The effects of retinoids on cartilage differentiation in micromass cultures of chick facial primordia and the relationship to a specific facial defect

Retinoids produce facial defects in chicken embryos. Outgrowth of the frontonasal mass with accompanying cartilage differentiation and pattern formation is inhibited. In contrast, the development of the mandibular primordia that give rise to the lower beak proceeds normally. To investigate whether the upper beak defect is based on the inhibition of cartilage differentiation specifically in the frontonasal mass, the effects of retinoids on chondrogenesis in micromass (high density) cultures of cells from facial primordia have been studied. When either 10(-6) M retinoic acid or 10(-8) M (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl-1- propenyl]benzoic acid (TTNPB; a stable retinoid) is added to the culture medium, cartilage differentiation is inhibited. Both frontonasal mass and mandible cultures are equally affected. The concentration of TTNPB found in both facial primordia in vivo, after a treatment that produces the defect, is also about 10(-8) M. This rules out preferential accumulation of the retinoid by the frontonasal mass as an explanation for the defect. In fact, the concentration of retinoid found in vivo, should, from the culture studies, be sufficient to markedly inhibit chondrogenesis in both the frontonasal mass and mandibles. The effects of exposure to retinoids in the intact face appear to be different to those in culture. Furthermore, when cells from retinoid-treated facial primordia are cultured in micromass, the extent and pattern of chondrogenesis in frontonasal mass cultures is identical to that of cells from untreated primordia. Cartilage differentiation in mandible cultures is slightly affected. These findings suggest that retinoids do not produce the specific facial defect by directly interfering with cartilage differentiation.     

Epithelial-mesenchymal interactions in the development of chick facial primordia and the target of retinoid action

The development of the chick face involves outgrowth of buds of tissue, accompanied by the differentiation of cartilage and bone in spatially defined patterns. To investigate the role of epithelial-mesenchymal interactions in facial morphogenesis, small fragments of facial tissue have been grafted to host chick wing buds to continue their development in isolation. Fragments of the frontonasal mass give rise to typical upper-beak-like structures: a long central rod of cartilage, the prenasal cartilage and an egg tooth. Meckel's cartilage, characteristic of the lower beak, develops from fragments of the mandible. Removal of the ectoderm prior to grafting leads to truncated development. In fragments of frontonasal mass mesenchyme only a small spur of cartilage differentiates and there is no outgrowth. The mandible is less affected; a rod of cartilage still forms but the amount of outgrowth is reduced. Retinoid treatment of chick embryos specifically affects the development of the upper beak and outgrowth and cartilage differentiation in the frontonasal mass are inhibited. The mandibles, however, are unaffected and develop normally. In order to investigate whether the epithelium or the mesenchyme of the frontonasal mass is the target of retinoid action, recombinations of retinoid-treated and untreated facial tissue have been grafted to host wing buds. Recombinations of retinoid-treated frontonasal mass ectoderm with untreated mesenchyme develop normally whereas recombinations of untreated ectoderm with retinoid-treated mesenchyme lead to truncations. The amount of outgrowth in fragments of mandibular tissue is slightly reduced when either the ectoderm or the mesenchyme has been treated with retinoids. These recombination experiments demonstrate that the mesenchyme of the frontonasal mass is the target of retinoid action. This suggests that retinoids interfere with the reciprocal epithelial-mesenchymal interactions necessary for outgrowth and normal upper beak development.     

