BMP receptor-activated Smads confer diverse functions during the development of the dorsal spinal cord.

Bone Morphogenetic Proteins (BMPs) have multiple activities in the developing spinal cord: they specify the identity of the dorsal-most neuronal populations and then direct the trajectories of dorsal interneuron (dI) 1 commissural axons. How are these activities decoded by dorsal neurons to result in different cellular outcomes? Our previous studies have shown that the diverse functions of the BMPs are mediated by the canonical family of BMP receptors and then regulated by specific inhibitory (I) Smads, which block the activity of a complex of Smad second messengers. However, the extent to which this complex translates the different activities of the BMPs in the spinal cord has remained unresolved. Here, we demonstrate that the receptor-activated (R) Smads, Smad1 and Smad5 play distinct roles mediating the abilities of the BMPs to direct cell fate specification and axon outgrowth. Smad1 and Smad5 occupy spatially distinct compartments within the spinal cord, with Smad5 primarily associated with neural progenitors and Smad1 with differentiated neurons. Consistent with this expression profile, loss of function experiments in mouse embryos reveal that Smad5 is required for the acquisition of dorsal spinal neuron identities whereas Smad1 is critical for the regulation of dI1 axon outgrowth. Thus the R-Smads, like the I-Smads, have discrete roles mediating BMP-dependent cellular processes during spinal interneuron development.

Amnion formation in the mouse embryo: the single amniochorionic fold model.

BACKGROUND: Despite the detailed knowledge obtained over the last decade on the molecular regulation of gastrulation in amniotes, the process of amnion development has been poorly described and illustrated in mice, and conflicting descriptions exist. Understanding the morphogenesis and development not only of the early mouse embryo, but also of its extraembryonic tissues, is crucial for correctly interpreting fate-mapping data and mouse mutants with gastrulation defects. Moreover, the recent isolation from amnion of cells with stem cell features further argues for a better understanding of the process of amnion formation. Here, we revisit the highly dynamic process of amnion formation in the mouse. Amnion development starts early during gastrulation and is intimately related to the formation of the exocoelom and the expansion of the amniotic fold. The authoritative description involves the fusion of two amniotic folds, a big posterior and a smaller anterior fold. We challenged this 'two amniotic folds' model by performing detailed histomorphological analyses of dissected, staged embryos and 3D reconstructions using historical sections. RESULTS: A posterior fold of extraembryonic ectoderm and associated epiblast is formed early during gastrulation by accumulation of extraembryonic mesoderm posterior to the primitive streak. Previously called the "posterior amniotic fold", we rename it the "amniochorionic fold" (ACF) because it forms both amnion and chorion. Exocoelom formation within the ACF seems not to involve apoptosis within the mesoderm. The ACF and exocoelom expand without disrupting the anterior junction of epiblast, extraembryonic ectoderm and visceral endoderm. No separate anterior fold is formed; its absence was confirmed in 3D reconstructions. Amnion and chorion closure is eccentric, close to the anterior margin of the egg cylinder: we name it the "anterior separation point". CONCLUSIONS: Here, we reconcile previous descriptions of amnion formation and provide new nomenclature, as well as an animation, that clarify and emphasize the arrangement of the tissues that contribute to amnion development and the dynamics of the process. According to our data, the amnion and the chorion are formed by a single amniochorionic fold initiated posteriorly. Finally, we give an overview on mutant mouse models with impaired amnion development.

Fetal mesenchymal stem cells: isolation, properties and potential use in perinatology and regenerative medicine.

The fetus is a source of nonembryonic stem cells (SC), with potential applications in perinatal medicine. Cells derived from the placenta, membranes, amniotic fluid or fetal tissues are higher in number, expansion potential and differentiation abilities compared with SC from adult tissues. Although some obstacles keep SC biology at distance from clinical application, the feasibility of using (homologous) SC for tissue engineering for the fetus with a congenital birth defect has been demonstrated. Also, other pathologies may benefit from SC technology.

Functional redundancy of TGF-beta family type I receptors and receptor-Smads in mediating anti-Mullerian hormone-induced Mullerian duct regression in the mouse.

