BMP4 modulates fibroblast growth factor-mediated induction of proximal and distal lung differentiation in mouse embryonic tracheal epithelium in mesenchyme-free culture.

Lung morphogenesis and differentiation require interaction between the epithelium and mesenchyme, which is mediated by diffusible molecules such as fibroblast growth factors (FGFs), bone morphogenetic protein 4 (BMP4), and Shh. We have used mesenchyme-free culture to study the effects of these molecules on lung epithelial differentiation. We have tested the individual abilities of FGF1, FGF2, FGF7, FGF9, FGF10, and FGF18, as well as BMP4 and Shh to promote growth and specify distal lung differentiation in mouse tracheal epithelium. The different FGFs exhibited distinct abilities to induce epithelial growth and the expression of the distal lung epithelial marker, surfactant protein C (SP-C), although all FGFs were able to induce expression of BMP4. Tracheal epithelium treated with FGF10 showed little growth and failed to express SP-C as measured by whole-mount in situ hybridization and quantitative real-time polymerase chain reaction. FGF1 treatment resulted in the strongest induction of SP-C. Treatment with BMP4 inhibited epithelial growth and differentiation and antagonized the stimulatory effects of FGF1. In contrast, inhibition of endogenous BMP4 signaling with Noggin protein did not inhibit growth or expression of SP-C but did increase the expression of the proximal lung markers CCSP and HFH4. Expression of Shh was not affected by any of the conditions tested. These results suggest that BMP4 does not signal epithelial cells to adopt a distal fate but may regulate the expansion of proximal epithelial cells in the lung.     

Differential response of Shh expression between chick forelimb and hindlimb buds by FGF-4.

The interactions of Sonic hedgehog (Shh) and fibroblast growth factor (FGF) play important roles in vertebrate limb pattern formation. In the posterior region of the chick limb bud, Shh and FGF-4 each maintain expression in a positive feedback loop. In the anterior region, Shh can also induce Fgf-4 expression in the anterior apical ectodermal ridge. However, the possibility of Shh induction by FGF protein is unclear. Because many experiments to analyze gene expression have been carried out by using the forelimb bud of the chick embryo, we investigated gene expression of the cells in the anterior region of the chick hindlimb bud after FGF-4 application and compared the results with those for the forelimb bud. When an FGF-4-containing bead was implanted into the anterior region of a stage 20 hindlimb bud, ectopic expression of Shh was induced in the mesenchyme beneath the anterior end of the apical ectodermal ridge at 36 hr after implantation. Subsequent to Shh activation, Hoxd13 was also observed in the anterior-distal region of the limb bud. Furthermore, FGF-4 implantation to the hindlimb bud caused additional digit formation accompanying respecification of positional value in the anterior tissue. Ectopic Shh was induced in cells located distal to the FGF-4 bead, and the cells of the flank region did not contribute to ectopic Shh induction. On the other hand, no ectopic Shh and Hoxd13 expression was detected by grafting an FGF-4 bead into the forelimb bud. Although FGF-4 implantation to the forelimb bud occasionally induced extra digit 2 formation, no embryos had an extra digit 3 or digit 4, and many specimens exhibited normal skeletal pattern. These results demonstrate the difference between the fore- and hindlimb buds in the cell competence of Shh induction in response to FGF-4, suggesting the possibility that the responsiveness of mesenchymal cells in signaling molecules is not the same in the fore- and hindlimb buds.     

Retinoic acid induces prostatic bud formation.

Formation of prostatic buds from the urogenital sinus (UGS) to initiate prostate development requires localized action of several morphogenetic factors. This report reveals all-trans-retinoic acid (RA) to be a powerful inducer of mouse prostatic budding that is associated with reciprocal changes in expression of two regulators of budding: sonic hedgehog (Shh) and bone morphogenetic protein 4 (Bmp4). Localization of retinoid signaling and expression of RA synthesis, metabolism, and receptor genes in the UGS on embryonic days 14.5-17.5 implicate RA in the mechanism of bud initiation. In UGS organ culture, RA increased prostatic budding, increased Shh expression, and decreased Bmp4. Prostatic budding was stimulated in the absence of RA by recombinant SHH, by blocking BMP4 signaling with NOGGIN, or by combined treatment with SHH and NOGGIN in UGS organ culture media. These observations suggest that reciprocal changes in hedgehog and BMP signaling by RA may regulate bud initiation.     

