Ventral vs. dorsal chick dermal progenitor specification

The dorsal and the ventral trunk integuments of the chick differ in their dermal cell lineage (originating from the somatic and somatopleural mesoderm respectively) and in the distribution of their feather fields. The dorsal macropattern has a large spinal pteryla surrounded by semi-apteria, whereas the ventral skin has a true medial apterium surrounded by the ventral pterylae. Comparison of the results of heterotopic transplantations of distal somatopleure in place of somatic mesoderm (Mauger 1972) or in place of proximal somatopleure (our data), leads to two conclusions. These are that the fate of the midventral apterium is not committed at day 2 of incubation and that the signals from the environment which specify the ventral and dorsal featherforming dermal progenitors are different. Effectively, Shh, but not Wnt -1 signalling can induce the formation of feather forming dermis from the embryonic somatopleure. Shh is not able, however, to trigger the formation of a feather forming dermis from the extra embryonic somatopleure. This brief report constitutes the first attempt, by comparing old and new preliminary results, to understand whether dermal progenitors at different sites are specified by different signalling pathways.     

Axes establishment during eye morphogenesis in Xenopus by coordinate and antagonistic actions of BMP4, Shh,and RA

We have examined the roles of BMP4, Shh, and retinoic acid in establishing the proximal-distal and dorsal-ventral axes in the developing Xenopus eye. Misexpression of BMP4 caused the absence of an optic stalk and the expansion of dorsal and distal markers, tbx2/3/5, and pax6, at the expense of ventral and proximal markers vax2 and pax2. When Shh or Noggin, an antagonist of BMPs, was misexpressed, the reverse expression patterns of these marker genes were observed. These results suggest that BMP4 is involved in the specification of not only dorsal in the optic cup but also distal in the optic vesicle. Because Shh did not suppress bmp4 expression, unlike Noggin, Shh and BMP4 may antagonistically regulate common downstream genes in developing eye. We also found the difference between the effects of Shh and retinoic acid, another possible ventralizing factor, suggesting that Shh could promote ventralization independently of retinoic acid. These findings provide important clues to the coordinate and antagonistic actions of BMP4, Shh, and retinoic acid in axes specifications of Xenopus eyes.     

Inductive signals from the somatopleure mediated by bone morphogenetic proteins are essential for the formation of the sternal component of avian ribs

The posterior five pairs of avian ribs are composed of vertebral and sternal components, both derived from the somitic mesoderm. For the patterning of the rib cartilage, inductive signals from neighboring tissues on the somitic mesoderm have been suggested to play critical roles. The notochord and surface ectoderm overlying the somitic mesoderm are essentially required for the development of proximal and distal regions of the ribs, respectively. Involvement of the somatopleure in rib development has already been suggested but is less understood than those of the notochord and surface ectoderm. In this study, we reinvestigated the role of the somatopleure during rib development. We first identified the chicken homologue of the mouse Mesenchymal forkhead-1 (cMfh-1) gene based on sequence similarities. cMfh-1 was observed to be expressed in the nonaxial mesoderm, including the somitic mesoderm, and, subsequently, in cartilage forming the ribs, vertebrae, and appendicular skeletal system. In the interlimb region, corresponding to somites 21-25 (or 26), cMfh-1-positive somitic mesoderm was seen penetrating the somatopleure of E4 embryos, and cMfh-1 was used as a molecular marker demarcating prospective rib cartilage. A series of experiments affecting the penetration of the somitic mesoderm into the somatopleure was performed in the present study, resulting in defects in sternal rib formation. The inductive signals emanating from the somatopleure mediated by BMP family proteins were observed to be essentially involved in the ingrowth of the somitic mesoderm. BMP4 alone, however, could not completely replace inductive signals from the somatopleure, suggesting the involvement of additional signals for rib formation.     

The formation of the avian scapula blade takes place in the hypaxial domain of the somites and requires somatopleure-derived BMP signals

The avian scapula is a long bone located dorsally on the thorax. The cranial part that articulates with the upper limb is derived from the somatopleure of the forelimb field, while the caudal part, the scapula blade, originates from the dermomyotomes of brachial and thoracic somites. In previous studies, we have shown that scapula blade formation is intrinsically controlled by segment-specific information as well as extrinsically by ectoderm-derived signals. Here, we addressed the role of signals derived from the lateral plate mesoderm on scapula development. Chick-quail chimera experiments revealed that scapula precursor cells are located within the hypaxial domain of the dermomyotome adjacent to somatopleural cells. Barrier implantation between these two cell populations inhibited scapula blade formation. Furthermore, we identified BMPs as scapula-inducing signals from the somatopleure using injection of Noggin-producing cells into the hypaxial domain of scapula-forming dermomyotomes. We found that inhibition of BMP activity interfered with scapula-specific Pax1 expression and scapula blade formation. Taken together, we demonstrate that the scapula-forming cells located within the hypaxial somitic domain require BMP signals derived from the somatopleure for their specification and differentiation.     

