Induced Repatterning of Type XVIII Collagen Associates with Ectopic Sonic Hedgehog and Lung Surfactant C Gene Expression and Changes in Epithelial Epigenesis in the Ureteric Bud
Seppo Vainio*,
Yanfeng Lin*, and
Taina Pihlajaniemi
*Biocenter Oulu and Department of Biochemistry, Faculties of Science and Medicine, University of Oulu, Oulu, Finland; Division of Nephrology, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts; and Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, University of Oulu, Oulu, Finland.
Correspondence to Dr. Seppo Vainio, Department of Biochemistry, P.O. Box 3000, FIN-90014 University of Oulu, Oulu, Finland; Phone: +358-8-553-1190; Fax: +358-8-553-1141;
ABSTRACT. How cell and tissue interactions lead to complex organstructures and differentiated cell types during organogenesisis one of the most fundamental questions in developmental biology.The embryonic lung and kidney of the mouse are useful modelsfor studying the molecular mechanisms of morphogenesis, andin both of these organs, the epithelial bud undergoes a characteristicbranching process. This review discusses the potential roleof an extracellular matrix molecule, type XVIII collagen, inthe generation of the branching patterns in the lung and kidneyand how its experimental respecification in tissue recombinantsbetween the ureteric bud and lung mesenchyme correlates withchanges in expression of signaling molecules such as sonic hedgehogand changes in cell fate as judged by ectopic expression ofthe lung surfactant C gene. E-mail: seppo.vainio@.oulu.fi
Interactions between epithelial and mesenchymal tissues induceproliferation and branching of the epithelium into the mesenchymein many organs such as the lung, kidney, pancreas, and toothbud. Epithelial branching morphogenesis is crucial for the establishmentof organ-specific structures, and the patterns of branchingare not random but under developmental control, at least duringthe early branching generations (1). In the early mammalianlung, for instance, the epithelial bud sprouts and sends outlateral branches in typical, invariant positions.
Epithelial branching may be inherent to the epithelium itselfor regulated by tissue interactions between the epithelium andmesenchyme (2,3). Studies of the mechanism of branching morphogenesisin lower organisms such as Drosophila melanogaster have suggestedthat a small number of signaling molecules may be responsiblefor the control of the epithelial branching process. Drosophilaserves as a useful system when screening for additional factorsinvolved in the branching process. The respiratory system ofthe Drosophila trachea, for example, is governed by the fibroblastgrowth factor (FGF) signaling pathway, which is reiterativelyregulated to contribute to patterning through successive roundsof branching. FGF signals are also expressed in the mammalianlung and implicated in its development (1,4). There are othersignals in addition to FGF, however, that have also been implicatedin the control of branching in the mammalian kidney (5), namelysignals from the TGF- superfamily, glial cell line–derivedneurotrophic factor (GDNF), which is expressed in the kidneymesenchyme and apparently binds to a RET receptor that is expressedin the ureteric bud and contributes to the control of branching(6,7). Roles have recently been demonstrated for pleiotrophin(HB-GAM) and Wnt2b as well in promoting epithelial branching(8,9).
The extracellular matrix (ECM) is an important component forregulating morphogenesis during embryogenesis. It not only providesa physical substratum for the spatial organization of cellsbut also may play a more active role in inductive tissue interactionsby controlling the activities of growth factors, for example(10). Type XVIII collagen is a recently identified ECM moleculeand basement membrane component that belongs to the collagenfamily of proteins and is expressed as three major variants(11–15) (Figure 1). It also functions as a part-time heparansulfate proteoglycan (PG) (14) and may regulate growth factorfunction in this way, as PGs have been implicated in growthfactor signaling (16). The longest of the three variant N-terminalnoncollagenous domains contains sequences with homology to thefrizzled proteins (11,17), and as the latter are Wnt receptors,this variant of collagen XVIII may also function as a putativeextracellular antagonist of Wnts. In addition, the C-terminalnon–triple-helical domain of collagen XVIII contains apotent peptide named endostatin that inhibits angiogenesis andpossibly thereby tumorigenesis (18,19). A knockout of the typeXVIII collagen gene performed recently in the mouse was shownto lead to delayed regression of blood vessels in the vitreousbody and impaired outgrowth of the retinal vessels, which suggeststhat collagen XVIII/endostatin is a critical factor for normalblood vessel formation in the developing eye in vivo (20). Inthis review, we discuss the role of type XVIII collagen in organogenesiswith reference to the kidney and lung and describe an experimentalsystem in which type XVIII collagen was found to be reorganizedin association with changes in epithelial epigenesis.
