Regulation of skeletal progenitor differentiation by the BMP and retinoid signaling pathways

The generation of the paraxial skeleton requires that commitment and differentiation of skeletal progenitors is precisely coordinated during limb outgrowth. Several signaling molecules have been identified that are important in specifying the pattern of these skeletal primordia. Very little is known, however, about the mechanisms regulating the differentiation of limb mesenchyme into chondrocytes. Overexpression of RARalpha in transgenic animals interferes with chondrogenesis and leads to appendicular skeletal defects (Cash, D.E., C.B. Bock, K. Schughart, E. Linney, and T.M. Underhill. 1997. J. Cell Biol. 136:445-457). Further analysis of these animals shows that expression of the transgene in chondroprogenitors maintains a prechondrogenic phenotype and prevents chondroblast differentiation even in the presence of BMPs, which are known stimulators of cartilage formation. Moreover, an RAR antagonist accelerates chondroblast differentiation as demonstrated by the emergence of collagen type II-expressing cells much earlier than in control or BMP-treated cultures. Addition of Noggin to limb mesenchyme cultures inhibits cartilage formation and the appearance of precartilaginous condensations. In contrast, abrogation of retinoid signaling is sufficient to induce the expression of the chondroblastic phenotype in the presence of Noggin. These findings show that BMP and RAR-signaling pathways appear to operate independently to coordinate skeletal development, and that retinoid signaling can function in a BMP-independent manner to induce cartilage formation. Thus, retinoid signaling appears to play a novel and unexpected role in skeletogenesis by regulating the emergence of chondroblasts from skeletal progenitors.     

Cellular and molecular mechanisms of development of the external genitalia

The limb and external genitalia are appendages of the body wall. Development of these structures differs fundamentally in that masculine development of the external genitalia is androgen dependent, whereas development of the limb is not. Despite this fundamental difference in developmental regulation, epithelial-mesenchymal interactions play key roles in the development of both structures, and similar regulatory molecules are utilized as mediators of morphogenetic cell-cell interactions during development of both the limb and external genitalia. Given the relatively high incidence of hypospadias, a malformation of penile development, it is appropriate and timely to review the morphological, endocrine, and molecular mechanisms of development of the genital tubercle (GT), the precursor of the penis in males and the clitoris in females. Morphological observations comparing development of the GT in humans and mouse emphasize the validity of the mouse as an animal model of GT development and validate the results of experimental studies. Accordingly, the use of mutant mice provides important insights into the roles of specific regulatory molecules in development of the external genitalia. While our current understanding of the morphological and molecular mechanisms of mammalian external genitalia development is still rudimentary, this review summarizes the current state of our knowledge and whenever possible draws from the rich experimental embryology literature on other relevant organs such as the developing limb. Future research on the hormonal and molecular mechanisms of GT development may yield strategies to prevent or reduce the incidence of hypospadias and to elucidate the molecular genetic mechanisms of GT morphogenesis, especially in relation to common organogenetic pathways utilized in other organ systems.     

Regulation of neural stem cell by bone morphogenetic protein (BMP) signaling during brain development

Neurogenesis is the process in which neurons are generated from neural stem/progenitor cells (NSCs/NPCs). It involves the proliferation and neuronal fate specification/differentiation of NSCs, as well as migration, maturation and functional integration of the neuronal progeny into neuronal network. NSCs exhibit the two essential properties of stem cells: self-renewal and multipotency. Contrary to previous dogma that neurogenesis happens only during development, it is generally accepted now that neurogenesis can take place throughout life in mammalian brains. This raises a new therapeutic potential of applying stem cell therapy for stroke, neurodegenerative diseases and other diseases. However, the maintenance and differentiation of NSCs/NPCs are tightly controlled by the extremely intricate molecular networks. Uncovering the underlying mechanisms that drive the differentiation, migration and maturation of specific neuronal lineages for use in regenerative medicine is, therefore, crucial for the application of stem cell for clinical therapy as well as for providing insight into the mechanisms of human neurogenesis. Here, we focus on the role of bone morphogenetic protein (BMP) signaling in NSCs during mammalian brain development.     

BMP signaling is required for development of the ciliary body

The ciliary body in the eye secretes aqueous humor and glycoproteins of the vitreous body and maintains the intraocular pressure. The ciliary muscle controls the shape of the lens through the ciliary zonules to focus the image onto the retina. During embryonic development, the ciliary epithelium is derived from the optic vesicle, but the molecular signals that control morphogenesis of the ciliary body are unknown. We report that lens-specific expression of a transgenic protein, Noggin, can block BMP signaling in the mouse eye and result in failure in formation of the ciliary processes. Co-expression of transgenic BMP7 restores normal development of the ciliary epithelium. Ectopic expression of Noggin also promotes differentiation of retinal ganglion cells. These results indicate that BMP signaling is required for development of the ciliary body and may also play a role in regulation of neuronal differentiation in the developing eye.     