Coordinate expression of Fgf8, Otx2, Bmp4, and Shh in the rostral prosencephalon during development of the telencephalic and optic vesicles.

Previous studies suggest that Fgf8 has a key role in regulating vertebrate development. In the rostral head of the embryonic chicken, there are increasing numbers of separate Fgf8 domains; these are present in tissues that appear to have previously expressed Otx2. As Fgf8 expression becomes established, Otx2 expression weakens, but remains in cells abutting the Fgf8 expression domain. These Fgf8 expression domains are closely associated with tissues expressing Bmp4 and Shh. Based on analogy with the embryonic limb, we suggest that Fgf8, Bmp4 and Shh function together in patterning regions of the embryonic head. Gene expression changes are particularly prominent in 14-21 somite stage embryos in the rostral forebrain, during early morphogenesis of the telencephalic and optic vesicles, when several new interfaces of Fgf8, Bmp4 and Shh are generated. To gain insights into the functions of fibroblast growth factor 8 (FGF8) in the embryonic forebrain, we studied the effects of implanting beads containing this protein in the dorsal prosencephalon of embryonic day 2 chicken embryos. Ectopic FGF8 had profound effects on morphogenesis of the telencephalic and optic vesicles. It disrupted formation of the optic stalk and caused a transformation of the pigment epithelium into neural retina. Within the telencephalon, FGF8 beads frequently induced a sulcus that had features of an ectopic rostral midline. The sulcus separated the telencephalon into rostral and caudal vesicles. Furthermore, we present evidence that FGF8 can regulate regionalization of the prosencephalon through inhibition of Otx2 and Emx2 expression. Thus, these experiments provide evidence that FGF8 can regulate both morphogenesis and patterning of the rostral prosencephalon (telencephalic and optic vesicles). FGF8 beads can induce midline properties (e.g. a sulcus) and can modulate the specification and differentiation of adjacent tissues. We suggest that some of these effects are through regulating the expression of homeobox genes (Otx2 and Emx2) that are known to participate in forebrain patterning.     

FGF signaling segregates biliary cell-lineage from chick hepatoblasts cooperatively with BMP4 and ECM components in vitro.

Intrahepatic bile ducts (IHBDs) are indispensable for transporting bile secreted from hepatocytes to the hepatic duct. The biliary epithelial cells (BECs) of the IHBD arise from bipotent hepatoblasts around the portal vein, suggesting the portal mesenchyme is essential for their development. However, except for Notch or Activin/TGF-beta signaling molecules, it is not known which molecules regulate IHBD development. Here, we found that FGF receptors and BMP4 are specifically expressed in the developing IHBD and the hepatic mesenchyme, respectively. Using a mesenchyme-free culture of liver bud, we showed that bFGF and FGF7 induce the hepatoblasts to differentiate into BECs, and that BMP4 enhances bFGF-induced BEC differentiation. The extracellular matrix (ECM) components in the hepatic mesenchyme induced BEC differentiation. Forced expression of a constitutively active form of the FGF receptor partially induced BEC differentiation markers in vivo. These data strongly suggest that bFGF and FGF7 promote BEC differentiation cooperatively with BMP4 and ECMs in vivo.     

Bmp7 mediates early signaling events during induction of chick epidermal organs.

