Thyroid hormone-induced expression of Sonic hedgehog correlates with adult epithelial development during remodeling of the Xenopus stomach and intestine

Sonic hedgehog (Shh) was isolated from the Xenopus laevis intestine as an early thyroid hormone (TH) response gene. To investigate possible roles of TH-upregulated expression of Shh during metamorphosis, we raised a polyclonal antibody against Xenopus Shh and immunohistochemically examined the relationship between Shh expression and the larval-to-adult intestinal remodeling at the cellular level. Our results indicate that the epithelial-specific expression of Shh in the intestine spatiotemporally correlates well with active proliferation and/or initial differentiation of the secondary (adult) epithelial primordia that originate from stem cells, but not with apoptosis of the primary (larval) epithelium. Given the similar transformations of the stomach during metamorphosis, we also analyzed Shh expression in this organ and found similar correlations in the stomach, although the position of the adult epithelial primordia and their final differentiation in the stomach are different from those in the intestine. Furthermore, we show here that Shh expression is organ-autonomously induced by TH and its correlation with the adult epithelial development is reproduced in vitro in both the intestine and the stomach. More importantly, addition of recombinant Shh protein to the culture medium results in developmental anomalies of both organs. However, differentiation of the adult epithelium is more severely inhibited by exogenous Shh in the intestine than in the stomach. These results suggest that TH-upregulated expression of Shh plays important roles in the postembryonic gastrointestinal remodeling, but its roles are at least partially different between the intestine and the stomach.     

NUDC expression during amphibian development.

To identify gene products important for gastrulation in the amphibian Pleurodeles waltl, a screen for regional differences in new protein expression at the early gastrula stage was performed. A 45 kDa protein whose synthesis was specific for progenitor endodermal cells was identified. Microsequencing and cDNA cloning showed that P45 is highly homologous to rat NUDC, a protein suggested to play a role in nuclear migration. Although PNUDC can be detected in all regions of the embryo, its de novo synthesis is tightly regulated spatially and temporally throughout oogenesis and embryonic development. New PNUDC synthesis in the progenitor endodermal cells depends on induction by the mesodermal cells in the gastrula. During development, PNUDC is localized in the egg cortical cytoplasm, at the cleavage furrow during the first embryonic division, around the nuclei and cortical regions of bottle cells in the gastrula, and at the basal region of polarized tissues in the developing embryo. These results show for the first time the expression and compartmentalization of PNUDC at distinct stages during amphibian development.     

Stage-specific polypeptide and villin expression during thyroid-hormone-induced substitution of the amphibian intestinal epithelium

Abstract. Treatment of anuran tadpoles with 5 n M 3,3',5-triiodo- l -thyronine (T₃) results in the complete substitution of the intestinal epithelium. We have examined the developmental pattern of protein synthesis in Alytes obstetricans intestinal epithelium using two-dimensional gel electrophoresis. Four different types of changes have been observed. The group I polypeptides (M_r:41 500; 44 500; 51 500; 55000 and 101000) are only synthesized during the first week of hormonal treatment. They are specific of the primary (larval) epithelium. On the other hand, polypeptides referred to as Group II (M_r: 47000; 48000; 58000; 66500, pl 5.2; 99500 and 102000) are not detected until day 8. They are characteristic of the secondary tissue. Polypeptides of Group III (M_r: 42000, pl 5.15 and 5.25; 42500, 47500, pl 5.25 and 5.55) expressed between the 6th and 8th day of T₃ treatment, are specific of growing stem cells. During this critical period, Group IV polypeptides (M_r: 63500; 66500, pl 6.35; 105000, pl 5.5 and 5.55) are not synthesized. The protein of M_r 105000 (pI 5.5 and 5.55) is immunologically related to villin, a core protein of intestinal microvilli. Expression of this protein has been analyzed by immunoreplica and immunocytochemical procedures during differentiation of basal stem cells into secondary absorptive epithelial cells. The results have been compared to that obtained during spontaneous metamorphosis [19].     

