Temporal and spatial effects of Sonic hedgehog signaling in chick eye morphogenesis

Proper dorsal--ventral pattern formation of the optic cup is essential for vertebrate eye morphogenesis and retinotectal topographic mapping. Previous studies have suggested that midline tissue-derived Sonic hedgehog (Shh) molecules play critical roles in establishing the bilateral eye fields and in determining the proximal--distal axis of the eye primordium. Here, we have examined the temporal requirements for Shh during the optic vesicle to optic cup transition and after early optic cup formation in chick embryos. Both misexpressing Shh by virus and blocking Shh activity by antibodies resulted in disruption of ventral ocular tissues. Decreasing endogenous Shh signals unexpectedly revealed a sharp morphological boundary subdividing dorsal and ventral portions of the optic cup. In addition, Shh signals differentially influenced expression patterns of genes involved in ocular tissue specification (Pax6, Pax2, and Otx2) and dorsal--ventral patterning (cVax) within the ventral but not dorsal optic cup. Ectopic Shh suppressed expression of Bone Morphogenetic Protein 4 (BMP4) in the dorsal retina, whereas reducing endogenous Sonic hedgehog activity resulted in a ventral expansion of BMP4 territory. These results demonstrate that temporal requirements for Shh signals persist after the formation of the optic cup and suggest that the early vertebrate optic primordium may be subdivided into dorsal and ventral compartments. We propose a model in which ventrally derived Shh signals and dorsally restricted BMP4 signals act antagonistically to regulate the growth and specification of the optic primordium.     

Sonic hedgehog is involved in formation of the ventral optic cup by limiting Bmp4 expression to the dorsal domain

Accumulating evidence suggests that Sonic hedgehog (Shh) signaling plays a crucial role in eye vesicle patterning in vertebrates. Shh promotes expression of Pax2 in the optic stalk and represses expression of Pax6 in the optic cup. Shh signaling contributes to establishment of both proximal–distal and dorsal–ventral axes by activating Vax1, Vax2, and Pax2. In the dorsal part of the developing retina, Bmp4 is expressed and antagonizes the ventralizing effects of Shh signaling through the activation of Tbx5 expression in chick and Xenopus. To examine the roles of Shh signaling in optic cup formation and optic stalk development, we utilized the Smoothened (Smo) conditional knockout (CKO) mouse line. Smo is a membrane protein which mediates Shh signaling into inside of cells. Cre expression was driven by Fgf15 enhancer. The ventral evagination of the optic cup deteriorated from E10 in the Smo-CKO, whereas the dorsal optic cup and optic stalk develop normally until E11. We analyzed expression of various genes such as Pax family (Pax2/Pax6), Vax family (Vax1/Vax2) and Bmp4. Bmp4 expression was greatly upregulated in the optic vesicle by the 21-somite stage. Then Vax1/2 expression was decreased at the 20- to 24-somite stages. Pax2/6 expression was affected at the 27- to 32-somite stages. Our data suggest that the effects of the absence of Shh signaling on Vax1/Vax2 are mediated through increased Bmp4 expression throughout the optic cup. Also unchanged patterns of Raldh2 and Raldh3 suggest that retinoic acid is not the downstream to Shh signaling to control the ventral optic cup morphology.     

Retinoic acid is required for specification of the ventral eye field and for Rathke's pouch in the avian embryo

We have investigated the role of retinoic acid (RA) in eye development using the vitamin A deficient quail model system, which overcomes problems of retinoic acid synthesising enzyme redundancy in the embryo. In the absence of retinoic acid, the ventral optic stalk and ventral retina are missing, whereas the dorsal optic stalk and dorsal retina develop appropriately. Other ocular abnormalities observed were a thinner retina and the lack of differentiation of the lens. In an attempt to explain this, we studied the expression of various dorsally and ventrally expressed genes such as Pax2, Pax6, Tbx6, Vax2, Raldh1 and Raldh3 and noted that they were unchanged in their expression patterns. In contrast, the RA catabolising enzymes Cyp26A1 and Cyp26B1 which are known to be RA-responsive were not expressed at all in the developing eye. At much earlier stages, the expression domain of Shh in the prechordal plate was reduced, as was Nkx2.1 and we suggest a model whereby the eye field is specified according to the concentration of SHH protein that is present. We also describe another organ, Rathke's pouch which fails to develop in the absence of retinoic acid. We attribute this to the down-regulation of Bmp2, Shh and Fgf8 which are known to be involved in the induction of this structure.     