Amniotes, regardless of genetic sex, develop two sets of genital ducts: the Wolffian and Müllerian ducts. For normal sexual development to occur, one duct must differentiate into its corresponding organs, and the other must regress. In mammals, the Wolffian duct differentiates into the male reproductive tract, mainly the vasa deferentia, epididymides, and seminal vesicles, whereas the Müllerian duct develops into the four components of the female reproductive tract, the oviducts, uterus, cervix, and upper third of the vagina. In males, the fetal Leydig cells produce testosterone, which stimulates the differentiation of the Wolffian duct, whereas the Sertoli cells of the fetal testes express anti-Müllerian hormone, which activates the regression of the Müllerian duct. Anti-Müllerian hormone is a member of the transforming growth factor-beta (TGF-beta) family of secreted signaling molecules and has been shown to signal through the BMP pathway. It binds to its type II receptor, anti-Müllerian hormone receptor 2 (AMHR2), in the Müllerian duct mesenchyme and through an unknown mechanism(s); the mesenchyme induces the regression of the Müllerian duct mesoepithelium. Using tissue-specific gene inactivation with an Amhr2-Cre allele, we have determined that two TGF-beta type I receptors (Acvr1 and Bmpr1a) and all three BMP receptor-Smads (Smad1, Smad5, and Smad8) function redundantly in transducing the anti-Müllerian hormone signal required for Müllerian duct regression. Loss of these genes in the Müllerian duct mesenchyme results in male infertility due to retention of Müllerian duct derivatives in an otherwise virilized male.

Functional redundancy of extracellular matrix protein 1 in epidermal differentiation.

BACKGROUND: Extracellular matrix protein 1 (ECM1) is a secreted protein expressed in skin. Its dermatological relevance has been highlighted by the discovery of loss-of-function mutations in ECM1 in patients with lipoid proteinosis (LiP). OBJECTIVES: To determine the role of ECM1 in epidermal differentiation by examining gene and protein expression of epidermal differentiation markers in individuals with LiP and histological assessment of transgenic mouse skin that overexpresses Ecm1a in basal or suprabasal epidermis. METHODS: Subconfluent, confluent and postconfluent LiP and control keratinocyte cultures were analysed by Northern and Western blotting for differences in expression of differentiation markers. Expression of these markers was analysed in skin of patients with LiP by immunohistochemistry. To study effects of Ecm1 overexpression on epidermal differentiation, transgenic mice were generated under control of either a keratin 14 or an involucrin promoter. RESULTS: No differential expression of the different markers analysed was observed in LiP keratinocytes compared with controls. No histological differences were found in Ecm1-overexpressing mouse skin compared with wild-type. CONCLUSIONS: Absence of ECM1 does not lead to differences in epidermal differentiation. Moreover, overexpression of Ecm1a in vivo does not exert dramatic effects on epidermal structure. Collectively, these findings suggest no role of ECM1 in epidermal differentiation.

Transforming growth factor beta signalling in vitro and in vivo: activin ligand-receptor interaction, Smad5 in vasculogenesis, and repression of target genes by the deltaEF1/ZEB-related SIP1 in the vertebrate embryo.

The identification and characterization of components of the transforming growth factor beta (TGFbeta) signalling pathway are proceeding at a very fast pace. To illustrate a number of our activities in this field, we first summarize our work aiming at the selection from a large collection of single residue substitution mutants of two activin A polypeptides in which D27 and K102, respectively, have been modified. This work has highlighted the importance of K102 and its positive charge for binding to activin type II receptors. Activin K102E, which did not bind to high-affinity receptor complexes, may be a valuable beta chain, when incorporated in recombinant inhibin to unambiguously detect novel inhibin binding sites at the cell surface. We then illustrate how Smad5 knockout mice and an overexpression approach with a truncated TGFbeta type II receptor in the mouse embryo can contribute to the identification of a novel TGFbeta-->TbetaRII/ALK1-->Smad5 pathway in endothelial cells in the embryo proper and the yolk sac vasculature. We conclude with a summary of our results with a Smad-interacting transcriptional repressor but focus on its biological significance in the vertebrate embryo.

