Embryonic mesoderm cells spread in response to platelet-derived growth factor and signaling by phosphatidylinositol 3-kinase.

Abnormal mesoderm movement, leading to defects in axial organization, is observed in mouse and Xenopus laevis embryos deprived of platelet-derived growth factor (PDGF) AA signaling. However, neither the cellular response to PDGF nor the signaling pathways involved are understood. Herein we describe an in vitro assay to examine the direct effect of PDGF AA on aggregates of Xenopus embryonic mesoderm cells. We find that PDGF AA stimulates aggregates to spread on fibronectin. This behavior is similar to that of migrating mesoderm cells in vivo that spread and form lamellipodia and filipodia on contact with fibronectin-rich extracellular matrix. We go on to show two lines of evidence that implicate phosphatidylinositol 3-kinase (PI3K) as an important component of PDGF-induced mesoderm cell spreading. (i) The fungal metabolite wortmannin, which inhibits signaling by PI3K, blocks mesoderm spreading in response to PDGF AA. (ii) Activation of a series of receptors with specific tyrosine-to-phenylalanine mutations revealed PDGF-induced spreading of mesoderm cells depends on PI3K but not on other signaling molecules that interact with PDGF receptors including phospholipase C gamma, Ras GTPase-activating protein, and phosphotyrosine phosphatase SHPTP2. These results indicate that a PDGF signal, medicated by PI3K, can facilitate embryonic mesoderm cell spreading on fibronectin. We propose that PDGF, produced by the ectoderm, influences the adhesive properties of the adjacent mesoderm cells during gastrulation.

A novel homeobox gene PV.1 mediates induction of ventral mesoderm in Xenopus embryos.

The formation of ventral mesoderm has been traditionally viewed as a result of a lack of dorsal signaling and therefore assumed to be a default state of mesodermal development. The discovery that bone morphogenetic protein 4 (BMP4) can induce ventral mesoderm led to the suggestion that the induction of the ventral mesoderm requires a different signaling pathway than the induction of the dorsal mesoderm. However, the individual components of this pathway remained largely unknown. Here we report the identification of a novel Xenopus homeobox gene PV.1 (posterior-ventral 1) that is capable of mediating induction of ventral mesoderm. This gene is activated in blastula stage Xenopus embryos, its expression peaks during gastrulation and declines rapidly after neurulation is complete. PV.1 is expressed in the ventral marginal zone of blastulae and later in the posterior ventral area of gastrulae and neurulae. PV.1 is inducible in uncommited ectoderm by the ventralizing growth factor BMP4 and counteracts the dorsalizing effects of the dominant negative BMP4 receptor. Overexpression of PV.1 yields ventralized tadpoles and rescues embryos partially dorsalized by LiCl treatment. In animal caps, PV.1 ventralizes induction by activin and inhibits expression of dorsal specific genes. All of these effects mimic those previously reported for BMP4. These observations suggest that PV.1 is a critical component in the formation of ventral mesoderm and possibly mediates the effects of BMP4.

Mutations in the rotated abdomen locus affect muscle development and reveal an intrinsic asymmetry in Drosophila.

In bilateral animals, the left and right sides of the body usually present asymmetric structures, the genetic bases of whose generation are still largely unknown [CIBA Foundation (1991) Biological Asymmetry and Handedness, CIBA Foundation Symposium 162 (Wiley, New York), pp. 1-327]. In Drosophila melanogaster, mutations in the rotated abdomen (rt) locus cause a clockwise helical rotation of the body. Even null alleles are viable but exhibit defects in embryonic muscle development, rotation of the whole larval body, and helical staggering of cuticular patterns in abdominal segments of the adult. rotated abdomen is expressed in the embryonic mesoderm and midgut but not in the ectoderm; it encodes a putative integral membrane glycoprotein (homologous to key yeast mannosyltransferases). Mesodermal cells defective in O-glycosylation lead to an impaired larval muscular system. We propose that the staggering of the adult abdominal segments would be a consequence of the relaxation of intrinsic rotational torque of muscle architecture, preventing the colateral alignment of the segmental histoblast cells during their proliferation at metamorphosis.