Expansion of intestinal epithelial stem cells during murine development

Murine small intestinal crypt development is initiated during the first postnatal week. Soon after formation, overall increases in the number of crypts occurs through a bifurcating process called crypt fission, which is believed to be driven by developmental increases in the number of intestinal stem cells (ISCs). Recent evidence suggests that a heterogeneous population of ISCs exists within the adult intestine. Actively cycling ISCs are labeled by Lgr5, Ascl2 and Olfm4; whereas slowly cycling or quiescent ISC are marked by Bmi1 and mTert. The goal of this study was to correlate the expression of these markers with indirect measures of ISC expansion during development, including quantification of crypt fission and side population (SP) sorting. Significant changes were observed in the percent of crypt fission and SP cells consistent with ISC expansion between postnatal day 14 and 21. Quantitative real-time polymerase chain reaction (RT-PCR) for the various ISC marker mRNAs demonstrated divergent patterns of expression. mTert surged earliest, during the first week of life as crypts are initially being formed, whereas Lgr5 and Bmi1 peaked on day 14. Olfm4 and Ascl2 had variable expression patterns. To assess the number and location of Lgr5-expressing cells during this period, histologic sections from intestines of Lgr5-EGFP mice were subjected to quantitative analysis. There was attenuated Lgr5-EGFP expression at birth and through the first week of life. Once crypts were formed, the overall number and percent of Lgr5-EGFP positive cells per crypt remain stable throughout development and into adulthood. These data were supported by Lgr5 in situ hybridization in wild-type mice. We conclude that heterogeneous populations of ISCs are expanding as measured by SP sorting and mRNA expression at distinct developmental time points.     

Decreased expression of Toll-like receptor-4 and MD-2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide

The lumenal surface of the colonic epithelium is continually exposed to Gram-negative commensal bacteria and LPS. Recognition of LPS by Toll-like receptor (TLR)-4 results in proinflammatory gene expression in diverse cell types. Normally, however, commensal bacteria and their components do not elicit an inflammatory response from intestinal epithelial cells (IEC). The aim of this study is to understand the molecular mechanisms by which IEC limit chronic activation in the presence of LPS. Three IEC lines (Caco-2, T84, HT-29) were tested for their ability to activate an NF-kappaB reporter gene in response to purified, protein-free LPS. No IEC line responded to LPS, whereas human dermal microvessel endothelial cells (HMEC) did respond to LPS. IEC responded vigorously to IL-1beta in this assay, demonstrating that the IL-1 receptor signaling pathway shared by TLRs was intact. To determine the reason for LPS hyporesponsiveness in IEC, we examined the expression of TLR4 and MD-2, a critical coreceptor for TLR4 signaling. IEC expressed low levels of TLR4 compared with HMEC and none expressed MD-2. To determine whether the low level of TLR4 expression or absent MD-2 was responsible for the LPS signaling defect in IEC, the TLR4 or MD-2 gene was transiently expressed in IEC lines. Transient transfection of either gene individually was not sufficient to restore LPS signaling, but cotransfection of TLR4 and MD-2 in IEC led to synergistic activation of NF-kappaB and IL-8 reporter genes in response to LPS. We conclude that IEC limit dysregulated LPS signaling by down-regulating expression of MD-2 and TLR4. The remainder of the intracellular LPS signaling pathway is functionally intact.     

Stage-specific polypeptides and villin expression during the intestinal epithelium substitution of the metamorphosing amphibian

The amphibian intestinal epithelium provides an excellent aid to study the developmental pattern of protein synthesis during cell life. The metamorphosing tissue demonstrates a kaleidoscope of cell degeneration, proliferation and differentiation. These events occur at specific period in a synchronized cell population. Two-dimensional gel electrophoresis, together with histological studies, has been used to examine the changes in the patterns of protein synthesis during intestinal epithelium substitution in metamorphosing Alytes obstetricians larvae. Of the approximately 280 polypeptides detected by this method, 24 show major changes in their patterns of synthesis. Five polypeptides are only synthesized during the larval period and are characteristic of the primary epithelium. Six polypeptides are characteristic of the secondary intestinal epithelium, as they are only detected in the newly-metamorphosed juvenile. Four polypeptides of Mr 81,000, 78,000, 42,000 (pI, 5.1 and 6.2) are characteristic of the epithelium crisis, as they are only detected during climax. They may represent molecular markers of growing stem cells. On the other hand, two polypeptides, of Mr 66,500 and 63,500, are not synthesized during this critical period, but are synthesized before and after metamorphosis. Seven polypeptides show changes in the relative rate of their synthesis during metamorphosis of the intestinal epithelium. Among them, the protein of Mr 105,000 which presents two isoelectric variants (pI 5.5 and 5.55) is immunologically related to villin. Expression of this protein has been studied using immunoblotting of cell extracts onto nitrocellulose and immunodetection in tissue sections. The protein is localized in the brush border of primary and secondary epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidative stress, tissue remodeling and regression during amphibian metamorphosis