Mice with a homozygous gene trap vector insertion in mgcRacGAP die during pre-implantation development.

In a phenotypic screen in mice using a gene trap approach in embryonic stem cells, we have identified a recessive loss-of-function mutation in the mgcRacGAP gene. Maternal protein is present in the oocyte, and mgcRacGAP gene transcription starts at the four-cell stage and persists throughout mouse pre-implantation development. Total mgcRacGAP deficiency results in pre-implantation lethality. Such E3.5 embryos display a dramatic reduction in cell number, but undergo compaction and form a blastocoel. At E3.0-3.5, binucleated blastomeres in which the nuclei are partially interconnected are frequently observed, suggesting that mgcRacGAP is required for normal mitosis and cytokinesis in the pre-implantation embryo. All homozygous mutant blastocysts fail to grow out on fibronectin-coated substrates, but a fraction of them can still induce decidual swelling in vivo. The mgcRacGAP mRNA expression pattern in post-implantation embryos and adult mouse brain suggests a role in neuronal cells. Our results indicate that mgcRacGAP is essential for the earliest stages of mouse embryogenesis, and add evidence that CYK-4-like proteins also play a role in microtubule-dependent steps in the cytokinesis of vertebrate cells. In addition, the severe phenotype of null embryos indicates that mgcRacGAP is functionally non-redundant and cannot be substituted by other GAPs during early cleavage of the mammalian embryo.

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.

Expression of the inhibitory Smad7 in early mouse development and upregulation during embryonic vasculogenesis.

SMAD proteins are downstream targets of serine/threonine kinase receptors of the transforming growth factor beta (TGF beta) superfamily. Ligands activating these receptors regulate cell growth, differentiation and development in many tissues of various organisms. In mammals eight different Smad genes are known, each with different roles in mediating signalling between plasma membrane and nucleus. Smad6 and Smad7 are inhibitors of TGF beta family signalling. They are both expressed in human adult vascular endothelial cells, particularly after these cells have been subjected to shear stress (Topper et al. [1997] Proc Natl Acad Sci USA 94:9314-9319). Here we show by reverse transcriptase polymerase chain reaction and in situ hybridization that Smad7 mRNA is highly expressed in the developing vascular system of the mouse embryo but is also detectable much earlier in preimplantation embryos and during gastrulation. We also demonstrate by transient transgenesis that overexpression of Smad7 in mouse zygotes inhibits development beyond the 2-cell stage. This confirms earlier conclusions of similar, but complementary, experiments using a dominant negative type II TGF beta receptor demonstrating that TGF beta signalling is required for normal preimplantation development.

Expression patterns of follistatin and two follistatin-related proteins during mouse development.

We compared the expression patterns of follistatin and two follistatin-related proteins (FRP and m7365) during early mouse development. m7365 is expressed continuously during preimplantation development, in contrast to FRP and follistatin. At early postimplantation stages, follistatin and 7365 are expressed from E6.0, while FRP is detected from E7.5 onwards. Although there is some overlap between the expression of these genes in the primitive streak and somites, their overall expression patterns are distinct.

Smad5 is essential for left-right asymmetry in mice.

Left-right (L-R) asymmetry of the vertebrate body plan is established from an originally morphologically symmetric embryo. Recent studies have implicated several TGF-beta family signaling proteins (i.e., nodal, lefty-1, lefty-2, activin receptor type IIB, and Smad2) in L-R axis determination in the mouse. However, the genetic pathways underlying L-R patterning are still unclear. Smad5 is a downstream component in the TGF-beta family signaling cascade, and lack of Smad5 results in embryonic lethality between E9.5 and E11.5. In this report, we demonstrate that Smad5 mutant embryos have defects in heart looping and embryonic turning which are the first signs of L-R asymmetry in mice. To gain more insights into the molecular basis of the laterality defects in the Smad5-deficient embryos, we examined the expression of lefty-1, lefty-2, nodal, and Pitx2 since the asymmetric expression of these genes always closely correlates with the direction of heart looping and embryonic turning. In the absence of Smad5, lefty-1 was expressed at very low or undetectable levels, while nodal, lefty-2, and Pitx2 were expressed bilaterally. These data suggest that Smad5 is upstream of lefty-1, nodal, and lefty-2, and as a consequence also of Pitx2, and Smad5 is essential for L-R axis determination.