Bone morphogenetic proteins produced by cells within embryoid bodies inhibit ventral directed differentiation by Sonic Hedgehog

Mouse embryoid bodies (EBs) differentiate into dorsal spinal cord neural progenitors in response to retinoic acid (RA). Our data demonstrate that the addition of Sonic Hedgehog (Shh) directs towards a ventral spinal cord neural tube fate, but only at extremely high concentrations. One possible explanation is the presence of dorsal directing factors. Bone morphogenetic proteins (BMPs), known to direct dorsal spinal cord neural differentiation, were expressed in RA-treated EBs. Shh more potently directed ventral differentiation when combined with the BMP inhibitor Noggin. Further, when BMP7 was added, the ability of Shh to direct ventral differentiation was further mitigated.     

Dorsalization of the neural tube by Xenopus tiarin, a novel patterning factor secreted by the flanking nonneural head ectoderm

We have isolated a novel secreted dorsalizing factor of the neural tube, Xenopus Tiarin, which belongs to the olfactomedin-related family. Tiarin expression starts at the late gastrula stage in the nonneural ectoderm adjacent to the anterior neural plate. Overexpression of Tiarin in the embryo causes expansion of dorsal neural markers and suppression of ventral markers. In the eye-forming field, Tiarin overexpression induces the retinal markers and represses optic stalk markers. Tiarin directly dorsalizes neural tissues in the absence of mesodermal tissues and antagonizes the ventralizing activity of Sonic hedghog (Shh). Unlike BMP4, another dorsalizing factor, Tiarin does not display antineuralizing activity on the ectoderm or mesoderm-ventralizing activity. These findings show that Tiarin is a novel patterning signal candidate acting in the specification of the dorsal neural tube.     

Coordinated regulation of dorsal bone morphogenetic protein 4 and ventral Sonic hedgehog signaling specifies the dorso‐ventral polarity in the optic vesicle and governs ocular morphogenesis through fibroblast growth factor 8 upregulation

Dorsal and ventral specification in the early optic vesicle plays a crucial role in vertebrate ocular morphogenesis, and proper dorsal‐ventral polarity in the optic vesicle ensures that distinct structures develop in separate domains within the eye primordium. The polarity is determined progressively during development by coordinated regulation of extraocular dorsal and ventral factors. In the present study, we cultured discrete portions of embryonic chick brains by preparing anterior cephalon, anterior dorsal cephalon and anterior ventral cephalon, and clearly demonstrate that bone morphogenetic protein 4 (BMP4) and Sonic hedgehog (Shh) constitute a dorsal‐ventral signaling system together with fibroblast growth factor 8 (FGF8). BMP4 and Shh upregulate Tbx5 and Pax2, as reported previously, and at the same time Shh downregulates Tbx5, while BMP4 affects Pax2 expression to downregulate similarly. Shh induces Fgf8 expression in the ventral optic vesicle. This, in turn, determines the distinct boundary of the retinal pigmented epithelium and the neural retina by suppressing Mitf expression. The lens develops only when signals from both the dorsal and ventral regions come across together. Inverted deposition of Shh and BMP4 signals in organ‐cultured optic vesicle completely re‐organized ocular structures to be inverted. Based on these observations we propose a novel model in which the two signals govern the whole of ocular development when they encounter each other in the ocular morphogenic domain.     