The induction and specification of a large number of vertebrate organs require reciprocal signaling between an epithelium and subjacent mesenchyme. In the formation of integumentary organs, the initial inductive signaling events leading to the formation of the organ primordia stem from the mesenchyme. However, the epithelium must have the capacity to respond to these signals. We demonstrate that bone morphogenetic protein 7 (Bmp7) is an early molecular marker for epidermal organ development during development of feathers and scales of the chick. Bmp7 is expressed broadly in the preplacode epidermis and subsequently becomes localized to the forming placodes of feathers and scales. An examination of Bmp7 expression in the scaleless mutant chicken integument indicates that Bmp7 expression in the epidermis is associated with the ability to form epidermal organs. We show that BMP7 function is necessary for the formation of epidermal placodes in both feather and scale forming epidermis. In addition, precocious expression of Bmp7 in the metatarsal epidermis of the Silkie mutant or treatment of the metatarsus with ectopic BMP7 protein results in feather development from scale forming integument. From these data, we propose that Bmp7 is necessary and sufficient, in a developmental context, to mediate the competence of an epithelium to respond to inductive signals from the underlying mesenchyme to form epidermal organs in the chick. We propose that regulation of Bmp7 in localized areas of the embryonic epidermis facilitates the development of regional formation of integumentary organs.     

Fibroblast growth factor signaling regulates Dach1 expression during skeletal development.

Dach1 is a mouse homologue of the Drosophila dachshund gene, which is a key regulator of cell fate determination during eye, leg, and brain development in the fly. We have investigated the expression and growth factor regulation of Dach1 during pre- and postnatal skeletal development in the mouse limb to understand better the function of Dach1. Dach1 was expressed in the distal mesenchyme of the early embryonic mouse limb bud and subsequently became restricted to the tips of digital cartilages. Dach1 protein was localized to postmitotic, prehypertrophic, and early hypertrophic chondrocytes during the initiation of ossification centers, but Dach1 was not expressed in growth plates that exhibited extensive ossification. Dach1 colocalized with Runx2/Cbfa1 in chondrocytes but not in the forming bone collar or primary spongiosa. Dach1 also colocalized with cyclin-dependent kinase inhibitors p27 (Kip1) and p57 (Kip2) in chondrocytes of the growth plate and in the epiphysis before the formation of the secondary ossification center. Because fibroblast growth factors (FGF), bone morphogenetic proteins (BMP), and hedgehog molecules (Hh) regulate skeletal patterning of the limb bud and chondrocyte maturation in developing endochondral bones, we investigated the regulation of Dach1 by these growth and differentiation factors. Expression of Dach1 in 11 days postcoitus mouse limb buds in organ culture was up-regulated by implanting beads soaked in FGF1, 2, 8, or 9 but not FGF10. BMP4-soaked beads down-regulated Dach1 expression, whereas Shh and bovine serum albumin had no effect. Furthermore, FGF4 or 8 could substitute for the apical ectodermal ridge in maintaining Dach1 expression in the limb buds. Immunolocalization of FGFR2 and FGFR3 revealed overlap with Dach1 expression during skeletal patterning and chondrocyte maturation. We conclude that Dach1 is a target gene of FGF signaling during limb skeletal development, and Dach1 may function as an intermediary in the FGF signaling pathway regulating cell proliferation or differentiation.     

Core binding factor beta functions in the maintenance of stem cells and orchestrates continuous proliferation and differentiation in mouse incisors

Rodent incisors grow continuously throughout life, and epithelial progenitor cells are supplied from stem cells in the cervical loop. We report that epithelial Runx genes are involved in the maintenance of epithelial stem cells and their subsequent continuous differentiation and therefore growth of the incisors. Core binding factor β (Cbfb) acts as a binding partner for all Runx proteins, and targeted inactivation of this molecule abrogates the activity of all Runx complexes. Mice deficient in epithelial Cbfb produce short incisors and display marked underdevelopment of the cervical loop and suppressed epithelial Fgf9 expression and mesenchymal Fgf3 and Fgf10 expression in the cervical loop. In culture, FGF9 protein rescues these phenotypes. These findings indicate that epithelial Runx functions to maintain epithelial stem cells and that Fgf9 may be a target gene of Runx signaling. Cbfb mutants also lack enamel formation and display downregulated Shh mRNA expression in cells differentiating into ameloblasts. Furthermore, Fgf9 deficiency results in a proximal shift of the Shh expressing cell population and ectopic FGF9 protein suppresses Shh expression. These findings indicate that Shh as well as Fgf9 expression is maintained by Runx/Cbfb but that Fgf9 antagonizes Shh expression. The present results provide the first genetic evidence that Runx/Cbfb genes function in the maintenance of stem cells in developing incisors by activating Fgf signaling loops between the epithelium and mesenchyme. In addition, Runx genes also orchestrate continuous proliferation and differentiation by maintaining the expression of Fgf9 and Shh mRNA.     