Figure 1. Schematic model for the molecular structure of mouse type XVIII collagen. Type XVIII collagen is a member of the collagen superfamily of extracellular matrix molecules. It is expressed in three variants, with the amino acid sizes of the various portions of the molecule as indicated. The locations of noncollagenous and collagenous areas, the endostatin domain, regions homologous to the frizzled (fz) and thrombospondin-1 (tsp) motifs, putative attachment sites for glycosaminoglycan (GAG) and asparagine (N)-linked sugars, and the arginine-glycine-aspartate (RGD) sequence are indicated. The signal peptides for the variants are shown by hatching, and the locations of cysteine residues (C) are indicated.
Expression of Type XVIII Collagen in Embryonic Lung and Kidney Is Developmentally Regulated
At the initiation of lung and kidney organogenesis (E10.5),when the epithelial bud grows into the mesenchyme, the typeXVIII collagen mRNA and protein are uniformly present in theepithelial bud in both of these organs. During the formationof the first epithelial branches, expression of this collagentype in the lung is localized in the epithelial tips and islost from the stalk area, whereas the opposite pattern is observedin the kidney (21) (Figure 2). This differential expressionpersists during later developmental stages and suggests developmentalroles for type XVIII collagen.
Figure 2. Expression of type XVIII collagen in the embryonic lung bud and kidney. Type XVIII expression is localized to the tips of the epithelial bud of the lung (A), whereas the opposite expression pattern prevails in the kidney (B), where it is lacking in the ureteric tips but is expressed in the stalk region of the ureteric bud. (C) Recombination of ureteric bud with lung mesenchyme has repatterned type XVIII collagen expression from the stalk region (seen in B) into the epithelial tip (as in A). The E11.0 ureteric bud was preincubated with Glial cell line-derived neurotrophic factor (GDNF) before recombination to induce competence in it to branch with lung mesenchyme.
Lung Mesenchyme Is Sufficient to Repattern Expression of Type XVIII Collagen from Kidney to Lung Type in the Ureteric Bud
What specifies the differential expression of type XVIII collagenin the two organs, and does it play a role in branching morphogenesis?To answer these questions, we performed a tissue recombinationexperiment in which the lung mesenchyme was co-cultured withthe ureteric bud. As expected, homotypic recombination of theureteric bud with the kidney mesenchyme or of the lung epithelialbud with the lung mesenchyme restored the in vivo patterns oftype XVIII collagen expression, but, interestingly, when theureteric bud was recombined with the lung mesenchyme, this ledto a complete shift in type XVIII collagen expression from thekidney type to the lung type, i.e., from the stalk region tothe epithelial tip (Figures 2 and 3). When the lung epithelialbud was recombined with kidney mesenchyme, no branching wasinduced and type XVIII expression was lost from the epithelialtips, pointing to a correlation with induced epithelial branching.Hence, lung mesenchyme signaling is sufficient to repatterntype XVIII collagen expression in the ureteric bud to resemblea pattern of the lung type.
Figure 3. Experimental respecification of type XVIII collagen expression from kidney to lung type. Type XVIII collagen is initially expressed uniformly in the epithelial buds of the lung epithelium (LE) and the ureteric bud (U). During the early stages of epithelial branching in the lung, it becomes localized in the epithelial tip region (yellow area), whereas the expression pattern in the kidney is the opposite, expression being lost from the ureteric tip and localized in the epithelial stalk region (yellow area). When the lung mesenchyme is experimentally recombined with the ureteric bud, the type XVIII collagen is repatterned from the stalk region to the tips, i.e., its expression pattern changes from the kidney to the lung type. This reorganization is accompanied by induced expression of Sonic hedgehog and the lung surfactant C gene, and the ureteric bud also changes its epigenesis toward the lung type. GDNF is important for generating the competence for the epithelium to branch with the lung mesenchyme, and Wnt2 is a candidate lung mesenchyme-derived signal that may contribute to the repatterning process.
