Extraembryonic Endoderm Cells Research Articles

Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts

The extra-embryonic endoderm lineage plays a major role in the nutritive support of the embryo and is required for several inductive events, such as anterior patterning and blood island formation. Blastocyst-derived embryonic stem (ES) and trophoblast stem (TS) cell lines provide good models with which to study the development of the epiblast and trophoblast lineages, respectively. We describe the derivation and characterization of cell lines that are representative of the third lineage of the blastocyst -extra-embryonic endoderm. Extra-embryonic endoderm (XEN) cell lines can be reproducibly derived from mouse blastocysts and passaged without any evidence of senescence. XEN cells express markers typical of extra-embryonic endoderm derivatives, but not those of the epiblast or trophoblast. Chimeras generated by injection of XEN cells into blastocysts showed exclusive contribution to extra-embryonic endoderm cell types. We used female XEN cells to investigate the mechanism of X chromosome inactivation in this lineage. We observed paternally imprinted X-inactivation, consistent with observations in vivo. Based on gene expression analysis, chimera studies and imprinted X-inactivation, XEN cell lines are representative of extra-embryonic endoderm and provide a new cell culture model of an early mammalian lineage.

Identification of an Enhancer That Controls Up-regulation of Fibronectin during Differentiation of Embryonic Stem Cells into Extraembryonic Endoderm

The extraembryonic endoderm is derived from inner cell mass cells of the blastocyst during early mouse embryogenesis. Formation of the extraembryonic endoderm, which later contributes to the yolk sac, appears to be a prerequisite for subsequent differentiation of the inner cell mass. While embryonic stem cells can be induced to differentiate into extraembryonic endoderm cells in vitro, the molecular mechanisms underlying this process are poorly understood. We used a promoter trap approach to search for genes that are expressed in embryonic stem cells and are highly up-regulated during differentiation to the extraembryonic endoderm fate. We showed that fibronectin fits this expression profile. Moreover we identified an enhancer in the 12th intron of the fibronectin locus that recapitulated the endogenous pattern of fibronectin expression. This enhancer carries Sox protein-binding sequences, and our analysis demonstrated that Sox7 and Sox17, which are highly expressed in the extraembryonic endoderm, were involved in enhancer activity.

Targeted Deletion of Fatty Acid Transport Protein-4 Results in Early Embryonic Lethality

Fatty acid transport protein-4 (FATP4) is the major FATP in the small intestine. We previously demonstrated, using in vitro antisense experiments, that FATP4 is required for fatty acid uptake into intestinal epithelial cells. To further examine the physiological role of FATP4, mice carrying a targeted deletion of FATP4 were generated. Deletion of one allele of FATP4 resulted in 48% reduction of FATP4 protein levels and a 40% reduction of fatty acid uptake by isolated enterocytes. However, loss of one FATP4 allele did not cause any detectable effects on fat absorption on either a normal or a high fat diet. Deletion of both FATP4 alleles resulted in embryonic lethality as crosses between heterozygous FATP4 parents resulted in no homozygous offspring; furthermore, no homozygous embryos were detected as early as day 9.5 of gestation. Early embryonic lethality has been observed with deletion of other genes involved in lipid absorption in the small intestine, namely microsomal triglyceride transfer protein and apolipoprotein B, and has been attributed to a requirement for fat absorption early in embryonic development across the visceral endoderm. In mice, the extraembryonic endoderm supplies nutrients to the embryo prior to development of a chorioallantoic placenta. In wild-type mice we found that FATP4 protein is highly expressed by the epithelial cells of the visceral endoderm and localized to the brush-border membrane of extraembryonic endodermal cells. This localization is consistent with a role for FATP4 in fat absorption in early embryogenesis and suggests a novel requirement for FATP4 function during development.

Regulation of the differentiation and behaviour of extra-embryonic endodermal cells by basement membranes.

