Expression of the follistatin/EGF-containing transmembrane protein M7365 (tomoregulin-1) during mouse development

A novel transmembrane protein (designated X7365) containing two follistatin modules and an epidermal growth factor (EGF) domain has been described in the hypothalamic-pituitary axis of Xenopus laevis. We have now cloned the highly conserved mouse orthologue (M7365), and its mRNA was detected in many mesodermal and (neuro)ectodermal tissues in 8.5-day-old mouse embryos. During further development, M7365 mRNA expression became restricted to certain regions in the brain and to ganglia. In the adult mouse, the brain is the major site of M7365 expression.     

Regulation of nodal and BMP signaling by tomoregulin-1 (X7365) through novel mechanisms

During early vertebrate development, members of the transforming growth factor beta (TGFbeta) family play important roles in a variety of processes, including germ layer specification, patterning, cell differentiation, migration, and organogenesis. The activities of TGFbetas need to be tightly controlled to ensure their function at the right time and place. Despite identification of multiple regulators of Bone Morphogenetic Protein (BMP) subfamily ligands, modulators of the activin/nodal class of TGFbeta ligands are limited, and include follistatin, Cerberus, and Lefty. Recently, a membrane protein, tomoregulin-1 (TMEFF1, originally named X7365), was isolated and found to contain two follistatin modules in addition to an Epidermal Growth Factor (EGF) domain, suggesting that TMEFF1 may participate in regulation of TGFbeta function. Here, we show that, unlike follistatin and follistatin-related gene (FLRG), TMEFF1 inhibits nodal but not activin in Xenopus. Interestingly, both the follistatin modules and the EGF motif contribute to nodal inhibition. A soluble protein containing the follistatin and the EGF domains, however, is not sufficient for nodal inhibition; the location of TMEFF1 at the membrane is essential for its function. These results suggest that TMEFF1 inhibits nodal through a novel mechanism. TMEFF1 also blocks mesodermal, but not epidermal induction by BMP2. Unlike nodal inhibition, regulation of BMP activities by TMEFF1 requires the latter's cytoplasmic tail, while deletion of either the follistatin modules or the EGF motif does not interfere with the BMP inhibitory function of TMEFF1. These results imply that TMEFF1 may employ different mechanisms in the regulation of nodal and BMP signals. In Xenopus, TMEFF1 is expressed from midgastrula stages onward and is enriched in neural tissue derivatives. This expression pattern suggests that TMEFF1 may modulate nodal and BMP activities during neural patterning. In summary, our data demonstrate that tomoregulin-1 is a novel regulator of nodal and BMP signaling during early vertebrate embryogenesis.     

Expression of mRNA for activin-binding protein (follistatin) during early embryonic development of Xenopus laevis.

Follistatin is a specific activin-binding protein and is supposed to control activin functions. During Xenopus embryonic development, activin is thought to act as a natural mesoderm-inducing factor. We isolated here the Xenopus follistatin cDNA from Xenopus ovary cDNA library and studied the expression of Xenopus follistatin gene during the course of early embryonic development. The Xenopus follistatin has an 84% homology at the level of deduced amino acid sequence with human and porcine follistatin. Its 3.5 kb mRNA is first expressed at the gastrula stage, when the expression of activin mRNA becomes first detectable, and increased thereafter. Another species of 2 kb mRNA become detectable from early neurula and also increased dramatically in tadpole. These results suggest that the follistatin acts also as a regulator of activin in inductive interactions during amphibian embryonic development.     

Differential expression of inhibin subunits and follistatin, but not of activin receptor type II, during early murine embryonic development.