The effects of local application of retinoids to different positions along the proximo-distal axis of embryonic chick wings

Summary Two retinoids, all-trans-retinoic acid and a synthetic analog, TTNPB, were locally applied to different positions along the proximo-distal axis of embryonic chick wing buds using controlled release carriers. Truncations or limbs with duplicated structures across the antero-posterior axis develop after retinoid application to distal positions in buds from stage 20–24 embryos. Phocomelic limbs develop when the retinoids are applied more proximally to buds of stage 23–24 embryos. Duplications of the pattern of structures along the proximo-distal axis never occur.Using TTNPB that is relatively stable, the amount of retinoid in the wing tissue when phocomelia is induced was measured. There is twice as much retinoid per cell in the proximal half of the bud as in the distal half of the bud. The concentration of TTNPB in proximal tissue is estimated to be three times higher than in distal tissue in which pattern formation and cartilage morphogenesis are relatively normal.At early stages in the development of phocomelia, the shape of the bud changes and the indentation that marks the elbow does not arise. Neither retinoid-induced cell killing nor effects on the pattern of programmed cell death were detected.The induction of phocomelia by retinoids appears to be based on effects on proximal cells, whereas retinoids produce pattern changes by acting on distal cells. Furthermore, compared with pattern changes, higher concentrations of retinoid in the bud tissue are required to produce phocomelia.     

INDUCTIVE TISSUE INTERACTIONS IN THE BEAK OF A CHICK EMBRYO *

Series of homologous and heterologous recombination experiments were made between the beak and other skin derivatives, by means of a modified chorioallantoic membrane grafting, to investigate inductive tissue interactions involved in the upper beak of a developing embryo and a hatched chick. 6-day beak epidermis, as well as 6-day cephalic skin epidermis, differentiated into typically normal epidermis of the beak, when they were associated with the mesenchyme taken from 6-day upper beak region. These epithelia, when grafted without association with beak mesenchyme, failed to differentiate into the beak epidermis. 6-day beak epidermis differentiated into typical down feathers when combined with 7-day back skin dermis, prospective feathered area. The inductive potency of the beak mesenchyme was not limited in embryonic life, but persisted even after hatching. These findings were discussed in relation to the role of inductive tissue interactions involved in the expression and stabilization of the differentiated characters of the epidermis in both embryos and adults.     

Distribution of retinoids in the chick limb bud: analysis with monoclonal antibody

Retinoic acid induces anteroposterior duplicate formation in developing chick limb bud, and it may be a natural morphogen involved in limb pattern formation. Retinoic acid is produced from retinol locally in the limb bud via retinal, and thus, to elucidate the distribution of these retinoids in the limb bud seems to be important for the understanding of the morphogen formation. We produced a monoclonal antibody against the retinoids with BSA-RA (bovine serum albumin-retinoic acid) conjugate for antigen, and investigated the distribution of retinoids in the chick limb bud. The antibody predominantly bound to retinoic acid, but weakly to retinol and retinal. Retinoids appeared in the limb bud at stage 18 and were distributed through stages 20-24, when the pattern formation in distal mesoderm was in progress. Initially they were found evenly in the whole mesoderm, but disappeared gradually from core mesoderm and remained only in the region of peripheral mesoderm at stage 24. At stage 26, retinoids were detected only in ectoderm. These results support the idea that the retinoids actually play roles in limb pattern formation and suggest that the retinoids in the peripheral mesoderm are important for pattern formation. Further, the role of retinoids in epidermis development at later limb bud stages is also suggested.     

Altered Sensitivity to Retinoid-Induced Apoptosis Associated with Changes in the Subcellular Distribution of Bcl-2

In the acute promyelocytic leukemia cell line NB4, Bcl-2 downregulation occurred as a late event of retinoid-induced differentiation. In the maturation-resistant NB4-R1 subclone, retinoids failed to downregulate Bcl-2 even in the situation of apoptosis massively induced by pan-agonists and RXR-selective agonists. We observed that NB4 and NB4-R1 cells differed with respect to the intracellular localization of Bcl-2 which showed a perinuclear localization in NB4-R1 cells, while Bax was broadly expressed in the cytoplasm and to only a minor extent in the perinuclear area. Therefore, the distinct intracellular localization of Bcl-2 and Bax was in general nonoverlaping. Bcl-2 remained massively expressed until cell disruption. Bax was not significantly upregulated in cells committed to death. However, Bax localization changed from a diffuse pattern to concentrate in few specific cytoplasmic area at a stage preceding the formation of apoptotic bodies. A human Bcl-2 transgene was transiently overexpressed in NB4-R1 cells which showed increased resistance to apoptosis induced by retinoids. Stably transfected clones of NB4-R1 cells showed an increased expression of Bcl-2 and a marked resistance to apoptosis. Interestingly, the overexpression of Bcl-2 restored a pattern of uniform Bcl-2 labeling in the cytoplasm and, remarkably, the colocalization of Bcl-2 with Bax. This work demonstrates that the ability of retinoid-induced cells to undergo apoptosis depends on the level of expression and the functional interaction between Bcl-2 and Bax.     