Early molecular effects of ethanol during vertebrate embryogenesis

Fetal alcohol spectrum disorder (FASD) is the combination of developmental, morphological, and neurological defects that result from exposing human embryos to ethanol (EtOH). Numerous embryonic structures are affected, leading to a complex viable phenotype affecting among others, the anterior/posterior axis, head, and eye formation. Recent studies have provided evidence suggesting that EtOH teratogenesis is mediated in part through a reduction in retinoic acid (RA) levels, targeting mainly the embryonic organizer (Spemann's organizer) and its subsequent functions. EtOH-treated Xenopus embryos were subjected to an analysis of gene expression patterns. Analysis of organizer-specific genes revealed a transient delay in the invagination of gsc- and chordin-positive cells that eventually reach their normal rostro-caudal position. Dorsal midline genes show defects along the rostro-caudal axis, lacking either their rostral (Xbra and Xnot2) or caudal (FoxA4b and Shh) expression domains. Head-specific markers like Otx2, en2, and Shh show abnormal expression patterns. Otx2 exhibits a reduction in expression levels, while en2 becomes restricted along the dorsal/ventral axis. During neurula stages, Shh becomes up-regulated in the rostral region and it is expressed in an abnormal pattern. These results and histological analysis suggest the existence of malformations in the brain region including a lack of the normal fore brain ventricle. An increase in the size of both the prechordal plate and the notochord was observed, while the spinal cord is narrower. The reduction in head and eye size was accompanied by changes in the eye markers, Pax6 and Tbx3. Our results provide evidence for the early molecular changes induced by EtOH exposure during embryogenesis, and may explain some of the structural changes that are part of the EtOH teratogenic phenotype also in FASD individuals.     

Exploration of Shh and BMP paracrine signaling in a prostate cancer xenograft

Stromal–epithelial signaling is a critical regulator of normal prostate development and has been speculated to play an equally important role in the development and progression of prostate cancer. Sonic hedgehog (Shh) and bone morphogenetic proteins (BMP-4, BMP-7), expressed by the urogenital sinus epithelium and mesenchyme, exert reciprocal and coordinate effects on outgrowth of nascent prostate ducts. Over-expression of Shh in the LNCaP xenograft was shown previously to accelerate tumor growth by a paracrine mechanism. A survey of BMP regulators expressed in the developing prostate revealed increased Noggin and BMP-7 mRNA in the stromal component of Shh over-expressing xenografts. In vitro studies demonstrated that treatment of LNCaP cells with BMP-4 and BMP-s7 induced Id-1 expression and inhibited tumor cell proliferation. The activity of BMP-4 was abrogated by co-addition of Noggin; the activity of BMP-7 was not. Quantitative analysis of BMP signaling revealed ambivalent results: decreased tumor cell expression of the BMP response gene Id-1 but increased staining for phospho-SMAD 1,5, 8. To directly test whether increased xenograft tumor growth could be explained by Noggin-mediated blockade of BMP-2/4 effects on tumor cell proliferation, we generated LNCaP xenografts containing stromal cells over-expressing Noggin. Tumor cells in these xenografts exhibited decreased Id-1 and reduced SMAD phosphorylation, but tumor growth was not altered. We conclude that tumor cell Shh expression can induce significant changes in expression of BMP ligands and inhibitors in the stromal microenvironment but that acceleration of LNCaP xenograft tumor growth by Shh over-expression cannot be attributed solely to increased Noggin expression in the tumor stroma.     

Keeping an eye on retinoic acid signaling during eye development

Retinoic acid is a metabolic derivative of vitamin A that plays an essential function in cell-cell signaling by serving as a ligand for nuclear receptors that directly regulate gene expression. The final step in the conversion of retinol to retinoic acid is carried out by three retinaldehyde dehydrogenases encoded by Raldh1 (Aldh1a1), Raldh2 (Aldh1a2), and Raldh3 (Aldh1a3). Mouse Raldh gene knockout studies have been instrumental in understanding the mechanism of retinoic acid action during eye development. Retinoic acid signaling in the developing eye is particularly complex as all three Raldh genes contribute to retinoic acid synthesis in non-overlapping locations. During optic cup formation Raldh2 is first expressed transiently in perioptic mesenchyme, then later Raldh1 and Raldh3 expression begins in the dorsal and ventral retina, respectively, and these sources of retinoic acid are maintained in the fetus. Retinoic acid is not required for dorsoventral patterning of the retina as originally thought, but it is required for morphogenetic movements that form the optic cup, ventral retina, cornea, and eyelids. These findings will help guide future studies designed to identify retinoic acid target genes during eye organogenesis.     