Activins are known to be potentially important regulators of early developmental processes in amphibians, birds, and mammalians. In this study we report the expression of the inhibin subunits, including those that make up activin, the activin-binding protein follistatin, and activin receptor type II in several in vitro systems that model early murine embryonic development, namely embryonic stem (ES) cells, embryonal carcinoma (EC) cells, and their differentiated derivatives. In addition, we examine the expression pattern of these factors in different stages of the mouse embryo itself. Expression of inhibin alpha and beta A subunits is restricted to certain differentiated cell types, while beta B subunits are expressed in both differentiated and undifferentiated cells. Our results further indicate a change in the expression pattern of inhibin subunits during early development from beta B at the blastocyst stage largely to beta A in postgastrulation embryos. This is similar to the expression pattern at equivalent stages of Xenopus and chick development. Expression of the activin-binding protein follistatin is altered by the induction of differentiation of P19 EC and ES cells by several factors, including retinoic acid. In contrast to the inhibin subunits and follistatin, activin receptor levels are not influenced by differentiation in these cell types. The results of this study demonstrate that the inhibin subunits and follistatin, but not the activin receptor type II, are differentially expressed during early murine development and suggest that the different forms of activin/inhibin are involved in the regulation of different developmental processes.     

Uterine-embryonic interaction in pig: activin, follistatin, and activin receptor II in uterus and embryo during early gestation

The mRNA expression patterns of activin beta(A) and follistatin in the uterus and embryo, the mRNA expression of the activin receptor II in the embryo, and the localization in the uterus of the immunoreactive activin beta(A) and the receptor II proteins in the uterus were examined at gestation days 7-12 after ovulation in pig. Activin was located predominantly at the mesometrial side of the uterus during all stages of pregnancy studied. Follistatin mRNA was absent in the uterus during these stages, suggesting that activin of uterine origin is not inhibited by intra-uterine follistatin. The receptor was localized throughout the glandular and luminal epithelium of the uterus. In the embryo, activin was expressed predominantly in the epiblast before unfolding, but after unfolding of the epiblast activin expression shifted to the trophoblast. The expression pattern of follistatin mRNA was contrarily to that of activin, i.e., before unfolding predominantly in the trophoblast (days 8-9), and shifted to the epiblast at day 10. During streak stages, follistatin was detected in the node and primitive streak. Activin receptor II mRNA was first detected at day 8 in the embryoblast. At day 11, it was expressed in trophoblast cells near the epiblast, and in the first ingressing mesoderm cells. During the streak stages, it was expressed predominantly in the trophoblast. The presence of activin and its receptor in uterine epithelium and early embryonic tissues indicate that both embryonic and uterine activin are involved in intra-uterine processes, such as attachment and early embryonic development. Mol. Reprod. Dev. 59: 390-399, 2001.     

Cloning,sequencing, and expressional analysis of the chick homologue of follistatin

Follistatin, a secreted glycoprotein, has been shown to act as a potent neural inducer during early amphibian development. The function of this protein during embryogenesis in higher vertebrates is unclear, and to further our understanding of its role we have cloned, sequenced, and performed an in-depth expressional analysis of the chick homologue of follistatin. In addition we also describe the expression pattern of activin βA and activin β B, proteins that have previously been shown to be able to interact with follistatin. In this study we show that the expression of follistatin and the activins do not always overlap. Follistatin was first detected in Hensen's node and subsequently in the region described by Spratt [1952] as the neuralising area. In older embryos it was also expressed in a highly dynamic manner in the hind-brain as well as in the somites. We also present evidence that follistatin may have a later role in the resegmentation of the somites. We were unable to detect the expression of activin βA during early embryogenesis, whereas activin βB was first expressed in the extending primitive streak and subsequently in the neural folds. The results from this study are consistent with a role for follistatin in neural induction but suggest it has additional functions unrelated to its inhibitory actions on activins. © 1995 Wiley-Liss, Inc.     