Vimentin Expression Pattern is Different between the Flank Region and Limb Regions of Somatopleural Mesoderm in the Chicken Embryo

It is known that the chicken flank somatopleure also has a limb-forming potential at early stages of development, but loses this potential later. Molecular changes during this process is, however, not well known. We obtained a monoclonal antibody which reacts to the flank somatopleure, but not to the wing bud, the leg bud and the neck somatopleure in the stage 22 chicken embryo. Further study revealed that this antibody is specific to vimentin. Time course of vimentin expression in the somatopleural mesoderm during the development was studied. It was revealed to be biphasic. Somatopleural mesoderm expressed vimentin at stage 10, but not at stage 16. Flank somatopleural mesoderm began to express vimentin again at stage 18, whereas limb bud mesenchymal cells did not until stage 27. The earlier re-expression of vimentin at the flank somatopleure suggests that certain physiological changes take place in cells at this region.     

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.     

Role of Islet1 in the patterning of murine dentition

It is believed that mouse dentition is determined by a prepatterning of the oral epithelium into molar (proximal) and incisor (distal) regions. The LIM homeodomain protein Islet1 (ISL1) is involved in the regulation of differentiation of many cell types and organs. During odontogenesis, we find Islet1 to be exclusively expressed in epithelial cells of the developing incisors but not during molar development. Early expression of Islet1 in presumptive incisor epithelium is coincident with expression of Bmp4, which acts to induce Msx1 expression in the underlying mesenchyme. To define the role of ISL1 in the acquisition of incisor shape, we have analysed regulation of Islet1 expression in mandibular explants. Local application of bone morphogenetic protein 4 (BMP4) in the epithelium of molar territories either by bead implantation or by electroporation stimulated Islet1 expression. Inhibition of BMP signalling with Noggin resulted in a loss of Islet1 expression. Inhibition of Islet1 in distal epithelium resulted in a loss of Bmp4 expression and a corresponding loss of Msx1 expression, indicating that a positive regulatory loop exists between ISL1 and BMP4 in distal epithelium. Ectopic expression of Islet1 in proximal epithelium produces a loss of Barx1 expression in the mesenchyme and resulted in inhibition of molar tooth development. Using epithelial/mesenchymal recombinations we show that at E10.5 Islet1 expression is independent of the underlying mesenchyme whereas at E12.5 when tooth shape specification has passed to the mesenchyme, Islet1 expression requires distal (presumptive incisor) mesenchyme. Islet1 thus plays an important role in regulating distal gene expression during jaw and tooth development.     

A new function of BMP4: dual role for BMP4 in regulation of Sonic hedgehog expression in the mouse tooth germ

The murine tooth development is governed by sequential and reciprocal epithelial-mesenchymal interactions. Multiple signaling molecules are expressed in the developing tooth germ and interact each other to mediate the inductive tissue interactions. Among them are Sonic hedgehog (SHH), Bone Morphogenetic Protein-2 (BMP2) and Bone Morphogenetic Protein-4 (BMP4). We have investigated the interactions between these signaling molecules during early tooth development. We found that the expression of Shh and Bmp2 is downregulated at E12.5 and E13.5 in the dental epithelium of the Msx1 mutant tooth germ where Bmp4 expression is significantly reduced in the dental mesenchyme. Inhibition of BMP4 activity by noggin resulted in repression of Shh and Bmp2 in wild-type dental epithelium. When implanted into the dental mesenchyme of Msx1 mutants, beads soaked with BMP4 protein were able to restore the expression of both Shh and Bmp2 in the Msx1 mutant epithelium. These results demonstrated that mesenchymal BMP4 represents one component of the signal acting on the epithelium to maintain Shh and Bmp2 expression. In contrast, BMP4-soaked beads repressed Shh and Bmp2 expression in the wild-type dental epithelium. TUNEL assay indicated that this suppression of gene expression by exogenous BMP4 was not the result of an increase in programmed cell death in the tooth germ. Ectopic expression of human Bmp4 to the dental mesenchyme driven by the mouse Msx1 promoter restored Shh expression in the Msx1 mutant dental epithelium but repressed Shh in the wild-type tooth germ in vivo. We further demonstrated that this regulation of Shh expression by BMP4 is conserved in the mouse developing limb bud. In addition, Shh expression was unaffected in the developing limb buds of the transgenic mice in which a constitutively active Bmpr-IB is ectopically expressed in the forelimb posterior mesenchyme and throughout the hindlimb mesenchyme, suggesting that the repression of Shh expression by BMP4 may not be mediated by BMP receptor-IB. These results provide evidence for a new function of BMP4. BMP4 can act upstream to Shh by regulating Shh expression in mouse developing tooth germ and limb bud. Taken together, our data provide insight into a new regulatory mechanism for Shh expression, and suggest that this BMP4-mediated pathway in Shh regulation may have a general implication in vertebrate organogenesis.     

