Demonstration of a phagocytic cell system belonging to the hemopoietic lineage and originating from the yolk sac in the early avian embryo.

It is well established that hemopoietic cells arising from the yolk sac invade the avian embryo. To study the fate and role of these cells during the first 2.5-4.5 days of incubation, we constructed yolk sac chimeras (a chick embryo grafted on a quail yolk sac and vice versa) and immunostained them with antibodies specific to cells of quail hemangioblastic lineage (MB1 and QH1). This approach revealed that endothelial cells of the embryonic vessels are of intraembryonic origin. In contrast, numerous hemopoietic cells of yolk sac origin were seen in embryos ranging from 2.5 to 4.5 days of incubation. These cells were already present within the vessels and in the mesenchyme at the earliest developmental stages analyzed. Two hemopoietic cell types of yolk sac origin were distinguishable, undifferentiated cells and macrophage-like cells. The number of the latter cells increased progressively as development proceeded, and they showed marked acid phosphatase activity and phagocytic capacity, as revealed by the presence of numerous phagocytic inclusions in their cytoplasm. The macrophage-like cells were mostly distributed in the mesenchyme and also appeared within some organ primordia such as the neural tube, the liver anlage and the nephric rudiment. Comparison of the results in the two types of chimeras and the findings obtained with acid phosphatase/MB1 double labelling showed that some hemopoietic macrophage-like cells of intraembryonic origin were also present at the stages considered. These results support the existence in the early avian embryo of a phagocytic cell system of blood cell lineage, derived chiefly from the yolk sac. Cells belonging to this system perform phagocytosis in cell death and may also be involved in other morphogenetic processes.     

Developmental Plasticity of the Prospective Dermatome and the Prospective Sclerotome Region of an Avian Somite

The ventro-medial wall of a somite gives rise to the sclerotome and then to cartilaginous axial skeleton, while the dorso-lateral wall differentiates into the dermomyotome to form dermal mesenchyme and muscle. Although previous studies suggested pluri-potency of somite cell differentiation, apparent pluri-potency may be the result of migration of predetermined cells. To investigate whether the developmental fate of any region is determined, I isolated fragments of a region of a quail somite and transplanted them into chick embryos. When a fragment of the ventral wall of a quail somite, the prospective sclerotome, was transplanted into a chick embryo between the ectoderm and a newly formed somite, the transplanted quail cells were shown to form myotome and mesenchyme in 4-day chimera embryos and to form muscle and dermal tissue in 9-day chimeras. On the other hand, when a fragment of the dorsal wall of a quail somite, the prospective dermomyotome, was transplanted into a chick embryo between the neural tube and a newly formed somite, the graft gave rise to mesenchyme around the neural tube and notochord and then to vertebral cartilage. Thus the developmental fate of a region of a somite was shown not to be determined at the time of somite segmentation, confirming previous observations.     

FGF signals induce Caprin2 expression in the vertebrate lens

The lens of the eye is derived from the non-neural ectoderm situated next to the optic vesicle. Fibroblast growth factor (FGF) signals play a major role at various stages of vertebrate lens development ranging from induction and proliferation to differentiation. Less is however known about the identity of genes that are induced by FGF activity within the lens. We have isolated and characterized mouse cytoplasmic activation/proliferation-associated protein-2 (Caprin2), with domains belonging to both the Caprin family and the C1q and tumour necrosis factor (TNF) super-family. Here we show that Caprin2 is expressed in the developing vertebrate lens in mouse and chick, and that Caprin2 expression is up-regulated in primary lens fiber cells, after the induction of crystallins the earliest known markers for differentiated lens fiber cells. Caprin2 is subsequently down-regulated in the centre of the lens at the time and at the position of the first fiber cell denucleation and terminal differentiation. In vitro analyses of lens fiber cell differentiation provide evidence that FGF activity emanating from neighboring prospective retinal cells is required and that FGF8 activity is sufficient to induce Caprin2 in lens fiber cells. These results not only provide evidence that FGF signals induce the newly characterized protein Caprin2 in the lens, but also support the general idea that FGF signals are required for lens fiber cell differentiation.     