Expression of Frizzleds and secreted frizzled-related proteins (Sfrps) during mammalian lens development

Recent studies indicate a role for Wnt signaling in regulating lens cell differentiation (Stump et al., 2003). Here we investigated expression patterns of Wnt receptors, the Frizzleds (Fzs) and the Wnt signaling regulators, the secreted frizzled-related proteins (Sfrps), during rodent lens development. RT-PCR showed that Fz receptors, Fz1-Fz8 are expressed in lens. In situ hybridization showed that all the Fz genes examined have similar expression patterns. Fzs are expressed throughout the early lens primordium. At embryonic day 14.5 (E14.5), Fz gene expression is predominantly localized to the epithelium and elongating cells at the lens equator. Fz expression is absent from lens fibers. This pattern of Fz gene expression continues throughout early postnatal development. Immunolocalization studies showed that Fz protein distribution closely follows that of the mRNAs. In addition, epithelial cells in FGF-treated explants show strongest Fz reactivity in cellular protrusions as they migrate and elongate. Sfrp1- Sfrp5 are expressed and all, except Sfrp2, have similar patterns of expression to each other and to the Fzs during lens development. Sfrp2 is strongly expressed in all lens pit cells but becomes restricted to the presumptive epithelial cells of the lens vesicle. By E14.5, Sfrp2 is only present in a few cells above the lens equator. Sfrp2 is not detected in the lens at E18.5 or at later stages. This study shows that multiple Fz and Sfrp genes are expressed during lens morphogenesis and differentiation. This is consistent with a role for Wnt-Fz signaling during both embryonic and postnatal lens development.     

Follistatin attenuates early liver fibrosis: effects on hepatic stellate cell activation and hepatocyte apoptosis

Activin A, a member of the transforming growth factor-beta superfamily, is constitutively expressed in hepatocytes and regulates liver mass through tonic inhibition of hepatocyte DNA synthesis. Follistatin is the main biological inhibitor of activin bioactivity. These molecules may be involved in hepatic fibrogenesis, although defined roles remain unclear. We studied activin and follistatin gene and protein expression in cultured rat hepatic stellate cells (HSCs) and in rats given CCl4 for 8 wk and examined the effect of follistatin administration on the development of hepatic fibrosis. In activated HSCs, activin mRNA was upregulated with high expression levels, whereas follistatin mRNA expression was unchanged from baseline. Activin A expression in normal lobular hepatocytes redistributed to periseptal hepatocytes and smooth muscle actin-positive HSCs in the fibrotic liver. A 32% reduction in fibrosis, maximal at week 4, occurred in CCl4-exposed rats treated with follistatin. Hepatocyte apoptosis decreased by 87% and was maximal at week 4 during follistatin treatment. In conclusion, activin is produced by activated HSCs in vitro and in vivo. Absence of simultaneous upregulation of follistatin gene expression in HSCs suggests that HSC-derived activin is biologically active and unopposed by follistatin. Our in vivo and in vitro results demonstrate that activin-mediated events contribute to hepatic fibrogenesis and that follistatin attenuates early events in fibrogenesis by constraining HSC proliferation and inhibiting hepatocyte apoptosis.     

Expression of BMP signalling pathway members in the developing zebrafish inner ear and lateral line

In this paper we describe the mRNA expression patterns of members of the bone morphogenetic protein (BMP) signalling pathway in the developing zebrafish ear. bmp2b, 4, and 7 are expressed in discrete areas of otic epithelium, some of which correspond to sensory patches. bmp2b and 4 mark the developing cristae before and during the appearance of differentiated hair cells. bmp4 is also expressed in a dorsal, non-sensory region of the ear. Expression of bmps in cristae is conserved between zebrafish, chick, and mouse, but there are also notable differences in ear expression patterns between these species. Of five zebrafish BMP antagonists, only one (follistatin) shows significant expression in the otic epithelium. The type I receptor bmpr-IB shows localised expression in the ear epithelium. Mediators of BMP signalling, smad1 and smad5, are expressed in statoacoustic and lateral line ganglia; smad5 is also expressed at low levels throughout the ear epithelium. An inhibitory smad, smad6, is expressed laterally in the ear epithelium. Lateral line primordia and neuromasts also express bmp2b, 4, follistatin, smad1, and smad5. The conservation of bmp expression in cristae among different species adds weight to the growing evidence that BMPs are required for the development of the vertebrate ear.     