Both the extracellular matrix and parathyroid hormone-related peptide (PTHrP) have been implicated in the differentiation and migration of extra-embryonic endodermal cells in the pre-implantation mammalian blastocyst. In order to define the individual roles and interactions between these factors in endodermal differentiation, we have used embryoid bodies derived from Lamc1(-/-) embryonic stem cells that lack basement membranes. The results show that in the absence of basement membranes, increased numbers of both visceral and parietal endodermal cells differentiate, but they fail to form organised epithelia. Furthermore, although parietal endodermal cells only migrate away from control embryoid bodies in the presence of PTHrP, they readily migrate from Lamc1(-/-) embryoid bodies in the absence of PTHrP, and this migration is unaffected by PTHrP. Thus, the basement membrane between epiblast and extra-embryonic endoderm is required for the proper organisation of visceral and parietal endodermal cells and also restricts their differentiation to maintain the population of primitive endodermal stem cells. Moreover, this basement membrane inhibits migration of parietal endodermal cells, the role of PTHrP being to stimulate delamination of parietal endodermal cells from the basement membrane rather than promoting migration per se.

Regulation of laminin and COUP-TF expression in extraembryonic endodermal cells.

Laminin expression and the subsequent deposition of a basement membrane by primitive endoderm cells is necessary for early mammalian development. We demonstrate that the transcription factors COUP-TF I and II are up-regulated in primitive endoderm cells faster than LAMB1 and LAMC1, and that either COUP-TF is sufficient to induce expression of these laminin genes.

Regulation of programmed cell death by basement membranes in embryonic development.

The formation of the proamniotic cavity in the mammalian embryo is the earliest of many instances throughout development in which programmed cell death and the formation of epithelia play fundamental roles (Coucouvanis, E., and G.R. Martin. 1995. Cell. 83:279-287). To determine the role of the basement membrane (BM) in cavitation, we use embryoid bodies derived from mouse embryonic stem cells in which the LAMC1 genes have been inactivated to prevent BM deposition (Smyth, N., H.S. Vatansever, P. Murray, M. Meyer, C. Frie, M. Paulsson, and D. Edgar. 1999. J. Cell Biol. 144:151-610). We demonstrate here that LAMC1-/- embryoid bodies are unable to cavitate, and do not form an epiblast epithelium in the absence of a BM, although both embryonic ectodermal cells and extraembryonic endodermal cells do differentiate, as evidenced by the expression of cell-specific markers. Acceleration or rescue of BM deposition by exogenous laminin in wild-type or LAMC1-/- embryoid bodies, respectively, results in cavitation that is temporally and spatially associated with restoration of epiblast epithelial development. We conclude that the BM not only directly regulates development of epiblast epithelial cells, but also indirectly regulates the programmed cell death necessary for cavity formation.

Expression of fibroblast growth factor receptors in peri-implantation mouse embryos.

FGF receptor (FGFR) function is essential during peri-implantation mouse development. To understand which receptors are functioning, we tested for the expression of all four FGF receptors in peri-implantation blastocysts. By RT-PCR, FGFR-3 and FGFR-4 were detected at high levels, FGFR-2 at lower levels, and FGFR-1 was detected at background levels compared to control tissues. Because FGFR-3 and FGFR-4 were detected at the highest levels, we studied these in detail. Between 3.5 days after fertilization (E3.5) and E6.0, FGFR-4 mRNA was detected ubiquitously in the peri-implantation embryo, restricted to the inner cell mass (ICM) and its derivatives and primitive endoderm by E6.0, and was not detected at E6.5. FGFR-3 mRNA was detected ubiquitously in the peri-implantation embryo with a tendency towards extraembryonic cells. We tested blastocyst outgrowths, a model for implantation, for FGFR-3 and FGFR-4 protein. FGFR-3 protein was detected in all cells early during the outgrowth. Later, FGFR-3 was detected in the extraembryonic endoderm and trophoblast giant cells (TGC), but not in the ICM. FGFR-4 protein was detected in all cells of the implanting embryo, but was restricted to the ICM/primitive endoderm in later stage outgrowths. The distribution of the receptor proteins in the blastocyst outgrowths is similar to the distribution of the mRNA detected by in situ hybridization of sections of embryos. The data suggest roles for FGFR-3 and FGFR-4 in peri-implantation development.