Anuran metamorphosis is characterized by rapid and drastic changes in the body form and function under the influence of thyroid hormones. We evaluated the involvement of reactive oxygen species and antioxidant defenses during intestinal remodeling and tail regression of tadpoles of Xenopus laevis. Oxidative stress resulting from depletion in catalase and reduced glutathione, and simultaneous increase in lipid peroxidation during intestinal remodeling as well as tail regression are probably responsible for cell death and differentiation in these organs. Gene expression data for superoxide dismutase and catalase supports this contention. A dramatic increase in another antioxidant, ascorbic acid content of both these organs during metamorphic climax indicates its multifactor role such as collagen synthesis in intestine and controlled tail regression. These findings suggest that the cellular environment in the intestine and tail becomes progressively more oxidizing during its remodeling and regression respectively.     

Temporal and spatial expression of an intestinal Na+/PO43− cotransporter correlates with epithelial transformation during thyroid hormone-dependent frog metamorphosis

The amphibian intestine has two morphologically distinct structures during development. Early embryogenesis generates a simple tube-like intestine in the tadpole whereas after thyroid hormone (T₃)-dependent metamorphosis a newly remodeled adult intestine is formed similar to that of higher vertebrates. This change requires a drastic transformation of the epithelial layer. We have isolated a Na⁺/PO₄³⁻ cotransporter gene that may contribute to this transformation. The deduced amino acid sequence of this gene shows a high degree of homology to the mammalian renal NA⁺/PO₄³⁻ cotransporters, which have little or no expression in organs other than the kidney. The frog gene is highly expressed and regulated by T₃ in the intestine with little expression and/or regulation by T₃ in most other organs. Its mRNA is restricted to the differentiated epithelial cells both in tadpoles and postmetamorphic frogs. Interestingly, its expression is low in premetamorphic tadpoles, but up-regulated when metamorphosis is initiated by endogenous T₃. As the larval epithelium undergoes programmed cell death (apoptosis), the mRNA level drops to a minimum. Subsequently, the gene is reactivated at the tip region of the newly formed adult intestinal folds and a crest-trough polarity of expression is established by the end of metamorphosis. This temporal regulation profile is also reproduced when premetamorphic tadpoles are treated with T₃ to induce precocious intestinal remodeling. These results suggest a possible role of the Na⁺/PO₄³⁻ cotransporter during metamorphosis and demonstrate that the adult epithelial cell differentiation pattern is established in the direction of crest-to-trough of the intestinal fold, concurrent with the epithelial morphogenic process. Dev Genet 20:53–66, 1997. © 1997 Wiley-Liss, Inc.     

GATA-6 maintains BMP-4 and Nkx2 expression during cardiomyocyte precursor maturation

GATA-6 is expressed in presumptive cardiac mesoderm before gastrulation, but its role in heart development has been unclear. Here we show that Xenopus and zebrafish embryos, injected with antisense morpholino oligonucleotides designed specifically to knock-down translation of GATA-6 protein, are severely compromised for heart development. Injected embryos express greatly reduced levels of contractile machinery genes and, at the same stage, of regulatory genes such as bone morphogenetic protein-4 (BMP-4) and the Nkx2 family. In contrast, initial BMP and Nkx2 expression is normal, suggesting a maintenance role for GATA-6. Endoderm is critical for heart formation in several vertebrates including Xenopus, and separate perturbation of GATA-6 expression in the deep anterior endoderm and in the overlying heart mesoderm shows that GATA-6 is required in both for cardiogenesis. The GATA-6 requirement in cardiac mesoderm was confirmed in zebrafish, an organism in which endoderm is thought not to be necessary for heart formation. We therefore conclude that proper maturation of cardiac mesoderm requires GATA-6, which functions to maintain BMP-4 and Nkx2 expression.     