The role of BMP signaling in outgrowth and patterning of the Xenopus tail bud.

Tail bud formation in Xenopus depends on interaction between a dorsal domain (dorsal roof) expressing lunatic fringe and Notch, and a ventral domain (posterior wall) expressing the Notch ligand Delta. Ectopic expression of an activated form of Notch, Notch ICD, by means of an animal cap graft into the posterior neural plate, results in the formation of an ectopic tail-like structure containing a neural tube and fin. However, somites are never formed in these tails. Here, we show that BMP signaling is activated in the posterior wall of the tail bud and is involved in the formation of tail somites from this region. Grafts into the posterior neural plate, in which BMP signaling is activated, will form tail-like outgrowths. Unlike the Notch ICD tails, the BMP tails contain well-organized somites as well as neural tube and fin, with the graft contributing to both somites and neural tube. Through a variety of epistasis-type experiments, we show that the most likely model involves a requirement for BMP signaling upstream of Notch activation, resulting in formation of the secondary neural tube, as well as a Notch-independent pathway leading to the formation of tail somites from the posterior wall.     

BMP and Hedgehog signaling during the development of scleral ossicles

Bone development is a complex process, involving multiple tissues and hierarchical inductive interactions. The study of skeletal development has largely focused on endochondral bones while intramembranous bones, such as the scleral ossicles within the avian eye, have received less attention. Our previous research directly demonstrated the involvement of sonic hedgehog and suggested the involvement of bmp2 and 4 during the development of scleral ossicles. The bones of the sclerotic ring are induced by overlying conjunctival papillae at HH 35 and 36. Here, we examine the spatial and temporal expression patterns of ptc1, ihh, bmp2, bmp4 and bmp7. We show that the cells of conjunctival papillae express ptc1, ihh and bmp2 at these stages; coincident with shh expression previously described. Interestingly, both ihh and ptc1 are also expressed in the mesenchyme underlying the papillae unlike shh and bmp2. Bmp4 and bmp7 are not expressed in these regions at any stages examined. Furthermore, using Noggin soaked beads implanted adjacent to papillae, we provide direct evidence that the BMP family of genes are important factors in the development of scleral ossicles. Localized inhibition of BMPs in this way causes a reduced expression of ihh in the surrounding tissue demonstrating that the BMP and Hedgehog pathways interact. Our data also demonstrates that the sclerotic ring has an intrinsic ability to compensate for missing elements. The scleral ossicle system provides a unique opportunity to investigate the epithelial-mesenchymal induction of intramembranous bones of the vertebrate skull.     

BMP signaling is essential for development of skeletogenic and neurogenic cranial neural crest

BMP signaling is essential for a wide variety of developmental processes. To evaluate the role of Bmp2/4 in cranial neural crest (CNC) formation or differentiation after its migration into the branchial arches, we used Xnoggin to block their activities in specific areas of the CNC in transgenic mice. This resulted in depletion of CNC cells from the targeted areas. As a consequence, the branchial arches normally populated by the affected neural crest cells were hypomorphic and their skeletal and neural derivatives failed to develop. In further analyses, we have identified Bmp2 as the factor required for production of migratory cranial neural crest. Its spatial and temporal expression patterns mirror CNC emergence and Bmp2 mutant embryos lack both branchial arches and detectable migratory CNC cells. Our results provide functional evidence for an essential role of BMP signaling in CNC development.     

BMP signaling in the control of skin development and hair follicle growth

Bone morphogenetic proteins (BMPs), their antagonists, and BMP receptors are involved in controlling a large number of biological functions including cell proliferation, differentiation, cell fate decision, and apoptosis in many different types of cells and tissues during embryonic development and postnatal life. BMPs exert their biological effects via using BMP-Smad and BMP-MAPK intracellular pathways. The magnitude and specificity of BMP signaling are regulated by a large number of modulators operating on several levels (extracellular, cytoplasmic, nuclear). In developing and postnatal skin, BMPs, their receptors, and BMP antagonists show stringent spatio-temporal expressions patterns to achieve proper regulation of cell proliferation and differentiation in the epidermis and in the hair follicle. Genetic studies assert an essential role for BMP signaling in the control of cell differentiation and apoptosis in developing epidermis, as well as in the regulation of key steps of hair follicle development (initiation, cell fate decision, cell lineage differentiation). In postnatal hair follicles, BMP signaling plays an important role in controlling the initiation of the growth phase and is also involved in the regulation of apoptosis-driven hair follicle involution. However, additional efforts are required to fully understand the mechanisms and targets involved in the realization of BMP effects on distinct cell population in the skin and hair follicle. Progress in this area of research will hopefully lead to the development of new therapeutic approaches for using BMPs and BMP antagonists in the treatment of skin and hair growth disorders.     