Epidermal differentiation does not involve the pro-apoptotic executioner caspases, but is associated with caspase-14 induction and processing.

The epidermis is a stratified squamous epithelium in which keratinocytes progressively undergo terminal differentiation towards the skin surface leading to programmed cell death. In this respect we studied the role of caspases. Here, we show that caspase-14 synthesis in the skin is restricted to differentiating keratinocytes and that caspase-14 processing is associated with terminal epidermal differentiation. The pro-apoptotic executioner caspases-3, -6, and -7 are not activated during epidermal differentiation. Caspase-14 does not participate in apoptotic pathways elicited by treatment of differentiated keratinocytes with various death-inducing stimuli, in contrast to caspase-3. In addition, we show that non-cornifying oral keratinocyte epithelium does not express caspase-14 and that the parakeratotic regions of psoriatic skin lesions contain very low levels of caspase-14 as compared to normal stratum corneum. These observations strongly suggest that caspase-14 is involved in the keratinocyte terminal differentiation program leading to normal skin cornification, while the executioner caspases are not implicated. Cell Death and Differentiation (2000) 7, 1218 - 1224

Presenilin 2 deficiency causes a mild pulmonary phenotype and no changes in amyloid precursor protein processing but enhances the embryonic lethal phenotype of presenilin 1 deficiency.

Mutations in the homologous presenilin 1 (PS1) and presenilin 2 (PS2) genes cause the most common and aggressive form of familial Alzheimer's disease. Although PS1 function and dysfunction have been extensively studied, little is known about the function of PS2 in vivo. To delineate the relationships of PS2 and PS1 activities and whether PS2 mutations involve gain or loss of function, we generated PS2 homozygous deficient (-/-) and PS1/PS2 double homozygous deficient mice. In contrast to PS1(-/-) mice, PS2(-/-) mice are viable and fertile and develop only mild pulmonary fibrosis and hemorrhage with age. Absence of PS2 does not detectably alter processing of amyloid precursor protein and has little or no effect on physiologically important apoptotic processes, indicating that Alzheimer's disease-causing mutations in PS2, as in PS1, result in gain of function. Although PS1(+/-) PS2( -/-) mice survive in relatively good health, complete deletion of both PS2 and PS1 genes causes a phenotype closely resembling full Notch-1 deficiency. These results demonstrate in vivo that PS1 and PS2 have partially overlapping functions and that PS1 is essential and PS2 is redundant for normal Notch signaling during mammalian embryological development.

Transforming growth factor-beta signalling in extraembryonic mesoderm is required for yolk sac vasculogenesis in mice.

We have analysed the function of transforming growth factor beta (TGF-beta) in yolk sac development in mice by generating somatic chimaeras in which the extraembryonic mesoderm, which gives rise to the endothelial and haematopoietic cells of the yolk sac vasculature, is derived from embryonic stem (ES) cells. The ES cells were stably transfected and express either the full-length type II binding receptor or a kinase-deficient mutant of this receptor. Examination of yolk sacs from chimaeras between E8.5 and 9.5, and analysis of marker expression in embryoid bodies from these mutant ES cell lines in prolonged suspension culture demonstrated that (1) a major function of TGF-beta in yolk sac mesoderm is to regulate production and deposition of fibronectin in the extracellular matrix that maintains yolk sac integrity, (2) TGF-beta signalling is not required for differentiation of extraembryonic mesoderm into endothelial cells but is necessary for their subsequent organisation into robust vessels, and (3) TGF-beta signalling must be tightly regulated for the differentiation of primitive haematopoietic cells to take place normally. Together, these results show that defective TGF-beta signalling in the extraembryonic mesoderm alone is sufficient to account for the extraembryonic phenotype reported previously in TGF-beta1(-/-) mice (Dickson, M. C., Martin, J. S., Cousins, F. M., Kulkarni, A. B., Karlsson, S. and Akhurst, R. J. (1995) Development 121, 1845-1854).