Expression of fibroblast growth factor receptors in peri‐implantation mouse embryos

FGF receptor (FGFR) function is essential during peri-implantation mouse development. To understand which receptors are functioning, we tested for the expression of all four FGF receptors in peri-implantation blastocysts. By RT-PCR, FGFR-3 and FGFR-4 were detected at high levels, FGFR-2 at lower levels, and FGFR-1 was detected at background levels compared to control tissues. Because FGFR-3 and FGFR-4 were detected at the highest levels, we studied these in detail. Between 3.5 days after fertilization (E3.5) and E6.0, FGFR-4 mRNA was detected ubiquitously in the peri-implantation embryo, restricted to the inner cell mass (ICM) and its derivatives and primitive endoderm by E6.0, and was not detected at E6.5. FGFR-3 mRNA was detected ubiquitously in the peri-implantation embryo with a tendency towards extraembryonic cells. We tested blastocyst outgrowths, a model for implantation, for FGFR-3 and FGFR-4 protein. FGFR-3 protein was detected in all cells early during the outgrowth. Later, FGFR-3 was detected in the extraembryonic endoderm and trophoblast giant cells (TGC), but not in the ICM. FGFR-4 protein was detected in all cells of the implanting embryo, but was restricted to the ICM/primitive endoderm in later stage outgrowths. The distribution of the receptor proteins in the blastocyst outgrowths is similar to the distribution of the mRNA detected by in situ hybridization of sections of embryos. The data suggest roles for FGFR-3 and FGFR-4 in peri-implantation development. Mol. Reprod. Dev. 51:254–264, 1998. © 1998 Wiley-Liss, Inc.

Mouse heparin binding protein-44 (HBP-44) associates with brushin, a high-molecular-weight glycoprotein antigen common to the kidney and teratocarcinomas.

Heparin binding protein-44 (HBP-44) is a heparin binding protein of 44 kDa, found by cDNA cloning using antibodies against teratocarcinoma glycoproteins [Furukawa, T. et al. (1990) J. Biochem. 108, 297-302]. The N-terminal sequence analysis reported in this publication establishes the structure of its mature form. Immunohistochemical staining revealed that HBP-44 was located in the tubular brush border of the kidney. HBP-44 formed a complex with brushin, a high molecular weight (450 kDa) glycoprotein antigen common to the kidney and teratocarcinoma, but not with OR8 antigen, another antigen (350 kDa) of the same category. Brushin was shown to be the mouse counterpart of rat Heymann nephritis antigen, called gp330. The association between HBP-44 and brushin was revealed not only by co-precipitation upon indirect immunoprecipitation, but also by ligand blotting with HBP-44-maltose binding protein fusion protein. Calcium ion stabilized the association. Disulfide bonds in brushin seemed to be necessary for the complex formation, since reductive cleavage of the bonds resulted in failure of the protein to associate with HBP-44 in a ligand blotting experiment. Association of HBP-44 with brushin occurred both in teratocarcinoma cells, in which these molecules are mainly located in extraembryonic endoderm cells, and in the kidney, suggesting that the complex has an unknown common function in the renal tubular brush border and the extraembryonic endoderm.

Effects of Different Conditions of Retinoic Acid Treatment on Expression of MK Gene, which is Transiently Activated during Differentiation of Embryonal Carcinoma Cells1 : (retinoic acid/teratocarcinoma/embryonal carcinoma cells/MK gene).

MK gene was intensely expressed, when aggregates of HM-1 embryonal carcinoma (EC) cells were treated with retinoic acid for 2 days to induce the differntiation to nerve cells, myoblasts and extraembryonic endoderm cells. The conditions inhibiting nerve cell diffrentiation or extraembryonic endoderm cell differentiation affected MK gene expression only slightly. The maximum level of MK RNA was detected 2 days after initiation of retionic acid treatment, when cells were morphologically indistinguishable from undifferentiated EC cells. Thus, MK gene appears to be expressed in differentiating EC cells irrespective of the direction of differentiation. The degree of MK gene expression in sparsely cultured HM-1 cells correlated with the concentration of retinoic acid, especially between 10-8 and 10-7 M. When retinoic acid treatment was terminated after 1 day, the amount of MK RNA started to decrease. These two results are consistent with the view that retionic acid complexed with the receptor is directly involved in expression of MK gene.

A retinoic acid-responsive element is present in the 5' flanking region of the laminin B1 gene.

