointestinal tracts of tadpoles at different stages were fixed in 4% paraformalde- hyde, embedded in OCT compound, and cryosectioned as described (10, 11). Sections were processed for in situ hybridization (10, 11) by using digoxigenin-labeled antisense probes against Musashi-1 (GenBank accession no. BI447711), intestinal fatty-acid binding protein (a gift of Yun-Bo Shi, National Institute of Child Health and Human Development, Bethesda), and TH-controlled basic leucine zipper (12). Sections were also processed by using immunohistochemistry (10, 11) with mono- clonal antibodies against smooth muscle actin (Sigma), active caspase-3 (Pharmingen), and epithelial cadherin (product number 5D3, University of Iowa Developmental Studies Hybridoma Bank, Iowa City), or a polyclonal antibody against fibronectin (a gift of D. W. DeSimone, University of Virginia, Charlottesville) by using methods previously described (13–15). Antibodies were detected by using Alexa Fluor 488- and 568-conjugated secondary antibodies (Molecular Probes). Intestines of some NF stage 50 (NF 50) and NF 54 tadpoles were processed in whole-mount by immunohistochemistry as described (13, 14). These intestines were sliced open longitudinally and flat-mounted onto slides with either the serosa or mucosa side facing the coverslip. To identify proliferating cells, live tadpoles were injected i.p. with 10 l of BrdUrd (10 mM) 12 h before fixation with 2% trichloroacetic acid for 2 h at room temperature. Intestines were rinsed in PBS three times for 15 min, embedded in OCT compound, cryosectioned, and then counterimmunostained with a primary antibody against fibronectin (a mesenchyme-specific marker) and Alexa Fluor 568-conjugated secondary antibody. Slides were then incubated in 4 M HCl for 1 h at room temperature, rinsed three times for 5 min in PBS, incubated in ice-cold ethanol ͞ acetic acid (2:1) for 8 min, rinsed three times for 5 min in PBS, blocked for 30 min in PBS with 10% normal goat serum, incubated for 1 h at room temperature with Alexa Fluor 488-conjugated-anti-BrdUrd (Molecular Probes) (1:30) in PBS with 10% NGS, rinsed in PBS three times for 15 min, and mounted with a coverslip. NF 50 and 54 tadpoles were induced to metamorphose with 10 nM T3 as described (13, 14). Statistics and Experimental Design. To measure intestine shortening during spontaneous metamorphosis, the gastrointestinal tracts of sibling tadpoles at various stages ( n ϭ 8 –10) were excised, and lengths were measured for the anterior intestine (duodenum ϩ anterior ileum) and the posterior intestine (posterior ileum ϩ colon ϩ rectum). The junction between the anterior and posterior ileum is the ileum’s switchback point (see Fig. 1). Differences in mean length with developmental stage were assessed statistically by one-factor ANOVA (Su- perANOVA, Abacus Concepts, Berkeley, CA), followed by Fisher’s pairwise comparisons. P values of Ͻ 0.01 were considered significant. To measure precocious intestinal shortening with T3 treatment, NF 54 tadpoles were induced with 10 nM T3 for 7 days. Tadpoles were sampled every 24 h ( n ϭ 6), and total intestine lengths (duodenum to rectum) were measured. Changes in length with time of T3 treatment were assessed statistically by one-factor ANOVA. P values of Ͻ 0.01 were considered significant. Before metamorphosis in X. laevis , the tadpole intestine consists of two spiral coils: the outer coil (duodenum and anterior ileum) reverses direction at the switchback point and is followed by the inner coil (posterior ileum and colon), which terminates at the rectum (Fig. 1 A and B ). The climax of metamorphosis (NF 59–65) is the period of maximal change when the endogenous TH is at its highest concentration (16). During climax, which lasts Ϸ 8 days, the intestine shortens by Ϸ 75% (Fig. 1 C and D and Table 1). From the start (NF 59) to the end (NF 66) of metamorphic climax, the percentage of shortening of the anterior and posterior intestine is similar (78.5% and 71.9%, respectively) (Table 1), supporting the findings of Pretty et al. (2) that the intestine shortens equally along its entire length. The premetamorphic tadpole gut (NF 46–54) can be induced to shorten precociously by treatment with 10 nM T3 for 3–7 days (Table 2). Shortening of the intestine during spontaneous metamorphosis is accompanied by a change in cross-sectional morphol- ogy. The premetamorphic tadpole duodenum (NF 54) is a simple thin tube with one large involution (the typhlosole) in the anterior intestine (Fig. 2 A ). At the end of metamorphosis (NF 66), the epithelium is structured into crypts and villi (Fig. 2 D ). As the mesenchyme and muscle layers thicken at metamorphic climax (NF 62 and NF 63, Fig. 2 B and C , respectively), the intestinal epithelium temporarily thickens to five- to eight-cell layers (Figs. 2 B Ј and C Ј ), compared with a thickness of one to two cells before (NF 54, Fig. 2 A Ј ) and after (NF 66, Fig. 2 D Ј ) metamorphosis. The expression of genes with different developmental profiles in the anterior intestinal (duodenal) epithelium has been assessed (Fig. 3). We chose the neural stem cell marker Musashi (17) because nests of adult epithelial precursor cells in the intestine were reported to express specifically this gene (18). Musashi mRNA is expressed constitutively in all cells of the epithelium at every developmental stage from NF 54–66 (Fig. 3 A ). Intestinal fatty acid binding protein is expressed in the anterior tadpole intestinal epithelium, shut off (eclipsed) during climax, and then expressed again after climax (5, 19). Every epithelial cell follows this gene expression pattern (Fig. 3 B ). TH ͞ basic leucine zipper (12) is a direct response gene expressed in the intestinal epithelium and the mesenchyme. The expression of this gene rises and falls with the endogenous TH concentration (Fig. 3 C ). Our in situ hybridization experiments demonstrate the uniformity of these gene expression profiles in the intestinal epithelium at each stage. Intestinal epithelial cell proliferation is very low before climax (20) (Fig. 4 A ), but increases greatly in all layers of the heaped epithelium at climax and throughout the epithelium at a high