Localisation of laminin and fibronectin during rat lens morphogenesis

Abstract. Immunofluorescence clearly localised laminin and fibronectin in the basement membranes of ocular epithelia through all stages of rat lens differentiation. Some fibronectin is also localised around the mesodermal cells associated with the epithelia. At 10 days of embryonic development, the presumptive lens ectoderm and optic veiscle are closely associated, and the "interspace" between the two tissues contains only a few mesodermal cells. Later, as the mesoderm is excluded and the lens palcode invaginates to form the lens pit, there is a marked increase in the concentration of both laminin and fibronectin in the interspace. At about 13 days, the interspace widens, and there is fluorescence for both glycoproteins in the basement membranes of the optic cup and lens vesicle; as the lens capsule thickens, the fluorescence for laminin increases in the latter. The unlabelled peroxidase anti-peroxidase (PAP) method shows that 'blebs' and 'blisters' of basement membranes, particularly from the optic vesicle, appear to give rise to cords of fibronectin- and laminin-positive material. These cords extend into the interspace and are associated with flocculent and fibrillar material. Therefore, the glycoproteins probably combine with other extracellular matrix (ECM) constituents, e.g. collagen, to form a network of fibrils in the interspace. This network must provide good adhesion between the lens placode and the optic vesicle so that invagination is co-ordinated to form the lens pit and the optic cup, respectively. It is suggested that, in addition to providing good adhesion between the tissues, this laminin- and fibronectin-rich ECM may stimulate the formation of basal extensions and cytoplasmic processes, particularly from the lens placode, and therefore, initiate the ectoderm to form lens placode.     

A re-examination of lens induction in chicken embryos: in vitro studies of early tissue interactions

Early studies on lens induction suggested that the optic vesicle, the precursor of the retina, was the primary inducer of the lens; however, more recent experiments with amphibians establish an important role for earlier inductive interactions between anterior neural plate and adjacent presumptive lens ectoderm in lens formation. We report here experiments assessing key inductive interactions in chicken embryos to see if features of amphibian systems are conserved in birds. We first examined the issue of specification of head ectoderm for a lens fate. A large region of head ectoderm, in addition to the presumptive lens ectoderm, is specified for a lens fate before the time of neural tube closure, well before the optic vesicle first contacts the presumptive lens ectoderm. This positive lens response was observed in cultures grown in a wide range of culture media. We also tested whether the optic vesicle can induce lenses in recombinant cultures with ectoderm and find that, at least with the ectodermal tissues we examined, it generally cannot induce a lens response. Finally, we addressed how lens potential is suppressed in non-lens head ectoderm and show an inhibitory role for head mesenchyme. This mesenchyme is infiltrated by neural crest cells in most regions of the head. Taken together, these results suggest that, as in amphibians, the optic vesicle cannot be solely responsible for lens induction in chicken embryos; other tissue interactions must send early signals required for lens specification, while inhibitory interactions from mesenchyme suppress lens-forming ability outside of the lens area.     

Embryonic macrophages of early rat yolk sac: Immunohistochemistry and ultrastructure with reference to endodermal cell layer

Macrophages are multifunctional cells that participate in numerous biological processes; they actively phagocytose foreign particles and cell debris. Embryonic tissue macrophages are present at early stages of mammalian development; their ontogeny and function is still under investigation. Our study used immunohistochemistry and electron microscopy to investigate early rat yolk sac macrophages using mouse antirat macrophage monoclonal antibodies (mAb) Mar 1 and Mar 3 produced by our laboratory. Mar 3 mAb revealed the first emergence of immature macrophages in the rat yolk sac at fetal day nine coinciding with the beginning of yolk sac haemopoiesis that consisted mainly of erythropoiesis, while Mar 1 mAb detected specifically rat yolk sac macrophages at about the 13th to 14th day of gestation. Immunoreactivity against Mar mAbs was mainly located in the yolk sac endodermal cell layer, which may signify endodermal origin of the yolk sac macrophages. Ultrastructurally mature yolk sac macrophages contained numerous endocytic vesicles or vacuoles, well-developed Golgi saccules and many electron dense granules in their cytoplasm and a number of microvillous projections from the cell surface. After establishment of the circulation between yolk sac and embryo, Mar 3 positive cells were also demonstrated inside fetal undifferentiated mesenchymal tissue at fetal day 12. The study demonstrated the first emergence of immature yolk sac macrophages being among the earliest haemopoietic cells formed in mammalian development. Thus, Mar mAbs managed to detect macrophage differentiation antigens through their development early in the rat yolk sac.     