The retinoic acid (RA)-associated differentiation of murine F9 teratocarcinoma stem cells results in dramatic changes in gene expression. The cellular gene encoding the B1 subunit of the extracellular matrix protein laminin is transcriptionally activated by RA, and its transcription is further enhanced by N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) during the differentiation of F9 stem cells into extraembryonic parietal endoderm cells. We now report that expression vectors encoding the human RA receptors RAR-alpha, RAR-beta, and RAR-gamma can activate chloramphenicol acetyltransferase (CAT) expression from laminin B1 promoter/CAT expression vectors (e.g., p1.6LAMCAT) in RA-treated F9 cells, as measured in a transient transfection assay. Bt2cAMP does not further enhance the RA-associated increase in CAT activity. Through the use of deletion and mutation analyses, the RA-responsive element (RARE) of the murine laminin B1 gene has been defined as a 46-base-pair element between -477 and -432 of the laminin B1 5' flanking region. Insertion of a region of DNA containing this RARE in either orientation into a thymidine kinase promoter/CAT expression vector causes CAT expression to be activated 5- to 9-fold by the cotransfected human RAR-alpha or RAR-beta constructs in RA-treated F9 cells, and this RARE also functions in human HeLa cells. In contrast, this RARE in the p1.6LAMCAT vector does not activate CAT expression when cotransfected into F9 stem cells with the c-erbA gene in the presence of thyroid hormone. This suggests that the laminin B1 gene is activated by RA but not by thyroid hormone in vivo.

Actin cytoskeleton of extraembryonic endoderm and teratocarcinoma-derived endoderm cells

Distinct F-actin- and myosin-containing stress fibers were observed in situ in many endoderm cells of parietal yolk sacs from 11-day mouse embryos. In visceral endoderm (VE) such fibers were not seen, and F-actin was concentrated in the cell periphery. Correspondingly, in electron microscopy ventral cell membrane-associated bundles of microfilaments were revealed in the periphery of parietal endoderm (PE) cells but not in VE cells. Both PE and VE cells formed stress fibers in primary cultures. Undifferentiated F9 embryonal carcinoma cells formed only short actin spikes and fibrils irrespective of growth substratum. In PE-like derivatives of F9 cells, on the other hand, distribution of F-actin was markedly affected by the growth substratum: They formed distinct stress fibers when plated on fibronectin but did not when plated on gelatin. Similarly, in teratocarcinoma-derived PE cells (PYS-2) adhesion to fibronectin induced the formation of distinct bundles of F-actin and plaques of vinculin. The results suggest that the susceptibility of teratocarcinoma cell actin cytoskeleton to the influence of molecular composition of surrounding matrix is developmentally regulated. On the other hand, the reason for the presence of stress fibers in PE cells and for their absence in VE cells is unclear.

Synthesis of serum proteins by cultured aggregates from endodermal cells of the area opaca of the primitive streak chick embryo.

The pattern of serum protein synthesis and secretion in aggregates of extraembryonic endoderm cells (EEC) from the area opaca of primitive streak chick embryos was studied. EEC aggregates were cultured for various time intervals and serum proteins were detected using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Serum proteins were identified based on their comigration with reference proteins from 4 day chick embryo serum and with reference proteins from egg white albumen and chicken serum. A number of serum proteins were detected in EEC aggregates including: two variants of immunoglobulin (IgG), four variants of transferrin, a protein with a molecular weight of 66 500 which may correspond to α globulins, prealbumin, and a protein with a molecular weight of 38 600 (serum protein 11) which remains unidentified. These proteins were also detected in the culture medium. The banding profiles of EEC extracts and culture medium were compared over various time intervals of culture (6, 18 and 30 h). The IgGs, transforms and serum protein 11 decreased in concentration in EEC extracts over the culture interval. These proteins as well as prealbumin, were detected in the culture medium. A number of proteins were synthesized by EEC, as determined by radiolabelled amino acid incorporation. All of the labelled serum proteins were detected in the culture medium, not in EEC extracts. These results suggest that serum proteins are synthesized by EEC then rapidly released into the medium. Labelled serum proteins detected in the culture medium include prealbumin and an unidentified serum protein (serum protein 14) which migrates with the tracking dye, both synthesized early in culture (6 h), and transferrin which was synthesized later (18 h) during culture.

Developmentally regulated expression of cell surface carbohydrates during mouse embryogenesis.