level even late in metamorphosis after the crypts and villi have been formed (NF 65, Fig. 4 E ). Proliferation in the mesenchyme increases during climax, reaches a peak at NF 61, and drops by NF 66. Because BrdUrd is a nuclear marker, and mesenchyme cells are mixed with muscle cells, we could not reliably evaluate muscle proliferation. Apoptosis within the anterior intestine, as measured by active caspase-3 immunoreactivity, is present in a small number of epithelial cells before climax (20) (Fig. 5 A ). By NF 61, when the epithelial cells are heaped, apoptosis occurs mainly in the cells that line the lumen (Fig. 5 B ). Many radial, but not longitudinal, muscle fibers in the ileum are immunoreactive for active caspase-3, but fewer are labeled in the duodenum (data not shown). By the end of metamorphosis (NF 66), apoptosis is confined primarily to epithelial cells located at the tips of the intestinal villi (Fig. 5 C ). Individual longitudinal smooth muscle fibers in the ileum of a NF 54 tadpole are Ϸ 160 m in length (Fig. 6 A ). After 72 h of T3 treatment, muscle fibers are much shorter ( Ϸ 90 m in length) than untreated fibers (Fig. 6 B ). This decrease in length by Ϸ 44% corresponds to the decrease in total intestine length after 72 h of T3 treatment (Table 2). Intestinal muscle fibers also shorten during spontaneous metamorphosis, although muscle fiber lengths become difficult to measure in vitro beyond NF 61, as individual fiber ends are not easily distinguished (data not shown). In a sagittal view of similarly treated tadpole intestine, the duodenal diameter decreases by one-third, and the epithelium collapses into folds (presumably because of longitudinal compression by the muscle fibers), but remains as a single-cell layer (Fig. 6 C and D ). Intestinal Shortening. The abrupt shortening of the anuran gut during metamorphosis has been well documented. The amount of shortening varies among species, but reports range from 58% for Rana temporaria (21) to as high as 90% in Alytes obstetricans (22). In our experiments, the X. laevis intestine shortens by 76% from the beginning of metamorphosis at NF 60 through its completion by NF 66, and most of the shortening is completed by the start of NF 62 (Table 1). The premetamorphic tadpole gut (NF 50–54) can be induced to shorten precociously by the same amount after treatment with TH ...
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... expressed specifically in the epithelium of the posterior intestine (6) and in the epithelium of the stomach (8). To date, no gene that is expressed exclusively in the larval intestine has been identified. This paper addresses two features of intestinal remodeling: the phenomenon of shortening and narrowing, and the remodeling of the intestinal epithelium. We describe the TH-induced contraction of longitudinal muscle fibers that accompanies intestinal shortening and the contraction and death of radial muscles as the diameter narrows. During this time, epithelial cells facing the lumen undergo cell death. A net effect of these size changes is transient ‘‘heaping’’ of the epithelium into many layers. In situ hybridization of genes that are expressed in the epithelium reveals a uniform change in all of the epithelial cells as the intestine transforms into its convoluted state of crypts and villi. Similarly, DNA replication occurs uniformly throughout the larval epithelium until days after metamorphosis is completed, when it localizes to the intestinal crypts. chemistry, and in Situ Hybridization. Tadpoles were staged by the tables of Nieuwkoop and Faber (NF) (9). Gastrointestinal tracts of tadpoles at different stages were fixed in 4% paraformalde- hyde, embedded in OCT compound, and cryosectioned as described (10, 11). Sections were processed for in situ hybridization (10, 11) by using digoxigenin-labeled antisense probes against Musashi-1 (GenBank accession no. BI447711), intestinal fatty-acid binding protein (a gift of Yun-Bo Shi, National Institute of Child Health and Human Development, Bethesda), and TH-controlled basic leucine zipper (12). Sections were also processed by using immunohistochemistry (10, 11) with mono- clonal antibodies against smooth muscle actin (Sigma), active caspase-3 (Pharmingen), and epithelial cadherin (product number 5D3, University of Iowa Developmental Studies Hybridoma Bank, Iowa City), or a polyclonal antibody against fibronectin (a gift of D. W. DeSimone, University of Virginia, Charlottesville) by using methods previously described (13–15). Antibodies were detected by using Alexa Fluor 488- and 568-conjugated secondary antibodies (Molecular Probes). Intestines of some NF stage 50 (NF 50) and NF 54 tadpoles were processed in whole-mount by immunohistochemistry as described (13, 14). These intestines were sliced open longitudinally and flat-mounted onto slides with either the serosa or mucosa side facing the coverslip. To identify proliferating cells, live tadpoles were injected i.p. with 10 l of BrdUrd (10 mM) 12 h before fixation with 2% trichloroacetic acid for 2 h at room temperature. Intestines were rinsed in PBS three times for 15 min, embedded in OCT compound, cryosectioned, and then counterimmunostained with a primary antibody against fibronectin (a mesenchyme-specific marker) and Alexa Fluor 568-conjugated secondary antibody. Slides were then incubated in 4 M HCl for 1 h at room temperature, rinsed three times for 5 min in PBS, incubated in ice-cold ethanol ͞ acetic acid (2:1) for 8 min, rinsed three times for 5 min in PBS, blocked for 30 min in PBS with 10% normal goat serum, incubated for 1 h at room temperature with Alexa Fluor 488-conjugated-anti-BrdUrd (Molecular Probes) (1:30) in PBS with 10% NGS, rinsed in PBS three times for 15 min, and mounted with a coverslip. NF 50 and 54 tadpoles were induced to metamorphose with 10 nM T3 as described (13, 14). Statistics and Experimental Design. To measure intestine shortening during spontaneous metamorphosis, the gastrointestinal tracts of sibling tadpoles at various stages ( n ϭ 8 –10) were excised, and lengths were measured for the anterior intestine (duodenum ϩ anterior ileum) and the posterior intestine (posterior