Role of FGF10/FGFR2b signaling during mammary gland development in the mouse embryo.

The mouse develops five pairs of mammary glands that arise during mid-gestation from five pairs of placodes of ectodermal origin. We have investigated the molecular mechanisms of mammary placode development using Lef1 as a marker for the epithelial component of the placode, and mice deficient for Fgf10 or Fgfr2b, both of which fail to develop normal mammary glands. Mammary placode induction involves two different signaling pathways, a FGF10/FGFR2b-dependent pathway for placodes 1, 2, 3 and 5 and a FGF10/FGFR2b-independent pathway for placode 4. Our results also suggest that FGF signaling is involved in the maintenance of mammary bud 4, and that Fgf10 deficient epithelium can undergo branching morphogenesis into the mammary fat pad precursor.     

Canonical WNT signaling promotes mammary placode development and is essential for initiation of mammary gland morphogenesis

Mammary glands, like other skin appendages such as hair follicles and teeth, develop from the surface epithelium and underlying mesenchyme; however, the molecular controls of embryonic mammary development are largely unknown. We find that activation of the canonical WNT/beta-catenin signaling pathway in the embryonic mouse mammary region coincides with initiation of mammary morphogenesis, and that WNT pathway activity subsequently localizes to mammary placodes and buds. Several Wnt genes are broadly expressed in the surface epithelium at the time of mammary initiation, and expression of additional Wnt and WNT pathway genes localizes to the mammary lines and placodes as they develop. Embryos cultured in medium containing WNT3A or the WNT pathway activator lithium chloride (LiCl) display accelerated formation of expanded placodes, and LiCl induces the formation of ectopic placode-like structures that show elevated expression of the placode marker Wnt10b. Conversely, expression of the secreted WNT inhibitor Dickkopf 1 in transgenic embryo surface epithelium in vivo completely blocks mammary placode formation and prevents localized expression of all mammary placode markers tested. These data indicate that WNT signaling promotes placode development and is required for initiation of mammary gland morphogenesis. WNT signals play similar roles in hair follicle formation and thus may be broadly required for induction of skin appendage morphogenesis.     

Splitting placodes: effects of bone morphogenetic protein and Activin on the patterning and identity of mouse incisors

SUMMARY The single large rodent incisor in each jaw quadrant is evolutionarily derived from a mammalian ancestor with many small incisors. The embryonic placode giving rise to the mouse incisor is considerably larger than the molar placode, and the question remains whether this large incisor placode is a developmental requisite to make a thick incisor. Here we used in vitro culture system to experiment with the molecular mechanism regulating tooth placode development and how mice have thick incisors. We found that large placodes are prone to disintegration and formation of two to three small incisor placodes. The balance between one large or multiple small placodes was altered through the regulation of bone morphogenetic protein (BMP) and Activin signaling. Exogenous Noggin, which inhibits BMP signaling, or exogenous Activin cause the development of two to three incisors. These incisors were more slender than normal incisors. Additionally, two inhibitor molecules, Sostdc1 and Follistatin, which regulate the effects of BMPs and Activin and have opposite expression patterns, are likely to be involved in the incisor placode regulation in vivo. Furthermore, inhibition of BMPs by recombinant Noggin has been previously suggested to cause a change in the tooth identity from the incisor to the molar. This evidence has been used to support a homeobox code in determining tooth identity. Our work provides an alternative interpretation, where the inhibition of BMP signaling can lead to splitting of the large incisor placode and the formation of partly separate incisors, thereby acquiring molar‐like morphology without a change in tooth identity.     

Development and loss of androgen responsiveness in the embryonic rudiment of the mouse mammary gland

The androgen-responsive phase in the development of the mammary gland was determined by exposing rudiments of various developmental stages to testosterone in vitro. Although testosterone causes destruction of the mammary epithelium in 14-day male fetuses, it failed to prevent formation of mammary buds in explanted 11-day skin. It was found that mammary rudiments become responsive to androgens only late in Day 13 of gestation, and that they are no longer responsive on Day 15 and later. Both acquisition and loss of androgen responsiveness do occur on time in explanted glands, indicating intrinsic developmental changes in the rudiment. The experiments and their results are schematically summarized in Fig. 2.     