Developmental profiles of activin betaA, betaB, and follistatin expression in the zebrafish ovary: evidence for their differential roles during sexual maturation and ovulatory cycle

Our recent experiments showed that gonadotropin(s) stimulated activin betaA and follistatin expression through the cAMP-PKA pathway but suppressed betaB via a cAMP-dependent but PKA-independent pathway in cultured zebrafish follicle cells. Given that pituitary gonadotropins are the major hormones controlling the development and function of the ovary, the differential expression of activin betaA and betaB as well as follistatin in response to gonadotropin(s) raises an interesting question about the temporal expression patterns of these molecules in vivo during sexual maturation and ovulatory cycle. Three experiments were performed in the present study. In the first experiment using sexually immature zebrafish, we followed the expression of activin betaA, betaB, and follistatin at the whole ovary level during a 10-day period in which the ovary developed from the primary growth stage to the one with nearly full-grown follicles. Activin betaA expression was very low at the primary growth stage but significantly increased with the growth of the ovary, and its rise was accompanied by an increase in follistatin expression. In contrast, the expression of activin betaB could be easily detected in the ovary of all stages; however, it did not exhibit an obvious trend of variation during the development. The second experiment examined the stage-dependent expression of activin betaA, betaB, and follistatin at the follicle level in the adult mature zebrafish. The expression of activin betaA was again low in the follicles during the primary growth stage, but exhibited a phenomenal increase after the follicles entered vitellogenesis with the peak level reached at midvitellogenic stage; in contrast, activin betaB mRNA could be easily detected at all stages with a slight increase during follicle growth. The expression of follistatin, on the other hand, also increased significantly during vitellogenesis; however, its level dropped sharply after reaching the peak at the midvitellogenic stage. In the third experiment, we investigated the dynamic changes of the ovarian activin betaA, betaB, and follistatin expression during the daily ovulatory cycle. The expression of activin betaA and follistatin gradually increased from 1800 h onward and reached the peak level around 0400 h when the germinal vesicles had migrated to the periphery in the full-grown oocytes. In contrast, activin betaB expression steadily declined, although not statistically significant, during the same period, but increased sharply at 0700 h when mature oocytes started to appear in most of the ovaries collected. In conclusion, activin betaA and betaB exhibit distinct expression patterns during the development of the ovary and the daily ovarian cycle of the zebrafish. It seems that activin betaA is involved in promoting ovary and follicle growth, whereas activin betaB may have a tonic role throughout follicle development but becomes critical at the late stage of oocyte maturation and/or ovulation.     

Sexually dimorphic gene expression in the developing mouse gonad

Over the course of a few days, the bipotential embryonic mouse gonad differentiates into either a testis or an ovary. Though a few gene expression differences that underlie gonadal sex differentiation have been identified, additional components of the testicular and ovarian developmental pathways must be identified to understand this process. Here we report the use of a PCR-based cDNA subtraction to investigate expression differences that arise during gonadal sex differentiation. Subtraction of embryonic day 12.5 (E12.5) XY gonadal cDNA with E12.5 XX gonadal cDNA yielded 19 genes that are expressed at significantly higher levels in XY gonads. These genes display a variety of expression patterns within the embryonic testis and encode a broad range of proteins. A reciprocal subtraction (of E12.5 XX gonadal cDNA with E12.5 XY gonadal cDNA) yielded two genes, follistatin and Adamts19, that are expressed at higher levels in XX gonads. Follistatin is a well-known antagonist of TGFbeta family members while Adamts19 encodes a new member of the ADAMTS family of secreted metalloproteases.     