ileum ϩ colon ϩ rectum). The junction between the anterior and posterior ileum is the ileum’s switchback point (see Fig. 1). Differences in mean length with developmental stage were assessed statistically by one-factor ANOVA (Su- perANOVA, Abacus Concepts, Berkeley, CA), followed by Fisher’s pairwise comparisons. P values of Ͻ 0.01 were considered significant. To measure precocious intestinal shortening with T3 treatment, NF 54 tadpoles were induced with 10 nM T3 for 7 days. Tadpoles were sampled every 24 h ( n ϭ 6), and total intestine lengths (duodenum to rectum) were measured. Changes in length with time of T3 treatment were assessed statistically by one-factor ANOVA. P values of Ͻ 0.01 were considered significant. Before metamorphosis in X. laevis , the tadpole intestine consists of two spiral coils: the outer coil (duodenum and anterior ileum) reverses direction at the switchback point and is followed by the inner coil (posterior ileum and colon), which terminates at the rectum (Fig. 1 A and B ). The climax of metamorphosis (NF 59–65) is the period of maximal change when the endogenous TH is at its highest concentration (16). During climax, which lasts Ϸ 8 days, the intestine shortens by Ϸ 75% (Fig. 1 C and D and Table 1). From the start (NF 59) to the end (NF 66) of metamorphic climax, the percentage of shortening of the anterior and posterior intestine is similar (78.5% and 71.9%, respectively) (Table 1), supporting the findings of Pretty et al. (2) that the intestine shortens equally along its entire length. The premetamorphic tadpole gut (NF 46–54) can be induced to shorten precociously by treatment with 10 nM T3 for 3–7 days (Table 2). Shortening of the intestine during spontaneous metamorphosis is accompanied by a change in cross-sectional morphol- ogy. The premetamorphic tadpole duodenum (NF 54) is a simple thin tube with one large involution (the typhlosole) in the anterior intestine (Fig. 2 A ). At the end of metamorphosis (NF 66), the epithelium is structured into crypts and villi (Fig. 2 D ). As the mesenchyme and muscle layers thicken at metamorphic climax (NF 62 and NF 63, Fig. 2 B and C , respectively), the intestinal epithelium temporarily thickens to five- to eight-cell layers (Figs. 2 B Ј and C Ј ), compared with a thickness of one to two cells before (NF 54, Fig. 2 A Ј ) and after (NF 66, Fig. 2 D Ј ) metamorphosis. The expression of genes with different developmental profiles in the anterior intestinal (duodenal) epithelium has been assessed (Fig. 3). We chose the neural stem cell marker Musashi (17) because nests of adult epithelial precursor cells in the intestine were reported to express specifically this gene (18). Musashi mRNA is expressed constitutively in all cells of the epithelium at every developmental stage from NF 54–66 (Fig. 3 A ). Intestinal fatty acid binding protein is expressed in the anterior tadpole intestinal epithelium, shut off (eclipsed) during climax, and then expressed again after climax (5, 19). Every epithelial cell follows this gene expression pattern (Fig. 3 B ). TH ͞ basic leucine zipper (12) is a direct response gene expressed in the intestinal epithelium and the mesenchyme. The expression of this gene rises and falls with the endogenous TH concentration (Fig. 3 C ). Our in situ hybridization experiments demonstrate the uniformity of these gene expression profiles in the intestinal epithelium at each stage. Intestinal epithelial cell proliferation is very low before climax (20) (Fig. 4 A ), but increases greatly in all layers of the heaped epithelium at climax and throughout the epithelium at a high level even late in metamorphosis after the crypts and villi have been formed (NF 65, Fig. 4 E ). Proliferation in the mesenchyme increases during climax, reaches a peak at NF 61, and drops by NF 66. Because BrdUrd is a nuclear marker, and mesenchyme cells are mixed with muscle cells, we could not reliably evaluate muscle proliferation. Apoptosis within the anterior intestine, as measured by active caspase-3 immunoreactivity, is present in a small number of epithelial cells before climax (20) (Fig. 5 A ). By NF 61, when the epithelial cells are heaped, apoptosis occurs mainly in the cells that line the lumen (Fig. 5 B ). Many radial, but not longitudinal, muscle fibers in the ileum are immunoreactive for active caspase-3, but fewer are labeled in the duodenum (data not shown). By the end of metamorphosis (NF 66), apoptosis is confined primarily to epithelial cells located at the tips of the intestinal villi (Fig. 5 C ). Individual longitudinal smooth muscle fibers in the ileum of a NF 54 tadpole are Ϸ 160 m in length (Fig. 6 A ). After 72 h of T3 treatment, muscle fibers are much shorter ( Ϸ 90 m in length) than untreated fibers (Fig. 6 B ). This decrease in length by Ϸ 44% corresponds to the decrease in total intestine length after 72 h of T3 treatment (Table 2). Intestinal muscle fibers also shorten during spontaneous metamorphosis, although muscle fiber lengths become difficult to measure in vitro beyond NF 61, as individual fiber ends are not easily distinguished (data not shown). In a sagittal view of similarly treated tadpole intestine, the duodenal diameter decreases by one-third, and the epithelium collapses into folds (presumably because of longitudinal compression by the muscle fibers), but remains as a single-cell layer (Fig. 6 C and D ). Intestinal Shortening. The abrupt shortening of the anuran gut during metamorphosis has been well documented. The amount of shortening varies among species, but reports range from 58% for Rana temporaria (21) to as high as 90% in Alytes obstetricans (22). In our experiments, the X. laevis intestine shortens by 76% from the beginning of metamorphosis at NF 60 through its completion by NF 66, and most of the shortening is completed by the start of NF 62 (Table 1). The premetamorphic tadpole gut (NF 50–54) can be induced to shorten precociously by the same amount after treatment with TH for 7 days (Table 2), the approximate amount of time it takes for a tadpole to undergo spontaneous metamorphosis (23). By the time the gut has completed shortening, the cross-sectional diameter has decreased by about ...