Early development of the cranial sensory nervous system: from a common field to individual placodes

Sensory placodes are unique columnar epithelia with neurogenic potential that develop in the vertebrate head ectoderm next to the neural tube. They contribute to the paired sensory organs and the cranial sensory ganglia generating a wide variety of cell types ranging from lens fibres to sensory receptor cells and neurons. Although progress has been made in recent years to identify the molecular players that mediate placode specification, induction and patterning, the processes that initiate placode development are not well understood. One hypothesis suggests that all placode precursors arise from a common territory, the pre-placodal region, which is then subdivided to generate placodes of specific character. This model implies that their induction begins through molecular and cellular mechanisms common to all placodes. Embryological and molecular evidence suggests that placode induction is a multi-step process and that the molecular networks establishing the pre-placodal domain as well as the acquisition of placodal identity are surprisingly similar to those used in Drosophila to specify sensory structures.     

An experimental analysis of factors affecting the localization of embryonic bone marrow

Summary The present investigations have been concerned with factors which determine and influence the localization and development of hemopoietic bone marrow in the embryo mouse and the adult. These studies, which have employed organ cultures and the transplantation of mouse embryo femur and tail rudiments, indicate that the surrounding mesenchyme is required for the normal development of the cartilage rudiment and its ossification, and for the formation and colonization of the marrow cavity. It was suggested that hemopoiesis results from the colonization of the “prepared” marrow cavity by stem cells arising from sources external to the rudiment. The addition of erythropoietin and L-thyroxine produced distinct erythropoietic differentiation in the normally myelocytic embryonic marrow cavity. The significance of the microenvironment present in developing bone rudiments and the initiation of hemopoiesis in stem cells was discussed. A hypothesis was developed to explain marrow localization in adults based on the colonization of bone rudiments which are developing their marrow sites at a time when the blood contains large numbers of colony-forming units.     

Vertebrate cranial placodes I. Embryonic induction

Cranial placodes are focal regions of thickened ectoderm in the head of vertebrate embryos that give rise to a wide variety of cell types, including elements of the paired sense organs and neurons in cranial sensory ganglia. They are essential for the formation of much of the cranial sensory nervous system. Although relatively neglected today, interest in placodes has recently been reawakened with the isolation of molecular markers for different stages in their development. This has enabled a more finely tuned approach to the understanding of placode induction and development and in some cases has resulted in the isolation of inducing molecules for particular placodes. Both morphological and molecular data support the existence of a preplacodal domain within the cranial neural plate border region. Nonetheless, multiple tissues and molecules (where known) are involved in placode induction, and each individual placode is induced at different times by a different combination of these tissues, consistent with their diverse fates. Spatiotemporal changes in competence are also important in placode induction. Here, we have tried to provide a comprehensive review that synthesises the highlights of a century of classical experimental research, together with more modern evidence for the tissues and molecules involved in the induction of each placode.     

鸟类仍保留牙齿发生定位的分子机制(英文)

众所周知现代鸟类不长牙齿,而其侏罗纪和白垩纪的祖先则长有牙齿。然而,在发育中鸡胚口腔中却残留着牙齿发生的原基,在形态上与哺乳动物臼牙牙原基极为相似。现代鸟类的胚胎组织是否具有牙齿发育的潜能,目前已有不少研究者对这一问题进行了探讨。Kollar和Fisher 等人将鸡胚胎下颌靠近口腔面的上皮与小鼠的牙间充质进行组织重组实验,并植入小鼠眼球中作intraocular grafting培养。他们的实验结果表明重组后的组织块可以发育形成牙齿的结构,包括形成成釉细胞(ameloblast),并能分泌釉质。Kollar等认为在进化过程中鸟类牙齿的消失并非由于口腔上皮中有关釉质合成的遗传信息的丢失,而是牙齿发育过程中的组织之间所必须的相互作用(次级诱导)受阻而造成的。Lemus和Fuenzalida等人的实验结果进一步证实了这一结论。他们用鹌鹑胚胎躯体的上皮组织与蜥蚁或兔子的牙间充质重组后,用鸡胚绒毛膜法进行培养,得到了发育很好的牙齿结构。发现鹌鹑的上皮细胞也可以分化形成釉质细胞,并分泌牙釉质。Cummings 将鹌鹑胚胎的牙上皮组织与小鼠胚胎的牙间充质组织重组后也得到类似的结果。根据小白鼠牙齿发育中已知的调控分子信号通路,我们曾对鸟类不长牙齿的分子机制进行了研究。我们的研究发现鸟类牙胚组织仍保留     