Smad5 knockout mice die at mid-gestation due to multiple embryonic and extraembryonic defects.

Smad5 has been implicated as a downstream signal mediator for several bone morphogenetic proteins (BMPs). To understand the in vivo function of Smad5, we generated mice deficient in Smad5 using embryonic stem (ES) cell technology. Homozygous mutant embryos die between E9.5 and E11.5, and display variable phenotypes. Morphological defects are first detected at E8.0 in the developing amnion, gut and heart (the latter defect being similar to BMP-2 knockout mice). At later stages, mutant embryos fail to undergo proper turning, have craniofacial and neural tube abnormalities, and are edematous. In addition, several extraembryonic lesions are observed. After E9.0, the yolk sacs of the mutants contain red blood cells but lack a well-organized vasculature, which is reminiscent of BMP-4, TGF-beta1 and TGF-beta type II receptor knockout mice. In addition, the allantois of many Smad5 mutants is fused to the chorion, but is not well-elongated. A unique feature of the Smad5 mutant embryos is that ectopic vasculogenesis and hematopoiesis is observed in the amnion, likely due to mislocation of allantois tissue. Despite the expression of Smad5 from gastrulation onwards, and in contrast to knockouts of Smad2 and Smad4, Smad5 only becomes essential later in extraembryonic and embryonic development.

Ectopic expression of the transforming growth factor beta type II receptor disrupts mesoderm organisation during mouse gastrulation.

Transforming growth factor beta (TGFbeta) regulates the cell cycle and extracellular matrix (ECM) deposition of many cells in vitro. We have analysed chimaeric mouse embryos generated from embryonic stem cells with abnormal receptor expression to study the effect of TGFbeta on these processes in vivo and the consequences for normal development. The binding receptor for TGFbeta, TbetaRII, is first detected in the embryo proper around day 8.5 in the heart. Ectopic expression of TbetaRII from the blastocyst stage onward resulted in an embryonic lethal around 9.5 dpc. Analysis of earlier stages revealed that the primitive streak of TbetaRII chimaeras failed to elongate. Furthermore, although cells passed through the streak and initially formed mesoderm, they tended to accumulate within the streak. These defects temporally and spatially paralleled the expression of the TGFbeta type I receptor, which is first expressed in the node and primitive streak. We present evidence that classical TGFbeta-induced growth inhibition was probably the cause of insufficient mesoderm being available for paraxial and axial structures. The results demonstrate that (1) TGFbeta mRNA and protein detected previously in early postimplantation embryos is present as a biologically active ligand; and (2) assuming that ectopic expression of TbetaRII results in no other changes in ES cells, the absence of TbetaRII is the principle reason why the embryo proper is unresponsive to TGFbeta ligand until after gastrulation.

Mouse embryonic stem cells with aberrant transforming growth factor beta signalling exhibit impaired differentiation in vitro and in vivo.