Transformation of amnion epithelium into skin and hair follicles

There is increasing interest into the extent to which epithelial differentiation can be altered by mesenchymal influence, and the molecular basis for these changes. In this study, we investigated whether amnion epithelium could be transformed into skin and hair follicles by associating E12.5 to E14.5 mouse amnion from the ROSA 26 strain, with mouse embryonic hair-forming dermis from a wild-type strain. These associations were able to produce fully formed hair follicles with associated sebaceous glands, and skin epidermis. Using beta-galactosidase staining we were able to demonstrate that the follicular epithelium and skin epidermis, but not the associated dermal cells, originated from the amnion. As Noggin and Sonic hedgehog (Shh) were recently shown to be required for early chick ventral skin formation, and able to trigger skin and feather formation from chick amnion, we associated cells engineered to produce those two factors with mouse amnion. In a few cases, we obtained hair buds connected to a pluristratified epithelium; however, the transformation of the amnion was impeded by uncontrolled fibroblastic proliferation. In contrast to an earlier report, none of our control amnion specimens autonomously transformed into skin and hair follicles, indicating that specific influences are necessary to elicit follicle formation from the mouse amnion. The ability to turn amnion into skin and its appendages has practical potential for the tissue engineering of replacement skin, and related biotechnological approaches.     

BMP signaling positively regulates Nodal expression during left right specification in the chick embryo

Exogenous application of BMP to the lateral plate mesoderm (LPM) of chick embryos at the early somite stage had a positive effect on Nodal expression. BMP applications into the right LPM were followed by a rapid activation of Nodal, while applications into the left LPM resulted in expansion of the normal domain of Nodal expression. Conversely, blocking of BMP signaling by Noggin in the left LPM interfered with the activation of Nodal expression. These results support a positive role for endogenous BMP on Nodal expression in the LPM. We also report that BMP positively regulates the expression of Caronte, Snail and Cfc in both the left and right LPM. BMP-treated embryos had molecular impairment of the midline with downregulation of Lefty1, Brachyury and Shh but we also show that the midline defect was not sufficient to induce ectopic Nodal expression. We discuss our findings in the context of the known molecular control of the specification of left-right asymmetry.     

The expression of the homeobox gene Msx1 reveals two populations of dermal progenitor cells originating from the somites

Experimental manipulation in birds has shown that trunk dermis has a double origin: dorsally, it derives from the somite dermomyotome, while ventrally, it is formed by the somatopleure. Taking advantage of an nlacZ reporter gene integrated into the mouse Msx1 locus (Msx1(nlacZ) allele), we detected segmental expression of the Msx1 gene in cells of the dorsal mesenchyme of the trunk between embryonic days 11 and 14. Replacing somites from a chick host embryo by murine Msx1(nlacZ )somites allowed us to demonstrate that these Msx1-(beta)-galactosidase positive cells are of somitic origin. We propose that these cells are dermal progenitor cells that migrate from the somites and subsequently contribute to the dorsalmost dermis. By analysing Msx1(nlacZ) expression in a Splotch mutant, we observed that migration of these cells does not depend on Pax3, in contrast to other migratory populations such as limb muscle progenitor cells and neural crest cells. Msx1 expression was never detected in cells overlying the dermomyotome, although these cells are also of somitic origin. Therefore, we propose that two somite-derived populations of dermis progenitor cells can be distinguished. Cells expressing the Msx1 gene would migrate from the somite and contribute to the dermis of the dorsalmost trunk region. A second population of cells would disaggregate from the somite and contribute to the dermis overlying the dermomyotome. This population never expresses Msx1. Msx1 expression was investigated in the context of the onset of dermis formation monitored by the Dermo1 gene expression. The gene is downregulated prior to the onset of dermis differentiation, suggesting a role for Msx1 in the control of this process.     