Induction of senescence-associated growth inhibitors in the tumor-suppressive function of retinoids

Retinoids, physiological regulators of cell growth and differentiation, are used in the treatment or chemoprevention of several malignant diseases. This class of compounds can induce growth arrest or apoptosis in tumor cells. Permanent growth arrest of retinoid-treated cells is often assumed to result from retinoid-induced differentiation. Recent studies in breast carcinoma and neuroblastoma cells demonstrated that retinoids can stop tumor cell growth through the program of senescence rather than differentiation. Retinoid-induced tumor suppression is associated with the induction of multiple intracellular and secreted growth-inhibitory proteins. Most of these proteins were also found to be upregulated in senescent cells. The induction of senescence-associated growth inhibitors appears to be an indirect effect of retinoids. Elucidation of the mechanisms responsible for the induction of growth-inhibitory genes in retinoid-treated cells should help in developing agents that would mimic the antiproliferative effect of retinoids in retinoid-insensitive cancers.     

Eph receptors and ephrins: effectors of morphogenesis

Eph receptor tyrosine kinases and their ligands, the ephrins, appear to lie functionally at the interface between pattern formation and morphogenesis. We review the role of Eph and ephrin signalling in the formation of segmented structures, in the control of axon guidance and cell migration and in the development of the vasculature. We address the question of how the specificity of response is achieved and discuss the specificity of ephrin-Eph interactions and the significance of structural domains in Eph receptors.     

Retinoid-induced apoptosis in normal and neoplastic tissues

Vitamin A and its derivatives (collectively referred to as retinoids) are required for many fundamental life processes, including vision, reproduction, metabolism, cellular differentiation, hematopoesis, bone development, and pattern formation during embryogenesis. There is also considerable evidence to suggest that natural and synthetic retinoids have therapeutical effects due to their antiproliferative and apoptosis-inducing effects in human diseases such as cancer. Therefore it is not surprising that a significant amount of research was dedicated to probe the molecular and cellular mechanisms of retinoid action during the past decade. One of the cellular mechanisms retinoids have been implicated in is the initiation and modulation of apoptosis in normal development and disease. This review provides a brief overview of the molecular basis of retinoid signaling, and focuses on the retinoid-regulation of apoptotic cell death and gene expression during normal development and in pathological conditions in vivo and in various tumor cell lines in vitro.     

Interaction between growth factors and retinoic acid in the induction of kidney tubulogenesis in tissue culture.