Roles of intracerebral activin,inhibin, and follistatin in the regulation of Kiss-1 gene expression: Studies using primary cultures of fetal rat neuronal cells

Hypothalamic kisspeptin, encoded by the Kiss-1 gene, governs the hypothalamic-pituitary-gonadal axis by directly regulating the release of gonadotropin-releasing hormone. In this study, we examined the roles of activin, inhibin, and follistatin in the regulation of Kiss-1 gene expression using primary cultures of fetal rat neuronal cells, which express the Kiss-1 gene and kisspeptin. Stimulation with activin significantly increased Kiss-1 gene expression in these cultures by 2.02 ± 0.39-fold. In contrast, a significant decrease in Kiss-1 gene expression was observed with inhibin A and follistatin treatment. Inhibin B did not modulate Kiss-1 gene expression. Activin, inhibin, and follistatin were also expressed in fetal rat brain cultures and their expression was controlled by estradiol (E2). The inhibin α, βA, and βB subunits were upregulated by E2. Similarly, follistatin gene expression was significantly increased by E2 in these cells. Our results suggest the possibility that activin, inhibin, and follistatin expressed in the brain participate in the E2-induced feedback control of the hypothalamic-pituitary-gonadal axis.     

Opposing Effects of Activin A and Follistatin on Developing Skeletal Muscle Cells

Activin and the activin-binding protein follistatin modulate a variety of biological processes and are abundant at sites of muscle development. Activin and follistatin were expressed in developing chick pectoral musclein vivoand in primary cell culture. Addition of recombinant activin inhibited muscle development in a dose-dependent manner as measured by the number of nuclei in myosin heavy chain positive cells and creatine phosphokinase activity. Conversely, follistatin potentiated muscle development. The effects of activin were found to be distinct from those of the related protein transforming growth factor (TGF) β1. Muscle development was repressed by activin at all time points investigated and did not recover with the removal of activin following a limited exposure. In contrast, while myogenic differentiation in TGFβ1 was initially repressed, muscle marker expression recovered to control levels—even in the continued presence of TGFβ1. Fibroblast growth factor (FGF) had little effect on inhibiton of muscle development caused by activin A. However, inhibition of development produced by TGFβ increased with increasing concentrations of FGF. Finally, early expression of myoD and myf5 mRNA by muscle cultures in the presence of activin and follistatin was analyzed. Activin-treated cultures expressed reduced myoD and myf5 levels at 1.5 days after plating. Myf5 levels in follistatin-treated cultures were elevated, but, surprisingly, these cultures showed a reduction in myoD levels. These data suggest that endogenously expressed activin and follistatin are important modulators of muscle development.     

Regulation of Hoxb3 expression in the hindbrain and pharyngeal arches by rae28, a member of the mammalian Polycomb group of genes

During animal development, Hox genes are expressed in characteristic, spatially restricted patterns and specify regional identities along the anterior-posterior (A-P) axis. Polycomb group (PcG) proteins in Drosophila repress Hox expression and maintain the expression patterns during development. Mice deficient for homologues of the Drosophila PcG genes, such as M33, bmi1, mel18, rae28 and eed, show altered Hox expression patterns. In this study, we examined the time course of Hoxb3 expression during late gastrulation and early segmentation of rae28-deficient mice. Hoxb3 was expressed ectopically in pharyngeal arch and hindbrain from embryonic day (E) 9.5 and 10.5, respectively. The anterior boundary of ectopic expression in the hindbrain extended gradually in the rostral direction as development proceeded from E10.5 to E12.5. Expression of kreisler and Krox20, which function as positive regulators of Hoxb3 expression, was not affected in rae28-deficient embryos. Analysis of a neural crest marker, p75, in rae28-deficient mice revealed that the neural crest cells begin to ectopically express Hoxb3 after leaving the hindbrain. Our results suggest that rae28 is not required for the establishment but maintenance of Hoxb3 expression.     