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... intestine are controlled by thyroid hormone (TH). There have been numerous studies on the cellular mechanism for this remodeling. Several researchers have described nests of adult cells in the tadpole epithelium that proliferate and expand at climax to replace the dying larval epithelium (1, 3–5). This scheme for remodeling of the epithelium requires two distinct populations of epithelial cells, larval and adult. One study considered that at least some adult cells are derived from larval epithelial cells (6). Subtractive hybridization identified a number of genes whose expression in the intestinal epithelium is altered by TH (7). Their expression patterns fell into different developmental profiles as measured by Northern blotting. One class of mRNAs rises in expression to a peak at climax and then falls, and the expression pattern parallels the concentration of endogenous TH. A second class is the inverse of the first; gene expression is high before climax, then drops at climax (‘‘eclipses’’) and rises again at the end of metamorphosis. This eclipse has also been described for genes expressed specifically in the epithelium of the posterior intestine (6) and in the epithelium of the stomach (8). To date, no gene that is expressed exclusively in the larval intestine has been identified. This paper addresses two features of intestinal remodeling: the phenomenon of shortening and narrowing, and the remodeling of the intestinal epithelium. We describe the TH-induced contraction of longitudinal muscle fibers that accompanies intestinal shortening and the contraction and death of radial muscles as the diameter narrows. During this time, epithelial cells facing the lumen undergo cell death. A net effect of these size changes is transient ‘‘heaping’’ of the epithelium into many layers. In situ hybridization of genes that are expressed in the epithelium reveals a uniform change in all of the epithelial cells as the intestine transforms into its convoluted state of crypts and villi. Similarly, DNA replication occurs uniformly throughout the larval epithelium until days after metamorphosis is completed, when it localizes to the intestinal crypts. chemistry, and in Situ Hybridization. Tadpoles were staged by the tables of Nieuwkoop and Faber (NF) (9). Gastrointestinal tracts of tadpoles at different stages were fixed in 4% paraformalde- hyde, embedded in OCT compound, and cryosectioned as described (10, 11). Sections were processed for in situ hybridization (10, 11) by using digoxigenin-labeled antisense probes against Musashi-1 (GenBank accession no. BI447711), intestinal fatty-acid binding protein (a gift of Yun-Bo Shi, National Institute of Child Health and Human Development, Bethesda), and TH-controlled basic leucine zipper (12). Sections were also processed by using immunohistochemistry (10, 11) with mono- clonal antibodies against smooth muscle actin (Sigma), active caspase-3 (Pharmingen), and epithelial cadherin (product number 5D3, University of Iowa Developmental Studies Hybridoma Bank, Iowa City), or a polyclonal antibody against fibronectin (a gift of D. W. DeSimone, University of Virginia, Charlottesville) by using methods previously described (13–15). Antibodies were detected by using Alexa Fluor 488- and 568-conjugated secondary antibodies (Molecular Probes). Intestines of some NF stage 50 (NF 50) and NF 54 tadpoles were processed in whole-mount by immunohistochemistry as described (13, 14). These intestines were sliced open longitudinally and flat-mounted onto slides with either the serosa or mucosa side facing the coverslip. To identify proliferating cells, live tadpoles were injected i.p. with 10 l of BrdUrd (10 mM) 12 h before fixation with 2% trichloroacetic acid for 2 h at room temperature. Intestines were rinsed in PBS three times for 15 min, embedded in OCT compound, cryosectioned, and then counterimmunostained with a primary antibody against fibronectin (a mesenchyme-specific marker) and Alexa Fluor 568-conjugated secondary antibody. Slides were then incubated in 4 M HCl for 1 h at room temperature, rinsed three times for 5 min in PBS, incubated in ice-cold ethanol ͞ acetic acid (2:1) for 8 min, rinsed three times for 5 min in PBS, blocked for 30 min in PBS with 10% normal goat serum, incubated for 1 h at room temperature with Alexa Fluor 488-conjugated-anti-BrdUrd (Molecular Probes) (1:30) in PBS with 10% NGS, rinsed in PBS three times for 15 min, and mounted with a coverslip. NF 50 and 54 tadpoles were induced to metamorphose with 10 nM T3 as described (13, 14). Statistics and Experimental Design. To measure intestine shortening during spontaneous metamorphosis, the gastrointestinal tracts of sibling tadpoles at various stages ( n ϭ 8 –10) were excised, and lengths were measured for the anterior intestine (duodenum ϩ anterior ileum) and the posterior intestine (posterior ileum ϩ colon ϩ rectum). The junction between the anterior and posterior ileum is the ileum’s switchback point (see Fig. 1). Differences in mean length with developmental stage were assessed statistically by one-factor ANOVA (Su- perANOVA, Abacus Concepts, Berkeley, CA), followed by Fisher’s pairwise comparisons. P values of Ͻ 0.01 were considered significant. To measure precocious intestinal shortening with T3 treatment, NF 54 tadpoles were induced with 10 nM T3 for 7 days. Tadpoles were sampled every 24 h ( n ϭ 6), and total intestine lengths (duodenum to rectum) were measured. Changes in length with time of T3 treatment were assessed statistically by one-factor ANOVA. P values of Ͻ 0.01 were considered significant. Before metamorphosis in X. laevis , the tadpole intestine consists of two spiral coils: the outer coil (duodenum