Coordinate regulation and synergistic actions of BMP4, SHH and FGF8 in the rostral prosencephalon regulate morphogenesis of the telencephalic and optic vesicles

We investigated the roles of bare morphogenetic protein (BMP), sonic hedgehog (SHH) and fibroblast growth factor (FGF)-expressing signaling centers in regulating the patterned outgrowth of the telencephalic and optic vesicles. Implantation of BMP4 beads in the anterior neuropore of stage 10 chicken embryos repressed FGF8 and SHH expression. Similarly, loss of SHH expression in Shh mutant mice leads to increased BMP signaling and loss of Fgf8 expression in the prosencephalon. Increased BMP signaling and loss of FGF and SHH expression was correlated with decreased proliferation, increased cell death, and hypoplasia of the telencephalic and optic vesicles. However, decreased BMP signaling, through ectopic expression of Noggin, a BMP-binding protein, also caused decreased proliferation and hypoplasia of the telencephalic and optic vesicles, but with maintenance of Fgf8 and Shh expression, and no detectable increase in cell death. These results suggest that optimal growth requires a balance of BMP, FGF8 and SHH signaling. We suggest that the juxtaposition of Fgf8, Bmp4 and Shh expression domains generate patterning centers that coordinate the growth of the telencephalic and optic vesicles, similar to how Fgf8, Bmp4 and Shh regulate growth of the limb bud. Furthermore, these patterning centers regulate regional specification within the forebrain and eye, as exemplified by the regulation of Emx2 expression by different levels of BMP signaling.In summary, we present evidence that there is cross-regulation between BMP-, FGF- and SHH-expressing signaling centers in the prosencephalon which regulate morphogenesis of, and regional specification within, the telencephalic and optic vesicles.     

Role of Sonic hedgehog in patterning of tracheal-bronchial cartilage and the peripheral lung.

Sonic hedgehog (Shh) was conditionally deleted in respiratory epithelial cells of the embryonic lung in vivo. Deletion of Shh before embryonic day (E) 13.5 resulted in respiratory failure at birth. While lobulation was not perturbed, the lungs were hypoplastic, with reduced branching of peripheral lung tubules, evident from E13.5. Smooth muscle and endothelial cells were absent or reduced, the latter in relationship to the loss of peripheral lung parenchyma. Tracheal-bronchial ring abnormalities occurred when Shh was deleted between E8.5 and E12.5. Deletion of Shh later in gestation (after E13.5) caused mild abrogation of peripheral branching morphogenesis but did not disrupt tracheal-bronchial development. Defects in branching morphogenesis and vascularization seen in Shh null mutant (Shh(-/-)) mice were substantially corrected when SHH was ectopically expressed in the respiratory epithelium; however, peripheral expression of SHH failed to correct cartilage abnormalities in the trachea and bronchi, indicating a spatial requirement for SHH expression near sites of cartilage formation. Expression of SHH by the respiratory epithelium plays an important role in the patterning of tracheal-bronchial mesenchyme required for formation of cartilage rings in conducting airways. SHH regulates branching morphogenesis and influences differentiation of the peripheral lung mesenchyme required for formation of bronchial and vascular smooth muscle.     