Spatial and temporal expression of Wnt and Dickkopf genes during murine lens development

Recent studies indicate a role for Wnt signalling in regulating lens cell differentiation (Stump et al., 2003). To further our understanding of this, we investigated the expression patterns of Wnts and Wnt signalling regulators, the Dickkopfs (Dkks), during murine lens development. In situ hybridisation showed that Wnt5a, Wnt5b, Wnt7a, Wnt7b, Wnt8a and Wnt8b genes are expressed throughout the early lens primordia. At embryonic day 14.5 (E14.5), Wnt5a, Wnt5b, Wnt7a, Wnt8a and Wnt8b are reduced in the primary fibres, whereas Wnt7b remains strongly expressed. This trend persists up to E15.5. At later embryonic stages, Wnt expression is predominantly localised to the epithelium and elongating cells at the lens equator. As fibre differentiation progresses, Wnt expression becomes undetectable in the cells of the lens cortex. The one exception is Wnt7b, which continues to be weakly expressed in cortical fibres. This pattern of expression continues through to early postnatal stages. However, by postnatal day 21 (P21), expression of all Wnts is distinctly weaker in the central lens epithelium compared with the equatorial region. This is most notable for Wnt5a, which is barely detectable in the central lens epithelium at P21. Dkk1, Dkk2 and Dkk3 have similar patterns of expression to each other and to the majority of the Wnts during lens development. This study shows that multiple Wnt and Dkk genes are expressed during lens development. Expression is predominantly in the epithelial compartment but is also associated, particularly in the case of Wnt7b, with early events in fibre differentiation.     

Expression of BMP2/4/7 during the odontogenesis of deciduous molars in miniature pig embryos

Bone morphogenetic proteins (BMPs) play important roles in tooth development. However, their expression has not been studied in miniature pigs, which have many anatomical similarities in oral and maxillofacial region compared to human. This study investigated BMP2/4/7 expression patterns during deciduous molar development in miniature pigs on embryonic days (E) 40, 50, and 60. The mandibles were fixed, decalcified, and embedded before sectioning. H&E staining, immunohistochemistry, in situ hybridization using specific radionuclide-labeled cRNA probes, and real-time PCR were used to detect the BMP expression patterns during morphogenesis of the third deciduous molar. H&E staining showed that for the deciduous third molar, E40 represented the cap stage, E50 represented the early bell stage, and E60 represented the late bell stage or secretory stage. BMP2 was expressed in both the enamel organ and in the dental mesenchyme on E40 and E50 and was expressed mainly in pre-odontoblasts on E60. BMP7 expression was similar to BMP2 expression, but BMP7 was also expressed in the inner enamel epithelium on E60. On E40, BMP4 was expressed mainly in the epithelium, with some weak expression in the mesenchyme. On E50, BMP4 expression was stronger in the mesenchyme but weaker in the epithelium. On E60, BMP4 was expressed mainly in the mesenchyme. These data indicated that BMP2/4/7 showed differential spatial and temporal expression during the morphogenesis and odontogenesis of deciduous molars, suggesting that these molecules were associated with tooth morphogenesis and cell differentiation.     

Distinct expression of midkine and pleiotrophin in the spinal cord and placental tissues during early mouse development

Midkine and pleiotrophin comprise a family of heparin-binding growth factors, and are expressed in overlapping tissues during the mid- to late-gestation periods of mouse development. Their distinct expression during early mouse development, as revealed by in situ hybridization, was reported. Midkine was expressed in the embryonic ectoderm from as early as embryonic day (E5.5). In the neural tube midkine was expressed specifically in the neuroepithelium, that is, in the whole area of the neural tube at E9.5, and in the ventricular zone from E10.5-13.5. At E15.5, when the neuroepithelium disappeared, midkine concomitantly became undetectable. In contrast, pleiotrophin expression started exclusively in the neural plate at E8.5, and in the lateral plate of the neural tube at E9.5. It then became restricted to a dorsal ventricular zone from E11.5-13.5, and finally to the central gray neurons at E15.5. Moreover, pleiotrophin was expressed in the ventral horns. Among placental tissues, midkine was detected in the chorion, the fetal component of the placenta, whereas pleiotrophin was found in the decidua basalis, the maternal component of the placenta. The distinct expression of midkine and pleiotrophin suggests their differential role in early development.