and anterior ileum) reverses direction at the switchback point and is followed by the inner coil (posterior ileum and colon), which terminates at the rectum (Fig. 1 A and B ). The climax of metamorphosis (NF 59–65) is the period of maximal change when the endogenous TH is at its highest concentration (16). During climax, which lasts Ϸ 8 days, the intestine shortens by Ϸ 75% (Fig. 1 C and D and Table 1). From the start (NF 59) to the end (NF 66) of metamorphic climax, the percentage of shortening of the anterior and posterior intestine is similar (78.5% and 71.9%, respectively) (Table 1), supporting the findings of Pretty et al. (2) that the intestine shortens equally along its entire length. The premetamorphic tadpole gut (NF 46–54) can be induced to shorten precociously by treatment with 10 nM T3 for 3–7 days (Table 2). Shortening of the intestine during spontaneous metamorphosis is accompanied by a change in cross-sectional morphol- ogy. The premetamorphic tadpole duodenum (NF 54) is a simple thin tube with one large involution (the typhlosole) in the anterior intestine (Fig. 2 A ). At the end of metamorphosis (NF 66), the epithelium is structured into crypts and villi (Fig. 2 D ). As the mesenchyme and muscle layers thicken at metamorphic climax (NF 62 and NF 63, Fig. 2 B and C , respectively), the intestinal epithelium temporarily thickens to five- to eight-cell layers (Figs. 2 B Ј and C Ј ), compared with a thickness of one to two cells before (NF 54, Fig. 2 A Ј ) and after (NF 66, Fig. 2 D Ј ) metamorphosis. The expression of genes with different developmental profiles in the anterior intestinal (duodenal) epithelium has been assessed (Fig. 3). We chose the neural stem cell marker Musashi (17) because nests of adult epithelial precursor cells in the intestine were reported to express specifically this gene (18). Musashi mRNA is expressed constitutively in all cells of the epithelium at every developmental stage from NF 54–66 (Fig. 3 A ). Intestinal fatty acid binding protein is expressed in the anterior tadpole intestinal epithelium, shut off (eclipsed) during climax, and then expressed again after climax (5, 19). Every epithelial cell follows this gene expression pattern (Fig. 3 B ). TH ͞ basic leucine zipper (12) is a direct response gene expressed in the intestinal epithelium and the mesenchyme. The expression of this gene rises and falls with the endogenous TH concentration (Fig. 3 C ). Our in situ hybridization experiments demonstrate the uniformity of these gene expression profiles in the intestinal epithelium at each stage. Intestinal epithelial cell proliferation is very low before climax (20) (Fig. 4 A ), but increases greatly in all layers of the heaped epithelium at climax and throughout the epithelium at a high level even late in metamorphosis after the crypts and villi have been formed (NF 65, Fig. 4 E ). Proliferation in the mesenchyme increases during climax, reaches a peak at NF 61, and drops by NF 66. Because BrdUrd is a nuclear marker, and mesenchyme cells are mixed with muscle cells, we could not reliably evaluate muscle proliferation. Apoptosis within the anterior intestine, as measured by active caspase-3 immunoreactivity, is present in a small number of epithelial cells before climax (20) (Fig. 5 A ). By NF 61, when the epithelial cells are heaped, apoptosis occurs mainly in the cells that line the lumen (Fig. 5 B ). Many radial, but not longitudinal, muscle fibers in the ileum are immunoreactive for active caspase-3, but fewer are labeled in the duodenum (data not shown). By the end of metamorphosis (NF 66), apoptosis is confined primarily to epithelial cells located at the tips of the intestinal villi (Fig. 5 C ). Individual longitudinal smooth muscle fibers in the ileum of a NF 54 tadpole are Ϸ 160 m in length (Fig. 6 A ). After 72 h of T3 treatment, muscle fibers are much shorter ( Ϸ 90 m in length) than untreated fibers (Fig. 6 B ). This decrease in length by Ϸ 44% corresponds to the decrease in total intestine length after 72 h of T3 treatment (Table 2). Intestinal muscle fibers also shorten during spontaneous metamorphosis, although muscle fiber lengths become difficult to measure in vitro beyond NF 61, as individual fiber ends are not easily ...
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Gene expression screens have been applied to a cultured cell line of Xenopus laevis, XL-177, to isolate genes that are up- and down-regulated in the first 8 h after thyroid hormone (TH) induction. At least 14 up-regulated genes were isolated from TH-induced cells grown in the presence or absence of cycloheximide, an inhibitor of protein synthesis....
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... The current knowledge of thyroid hormones in anurans, based in the model species Xenopus laevis (Daudin, 1802) (and X. tropicalis [Gray, 1864]), emphasizes their role in the events of metamorphosis, including analyses of TH levels, expression patterns and function of TH receptors (TR), the effects of THs on organogenesis, and the mechanisms underlying the pleiotropic actions of THs (Shi, 2000;Huang et al., 2001;Das et al., 2002;Schreiber et al., 2005;Fort et al., 2007;Buchholz, 2017;Yaoita, 2019;Paul et al., 2022;Tanizaki et al., 2022;among others). Those mechanistic approaches are complemented with studies exploring physiological, toxicological, ecological, developmental, and evolutionary aspects of the TH in few frog species (Michael and Al Adhami, 1974;Dodd and Dodd, 1976;Callery and Elinson, 2000;Opitz et al., 2005;Tata, 2006;Fernandez-Mongil et al., 2009;Grim et al., 2009;Vassilieva, 2009, 2014;Rose and Cahill, 2019;Fabrezi et al., 2019;Rose, 2021;Fabrezi and Cruz, 2021;Vassilieva and Smirnov, 2021;Poulsen et al., 2023). ...