Cell surface carbohydrates undergo marked alterations during mouse embryogenesis. In preimplantation embryos, many carbohydrate markers show stage-specific expression in diverse ways. In early postimplantation embryos, certain carbohydrate markers are localized in defined regions in the embryo. Important carriers of stage-specific carbohydrates are the lactoseries structure (Gal beta 1----4GlcNAc) and the globoseries structure (Gal alpha 1----4Gal). Notably, the glycoprotein-bound large carbohydrate of poly-N-acetyllactosamine-type ([Gal beta 1----4GlcNAc beta 1----3]n) carries a number of markers preferentially expressed in early embryonic cells. These markers are of practical value in analyzing embryogenesis and cell differentiation. For example, in order to monitor in vitro differentiation of multipotential embryonal carcinoma cells, stage-specific embryonic antigen-1 (SSEA-1) and the Lotus agglutinin receptor have been used as markers of the undifferentiated cells, and the Dolichos agglutinin receptor has been used as a marker of extraembryonic endoderm cells. Developmental control of cell surface carbohydrates is attained by controlled expression of activities of key glycosyltransferases; for example, the activity of N-acetylglucosaminide alpha 1----3 fucosyltransferase is lost during in vitro differentiation of embryonal carcinoma cells to parietal endoderm cells, in parallel to the disappearance of SSEA-1. Accumulating evidence suggests that poly-N-acetyllactosamine-type glycans that are abundant in early embryonic cells are involved in cell surface recognition of these cells.

Intermediate filaments of the midgestation rat trophoblast giant cell

Trophectoderm (TE) of the rodent blastocyst, the preimplantation precursor of the trophoblast giant cell (TGC), is the first embryonic cell to exhibit intermediate filaments (IF). The two IF proteins of TE (54K and 46K) have been variously described as trophectoderm specific, noncytokeratin, or cytokeratin and have been identified with Endo A and Endo B, IF proteins extracted from extraembryonic endodermal cells. IF proteins of midgestation rat TGC, the postimplantation descendant of TE, were compared to IF proteins of various rat simple epithelial cells by two-dimensional gel electrophoresis, partial proteolytic digest, antibody recognition on electrophoretic transfer, and antibody recognition by indirect immunofluorescence. The two TE IF proteins at 54K and 46K were identified in TGC IF and recognized by anti-Endo A, anti-Endo B, respectively, and anticytokeratins. TGC were found to possess additional cytokeratins at 52K, 45K, 43K, and 40K. The profile of TGC cytokeratins was qualitatively identical to that of various rat simple epithelial cells. The results suggest that (a) TE and TGC IF proteins are cytokeratins, (b) TE and TGC cytokeratins are characteristic of a simple epithelial cell, and (c) the morphologic and functional differentiation of TE to TGC is accompanied by elaboration of the cytokeratin profile.

Release of β- d-galactoside-binding lectins into the cavities of aggregates of chick extraembryonic endoderm cells

Dissociated cells from the extraembryonic endoderm of gastrulating chick embryos form aggregates when cultured in rotating flasks. The large cellular aggregates are initially solid but subsequently cavitate to form hollow, thin-walled vesicles. These cells also contain an endogenous β- d-galactoside-binding lectin. Previous work has shown that high extracellular concentrations of this lectin are associated with decreased cell—cell adhesion [Milos, N. and S.E. Zalik: Differentiation 21, 175–182 (1982)]. We have removed the fluid contents from aggregates cultured for 24 and 48 h and tested them for the presence of lectin activity. The results demonstrate that lectin activity is detectable in a higher number of aggregates cultured for 24 as opposed to 48 h (75% vs. 28%, respectively). The lectin activity per aggregate is also higher in aggregates cultured for 24 h (180 vs. 67 hemagglutinating units, respectively, for 24- and 48-h aggregates). Thus, at the time when cells are moving apart from one another during aggregate cavitation, detectable lectin activity is released into the vesicular contents of the aggregate.

Preimplantation mouse embryos and liver express the same type I keratin gene product.