Exogenous fibroblast growth factor 8 rescues development of mouse diastemal vestigial tooth ex vivo

Regression of vestigial tooth buds results in the formation of the toothless diastema, a unique feature of the mouse dentition. Revitalization of the diastemal vestigial tooth bud provides an excellent model for studying tooth regeneration and replacement. It has been previously shown that suppression of fibroblast growth factor (FGF) signaling in the diastema results in vestigial tooth bud regression. In this study, we report that application of exogenous FGF8 to the mouse embryonic diastemal region rescues diastemal tooth development. However, this rescue of diastemal tooth development occurs only in an isolated diastemal regions and not in the mandibular quadrant, which includes the incisor and molar germs. FGF8 promotes cell proliferation and inhibits apoptosis in diastemal tooth epithelium, and revitalizes the tooth developmental program, as evidenced by the expression of genes critical for normal tooth development. Our results also support the idea that the adjacent tooth germs contribute to the suppression of diastemal vestigial tooth buds by means of multiple signals. Developmental Dynamics 240:1344–1353, 2011. © 2011 Wiley‐Liss, Inc.     

Levels of Gli3 repressor correlate with Bmp4 expression and apoptosis during limb development.

Removal of the posterior wing bud leads to massive apoptosis of the remaining anterior wing bud mesoderm. We show here that this finding correlates with an increase in the level of the repressor form of the Gli3 protein, due to the absence of the Sonic hedgehog (Shh) protein signaling. Therefore, we used the anterior wing bud mesoderm as a model system to analyze the relationship between the repressor form of Gli3 and apoptosis in the developing limb. With increased Gli3R levels, we demonstrate a concomitant increase in Bmp4 expression and signaling in the anterior mesoderm deprived of Shh signaling. Several experimental approaches show that the apoptosis can be prevented by exogenous Noggin, indicating that Bmp signaling mediates it. The analysis of Bmp4 expression in several mouse and chick mutations with defects in either expression or processing of Gli3 indicates a correlation between the level of the repressor form of Gli3 and Bmp4 expression in the distal mesoderm. Our analysis adds new insights into the way Shh differentially controls the processing of Gli3 and how, subsequently, BMP4 expression may mediate cell survival or cell death in the developing limb bud in a position-dependent manner.     

Expression and Regulation of Hedgehog-Interacting Protein During Early Tooth Development

Sonic hedgehog ( Shh ) expression is highly localized to the epithelium at the future sites of tooth development. This restricted expression suggests that inhibition of Shh in areas where teeth do not form may be an important mechanism in tooth germ localization. Recently, Hip, a putative vertebrate antagonist of Shh, has been identified. We have investigated the expression of Hip during early tooth development and found it not to be localized in cells immediately adjacent to Shh -expressing cells, but rather at a distance, separated by cells expressing Ptc1 . Hip is also regulated by Shh in the first branchial arch. Shh-soaked agarose beads are able to induce the expression of Hip in odontogenic mesenchyme. A role for Hip might be to prevent the spread of excess Shh ligand beyond an immediate Ptc1-induced zone in odontogenic mesenchyme. This mechanism would therefore restrict Shh signaling specifically to those regions along the oral axis that are destined to form teeth.     

Molecular crosstalk in tracheal development and its recurrence in adult tissue regeneration

The trachea is a rigid air duct with some mobility, which comprises the upper region of the respiratory tract and delivers inhaled air to alveoli for gas exchange. During development, the tracheal primordium is first established at the ventral anterior foregut by interactions between the epithelium and mesenchyme through various signaling pathways, such as Wnt, Bmp, retinoic acid, Shh, and Fgf, and then segregates from digestive organs. Abnormalities in this crosstalk result in lethal congenital diseases, such as tracheal agenesis. Interestingly, these molecular mechanisms also play roles in tissue regeneration in adulthood, although it remains less understood compared with their roles in embryonic development. In this review, we discuss cellular and molecular mechanisms of trachea development that regulate the morphogenesis of this simple tubular structure and identities of individual differentiated cells. We also discuss how the facultative regeneration capacity of the epithelium is established during development and maintained in adulthood.