Analysis of FGF-Dependent and FGF-Independent Pathways in Otic Placode Induction

The inner ear develops from a patch of thickened cranial ectoderm adjacent to the hindbrain called the otic placode. Studies in a number of vertebrate species suggest that the initial steps in induction of the otic placode are regulated by members of the Fibroblast Growth Factor (FGF) family, and that inhibition of FGF signaling can prevent otic placode formation. To better understand the genetic pathways activated by FGF signaling during otic placode induction, we performed microarray experiments to estimate the proportion of chicken otic placode genes that can be up-regulated by the FGF pathway in a simple culture model of otic placode induction. Surprisingly, we find that FGF is only sufficient to induce about 15% of chick otic placode-specific genes in our experimental system. However, pharmacological blockade of the FGF pathway in cultured chick embryos showed that although FGF signaling was not sufficient to induce the majority of otic placode-specific genes, it was still necessary for their expression in vivo. These inhibitor experiments further suggest that the early steps in otic placode induction regulated by FGF signaling occur through the MAP kinase pathway. Although our work suggests that FGF signaling is necessary for otic placode induction, it demonstrates that other unidentified signaling pathways are required to co-operate with FGF signaling to induce the full otic placode program.     

Fgf8 and Fgf3 are required for zebrafish ear placode induction,maintenance and inner ear patterning

The vertebrate inner ear develops from initially 'simple' ectodermal placode and vesicle stages into the complex three-dimensional structure which is necessary for the senses of hearing and equilibrium. Although the main morphological events in vertebrate inner ear development are known, the genetic mechanisms controlling them are scarcely understood. Previous studies have suggested that the otic placode is induced by signals from the chordamesoderm and the hindbrain, notably by fibroblast growth factors (Fgfs) and Wnt proteins. Here we study the role of Fgf8 as a bona-fide hindbrain-derived signal that acts in conjunction with Fgf3 during placode induction, maintenance and otic vesicle patterning. Acerebellar (ace) is a mutant in the fgf8 gene that results in a non-functional Fgf8 product. Homozygous mutants for acerebellar (ace) have smaller ears that typically have only one otolith, abnormal semi-circular canals, and behavioral defects. Using gene expression markers for the otic placode, we find that ace/fgf8 and Fgf-signaling are required for normal otic placode formation and maintenance. Conversely, misexpression of fgf8 or Fgf8-coated beads implanted into the vicinity of the otic placode can increase ear size and marker gene expression, although competence to respond to the induction appears restricted. Cell transplantation experiments and expression analysis suggest that Fgf8 is required in the hindbrain in the rhombomere 4-6 area to restore normal placode development in ace mutants, in close neighbourhood to the forming placode, but not in mesodermal tissues. Fgf3 and Fgf8 are expressed in hindbrain rhombomere 4 during the stages that are critical for placode induction. Joint inactivation of Fgf3 and Fgf8 by mutation or antisense-morpholino injection causes failure of placode formation and results in ear-less embryos, mimicking the phenotype we observe after pharmacological inhibition of Fgf-signaling. Fgf8 and Fgf3 together therefore act during induction and differentiation of the ear placode. In addition to the early requirement for Fgf signaling, the abnormal differentiation of inner ear structures and mechanosensory hair cells in ace mutants, pharmacological inhibition of Fgf signaling, and the expression of fgf8 and fgf3 in the otic vesicle demonstrate independent Fgf function(s) during later development of the otic vesicle and lateral line organ. We furthermore addressed a potential role of endomesomerm by studying mzoep mutant embryos that are depleted of head endomesodermal tissue, including chordamesoderm, due to a lack of Nodal-pathway signaling. In these embryos, early placode induction proceeds largely normally, but the ear placode extends abnormally to midline levels at later stages, suggesting a role for the midline in restricting placode development to dorsolateral levels. We suggest a model of zebrafish inner ear development with several discrete steps that utilize sequential Fgf signals during otic placode induction and vesicle patterning.     