... While exposure to Bd metabolites has been shown to be protective for both tadpole and adult Cuban treefrogs , Nordheim et al. 2022, it remains unknown whether metabolite exposure can protect metamorphic frogs. During metamorphosis, amphibians completely rebuild important body systems, e.g. the gastrointestinal tract (Schreiber et al. 2005). Although some immunological memory persists through metamorphosis (Barlow & Cohen 1983), the developing larvae suppress their immune system possibly to prevent the immune system from attacking and destroying their own developing tissue during development (Rollins-Smith et al. 1997). ...
The pathogenic fungus Batrachochytrium dendrobatidis (Bd) is associated with drastic global amphibian declines. Prophylactic exposure to killed zoospores and the soluble chemicals they produce ( Bd metabolites) can induce acquired resistance to Bd in adult Cuban treefrogs Osteopilus septentrionalis . Here, we exposed metamorphic frogs of a second species, the Pacific chorus frog Pseudacris regilla , to one of 2 prophylactic treatments prior to live Bd exposures: killed Bd zoospores with metabolites, killed zoospores alone, or a water control. Prior exposure to killed Bd zoospores with metabolites reduced Bd infection intensity in metamorphic Pacific chorus frogs by 60.4% compared to control frogs. Interestingly, Bd intensity in metamorphs previously exposed to killed zoospores alone did not differ in magnitude relative to the control metamorphs, nor to those treated with killed zoospores plus metabolites. Previous work indicated that Bd metabolites alone can induce acquired resistance in tadpoles, and so these findings together indicate that it is possible that the soluble Bd metabolites may contain immunomodulatory components that drive this resistance phenotype. Our results expand the generality of this prophylaxis work by identifying a second amphibian species (Pacific chorus frog) and an additional amphibian life stage (metamorphic frog) that can acquire resistance to Bd after metabolite exposure. This work increases hopes that a Bd -metabolite prophylaxis might be widely effective across amphibian species and life stages.
... Some larval epithelial cells undergo dedifferentiation during metamorphosis to form clusters of cells that proliferate rapidly and express well-known adult intestinal stem cell markers such as Lgr5 by climax of metamorphosis, e.g., stage 61 (about 6-7 weeks of age) ( Figure 1A) (11,21,22). By the end of metamorphosis or stage 66 (about 2 months after fertilization), these proliferating stem cells differentiate to form a multi-folded epithelium surrounded by elaborate connective tissue and muscles (4,16,17,23,24). In the adult frog, the intestinal stem cells are localized at the bottom of the epithelial fold while cell death occurs mainly at the crest of the fold, similar to those taking place in the crypt-villus unit in adult mammalian intestine (16,25). ...
Amphibian metamorphosis resembles mammalian postembryonic development, a period around birth when many organs mature into their adult forms and when plasma thyroid hormone (T3) concentration peaks. T3 plays a causative role for amphibian metamorphosis. This and its independence from maternal influence make metamorphosis of amphibians, particularly anurans such as pseudo-tetraploid Xenopus laevis and its highly related diploid species Xenopus tropicalis, an excellent model to investigate how T3 regulates adult organ development. Studies on intestinal remodeling, a process that involves degeneration of larval epithelium via apoptosis and de novo formation of adult stem cells followed by their proliferation and differentiation to form the adult epithelium, have revealed important molecular insights on T3 regulation of cell fate during development. Here, we review some evidence suggesting that T3-induced activation of cell cycle program is important for T3-induced larval epithelial cell death and de novo formation of adult intestinal stem cells.
... For example, from early larval stages, tail muscle is formed and only experiences subtle increases in size until resorption during metamorphosis [26]. By contrast, the gut persists through metamorphosis but is transformed, shortening by 75% and undergoing extensive remodelling that includes the apoptosis and de-differentiation of epithelial cells to stem cells [27][28][29]. The heart and liver remain relatively unchanged in structure from middle larval stages, but greatly increase in size from metamorphosis onwards [11,30]. ...
... In our study, the telomeres of gut cells showed a remarkable change in length during metamorphosis, presumably linked to the degree of remodelling experienced by this tissue. During X. laevis metamorphosis, gut shortening and remodelling includes larval epithelial apoptosis and differentiation into stem cells [27,28,49], a phase of cell turnover that may explain longer telomeres in the gut cells of metamorphic individuals than in larvae. After metamorphosis, a selfrenewing system of stems cells is established in the intestinal epithelium and differentiated cells invaginate into connective Table 2. Summary of linear models testing for the effect of growth rate, temperature, and tissue on relative telomere length in Xenopus laevis from stage NF54 larvae to 7-month-old adults. ...
... Bold indicates significant effects (p < 0.05). tissue to form the intestine folds [27,28,49]. This system is present in adult but a large degree of differentiation may be behind the telomere shortening in gut tissue that occurs from the conclusion of metamorphosis through to the adult frog. ...
Telomere attrition is considered a useful indicator of cellular and whole-organism ageing rate. While approximately 80% of animal species undergo metamorphosis that includes extensive tissue transformations (involving cell division, apoptosis, de-differentiation and de novo formation of stem cells), the effect on telomere dynamics is unknown. We measured telomeres in Xenopus laevis developing from larvae to adults under contrasting environmental temperatures. Telomere dynamics were linked to the degree of tissue transformation during development. Average telomere length in gut tissue increased dramatically during metamorphosis, when the gut shortens by 75% and epithelial cells de-differentiate into stem cells. In the liver (retained from larva) and hindlimb muscle (newly formed before metamorphosis), telomeres gradually shortened until adulthood, likely due to extensive cell division. Tail muscle telomere lengths were constant until tail resorption, and those in heart (retained from larva) showed no change over time. Telomere lengths negatively correlated with larval growth, but for a given growth rate, telomeres were shorter in cooler conditions, suggesting that growing in the cold is more costly. Telomere lengths were not related to post-metamorphic growth rate. Further research is now needed to understand whether telomere dynamics are a good indicator of ageing rate in species undergoing metamorphosis.