The cytoskeletal B protein isolated from extraembryonic endodermal cells (Endo B) is a 50-kDa subunit of intermediate filaments that is expressed in trophoblast and extraembryonic endoderm of early mouse embryos. Endo B was compared to cytokeratin D of adult mouse liver by immunoprecipitation, two-dimensional gel electrophoresis, and peptide mapping. The two proteins were indistinguishable. A cDNA probe for Endo B mRNA identified mRNA species of similar size in liver and endoderm, and primer extension analysis indicates that the Endo B mRNAs from the two cell types have similar 5' ends. An internal fragment of the Endo B cDNA was found to cross-hybridize with a conservative domain of a human type I keratin cDNA under low stringency conditions, demonstrating that Endo B is related to type I keratins. However, under stringent conditions necessary for genomic Southern analysis, mouse and human genomic fragments homologous to the Endo B cDNA were distinct from those defined by hybridization with the type I keratin cDNA. These results indicate that Endo B is related to the type I keratin family and expands the number of type I keratin genes identified in both the mouse and human genomes. It is likely that extraembryonic endoderm, one of the first differentiated cell types of the mammalian embryo, and adult liver express the same Endo B gene.

A teratocarcinoma-derived endoderm stem cell line (1H5) that can differentiate into extra-embryonic endoderm cell types

We investigated the ability of the teratocarcinoma-derived, epithelial-type cell line 1H5 to differentiate into either of the two pathways to primary endoderm, and tested the hypothesis that 1H5 represents a state similar to primitive endoderm in the late 4th-day blastocyst. Like other endodermal cell types, 1H5 cells mixed with embryonal-carcinoma cells sort out into “embryoid bodies” or structures that resemble 4th-day mouse embryos. The epithelial line conforms morphologically and biochemically to the few known characteristics typical of primitive endoderm. The present study demonstrates that the formation in vitro of overt visceral endoderm is readily achieved. The spontaneous arrangement of the cells into a cystic form is followed by the appearance of several markers of visceral endoderm, most notably alphafetoprotein, which is detected when 1H5 cells are cultured either in the presence of retinoic acid or when the cells interact with embryonal-carcinoma cells in a specific spatial arrangement after sorting out. However, some less specific properties of visceral endoderm are not expressed. Although 1H5 differentiates histologically into parietal-like endoderm in the tumor form, parietal cells cannot yet be identified with certainty in vitro because of the paucity of parietal-specific markers. The 1H5 cell line could provide a useful system for studying the characteristics and mechanisms underlying visceral-endoderm differentiation in vitro, since it has the distinct advantage that homogeneous cultures are produced, in contrast to other teratocarcinoma cell lines such as F9 which differentiate into a mixture of cell types.

Le marquage immunologiquede l'α-ftoprotéine dans l'endoderme proximal et sa signification dans les différenciations extra-embryonnaires des tératocarcinomes de la souris

Summary The ABC technique using a specific anti-mouse α-fœtoprotein (AFP)serum permits identification of groups of extraembryonic proximal endoderm cells in certain teratocarcinomas. These AFP + cells are always associated with trophoblastic and parietal endodermal patterns and structures. These observations strongly support the hypothesis of a common precursor to the extraembryonic tissues which appears during the first phases of egg development.

Intercellular relationships during cavitation of aggregates of extraembryonic endoderm cells from gastrulating chick embryos

Extraembryonic endoderm cells from gastrulating chick embryos undergo epiboly and change from a multilayered cell group to a single cell layer surrounding the yolk. Single cell suspensions from this cell layer can aggregate in vitro to form aggregates that cavitate. To study the stages of cavitation aggregates were harvested after different times in culture, and fixed and processed for light and electron microscopy. In aggregates harvested at 75 min of culture cell contact consisted of areas of parallel and close membrane apposition and interdigitation. Desmosomes were occasionally observed. Aggregates in the early stages of cavitation (24 h) contained numerous intercellular spaces bordered by irregularly shaped cells which appeared to be digesting their yolk and releasing material extracellularly. Long cytoplasmic projections were extended into these spaces. In addition to regions of parallel membrane apposition and interdigitation, desmosomes and adherens junctions were observed. Cells closer to the periphery of the aggregates displayed fewer cell projections and also showed signs of release of material extracellularly. After 48 h of culture, a single smooth-walled central cavity was present and cells still exhibited signs of extracellular release of material. These same cell shapes and intercellular junctions were also observed when area opaca tissue dissected from gastrulating embryos was examined. Aggregates of different sizes were created and cultured. The results suggest that a critical tissue mass may be important for cavitation.