WNT signals are required for the initiation of hair follicle development

Hair follicle morphogenesis is initiated by a dermal signal that induces the development of placodes in the overlying epithelium. To determine whether WNT signals are required for initiation of follicular development, we ectopically expressed Dickkopf 1, a potent diffusible inhibitor of WNT action, in the skin of transgenic mice. This produced a complete failure of placode formation prior to morphological or molecular signs of differentiation, and blocked tooth and mammary gland development before the bud stage. This phenotype indicates that activation of WNT signaling in the skin precedes, and is required for, localized expression of regulatory genes and initiation of hair follicle placode formation.     

Differential requirements for FGF3, FGF8 and FGF10 during inner ear development

FGF signaling is required during multiple stages of inner ear development in many different vertebrates, where it is involved in induction of the otic placode, in formation and morphogenesis of the otic vesicle as well as for cellular differentiation within the sensory epithelia. In this study we have looked to define the redundant and conserved roles of FGF3, FGF8 and FGF10 during the development of the murine and avian inner ear. In the mouse, hindbrain-derived FGF10 ectopically induces FGF8 and rescues otic vesicle formation in Fgf3 and Fgf10 homozygous double mutants. Conditional inactivation of Fgf8 after induction of the placode does not interfere with otic vesicle formation and morphogenesis but affects cellular differentiation in the inner ear. In contrast, inactivation of Fgf8 during induction of the placode in a homozygous Fgf3 null background leads to a reduced size otic vesicle or the complete absence of otic tissue. This latter phenotype is more severe than the one observed in mutants carrying null mutations for both Fgf3 and Fgf10 that develop microvesicles. However, FGF3 and FGF10 are redundantly required for morphogenesis of the otic vesicle and the formation of semicircular ducts. In the chicken embryo, misexpression of Fgf3 in the hindbrain induces ectopic otic vesicles in vivo. On the other hand, Fgf3 expression in the hindbrain or pharyngeal endoderm is required for formation of the otic vesicle from the otic placode. Together these results provide important insights into how the spatial and temporal expression of various FGFs controls different steps of inner ear formation during vertebrate development.     

Gli3-mediated somitic Fgf10 expression gradients are required for the induction and patterning of mammary epithelium along the embryonic axes

Little is known about the regulation of cell fate decisions that lead to the formation of five pairs of mammary placodes in the surface ectoderm of the mouse embryo. We have previously shown that fibroblast growth factor 10 (FGF10) is required for the formation of mammary placodes 1, 2, 3 and 5. Here, we have found that Fgf10 is expressed only in the somites underlying placodes 2 and 3, in gradients across and within these somites. To test whether somitic FGF10 is required for the formation of these two placodes, we analyzed a number of mutants with different perturbations of somitic Fgf10 gradients for the presence of WNT signals and ectodermal multilayering, markers for mammary line and placode formation. The mammary line is displaced dorsally, and formation of placode 3 is impaired in Pax3ILZ/ILZ mutants, which do not form ventral somitic buds. Mammary line formation is impaired and placode 3 is absent in Gli3Xt-J/Xt-J and hypomorphic Fgf10 mutants, in which the somitic Fgf10 gradient is shortened dorsally and less overall Fgf10 is expressed, respectively. Recombinant FGF10 rescued mammogenesis in Fgf10(-/-) and Gli3Xt-J/Xt-J flanks. We correlate increasing levels of somitic FGF10 with progressive maturation of the surface ectoderm, and show that full expression of somitic Fgf10, co-regulated by GLI3, is required for the anteroposterior pattern in which the flank ectoderm acquires a mammary epithelial identity. We propose that the intra-somitic Fgf10 gradient, together with ventral elongation of the somites, determines the correct dorsoventral position of mammary epithelium along the flank.     