How and when the regional competence of chick epidermis is established: feathers vs. scutate and reticulate scales, a problem en route to a solution

Most of the chick body is covered with feathers, while the tarsometatarsus and the dorsal face of the digits form oblong overlapping scales (scuta) and the plantar face rounded nonoverlapping scales (reticula). Feathers and scuta are made of beta-keratins, while the epidermis of reticula and inter-appendage or apteria (nude regions) express a-keratins. These regional characteristics are determined in skin precursors and require an epidermal FGF-like signal to be expressed. Both the initiation of appendages, their outline and pattern depend on signals from the dermis, while their asymmetry and outgrowth depend on epidermal competence. For example, the plantar dermis of the central foot pad induces reticula in a plantar or feathers in an apteric epidermis, in a hexagonal pattern starting from the medial point. By manipulating Shh levels in the epidermis, the regional appendage type can be changed from scuta or reticula to feather, whereas the inhibition of Wnt7a, together with a downregulation of Shh gives rise to reticula and in extreme cases, apteria. During morphogenesis of plantar skin, the epidermal expression of En-1, acting as a repressor both of Wnt7a and Shh, is linked to the formation of reticula. Finally, in birds, the complex formation of feathers, which can be easily triggered, even in the extra-embryonic somatopleure, may result from a basic genetic program, whereas the simple formation of scales appears secondarily derived, as requiring a partial (scuta) or total (reticula) inhibition of epidermal outgrowth and beta-keratin gene expression, an inhibition lost for the scuta in the case of feathered feet breeds.     

Rôle du tube neural dans le développement du plumage dorsal de l'embryon de Poulet

Résumé Des expériences d'excision et de remplacement par divers implants d'une portion de tube neural et des expériences d'interposition d'un écran entre le tube neural et le mésoderme somitique ont été pratiquées chez l'embryon de Poulet de 2 à 2 jours 1/2 d'incubation, afin d'étudier le rôle du tube neural dans le développement du plumage dorsal.L'excision entraîne la non-différenciation d'une portion de la ptéryle spinale: absence de chevrons plumaires sans perturbation du patron hexagonal, ou formation d'une aptérie ou d'une zone irrégulièrement emplumée.Le remplacement par un fragment d'agar-agar ou de tantale ou par un autre tissu (tube digestif, mésoderme somatopleural, somites) aboutit aux mêmes types de déficience du plumage dorsal que ceux qui sont produits par les excisions. De même, le remplacement par un fragment de tube neural préalablement traité à la température de 100° C perturbe gravement le développement du plumage dorsal. Au contraire, si le tube neural a été exposé à la température de 60° C seulement, le patron plumaire spinal se développe normalement ou presque.L'interposition d'un écran solide de 0,8 à 2 mm de long (tantale, membrane coquillière, filtre Millipore) entre le tube neural et le mésoderme somitique provoque une échancrure triangulaire de la ptéryle spinale en empêchant le développement des plumes du territoire plumigène situé latéralement par rapport à l'écran.On en conclut que la présence du tube neural est indispensable à la différenciation du patron plumaire dorsal. Le tube neural ne peut être remplacé par un autre tissu, même plumigène, ni par des objets inanimés. Il est vraisemblablement nécessaire à la transformation du dermatome en cellules prédermiques. En son absence, le derme dense ne se constitue pas et, par conséquent, la ptéryle spinale ne peut se développer au niveau de l'opération.

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Role of neural tube in the development of the dorsal plumage in the chick embryo

Summary Experiments were performed on 2- to 2 1/2-day chick embryos in order to study the role of the neural tube in the development of the dorsal plumage. Pieces of neural tube were excised or replaced by various living or inanimate implants. In other experiments, a screen was interposed between neural tube and somitic mesoderm.The excision resulted in the non-differentiation of a portion of the spinal pteryla: absence of several feather chevrons without disruption of the hexagonal feather pattern, or formation of an apterium or an irregularly feathered area.The replacement by a piece of agar or tantalum or by another tissue (gut, somatopleural mesoderm, somites) led to the same type of dorsal plumage deficiencies as those which were produced by the excisions. Similarly, the replacement by a fragment of neural tube treated at 100° C severely interfered with the development of dorsal plumage. On the contrary, when the neural tube had been exposed to a temperature of 60° C only, the spinal feather pattern was normal or nearly so.The interposition of a solid screen 0.8 to 2 mm in length (tantalum, egg shell membrane, Millipore filter) between neural tube and somitic mesoderm resulted in the formation of a featherless triangular notch in one side of the spinal pteryla. The screen prevented the development of the feathers in the feather field lateral to the screen.These experiments show that the neural tube is indispensable for the differentiation of the dorsal feather pattern. The neural tube cannot be replaced by inanimate objects or by any of the tested tissues, not even by feather-forming ones like somites. Its presence is likely to be necessary for the transformation of dermatomes into predermal cells. When it is absent, dense feather-forming dermis does not form at that level and, consequently, the corresponding portion of the spinal pteryla cannot develop.