... Many tadpoles complete the dietary shift from herbivore to carnivore in this process (Gehrig et al. 2019;Zhang et al. 2020). At the same time, larval gut undergoes dramatic apoptosis and cell proliferation to transform into a complex adult form with multifolded epithelium, accompanying the extensive shortening of intestine (Schreiber et al. 2005;Heimeier et al. 2010;Sirakov and Plateroti 2011;Chai et al. 2018). In addition, the intestinal remodeling is high susceptible to external factors such as environmental pollutants and could be used as a model to evaluate the potentially negative effects of water pollution (Xie et al. 2020;Shen et al. 2022). ...
In amphibians, lead (Pb) exposure could alter the composition and structure of gut microbiota, but changes involving microbiota of several successive phases following Pb exposure have been less studied. In the present study, we compared the effects of Pb exposure on morphological parameters and gut microbiota of Bufo gargarizans at Gosner stage (Gs) 33, Gs36, and Gs42. Our results showed that total length (TL), snout-vent length (SVL), and body wet weight (TW) of B. gargarizans at Gs33, as well as TL and SVL at Gs42, were significantly increased after Pb exposure. In addition, high-throughput sequencing analysis indicated that gut microbiota has distinct responses to Pb exposure at different developmental stages. The diversity of gut microbiota was significantly reduced under Pb exposure at Gs33, while it was significantly increased at Gs42. In terms of community composition, Spirochaetota, Armatimonadota, and Patescibacteria appeared in the control groups at Gs42, but not after Pb treatment. Furthermore, functional prediction indicated that the relative abundance of metabolism pathway was significantly decreased at Gs33 and Gs36, and significantly increased at Gs42. Our results fill an important knowledge gap and provide comparative information on the gut microbiota of tadpoles at different developmental stages following Pb exposure.
... Over the metamorphosis process, the intestine of premetamorphic tadpoles consist in a simple thin tube which undergoes elongation, looping and rotation events (Bloom et al., 2013), as visible in unexposed larvae between T2 and T12. At the same time, gut complexity increases through crypt and villi structuration and shortening at climax (from stage 60) under the action of T3 hormone (Chalmers and Slack, 1998;Heimeier et al., 2010;Schreiber et al., 2005;Shi et al., 2001;Sterling et al., 2012). Exposure to increasing GO concentrations did not led to the alteration of larval development as indicated by the similar NF stage 57 reached following 12 days of exposure to any GO concentration. ...
Graphene-based nanomaterials such as graphene oxide (GO) possess unique properties triggering high expectations for the development of technological applications. Thus, GO is likely to be released in aquatic ecosystems. It is essential to evaluate its ecotoxicological potential to ensure a safe use of these nanomaterials. In amphibians, previous studies highlighted X. laevis tadpole growth inhibitions together with metabolic disturbances and genotoxic effects following GO exposure. As GO is known to exert bactericidal effects whereas the gut microbiota constitutes a compartment involved in host homeostasis regulation, it is important to determine if this microbial compartment constitutes a toxicological pathway involved in known GO-induced host physiological impairments. This study investigates the potential
link between gut microbial communities and host physiological alterations. For this purpose, X. laevis tadpoles were exposed during 12 days to GO. Growth rate was monitored every 2 days and genotoxicity was assessed through enumeration of micronucleated erythrocytes. Genomic DNA was also extracted from the whole intestine to quantify gut bacteria and to analyze the community composition. GO exposure led to a dose dependent growth inhibition and genotoxic effects were detected following exposure to low doses. A transient decrease of the total bacteria was noticed with a persistent shift in the gut microbiota structure in exposed animals. Genotoxic effects were associated to gut microbiota remodeling characterized by an increase of the relative abundance of Bacteroides fragilis. The growth inhibitory effects would be associated to a shift in the Firmicutes/Bacteroidetes ratio while metagenome inference
suggested changes in metabolic pathways and upregulation of detoxification processes. This work indicates that the gut microbiota compartment is a biological compartment of interest as it is integrative of host physiological alterations and should be considered for ecotoxicological studies as structural or functional impairments could lead to later life host fitness loss.
... During this postnatal maturation program, increased circulating TH levels are responsible for gut remodeling, including the first phase of apoptosis and shortening of the gut length followed by an increase in cell proliferation [4]. Interestingly, it has been reported that these maturation steps in gut tadpoles depend on complex signaling between different cell types, leading to the emergence of stem cells (SCs) and the establishment of the adult epithelium [5,6]. In the mammalian intestine, it is well established that continuous epithelium renewal and SC maintenance depends on complex cell interactions and on signaling pathways that globally define and contribute to the SC niche [7,8]. ...
... In amphibians, primary culture approaches have shown that the action of THs in non-epithelial cells is required for the appearance of adult epithelium and the emergence of the intestinal SCs [5,6]. These results underline the importance of TH-dependent epithelial-connective tissue interactions in the establishment of an intestinal SC niche in the developing amphibian intestine [83,84]. ...
Several studies emphasized the function of the thyroid hormones in stem cell biology. These hormones act through the nuclear hormone receptor TRs, which are T3-modulated transcription factors. Pioneer work on T3-dependent amphibian metamorphosis showed that the crosstalk between the epithelium and the underlying mesenchyme is absolutely required for intestinal maturation and stem cell emergence. With the recent advances of powerful animal models and 3D-organoid cultures, similar findings have now begun to be described in mammals, where the action of T3 and TRα1 control physiological and cancer-related stem cell biology. In this review, we have summarized recent findings on the multiple functions of T3 and TRα1 in intestinal epithelium stem cells, cancer stem cells and their niche. In particular, we have highlighted the regulation of metabolic functions directly linked to normal and/or cancer stem cell biology. These findings help explain other possible mechanisms by which TRα1 controls stem cell biology, beyond the more classical Wnt and Notch signaling pathways.