A type 1 serine/threonine kinase receptor that can dorsalize mesoderm in Xenopus.

We have cloned a type I serine/threonine kinase receptor, XTrR-I, from Xenopus. XTrR-I (Xenopus transforming growth factor beta-related receptor type I) is expressed in all regions of embryos throughout early development. Overexpression of this receptor does not affect ectoderm or endoderm but dorsalizes the mesoderm such that muscle appears in ventral mesoderm and notochord appears in lateral mesoderm normally fated to become muscle. In addition, overexpression of XTrR-I in UV-treated embryos is able to cause formation of a partial dorsal axis. These results suggest that XTrR-I encodes a receptor which responds in normal development to a transforming growth factor beta-like ligand so as to promote dorsalization. Its function would therefore be to direct mesodermalized tissue into muscle or notochord.

Endothelin 3 induces skin pigmentation in a keratin-driven inducible mouse model

Endothelin 3 (Edn3) is a ligand important to developing neural crest cells (NCC). Some NCC eventually migrate into the skin and give rise to the pigment-forming melanocytes found in hair follicles. Edn3's effects on NCC have been largely explored through spontaneous mutants and cell culture experiments. These studies have shown the Endothelin receptor B/Edn3 signaling pathway to be important in the proliferation/survival and differentiation of developing melanocytes. To supplement these investigations I have created doxycycline-responsive transgenic mice which conditionally over-express Edn3. These mice will help us clarify Edn3's role during the development of early embryonic melanoblasts, differentiating melanocyte precursors in the skin, and fully differentiated melanocytes in the hair follicle. The transgene mediated expression of Edn3 was predominantly confined to the roof plate of the neural tube and surface ectoderm in embryos and postnatally in the epidermal keratinocytes of the skin. Relative to littermate controls, transgenics develop increased pigmentation on most areas of the skin. My doxycycline-based temporal studies have shown that both embryonic and postnatal events are important for establishing and maintaining pigmented skin. The study of my Edn3 transgenic mice may offer some insight into the genetics behind benign dermal pigmentation and offer clues about the time periods important in establishing these conditions. This apparently abnormal development is echoed in a benign condition of human skin. Cases of dermal melanocytosis, such as common freckles, Mongolian spotting, and nevus of Ito demonstrate histological and etiological characteristics similar to those of the transgenic mice generated in this study.

Inducible Overexpression of Endothelin-3 in a Transgenic Mouse Leads to Hyperpigmentation That Decreases Over Time

Endothelin-3 (Edn3) has been shown to be an essential environmental cue in melanocyte development. Edn3 and its receptor, EdnrB, are allelic to mouse mutations occurring at the lethal spotting and piebald loci, respectively; these mutations result in hypopigmentation phenotypes. Mutations in the genes for both Edn3 and EdnrB are implicated in human pigmentation disorders such as Waardenburg-Shah syndrome, which is characterized by pigmentation defects, deafness, and megacolon. In this study, a tetracycline-inducible transgenic mouse model that overexpresses Edn3 under the control of the Keratin 5 promoter was shown to produce a hyperpigmentation phenotype that decreases over time. The expression pattern of transgenic Edn3 and its effects on the melanocyte population were examined in transgenic embryos, postnatal skin, and the skin of adult mice that exhibit faded hyperpigmentation. These studies suggest that overexpression of Edn3 in this model is restricted primarily to the roof plate of the neural tube and surface ectoderm in the developing embryo and to keratinocytes in the epidermis of postnatal mice. A decline in transgenic expression and a reduction in the dermal melanocytes and free melanin that characterize the phenotype in juvenile mice were shown to correlate with the fading of the hyperpigmentation phenotype. Transgenic mice in which transgenic expression was repressed (resulting in the disappearance of the hyperpigmentation phenotype) also exhibited a decrease in the dermal melanocyte population. The Edn3-overexpressing mice used in this study might be helpful m understanding human skin conditions characterized by dermal melanocytosis.