Conserved expression control and shared activity between cognate T-box genes Tbx2 and Tbx3 in connection with Sonic hedgehog signaling during Xenopus eye development

Tbx2 and Tbx3 are considered to be cognate genes within a Tbx2/3/4/5 subfamily of T-box genes and are expressed in closely overlapping areas in a variety of tissues, including the eye. Herein, we show that misexpression of Tbx2 and Tbx3 in Xenopus embryos gave rise to defective eye morphogenesis, which was reminiscent of the defect caused by attenuated Sonic hedgehog (Shh) signaling. Indeed, Tbx2/3 misexpression suppressed Gli1, Gli2, Ptc2 and Pax2, mediators or targets of Hedgehog (Hh) signals. From these data, Tbx2/3 may have a shared function in inhibiting Gli-dependent Shh signaling during eye development. Conversely, the expression of Tbx2/3 was severely affected by both Shh and a putative dominant negative form of Hh, as well as by both transactivator and transrepressor forms of Gli-fusion proteins, suggesting that the expression of Tbx2/3 may be regulated by a Gli-dependent Hh signal transduction pathway. Because the Shh signal has been considered to play crucial roles in the formation of the proximal-distal and dorsal-ventral axes in the eyes, these findings about the mutual regulatory mechanism between Tbx2/3 and Gli-dependent Hh signaling provide valuable insight into the cause of the localized expression of Tbx2/3 and their role during the formation of these axes. In addition, our findings also imply the conserved regulation and shared activity between the cognate genes of Tbx2 and Tbx3.     

Peptidyl-prolyl cis-trans isomerase xFKBP1B induces ectopic secondary axis and is involved in eye formation during Xenopus embryogenesis

Although Xenopus FKBP1A (xFKBP1A) induces an ectopic dorsal axis in Xenopus embryos, involvement of xFKBP1B, a vertebrate paralogue of FKBP1A, in embryogenesis remains undetermined. Here, we demonstrate that xFKBP1B induces ectopic dorsal axis and involves in eye formation of Xenopus embryos. Injection of the xFKBP1B mRNA in ventral blastomeres of 4-cell stage Xenopus embryos induced a secondary axis and showed multiplier effect to that of xFKBP1A on this when xFKBP1A was co-injected. In addition, BMP4 and Smad1 mRNAs did not affect the ability of xFKBP1B to induce the ectopic secondary axis when either was co-injected with xFKBP1B in ventral blastomeres, whereas they downed out that of xFKBP1A, suggesting that xFKBP1A and xFKBP1B induce the ectopic secondary axis through affecting different pathways from each other. On the other hand, the injection of the FKBP1B mRNA in dorsal blastomeres showed eye malformation, and suppressed almost completely the expression of Rx1, Mitf, and Vax2 mRNAs. xFKBP1B was expressed in the dorsal side of the embryo including the eye during embryogenesis at least until stage 46. Injection of morpholino of the xFKBP1B mRNA in dorsal blastomeres induced additional retina or failed to close tapetum nigrum in the ventral side within the optic cap, whereas it did not affect the dorsal organ development. The injection of the morpholino reduced the expression of Xotx2 and Rx1 mRNAs in the eye. These observations suggest that xFKBP1B is a key factor that regulates the expression levels of the genes involved in eye formation during Xenopus embryogenesis.     

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.     

Noggin expression in a mesodermal pluripotent cell line C1 and its regulation by BMP

Osteoblasts and chondrocytes are derived from mesodermal stem cells and their differentiation is under the control of coordinated interaction among signaling molecules. Noggin is one of the signaling molecules which bind to and inactivate BMPs to induce neural tissues and dorsal mesoderm in Xenopus. However, its expression and regulation in mammalian cells has not been known. In this study, we investigated expression of noggin in murine pluripotent mesodermal cell line, C1. Noggin expression was very low in these C1 cells before they were induced to differentiate. When C1 cells were induced to differentiate into chondrocytes in aggregate cultures in the presence of dexamethasone(dex), noggin expression was significantly increased. In a sharp contrast, when the C1 cells were induced to differentiate into osteoblastic cells by the treatment with beta glycerophosphate (betaGP) and ascorbic acid (AA), noggin mRNA expression remained to be barely detectable. Noggin expression was also observed in the developing cartilage of vertebrae in 15.5 dpc mouse embryos. The noggin mRNA level in C1 cells in monolayer cultures was enhanced significantly by the treatment with BMP4/7 in a dose-dependent manner with a maximal effect at 100 ng/ml. The BMP4/7 effect on noggin expression was time dependent starting within 12 h and peaked at 24 h. These results indicate that noggin is expressed in the pluripotent mesodermal cell line C1 and that its expression is regulated by BMP.     