... Our results showed that remodeling of the intestine is the one of the most dramatic changes during metamorphosis as noted previously 57 . Enterocytes, especially at later stages, exhibited high heterogeneity due to changes in diet during metamorphosis to form the complex adult intestine 56,58 . ...
The rapid development of high-throughput single-cell RNA sequencing technology offers a good opportunity to dissect cell heterogeneity of animals. A large number of organism-wide single-cell atlases have been constructed for vertebrates such as Homo sapiens, Macaca fascicularis, Mus musculus and Danio rerio. However, an intermediate taxon that links mammals to vertebrates of more ancient origin is still lacking. Here, we construct the first Xenopus cell landscape to date, including larval and adult organs. Common cell lineage-specific transcription factors have been identified in vertebrates, including fish, amphibians and mammals. The comparison of larval and adult erythrocytes identifies stage-specific hemoglobin subtypes, as well as a common type of cluster containing both larval and adult hemoglobin, mainly at NF59. In addition, cell lineages originating from all three layers exhibits both antigen processing and presentation during metamorphosis, indicating a common regulatory mechanism during metamorphosis. Overall, our study provides a large-scale resource for research on Xenopus metamorphosis and adult organs. Single-cell RNA sequencing technology offers a unique opportunity to dissect cell heterogeneity of animals. Here, the authors construct a Xenopus cell landscape including larval and adult organs to dissect cell heterogeneity of the amphibian.
... We also used an air reached developmental stage NF57 (i.e., all five toes separated;Nieuwkoop and 144 Faber, 1994), five tadpoles were randomly selected from each aquarium. At this pro-145 metamorphic stage, the gut is largest and longest in X. laevis(Schreiber et al. 2005). The146 collected tadpoles were washed with filtered water and immediately anesthetized with 200 mg 147 /Lof tricaine methanesulfonate (MS-222, Ethyl 3-aminobenzoate methanesulfonate; Sigma-148 Aldrich) buffered with 200 mg/L of sodium bicarbonate (Cecala et al., 2007), and subsequently 149 euthanized for prolonged exposure to this solution. ...
Global changes in temperature, predator introductions, and pollution might challenge animals by altering food conditions. A fast-growing source of environmental pollution are microplastics. If ingested with the natural food source, microplastics act as artificial fibers that reduce food quality by decreasing nutrient and energy density with possible ramifications for growth and development. Animals might cope with altered food conditions with digestive plasticity. We examined experimentally whether larvae of the African clawed frog (Xenopus laevis) exhibit digestive morphology plasticity (i.e., gut length, mass, and diameter) in response to microplastics ingestion. As natural systems contain non-digestible particles similar in size and shape to microplastics, we included cellulose as a natural fiber control group. Gut length and mass increased in response to microplastics and cellulose ingestion indicating that both types of fibers induced digestive plasticity. Body mass and body condition were similar across experimental groups, indicating that larvae fully compensated for low nutrient and energy density by developing longer intestines. The ability of a species to respond plastically to environmental variation, as X. laevis responded, indicates that this species might have the potential to cope with new conditions during global change, although it is uncertain whether this potential may be reduced in a multi-stressor environment.
... Amphibians complete the transformation of their digestive tracts from a long and simple non-acidic stomach to a short and complex acidic stomach through a metamorphosis process (Zhang et al., 2020). The close relationship between the structure of the digestive tract and dietary changes can lead to changes in the intestinal microbiomes (Schreiber et al., 2005). Studies have shown that the intestinal microbiota of frogs change from fish-like (Proteobacteria, Firmicutes) during the tadpole stage to an amniotic membrane (Firmicutes, Bacteroides) phenotype during the frog stage (Kohl et al., 2013;Zhang et al., 2018). ...
Intestinal microbiota play an important role in the life of amphibians and its composition may vary by developmental stage. In this study, 16S rRNA high‐throughput sequencing was used to profile the intestinal microbiota of Hynobius maoershanensis, which exclusively inhabit the Maoer Mountain swamp at an altitude of approximately 2,000 m. We characterized the bacterial composition, structure, and function of the microbiota of H. maoershanensis at different developmental stages. The alpha diversity was not markedly different for the Simpson, Shannon, Ace, and Sobs indices of microbes. The beta diversity revealed that there were age‐related differences in the structure of the intestinal microbes of H. maoershanensis, specifically, at the phylum level. Bacteroidetes and Proteobacteria were the dominant bacteria present in the adult stage, and the relative abundance of Bacteroidetes was significantly higher compared with that of tadpoles. Firmicutes and Proteobacteria were the dominant phylum during the tadpole stage and their relative abundance was significantly higher compared with the adult period. Functional analysis revealed that the pathways associated with organismal systems and metabolism were significantly enriched in the adults, whereas human diseases, genetic information processing, and cellular processes were more enriched in the hindlimb bud stage. Human diseases and environmental information processing were more enriched in the forelimb bud stage at KEGG pathway level 1. Possibilities for the observed discrepancies include the adaptation to eating habits and the remodeling of the intestines during development. We speculated that H. maoershanensis adults may be more suitable to a high‐fiber diet, whereas the tadpoles are associated with a carnivorous diet. Our study provides evidence of variations in the intestinal microbiota during development in amphibians, highlighting the influence of historical developments on the intestinal microbiota and an increased understanding of the importance of physiological characteristics in shaping the intestinal microbiota of amphibians. These data will help us formulate more effective protection measures for H. maoershanensis. Hynobius maoershanensis (Urodela: Hynobiidae). The larvae of H. Maoershanensis live an aquatic life, whereas the adults live amphibiously. Due to the sharp decline of its population, it is the key protection wildlife in national nature reserve in China.