Kidney tubulogenesis is the initial step in renal organogenesis. The precise molecular determinants of this pattern formation are presently unknown, although soluble factors, such as growth factors, and insoluble factors, such as extracellular matrix molecules, most likely play fundamental roles in this process. To define the molecular determinants of renal proximal tubule morphogenesis, primary cultures of rabbit renal proximal tubule cells in hormonally defined, serum-free media were treated with transforming growth factor-beta 1 (TGF-beta 1), epidermal growth factor (EGF), and the retinoid, all trans-retinoic acid (RA), singly or in combination. Utilizing phase contrast and light and transmission electron microscopy, the simultaneous administration of TGF-beta 1 (10 ng/ml), EGF (1 nM), and RA (0.1 nM) transformed a confluent monolayer of renal proximal tubule cells within 5 to 6 days into three-dimensional cell aggregates containing lumens within the interior of the cell clusters. The lumens were bordered by tubule cells possessing a polarized epithelial cell phenotype with extensive microvilli formation and tight junctional complexes along the luminal border. All three factors were necessary and sufficient to induce this phenotypic transformation. Further studies demonstrated that RA promoted the deposition of the A and B1 chains of laminin, a cell attachment protein of the basement membrane, in a small subset of proximal tubule cells in culture, as deduced by indirect immunofluorescent microscopy. Additional studies demonstrated that soluble purified laminin fully substituted for RA in this system to promote renal tubulogenesis when combined with TGF-beta 1 and EGF. These results demonstrate that the growth factors, TGF-beta 1 and EGF, and the retinoid, RA, promote tubulogenesis in adult renal proximal tubule cells in tissue culture in a manner reminiscent of inductive embryonic kidney morphogenesis. These observations define a coordinated interplay between growth factors and retinoids to induce pattern formation and morphogenesis. Furthermore, the demonstration of RA-induced laminin deposition as a critical event in this morphogenic process identifies laminin as a possible target protein for RA to act as a morphogen.     

Mechanics as a Means of Information Propagation in Development

New research demonstrates that mechanics can serve as a means of information propagation in developing embryos. Historically, the study of embryonic development has had a dichotomy between morphogens and pattern formation on the one hand and morphogenesis and mechanics on the other. Secreted signals are the preeminent means of information propagation between cells and used to control cell fate, while physical forces act downstream or in parallel to shape tissue morphogenesis. However, recent work has blurred this division of function by demonstrating that mechanics can serve as a means of information propagation. Adhesive or repulsive interactions can propagate through a tissue as a wave. These waves are rapid and directional and can be used to control the flux of cells through a developmental trajectory. Here, two examples are reviewed in which mechanics both guides and mediates morphogenesis and two examples in which mechanics intertwines with morphogens to regulate cell fate.     

Effects of retinoids on tooth morphogenesis and cytodifferentiations, in vitro.

The first embryonic lower mouse molar was used as a model system to investigate the effects of two retinoids, retinoic acid (RA) and a synthetic analogue, Ch55, on morphogenesis and cytodifferentiations in vitro. Exogenous retinoids were indispensable for morphogenesis of bud, cap and bell-stage molars in serum-free, chemically-defined, culture media. Transferrin and RA or transferrin and Ch55 acted synergistically in promoting morphogenesis from bud and cap-stage explants. Transferrin, per se, had no morphogenetic effect. Epithelial histogenesis, odontoblast functional differentiation and ameloblast polarization always occurred in RA-depleted explants. Comparison of the distributions of bromodeoxyuridine (BrdU) incorporation between explants cultured in the absence or presence of RA revealed that RA could modify the patterns of cell proliferation in the inner dental epithelium and dental mesenchyme. Inner dental epithelium cell proliferation is regulated by the dental mesenchyme through basement membrane-mediated interactions, and tooth morphogenesis is controlled by the dental mesenchyme. Laminin is a target molecule of retinoid action. Using a monospecific antibody, we immunolocalized laminin and/or structurally-related molecules sharing the laminin B chain in the embryonic dental mesenchyme and in the dental basement membrane and showed that RA could promote the synthesis or secretion of these molecules. Based on previous in situ hybridization data, it was speculated that CRABPs might regulate the effects of RA on embryonic dental cell proliferation. The fact that Ch55, a retinoid which does not bind to CRABPs, is 100 times more potent than RA in promoting tooth morphogenesis in vitro seems to rule out this hypothesis. On the other hand, the stage-specific inhibition of tooth morphogenesis by excess RA is consistent with the hypothesis that CRABPs might protect embryonic tissues against potentially teratogenic concentrations of free retinoids.     