Regulation of cell number by MAPK-dependent control of apoptosis: a mechanism for trophic survival signaling

Trophic mechanisms in which neighboring cells mutually control their survival by secreting extracellular factors play an important role in determining cell number. However, how trophic signaling suppresses cell death is still poorly understood. We now show that the survival of a subset of midline glia cells in Drosophila depends upon direct suppression of the proapoptotic protein HID via the EGF receptor/RAS/MAPK pathway. The TGFalpha-like ligand SPITZ is activated in the neurons, and glial cells compete for limited amounts of secreted SPITZ to survive. In midline glia that fail to activate the EGFR pathway, HID induces apoptosis by blocking a caspase inhibitor, Diap1. Therefore, a direct pathway linking a specific extracellular survival factor with a caspase-based death program has been established.     

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.     

Cyclopentenone prostaglandins induce lymphocyte apoptosis by activating the mitochondrial apoptosis pathway independent of external death receptor signaling

15-Deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) is a naturally occurring cyclopentenone metabolite of PGD(2) that possesses both peroxisome proliferator-activated receptor gamma (PPAR-gamma)-dependent and PPAR-gamma-independent anti-inflammatory properties. Recent studies suggest that cyclopentenone PGs may play a role in the down-regulation of inflammation-induced immune responses. In this study, we report that 15d-PGJ(2) as well as synthetic PPAR-gamma agonists inhibit lymphocyte proliferation. However, only 15d-PGJ(2), but not the specific PPAR-gamma activators, induce lymphocyte apoptosis. We found that blocking of the death receptor pathway in Fas-associated death domain(-/-) or caspase-8(-/-) Jurkat T cells has no effect on apoptosis induction by 15d-PGJ(2). Conversely, overexpression of Bcl-2 or Bcl-x(L) completely inhibits the initiation of apoptosis, indicating that 15d-PGJ(2)-mediated apoptosis involves activation of the mitochondrial pathway. In line with these results, 15d-PGJ(2) induces mitochondria disassemblage as demonstrated by dissipation of mitochondrial transmembrane potential (Deltapsi(m)) and cytochrome c release. Both of these events are partially inhibited by the broad spectrum caspase inhibitor benzyloxycarbonil-Val-Ala-Asp-fluoromethylketone, suggesting that caspase activation may amplify the mitochondrial alterations initiated by 15d-PGJ(2). We also demonstrate that 15d-PGJ(2) potently stimulates reactive oxygen species production in Jurkat T cells, and Deltapsi(m) loss induced by 15d-PGJ(2) is prevented by the reactive oxygen species scavenger N-acetyl-L-cysteine. In conclusion, our data indicate that cyclopentenone PGs like 15d-PGJ(2) may modulate immune responses even independent of PPAR-gamma by activating the mitochondrial apoptosis pathway in lymphocytes in the absence of external death receptor signaling.     

Regulation of gut and heart left-right asymmetry by context-dependent interactions between xenopus lefty and BMP4 signaling

The Lefty subfamily of TGFbeta signaling molecules has been implicated in early development in mouse, zebrafish, and chick. Here, we show that Xenopus lefty (Xlefty) is expressed both bilaterally in symmetric midline domains and unilaterally in left lateral plate mesoderm and anterior dorsal endoderm. To examine the roles of Xlefty in left-right development, we created a system for scoring gut asymmetry and examined the effects of unilateral Xlefty misexpression on gut development, heart development, and Xnr-1 and XPitx2 expression. In contrast to the unilateral effects of Vg1, Activin, Nodal, or BMPs, targeted expression of Xlefty in either the left or the right side of Xenopus embryos randomized the direction of heart looping, gut coiling, and left-right positioning of the gut and downregulated the asymmetric expression of Xnr-1 and XPitx2. It is currently thought that Lefty proteins act as feedback inhibitors of Nodal signaling. However, this would not explain the effects of right-sided Xlefty misexpression. Here, we show that Xlefty interacts with the signaling pathways of other members of the TGFbeta family during left-right development. Results from coexpression of Xlefty and Vg1 indicate that Xlefty can nullify the effects of Vg1 ectopic expression and that Xlefty is downstream of left-sided Vg1 signaling. Results from coexpression of Xlefty and XBMP4 indicate that XLefty and XBMP4 interact both synergistically and antagonistically in a context-dependent manner. We propose a model in which interactions of Xlefty with multiple members of the TGFbeta family enhance the differences between the right-sided BMP/ALK2/Smad pathway and the left-sided Vg1/anti-BMP/Nodal pathway, leading to left-right morphogenesis of the gut and heart.     