Signaling dynamics of feather tract formation from the chick somatopleure

In the chick, most feathers are restricted to specific areas of the skin, the feather tracts or pterylae, while other areas, such as the apteria, remain bare. In the embryo, the expansion and closure of the somatopleure leads to the juxtaposition of the ventral pteryla, midventral apterium and amnion. The embryonic proximal somatopleural mesoderm is determined to form a feather-forming dermis at 2 days of incubation (E2), while the embryonic distal and the extra-embryonic somatopleure remain open to determination. We found a progressive, lateral expression of Noggin in the embryonic area, and downregulation of Msx1, a BMP4 target gene, with Msx1 expression being ultimately restricted to the most distal embryonic and extra-embryonic somatopleural mesoderm. Msx1 downregulation thus correlates with the formation of the pterylae, and its maintenance to that of the apterium. Suspecting that the inhibition of BMP4 signaling might be linked to the determination of a feather-forming dermis, we grafted Noggin-expressing cells in the distal somatopleure at E2. This elicited the formation of a supplementary pteryla in the midventral apterium. Endogenous Noggin, which is secreted by the intermediate mesoderm at E2, then by the proximal somatopleure at E4, could be sufficient to suppress BMP4 signaling in the proximal somatopleural mesoderm and then in part of the distal somatopleure, thus in turn allowing the formation of the dense dermis of the future pterylae. The same result was obtained with the graft of Shh-producing cells, but Noggin and Shh are both required in order to change the future amnion into a feather-bearing skin. A possible synergistic role of endogenous Shh from the embryonic endoderm remains to be confirmed.     

Preparation and analysis of biological activity of the secreted proteins of the Noggin family

Methods for preparation of recombinant physiologically active proteins of the Noggin family (Noggin1 and Noggin2) of the clawed frog Xenopus laevis that are capable of interaction with BMP factors of the TGF-beta superfamily have been developed. Structures were designed for the expression of Noggin1 and Noggin2 proteins allowing us to produce them from the directly microinjected synthetic mRNA in the cells of developing embryos of the clawed frog, as well as in the prokaryotic expression system. Target proteins contained three Myc-epitopes at the N-terminal. Introduction of these’ tags’ allowed comparison of the expression level of the Noggin1 and Noggin2 proteins, isolation on the affinity immunosorbent, and demonstration of biological activity of the isolated Noggin-proteins through the analysis of their ability to bind the BMP4 factor of the TGF-beta superfamily with coimmunoprecipitation method.     

Expression of Sox1 during Xenopus early embryogenesis

Sox B1 group genes, Sox1, Sox2, and Sox3 (Sox1-3), are involved in neurogenesis in various species. Here, we identified the Xenopus homolog of Sox1, and investigated its expression patterns and neural inducing activity. Sox1 was initially expressed in the anterior neural plate of Xenopus embryos, with expression restricted to the brain and optic vesicle by the tailbud stage. Expression subsequently decreased in the eye region by the tadpole stage. Sox1 expression in animal cap explants was induced by inhibition of BMP signaling in the same manner as Sox2, Sox3, and SoxD. In addition, overexpression of Sox1 induced neural markers in ventral ectoderm and in animal caps. These results implicate Xenopus Sox1 in neurogenesis, especially brain and eye development.     