Extraembryonic origin of circulating endothelial cells

Circulating endothelial cells (CEC) are contained in the bone marrow and peripheral blood of adult humans and participate to the revascularization of ischemic tissues. These cells represent attractive targets for cell or gene therapy aimed at improving ischemic revascularization or inhibition of tumor angiogenesis. The embryonic origin of CEC has not been addressed previously. Here we use quail-chick chimeras to study CEC origin and participation to the developing vasculature. CEC are traced with different markers, in particular the QH1 antibody recognizing only quail endothelial cells. Using yolk-sac chimeras, where quail embryos are grafted onto chick yolk sacs and vice-versa, we show that CEC are generated in the yolk sac. These cells are mobilized during wound healing, demonstrating their participation to angiogenic repair processes. Furthermore, we found that the allantois is also able to give rise to CEC in situ. In contrast to the yolk sac and allantois, the embryo proper does not produce CEC. Our results show that CEC exclusively originate from extra-embryonic territories made with splanchnopleural mesoderm and endoderm, while definitive hematopoietic stem cells and endothelial cells are of intra-embryonic origin.     

FGF-mediated induction of ciliary body tissue in the chick eye

Upon morphogenesis, the simple neuroepithelium of the optic vesicle gives rise to four basic tissues in the vertebrate optic cup: pigmented epithelium, sensory neural retina, secretory ciliary body and muscular iris. Pigmented epithelium and neural retina are established through interactions with specific environments and signals: periocular mesenchyme/BMP specifies pigmented epithelium and surface ectoderm/FGF specifies neural retina. The anterior portions (iris and ciliary body) are specified through interactions with lens although the molecular mechanisms of induction have not been deciphered. As lens is a source of FGF, we examined whether this factor was involved in inducing ciliary body. We forced the pigmented epithelium of the embryonic chick eye to express FGF4. Infected cells and their immediate neighbors were transformed into neural retina. At a distance from the FGF signal, the tissue transitioned back into pigmented epithelium. Ciliary body tissue was found in the transitioning zone. The ectopic ciliary body was never in contact with the lens tissue. In order to assess the contribution of the lens on the specification of normal ciliary body, we created optic cups in which the lens had been removed while still pre-lens ectoderm. Ciliary body tissue was identified in the anterior portion of lens-less optic cups. We propose that the ciliary body may be specified at optic vesicle stages, at the same developmental stage when the neural retina and pigmented epithelium are specified and we present a model as to how this could be accomplished through overlapping BMP and FGF signals.     

A study of the development of the hemopoietic system using quail-chick chimeras obtained by blastoderm recombination

The anterior part of the area pellucida from quail blastoderms extending to the 10th or the 17th somite level was substituted for the corresponding region of chick blastoderms in ovo. Reciprocal grafts were also carried out. In external appearance the resulting chimeras had a composite body, one species contributing the head and neck or the head, neck, and wing regions while the other species provided the remainder. The chimeras were always grafted on a chick yolk sac. The cellular composition of hemopoietic organs according to species was analyzed by means of the quail-chick nuclear difference, in 39 viable chimeras at 11–13 days of incubation. The thymus composition depended on the level of the boundary between the two species. In chimeras in which the quail contributed head and neck, the thymic epithelial stroma was made of quail cells while the lymphoid population was of chick origin. In contrast, when the quail contribution also extended to the wings, both thymic stroma and lymphoid cells were of quail origin. In reciprocal combinations, in which head and neck were of chicken origin on a quail body, a different result was obtained: no lymphoid cells were present in the thymus which was reduced to its epithelial component and this was of chick origin. On the other hand, if the chick contribution extended to the wings, as in the reciprocal combination, all thymus components were of chick origin. The spleen and the bursa of Fabricius in most instances did not differ in their cellular composition from the surrounding tissues; however in some chimeras a minor admixture of cells of the other species was also found. Overall these results suggest that hemopoietic stem cells destined to colonize intraembryonic organs arise in territories derived from the whole area pellucida excluding the prospective head-neck region. Furthermore, each hemopoietic organ rudiment appears to be colonized by precursors derived from adjacent territories.     