Spatial and temporal expression pattern of a novel gene in the frog Xenopus laevis: correlations with adult intestinal epithelial differentiation during metamorphosis

We report the cloning of a novel gene (ID14) and its expression pattern in tadpoles and adults of Xenopus laevis. ID14 encodes a 315-amino acid protein that has a signal peptide and a nidogen domain. Even though several genes have a nidogen domain, ID14 is not the homolog of any known gene. ID14 is a late thyroid hormone (TH)-regulated gene in the tadpole intestine, and its expression in the intestine does not begin until the climax of metamorphosis, correlating with adult intestinal epithelial differentiation. In contrast, ID14 is expressed in tadpole skin and tail and is not regulated by TH. In situ hybridization revealed that this putative extracellular matrix protein is expressed in the epithelia of the tadpole skin and tail and in the intestinal epithelium after metamorphosis. In the adult, ID14 is found predominantly in the intestine with weak expression in the stomach, lung, and testis. Its exclusive expression in the adult intestinal epithelial cells makes it a useful marker for developmental studies and may give insights into cell/cell interactions in intestinal metamorphosis and adult intestinal stem cell maintenance.     

The development of the adult intestinal stem cells: Insights from studies on thyroid hormone-dependent amphibian metamorphosis

Adult organ-specific stem cells are essential for organ homeostasis and repair in adult vertebrates. The intestine is one of the best-studied organs in this regard. The intestinal epithelium undergoes constant self-renewal throughout adult life across vertebrates through the proliferation and subsequent differentiation of the adult stem cells. This self-renewal system is established late during development, around birth, in mammals when endogenous thyroid hormone (T3) levels are high. Amphibian metamorphosis resembles mammalian postembryonic development around birth and is totally dependent upon the presence of high levels of T3. During this process, the tadpole intestine, predominantly a monolayer of larval epithelial cells, undergoes drastic transformation. The larval epithelial cells undergo apoptosis and concurrently, adult epithelial stem/progenitor cells develop de novo, rapidly proliferate, and then differentiate to establish a trough-crest axis of the epithelial fold, resembling the crypt-villus axis in the adult mammalian intestine. We and others have studied the T3-dependent remodeling of the intestine in Xenopus laevis. Here we will highlight some of the recent findings on the origin of the adult intestinal stem cells. We will discuss observations suggesting that liganded T3 receptor (TR) regulates cell autonomous formation of adult intestinal progenitor cells and that T3 action in the connective tissue is important for the establishment of the stem cell niche. We will further review evidence suggesting similar T3-dependent formation of adult intestinal stem cells in other vertebrates.     

Atrial chamber-specific expression of sarcolipin is regulated during development and hypertrophic remodeling

Intracellular Ca2+ regulation is critical in the normal cardiac function and development of pathologic hearts. Phospholamban, an endogenous inhibitor of sarcoplasmic reticulum Ca2+ ATPase in the sarcoplasmic reticulum, plays an important role in Ca2+ cycling in heart. Recently, sarcolipin has been identified as having a similar function as phospholamban in skeletal muscle. Because phospholamban is differentially expressed in atrial and ventricular myocardia and its expression is often altered in diseased hearts, we investigated the cardiac chamber specificity of sarcolipin expression and its regulation during development and hypertrophic remodeling. Northern blot analysis revealed that the expression of mouse sarcolipin mRNA was most abundant in the atria and was undetectable in the ventricles, indicating an atrial chamber-specific expression pattern. Atrial chamber-specific expression of sarcolipin mRNA was increased during development. These findings were confirmed by in situ hybridization studies. In addition, sarcolipin expression was down-regulated in the atria of hypertrophic heart when induced by ventricular specific overexpression of the activated H-ras gene. In humans, sarcolipin mRNA was also expressed in the atria but not detected in the ventricles, although sarcolipin expression was most abundant in skeletal muscle. Taken together, sarcolipin is likely to be an atrial chamber-specific regulator of Ca2+ cycling in heart.     

pRb-mediated control of epithelial cell proliferation and Indian Hedgehog expression in mouse intestinal development

Background  

Self-renewal of the epithelium of the small intestine is a highly regulated process involving cell proliferation and differentiation of stem cells or progenitor cells located at the bottom of the crypt, ending ultimately with extrusion of the terminally differentiated cells at the tip of villus.