Type XVIII Is Essential for Ureteric Bud Branching with Lung Mesenchyme In Vitro
Repatterning of type XVIII collagen from the kidney to the lungtype is associated with changes in epithelial epigenesis towardthe lung type of branching (21), but is it essential for reshapingthe branching process? An anti–all-type XVIII collagen(ELQ) antibody recognizing all three type XVIII collagen variantpolypeptides was used to test the role of the repatterning processin reprogramming ureteric branching morphogenesis (21). Lungorganogenesis is normal after an addition of 120 µg/mlpreimmune IgG to the culture medium, so this can serve as acontrol. During the 3-d culture period, the number of lung epithelialtips increased in the preimmune IgG-treated explants, whereastreatment with the anti–all ELQ led on average to a 34%decrease in the number. We thus may conclude that type XVIIIcollagen is essential for normal lung morphogenesis in vitro.When the same antibody was used in heterotypic tissue recombinantsbetween the ureteric bud and lung mesenchyme, it completelyblocked ureteric branching, suggesting that type XVIII is ofimportance for epithelial branching in the ureteric bud in thepresence of lung mesenchyme.
Altered Ureteric Bud Branching as a Response to Lung Mesenchyme Signaling
It was of interest to analyze whether the ureteric bud changesits morphogenesis in the presence of lung mesenchyme. This wasmonitored by computer skeletonizing of the branches of uretericbuds seen in homotypic and heterotypic tissue recombinants.Analysis of the epithelial skeletons suggested a change in uretericepigenesis toward that of the early lung type in the heterotypicrecombinants, where the position of the first branches of thesecondary buds and the number of ureteric bud tips assumed valuesclose to those observed for lung epithelial branching (21,22).
GDNF as an Inducer of Ureteric Bud Competence to Branch with Lung Mesenchyme
Although earlier studies had shown that the ureteric bud ofthe E11-d-old mouse embryo is not able to branch with mesenchymeof nonkidney origin (23), more recent studies have revealedthat an E.11.5-stage ureteric bud that has branched once hasobtained a competence to develop with mesenchyme originatingfrom an E11.5 lung (24). Hence, branching of the epithelialbud with lung mesenchyme is stage dependent and is apparentlypotentiated by kidney mesenchyme-derived signals that triggerthe branching process. Our studies have pointed to a criticalrole for GDNF in the generation of competence for the E11 uretericbud to branch with lung mesenchyme (21). Beads soaked with GDNFinduce loss of type XVIII collagen from the ureteric tips andlead to subsequent induction of branching in the ureteric bud,hence the competence to branch correlates with loss of typeXVIII collagen from the ureteric tips. We conclude that GDNFis a critical inducer of this competence in the ureteric budin cross-talk with lung mesenchyme.
Repatterning of Type XVIII Collagen Expression in the Ureteric Bud Is Accompanied by Ectopic Induction of Lung Surfactant C and Sonic Hedgehog Expression
Although GDNF signaling leads to downregulation of type XVIIIcollagen expression in the ureteric tips, GDNF is not the factorthat induces subsequent repatterning of the expression of thiscollagen in the tips with the lung mesenchyme. To monitor forsignals that could be involved in inducing the expression oftype XVIII collagen in the ureteric tips and the repatterningepithelium, we screened for the expression of certain genesthat have been implicated in lung development after type XVIIIcollagen antibody blocking (21), including Sonic hedgehog (Shh)(25), Wnt2 (26), and FGF10 (27). Wnt2 expression was markedlyreduced in the lung mesenchyme after treatment with the anti–alltype XVIII collagen antibody, suggesting a role for Wnt2 signalingin the control of type XVIII collagen expression. Wnt2 expressionalso persisted in the mesenchyme in the tissue recombinantsbetween the lung mesenchyme and the ureteric bud, suggestinga role for it in lung-type signaling. A more direct experimentwith cells expressing the Wnt2 signal would be needed, however,to establish its role fully. Moreover, the analysis demonstrateda correlation between type XVIII collagen and Shh expressionin the epithelial tissue of the kidney and lung and also ectopicShh expression in ureteric bud tips in the lung mesenchyme-uretericbud tissue recombinants, suggesting that Shh could be a mediatorof type XVIII collagen repatterning and also contribute to changesin epithelial development.