     

Mouse epiblasts change responsiveness to BMP4 signal required for PGC formation through functions of extraembryonic ectoderm

Mouse primordial germ cells (PGCs) are initially identified as a cluster of alkaline phosphatase (AP)-positive cells within the extraembryonic mesoderm near the posterior part of the primitive streak at embryonic day (E) 7.25. Clonal analysis of epiblast cells has revealed that the putative precursors of PGCs are localized in the proximal epiblast, and we demonstrated that the conditions required for PGC formation are induced in the proximal region of epiblasts by extraembryonic ectoderm. Bone morphogenetic protein (BMP) 4 and BMP8b, which belong to the transforming growth factor-beta (TGF-beta) superfamily, might generate induction signals from extraembryonic ectoderm. Smad1 and Smad5, which are intracellular signaling molecules for BMP4, might also play a critical role in stimulating epiblasts to form PGC. However, how pluripotential epiblasts temporally and spatially respond to BMP signals to form PGCs remains unclear. The present study examines changes of responsiveness to BMP4 for PGC formation in epiblasts and their molecular mechanisms. We initially examined the effect of recombinant human (rh) BMP4 upon cultured epiblasts at different developmental stages, and found that they acquire the ability to respond to BMP4 signals for PGC formation between E5.25 and E5.5. In addition, such competence was conferred upon epiblasts by the extraembryonic ectoderm. We also showed that the increased expression of Smad1 and the onset of Smad5 expression induced by extraembryonic ectoderm might be responsible for quick acquisition of this competence. Furthermore, we show that only proximal epiblast cells maintain responsiveness to BMP4 for PGC formation at E6.0, and that this is associated with the proximal epiblast-specific expression of Smad5. These results explain why only the proximal region of epiblasts can sustain the ability to form PGCs.     

Use of retinoic acid for the analysis of dermal-epidermal interactions in the tarsometatarsal skin of the chick embryo

Feet of chicks are normally covered with scales. Injection of retinoic acid into the amniotic cavity of 10-day chick embryos causes the formation of feathers on the foot scales. To elucidate whether retinoic acid affects primarily the epidermis or the dermis, heterotypic dermal-epidermal recombinants of tarsometatarsal skin were tested as to their morphogenetic capacity, when grafted to the chick chorioallantoic membrane. Recombinants involving treated epidermis and untreated dermis formed feathered scales, while the reverse recombinants of untreated epidermis and treated dermis led to the formation of scales only. Likewise the association of treated tarsometatarsal dermis with untreated epidermis from a non-appendage-forming region (the midventral apterium) resulted in the formation of scales only. These results show that retinoic acid affects primarily the epidermis. Further insight into the mechanism of dermal-epidermal interaction was gained by heterotopic recombinations of early (8.5- and 10-day) untreated tarsometatarsal dermis with epidermis from the midventral apterium. These recombinants formed scales, proving that tarsometatarsal dermis is endowed with scale-forming properties as early as 8.5 days of incubation. Finally, it is concluded that retinoic acid acts on the chick foot epidermal cells by temporarily inhibiting their scale placode-forming properties, allowing their latent feather placode-forming properties to be expressed.     

Msx1 expressing mesoderm is important for the apical ectodermal ridge (AER)-signal transfer in chick limb development

The apical ectodermal ridge (AER) is a specialized thickening of the distal limb ectoderm, and its signals are known to support limb morphogenesis. The expression of a homeobox gene, Msx1 , in the distal limb mesoderm depends on signals from the AER. In the present paper it is reported that Msx1 expression in the distal mesoderm is necessary for the transfer of AER signals in chick limb buds. Interruption of AER-mesoderm interaction by insertion of a thick filter led to the inhibition of pattern specification in the mesoderm just under the filter. In such cases, the expression of Msx1 disappeared in the mesoderm under the filter, suggesting that AER is able to signal over short ranges. In advanced limb buds, Msx1 is also expressed in the proximal mesoderm under the anterior ectoderm. However, it was found that a grafted antero-proximal mesoderm shows no inhibitory effects on pattern specification of the host mesoderm, as is the case with the distal mesoderm. On the other hand, grafted mesoderms without potent Msx1 re-expression, even underneath AER, disturbed normal limb development. In such cases, the expression of Msx1 disappeared in the mesoderm under the grafts, whereas Fgf-8 expression was maintained in the AER above the graft. These results indicate that the expression of Msx1 in the mesoderm is important for the transfer of AER signals.