BMP2 is required for early heart development during a distinct time period

BMP2, like its Drosophila homologue dpp, is an important signaling molecule for specification of cardiogenic mesoderm in vertebrates. Here, we analyzed the time-course of BMP2-requirement for early heart formation in whole chick embryos and in explants of antero-lateral plate mesoderm. Addition of Noggin to explants isolated at stage 4 and cultured for 24 h resulted in loss of NKX2.5, GATA4, eHAND, Mef2A and vMHC expression. At stages 5-8 the individual genes showed differential sensitivity to Noggin addition. While expression of eHAND, NKX2.5 and Mef2A was clearly reduced by Noggin vMHC was only marginally affected. In contrast, GATA4 expression was enhanced after Noggin treatment. The developmental period during which cardiac mesoderm required the presence of BMP signaling in vivo was assessed by implantation of Noggin expressing cells into stage 4-8 embryos which were then cultured until stage 10-11. Complete loss of NKX2.5 and eHAND expression was observed in embryos implanted at stages 4-6, and expression was still suppressed in stages 7 and 8 implanted embryos. GATA4 expression was also blocked by Noggin at stage 4, however increased at stages 5, 6 and 7. Explants of central mesendoderm, that normally do not form heart tissue were employed to study the time-course of BMP2-induced cardiac gene expression. The induction of cardiac lineage markers in central mesendoderm of stage 5 embryos was distinct for different genes. While GATA4, -5, -6 and MEF2A were induced to maximal levels within 6 h after BMP2 addition, eHAND and dHAND required 12 h to reach maximum levels of expression. NKX2.5 was induced by 6 h and accumulated over 48 h. vMHC and titin were induced at significant levels only after 48 h of BMP2 addition. These results indicate that cardiac marker genes display distinct expression kinetics after BMP2 addition and differential response to Noggin treatment suggesting complex regulation of myocardial gene expression in the early tubular heart.     

The BMP antagonist Noggin promotes cranial and spinal neurulation by distinct mechanisms

Here we characterize the consequences of elevated bone morphogenetic protein (BMP) signaling on neural tube morphogenesis by analyzing mice lacking the BMP antagonist, Noggin. Noggin is expressed dorsally in the closing neural folds and ventrally in the notochord and somites. All Noggin-/- pups are born with lumbar spina bifida; depending on genetic background, they may also have exencephaly. The exencephaly is due to a primary failure of neurulation, resulting from a lack of mid/hindbrain dorsolateral hinge point (DLHP) formation. Thus, as previously shown for Shh signaling at spinal levels, BMP activity may inhibit cranial DLHP morphogenesis. However, the increased BMP signaling observed in the Noggin-/- dorsal neural tube is not sufficient to cause exencephaly; it appears to also depend on the action of a genetic modifier, which may act to increase dorsal Shh signaling. The spinal neural tube defect results from a different mechanism: increased BMP signaling in the mesoderm between the limb buds leads to abnormal somite differentiation and axial skeletal malformation. The resulting lack of mechanical support for the neural tube causes spina bifida. We show that this defect is due to elevated BMP4 signaling. Thus, Noggin is required for mammalian neurulation in two contexts, dependent on position along the rostrocaudal axis.     

Intrinsic and extrinsic inhibition of oligodendrocyte development by rat retina

Cell patterning in the vertebrate CNS reflects the combination of localized cell induction, migration and differentiation. A striking example of patterning is the myelination of visual system. In many species, retinal ganglion cell axons are myelinated in the optic nerve but are unmyelinated in the retina. Here, we confirm that rat and mouse retina lack oligodendrocytes and their precursors and identify multiple mechanisms that might contribute to their absence. Soluble cues from embryonic retina inhibit the induction of oligodendrocytes from neural stem cells and their differentiation from optic nerve precursors. This inhibition is mediated by retinal-derived BMPs. During development BMPs are expressed in the retina and addition of the BMP antagonist Noggin reversed retinal inhibition of oligodendrocyte development. The lack of retinal oligodendrocytes does not simply reflect expression of BMPs, since no oligodendrocytes or their precursors developed when embryonic retinal cells were grown in the presence of Noggin and/or inductive cues such as Shh and IGF-1. Similarly, injection of Noggin into the postnatal rat eye failed to induce oligodendrocyte differentiation. These data combined with the proposed inhibition of OPC migration by molecules selectively expressed at the nerve retina junction suggest that multiple mechanisms combine to suppress retinal myelination during development.