When expression of the lung surfactant C (SpC), which servesas a marker of the type II pneumocytes, was analyzed to monitorpotential changes in cell differentiation markers in the heterotypictissue recombinants, ectopic expression of SpC was observedin the tips of ureteric buds in the recombinants between theseand lung mesenchyme, as was the case with type XVIII collagenand Shh (21). Hence, lung mesenchyme signaling may have changedthe status of cell differentiation in the ureteric buds.
Role of Type XVIII Collagen in Epithelial Development
The lung mesenchyme seems to be able to reprogram the developmentof ureteric bud epigenesis in the tissue recombinants towardthe early lung type. This may be deduced on the grounds thatthe embryonic lung mesenchyme completely respecifies type XVIIIcollagen expression in the ureteric bud, shifting it from theepithelial stalk to the distal tip, so that it is identicalto that in the lung. This shift correlated with changes in theexpression of Shh and the SpC gene, both of which were alsolocalized in the epithelial tips. This suggests that the lungmesenchyme may also be able to respecify cell differentiationin the kidney-derived ureteric bud toward that of the embryoniclung, i.e., it seems to act on the ureteric bud in the mannerof an instructive inducer.
We do not know how the loss of type XVIII collagen from theureteric tips occurs or how expression is maintained in thestalk of the ureteric bud, but it is likely that constant remodelingof the ECM at the growing tips of the invading ureteric budis needed for branching to take place, and it is possible thatthe loss of type XVIII collagen from the tips allows epithelialbranching to occur. Matrix metalloproteases (MMP) and theirinhibitors, tissue metalloproteases (TIMP), are expressed inthe mesenchyme and epithelium, respectively, and their expressionis regulated by many cytokines and growth factors that are expressedin the embryonic lung and kidney, e.g., TGF- and EGF (for areview, see 28). MMP2 and MMP9 are also expressed in the kidneymesenchyme adjacent to the branching ureteric bud at E11.5 (29)and have been implicated in Wnt signaling (30). Experimentsshould be conducted in the future to test whether these MMPcould play a role in the degradation of type XVIII collagen.
When considering the potential developmental role of type XVIIIcollagen, we should remember that it could bind Wnts to thebronchial epithelial tips and thereby concentrate them and promotethe characteristic morphogenesis of the epithelial type in thelung. It also functions as a PG (14), and as PGs are necessaryfor Wnt signaling (31), it thus could play a role in this signaling,which is known to be important for kidney development, as demonstratedin the case of Wnt4 or Wnt11 in the kidney (24; for a review,see 3). Wnt2/2b is expressed in the mesenchyme of the lung adjacentto the tips, in common with type XVIII collagen, whereas Wnt7bis expressed in the epithelium (21). Hence, type XVIII collagenmay play a role in modulating Wnt signaling and thereby alsoin branching morphogenesis. Shh, in turn, could also regulatethe expression of Bmp4 and/or Wnt2 in the adjacent lung mesenchymeand contribute to branching in this way, a hypothesis also favoredby Bellusci et al. (32). Glypican 3 has recently been shownto be a receptor for endostatin, which has been found to promotebranching of the ureteric bud (33,34). Hence, proteolytic processingof type XVIII collagen at the ureteric tip may directly promoteureteric branching in the kidney via the resulting endostatin.Taken together, our data support a model in which differencesin morphogenesis between organs undergoing epithelial branchingmay be regulated by localized patterning cues in the epitheliumand mesenchyme. The matrix, including such locally expressedmolecules as type XVIII collagen, could play a role in localizinginductive signals that contribute to morphogenesis of the epitheliumby controlling cell division and adhesion, for example, whichare apparently critical for epithelial branching. The uretericbud and lung mesenchyme tissue recombination assay system mayserve as a useful experimental model to study the molecularmechanisms by which the differences in form appear during organogenesis.
Acknowledgments
This work was supported financially by the Academy of Finland(41001, 49986, 44843), European Union (QLK3-2001-01275), theSigrid Jusélius Foundation, and the Center for InternationalMobility (YL).
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Abstract
Introduction
superfamily, glial cell line–derived neurotrophic factor (GDNF), which is expressed in the kidney mesenchyme and apparently binds to a RET receptor that is expressed in the ureteric bud and contributes to the control of branching (6,7). Roles have recently been demonstrated for pleiotrophin (HB-GAM) and Wnt2b as well in promoting epithelial branching (8,9). 