Embryonic stem (ES) cells are resistant to transforming growth factor beta (TGF beta). We have shown previously that they lack type-II binding receptors (T beta RII) and in this respect resemble the inner cell mass and ectoderm cells of mouse embryos 4.5-7.5 days post coitum (dpc); they do however express type-I (alk-5) signalling receptors. Here we show that in contrast to several tumour cell lines, stable transfection of wtT beta RII is not sufficient for ES cells to become biologically sensitive to TGF beta. We analysed the expression of several down-stream molecules known to be involved in TGF beta signalling (Smads) and TGF beta-mediated cell cycle regulation (cyclins D) during the differentiation of control and wtT beta RII-expressing ES cells and showed that upregulation of these molecules correlated with (i) an increase in plasminogen activator inhibitor-1 (PAI-1) synthesis and (ii) growth inhibition, following addition of TGF beta 1. These TGF beta responses were reduced in an ES cell line expressing a dominant negative (truncated) T beta RII (delta T beta RII). The differentiation pattern of control and wtT beta RII-expressing ES cells was indistinguishable in monolayer culture and as embryoid bodies, but in delta T beta RII ES cells, the capacity to form mesodermal derivatives in monolayer cultures in response to the addition of retinoic acid (RA) and removal of leukemia inhibitory factor (LIF) was lost, and only endoderm-like cells formed. The T beta RII and delta T beta RII ES cells were, however, both distinguishable from control ES cells when allowed to differentiate in chimaeric embryos following aggregation with morula-stage hosts. Conceptuses containing mutant cells, recovered from pseudopregnant females at the equivalent of 9.5 dpc, exhibited highly defective yolk sac development; most strikingly, no blood vessels were present and in addition the yolk sacs with derivatives of ES cells containing wtT beta RII were blistered and lacked haematopoietic cells. The implications for understanding TGF beta signalling in early mouse development are discussed.

Identification of two distinct functions for TGF-beta in early mouse development.

In this study the function of transforming growth factor-beta (TGF-beta) in preimplantation mouse embryos was examined. By RT-PCR, mRNA for the signalling type I (T beta R-I) and type II (T beta R-II) receptors for TGF-beta was shown to be present in two distinct time windows: in fertilized oocytes and at the blastocyst stage. The function of TGF-beta at these times was analysed in two ways. Firstly, the TGF-beta signalling pathway was blocked by injecting a DNA construct encoding a truncated T beta R-II, that acts as a dominant-negative receptor, in fertilized oocytes, and the effect on development was determined. Secondly, inner cell masses isolated at the blastocyst stage were cultured in vitro with and without TGF-beta under conditions that favour the outgrowth of parietal endoderm. The results show that TGF-beta signalling mediated by maternally expressed receptors is important for development of preimplantation embryos beyond the two-cell stage, and suggest a regulatory role for TGF-beta in the outgrowth of parietal endoderm.

Cyclic AMP-induced differentiation increases the synthesis of extracellular superoxide dismutase in rat C6 glioma.

The effect of membrane permeable cAMP analogues on the expression of extracellular superoxide dismutase (EC-SOD) was studied in rat C6 glioma. EC-SOD is constitutively expressed but stimulation with cAMP analogues still increased the EC-SOD transcription and the secreted SOD activity. The potency to enhance EC-SOD expression is correlated with the ability of the cAMP analogue to induce cAMP-dependent differentiation in C6. The increase in EC-SOD mRNA and in secreted activity depended on the concentration of the cAMP analogues and on the cultivation time. Twenty-four hours after addition of 0.5 mM N6, O'2-dibutyryl cAMP (dbcAMP) or N6-monobutyryl cAMP (N6-mbcAMP) EC-SOD mRNA expression increased approximately twofold, while stimulation for 68 h with 0.5 mM N6-mbcAMP or 1 mM 8-Chloro cAMP (ClcAMP) and 1 mM dbcAMP enhanced the mean secreted activity/cell three- and fivefold, respectively. O'2-monobutyryl cAMP (O'2-mbcAMP) did not affect EC-SOD synthesis. The enhancement in EC-SOD activity did not require activation of protein kinase A. ATP, TGF-beta, IFN-gamma, and LPS did not affect EC-SOD synthesis. The presented data point to a cAMP-dependent pathway for the enhanced expression of EC-SOD by glial cells in brain.

Cadherin-dependent cell aggregation is affected by decapeptide derived from rat extracellular super-oxide dismutase.

A synthetic HAV-containing decapeptide homologous to the amino acid sequence 44R-Q53 in rat extracellular superoxide dismutase B affects cadherin-dependent cell aggregation. Cell lines, some of them transfected, expressing different types of cadherins were tested using in vitro cell aggregation and cell dissociation assays. A concentration-dependent inhibition of aggregation by the EC-SOD-derived HAV-containing peptide was detected only in N-cadherin expressing cells. These results suggest the localisation and possible protective role of EC-SOD B for cells expressing N-cadherin.