Analysis of four maize mutants arrested in early embryogenesis reveals an irregular pattern of cell division

The process that leads to embryo formation appears to follow a defined pattern, whose sequential developmental steps—under strict genetic control—can be analysed through the study of mutants affecting embryogenesis. We present the analysis of four embryo-specific (emb) mutants of maize, characterised by abnormal development not overcoming the proembryo or early transition stage, that define three separate genes on the basis of their chromosomal location and complementation pattern. A common feature emerging from histological analysis is that suppression of morphogenesis is accompanied by an uncontrolled pattern of cell division. The block in embryo development is associated with abnormal suspensor proliferation, possibly due to the absence of a signal elaborated by the embryo proper and required for suspensor cell identity maintenance. Mutant endosperm morphogenesis is not impaired, as shown by the formation of the expected domains, i.e. aleurone, starchy endosperm, embryo-surrounding region and basal endosperm transfer layer. The program of cell death appears impaired in the mutants, as expected if this process is essential in determining the shape and morphology of the developing organs. An unexpected result is obtained when mutant embryo rescue is attempted. Immature embryos transferred to a basal medium germinated, yielding small but otherwise normal seedlings, an observation not consistent with the histological evidence of a complete absence of morphogenetic potential. The analysis of emb mutants appears a promising tool to elucidate crucial points of embryo development such as the coupling of cell division with morphogenesis, cell-to-cell interactions, the relationship between embryo and endosperm development, and the interaction between embryo proper and suspensor.     

Tendon-muscle crosstalk controls muscle bellies morphogenesis, which is mediated by cell death and retinoic acid signaling

Vertebrate muscle morphogenesis is a complex developmental process, which remains quite yet unexplored at cellular and molecular level. In this work, we have found that sculpturing programmed cell death is a key morphogenetic process responsible for the formation of individual foot muscles in the developing avian limb. Muscle fibers are produced in excess in the precursor dorsal and ventral muscle masses of the limb bud and myofibers lacking junctions with digital tendons are eliminated via apoptosis. Microsurgical experiments to isolate the developing muscles from their specific tendons are consistent with a role for tendons in regulating survival of myogenic cells. Analysis of the expression of Raldh2 and local treatments with retinoic acid indicate that this signaling pathway mediates apoptosis in myogenic cells, appearing also involved in tendon maturation. Retinoic acid inhibition experiments led to defects in muscle belly segmentation and myotendinous junction formation. It is proposed that heterogeneous local distribution of retinoids controlled through Raldh2 and Cyp26A1 is responsible for matching the fleshy and the tendinous components of each muscle belly.     

Aberrant activation of p53 due to loss of MDM2 or MDMX causes early lens dysmorphogenesis

Although forming a heterodimer or heterooligomer is essential for MDM2 and MDMX to fully control p53 during early embryogenesis, deletion of either MDM2 or MDMX in specific tissues using the loxp-Cre system reveals phenotypic diversity during organ morphogenesis, which can be completely rescued by loss of p53, suggesting the spatiotemporal independence and specificity of the regulation of p53 by MDM2 and MDMX. In this study, we investigated the role of the MDM2–MDMX-p53 pathway in the developing lens that is a relatively independent region integrating cell proliferation, differentiation and apoptosis. Using the mice expressing Cre recombinase specifically in the lens epithelial cells (LECs) beginning at E9.5, we demonstrated that deletion of either MDM2 or MDMX induces apoptosis of LEC and reduces cell proliferation, resulting in lens developmental defect that finally progresses into aphakia. Specifically, the lens defect caused by MDM2 deletion was evident at E10, occurring earlier than that caused by MDMX deletion. These lens defects were completely rescued by loss of two alleles of p53, but not one allele of p53. These results demonstrate that both MDM2 and MDMX are required for monitoring p53 activity during lens development, and they may function independently or synergistically to control p53 and maintain normal lens morphogenesis.     