Mesenchymal/epithelial regulation of retinoic acid signaling in the olfactory placode

We asked whether mesenchymal/epithelial (M/E) interactions regulate retinoic acid (RA) signaling in the olfactory placode and whether this regulation is similar to that at other sites of induction, including the limbs, branchial arches, and heart. RA is produced by the mesenchyme at all sites, and subsets of mesenchymal cells express the RA synthetic enzyme RALDH2, independent of M/E interactions. In the placode, RA-producing mesenchyme is further distinguished by its coincidence with a molecularly distinct population of neural crest-associated cells. At all sites, expression of additional RA signaling molecules (RARalpha, RARbeta, RXR, CRABP1) depends on M/E interactions. Of these molecules, RA regulates only RARbeta, and this regulation depends on M/E interaction. Expression of Fgf8, shh, and Bmp4, all of which are thought to influence RA signaling, is also regulated by M/E interactions independent of RA at all sites. Despite these common features, RALDH3 expression is distinct in the placode, as is regulation of RARbeta and RALDH2 by Fgf8. Thus, M/E interactions regulate expression of RA receptors and cofactors in the olfactory placode and other inductive sites. Some aspects of regulation in the placode are distinct, perhaps reflecting unique roles for additional local signals in neuronal differentiation in the developing olfactory pathway.     

Podoplanin is expressed by a sub-population of human foetal rib and knee joint rudiment chondrocytes

The aim of this study was to determine if podoplanin was expressed by rudiment chondrocytes in human foetal cartilages. Podoplanin was immunolocalised in first trimester human foetal rib and knee joint rudiments to a sub-population of chondrocytes deep in the rib rudiments, tibial and femoral growth plates and cells associated with the cartilage canals of the foetal knee joint rudiments. Lymphatic vessels in the loose stromal tissues surrounding the developing rudiments were also demonstrated on the same histology slides using antipodoplanin (MAb D2-40) and anti-LYVE-1 and differentiated from CD-31 positive blood vessels confirming the discriminative capability of the antibody preparations used. The D2-40 positive rib and knee rudiment chondrocytes were not stained with antibodies to LYVE-1, CD-31 or CD-34 however perlecan was a prominent pericellular proteoglycan around these cells confirming their chondrogenic phenotype. Discernable differences were evident between the surface and deep rudiment chondrocytes in terms of their antigen reactivities detected with MAb D2-40 or antiperlecan antibodies. Binding of the cytoplasmic tail of PDPN to the ERM proteins ezrin, radixin and moeisin may result in changes in cytoskeletal organisation which alter the phenotype of this central population of rudiment cells. This may contribute to morphological changes in the rudiment cartilages which lead to establishment of the primary ossification centres and is consistent with their roles as transient developmental scaffolds during tissue development.     

Equivalent progenitor cells in the zebrafish anterior preplacodal field give rise to adenohypophysis, lens, and olfactory placodes

Embryonic organizing centers secrete signaling molecules that instruct target cells about their position and future identity. Information about cell position in relation to sources of instructive signals and about precursor cell lineages is key to our understanding of developmental processes that restrict cell potency and determine cell fate. We review adenohypophysis, lens, and olfactory placode formation and how gene expression patterns, cell positions, and cell fates in the anterior neural plate and anterior placodal field correlate in zebrafish and other vertebrates. Single cell lineage analysis in zebrafish suggests that the majority of preplacodal cells might be specified for pituitary, lens, or olfactory placode by the end of gastrulation.