Regulation of mammalian Notch signaling and embryonic development by the protein O-glucosyltransferase Rumi

Protein O-glucosylation is a conserved post-translational modification that occurs on epidermal growth factor-like (EGF) repeats harboring the C(1)-X-S-X-P-C(2) consensus sequence. The Drosophila protein O-glucosyltransferase (Poglut) Rumi regulates Notch signaling, but the contribution of protein O-glucosylation to mammalian Notch signaling and embryonic development is not known. Here, we show that mouse Rumi encodes a Poglut, and that Rumi(-/-) mouse embryos die before embryonic day 9.5 with posterior axis truncation and severe defects in neural tube development, somitogenesis, cardiogenesis and vascular remodeling. Rumi knockdown in mouse cell lines results in cellular and molecular phenotypes of loss of Notch signaling without affecting Notch ligand binding. Biochemical, cell culture and cross-species transgenic experiments indicate that a decrease in Rumi levels results in reduced O-glucosylation of Notch EGF repeats, and that the enzymatic activity of Rumi is key to its regulatory role in the Notch pathway. Genetic interaction studies show that removing one copy of Rumi in a Jag1(+/-) (jagged 1) background results in severe bile duct morphogenesis defects. Altogether, our data indicate that addition of O-glucose to EGF repeats is essential for mouse embryonic development and Notch signaling, and that Jag1-induced signaling is sensitive to the gene dosage of the protein O-glucosyltransferase Rumi. Given that Rumi(-/-) embryos show more severe phenotypes compared to those displayed by other global regulators of canonical Notch signaling, Rumi is likely to have additional important targets during mammalian development.     

Contribution of the conserved amino acids of the melanocortin-4 receptor in [corrected] [Nle4,D-Phe7]-alpha-melanocyte-stimulating [corrected] hormone binding and signaling

Melanocortin 4 receptor (MC4R) plays an important role in the regulation of food intake and body weight. To determine the molecular basis of human MC4R (hMC4R) responsible for alpha-melanocortin-stimulating hormone (alpha-MSH) binding, in this study, we utilized both receptor domain exchange and site-directed mutagenesis studies to investigate the molecular determinants of hMC4R responsible for alpha-MSH binding and signaling. alpha-MSH is a potent agonist at hMC4R but not at hMC2R. Cassette substitutions of the second, third, fourth, fifth, and sixth transmembrane regions (TM) of the hMC4R with the homologous regions of hMC2R were performed and alpha-MSH binding and signaling were examined. Our results indicate that each chimeric receptor was expressed at the cell surface and the expression levels remain similar to that of the wild-type receptor. The cassette substitutions of the second, fourth, fifth, and sixth TMs of the hMC4R with homologous regions of the hMC2R did not significantly alter alpha-MSH binding affinity and potency except substitution of the TM3 of the hMC4R, suggesting that the conserved residues in TMs of the hMC4R are crucial for alpha-MSH binding and signaling. Further mutagenesis studies indicate that conserved residues Glu(100) in TM2, Asp(122), Asp(126) in TM3 and Trp(258), Phe(261), His(264) in TM6 are involved in alpha-MSH binding and signaling. In conclusion, our results suggest that the conserved residues in the TM2, TM3, and TM6 of the hMC4R are responsible for alpha-MSH binding and signaling.     

Regulation of wingless signaling by the CKI family in Drosophila limb development

The Wingless (Wg)/Wnt signaling pathway regulates a myriad of developmental processes and its malfunction leads to human disorders including cancer. Recent studies suggest that casein kinase I (CKI) family members play pivotal roles in the Wg/Wnt pathway. However, genetic evidence for the involvement of CKI family members in physiological Wg/Wnt signaling events is lacking. In addition, there are conflicting reports regarding whether a given CKI family member functions as a positive or negative regulator of the pathway. Here we examine the roles of seven CKI family members in Wg signaling during Drosophila limb development. We find that increased CKIepsilon stimulates whereas dominant-negative or a null CKIepsilon mutation inhibits Wg signaling. In contrast, inactivation of CKIalpha by RNA interference (RNAi) leads to ectopic Wg signaling. Interestingly, hypomorphic CKIepsilon mutations synergize with CKIalpha RNAi to induce ectopic Wg signaling, revealing a negative role for CKIepsilon. Conversely, CKIalpha RNAi enhances the loss-of-Wg phenotypes caused by CKIepsilon null mutation, suggesting a positive role for CKIalpha. While none of the other five CKI isoforms can substitute for CKIalpha in its inhibitory role in the Wg pathway, several CKI isoforms including CG12147 exhibit a positive role based on overexpression. Moreover, loss of Gilgamesh (Gish)/CKIgamma attenuates Wg signaling activity. Finally, we provide evidence that several CKI isoforms including CKIalpha and Gish/CKIgamma can phosphorylate the Wg coreceptor Arrow (Arr), which may account, at least in part, for their positive roles in the Wg pathway.