Review: cornification,morphogenesis and evolution of feathers

Feathers are corneous microramifications of variable complexity derived from the morphogenesis of barb ridges. Histological and ultrastructural analyses on developing and regenerating feathers clarify the three-dimensional organization of cells in barb ridges. Feather cells derive from folds of the embryonic epithelium of feather germs from which barb/barbule cells and supportive cells organize in a branching structure. The following degeneration of supportive cells allows the separation of barbule cells which are made of corneous beta-proteins and of lower amounts of intermediate filament (IF)(alpha) keratins, histidine-rich proteins, and corneous proteins of the epidermal differentiation complex. The specific protein association gives rise to a corneous material with specific biomechanic properties in barbules, rami, rachis, or calamus. During the evolution of different feather types, a large expansion of the genome coding for corneous feather beta-proteins occurred and formed 3–4-nm-thick filaments through a different mechanism from that of 8–10 nm IF keratins. In the chick, over 130 genes mainly localized in chromosomes 27 and 25 encode feather corneous beta-proteins of 10–12 kDa containing 97–105 amino acids. About 35 genes localized in chromosome 25 code for scale proteins (14–16 kDa made of 122–146 amino acids), claws and beak proteins (14–17 kDa proteins of 134–164 amino acids). Feather morphogenesis is periodically re-activated to produce replacement feathers, and multiple feather types can result from the interactions of epidermal and dermal tissues. The review shows schematic models explaining the translation of the morphogenesis of barb ridges present in the follicle into the three-dimensional shape of the main types of branched or un-branched feathers such as plumulaceous, pennaceous, filoplumes, and bristles. The temporal pattern of formation of barb ridges in different feather types and the molecular control from the dermal papilla through signaling molecules are poorly known. The evolution and diversification of the process of morphogenesis of barb ridges and patterns of their formation within feathers follicle allowed the origin and diversification of numerous types of feathers, including the asymmetric planar feathers for flight.     

Control of retinoic acid synthesis and FGF expression in the nasal pit is required to pattern the craniofacial skeleton

Endogenous retinoids are important for patterning many aspects of the embryo including the branchial arches and frontonasal region of the embryonic face. The nasal placodes express retinaldehyde dehydrogenase-3 (RALDH3) and thus retinoids from the placode are a potential patterning influence on the developing face. We have carried out experiments that have used Citral, a RALDH antagonist, to address the function of retinoid signaling from the nasal pit in a whole embryo model. When Citral-soaked beads were implanted into the nasal pit of stage 20 chicken embryos, the result was a specific loss of derivatives from the lateral nasal prominences. Providing exogenous retinoic acid residue development of the beak demonstrating that most Citral-induced defects were produced by the specific blocking of RA synthesis. The mechanism of Citral effects was a specific increase in programmed cell death on the lateral (lateral nasal prominence) but not the medial side (frontonasal mass) of the nasal pit. Gene expression studies were focused on the Bone Morphogenetic Protein (BMP) pathway, which has a well-established role in programmed cell death. Unexpectedly, blocking RA synthesis decreased rather than increased Msx1, Msx2, and Bmp4 expression. We also examined cell survival genes, the most relevant of which was Fgf8, which is expressed around the nasal pit and in the frontonasal mass. We found that Fgf8 was not initially expressed along the lateral side of the nasal pit at the start of our experiments, whereas it was expressed on the medial side. Citral prevented upregulation of Fgf8 along the lateral edge and this may have contributed to the specific increase in programmed cell death in the lateral nasal prominence. Consistent with this idea, exogenous FGF8 was able to prevent cell death, rescue most of the morphological defects and was able to prevent a decrease in retinoic acid receptorbeta (Rarbeta) expression caused by Citral. Together, our results demonstrate that endogenous retinoids act upstream of FGF8 and the balance of these two factors is critical for regulating programmed cell death and morphogenesis in the face. In addition, our data suggest a novel role for endogenous retinoids from the nasal pit in controlling the precise downregulation of FGF in the center of the frontonasal mass observed during normal vertebrate development.