Roles of erbB4, rhombomere-specific, and rhombomere-independent cues in maintaining neural crest-free zones in the embryonic head

Within the developing vertebrate head, the migration of neural tube-derived neural crest cells (NCCs) through the cranial mesenchyme is patterned into three streams, with mesenchyme adjacent to rhombomeres (r)3 and r5 maintained NCC-free. The receptor tyrosine kinase erbB4 is expressed within r3 and r5 and is required to maintain the r3-adjacent NCC-free zone in mouse embryos. In this study, we demonstrate that the extent of r3 involvement in patterning mouse NCC migration is restricted to the same dorsolateral region regulated by erbB4. In chick embryos, we show that erbB4 signaling similarly maintains the r3-adjacent NCC-free zone. However, although r5 expresses erbB4, this is insufficient to maintain the r3-adjacent NCC-free zone in grafting experiments where r5 replaced r3, indicating that erbB4 requires additional factors at the A-P level of r3 to pattern NCC migration. Furthermore, we show that the r5-adjacent NCC-free zone is maintained independently of r5, but requires surface ectoderm. Finally, we demonstrate that avian cranial surface ectoderm is patterned molecularly, with dorsolateral surface ectoderm at the levels of r2/3 and r7 expressing the sulfatase QSulf1 in quail, or the orthologue CSulf1 in chick. Aberrant NCC migration into r3-adjacent mesenchyme correlated with more focused QSulf1 expression in r2/3 surface ectoderm.     

Optic cup morphogenesis requires pre-lens ectoderm but not lens differentiation

The formation of the vertebrate optic cup is a morphogenetic event initiated after the optic vesicle contacts the overlying surface/pre-lens ectoderm. Placodes form in both the optic neuroepithelium and lens ectoderm. Subsequently, both placodes invaginate to form the definitive optic cup and lens, respectively. We examined the role of the lens tissue in inducing and/or maintaining optic cup invagination in ovo. Lens tissue was surgically removed at various stages of development, from pre-lens ectoderm stages to invaginating lens placode. Removal of the pre-lens ectoderm resulted in persistent optic vesicles that initiated neural retinal differentiation but failed to invaginate. In striking contrast, ablation of the lens placode gave rise to optic vesicles that underwent invagination and formed the optic cup. The results suggest that: (1) the optic vesicle neuroepithelium requires a temporally specific association with pre-lens ectoderm in order to undergo optic cup morphogenesis; and (2) the optic cup can form in the absence of lens formation. If ectopic BMP is added, a neural retina does not develop and optic cup morphogenesis fails, although lens formation appears normal. FGF-induced neural retina differentiation in the absence of the pre-lens ectoderm is not sufficient to create an optic cup. We hypothesize the presence of a signal coming from the pre-lens ectoderm that induces the optic vesicle to form an optic cup.     

Monoclonal antibodies specific to quail embryo tissues: their epitopes in the developing quail embryo and their application to identification of quail cells in quail-chick chimeras.

Quail-chick chimeras have been used extensively in the field of developmental biology. To detect quail cells more easily and to detect cellular processes of quail cells in quail-chick chimeras, we generated four monoclonal antibodies (MAb) specific to some quail tissues. MAb QCR1 recognizes blood vessels, blood cells, and cartilage cells, MAb QB1 recognizes quail blood vessels and blood cells, and MAb QB2 recognizes quail blood vessels, blood cells, and mesenchymal tissues. These antibodies bound to those tissues in 3-9-day quail embryos and did not bind to any tissues of 3-9-day chick embryos. MAb QSC1 is specific to the ventral half of spinal cord and thymus in 9-day quail embryo. No tissue in 9-day chick embryo reacted with this MAb. This antibody binds transiently to a small number of brain vesicle cells in developing chick embryo as well as in quail embryo. A preliminary application of two of these MAb, QCR1 and QSC1, on quail-chick chimeras of neural tube and somites is reported here.     

Reliability of delta-crystallin as a marker for studies of chick lens induction

Induction of a lens by the optic vesicle of the brain was the first demonstration of how tissue interactions could influence cell fate during development. However, recent work with amphibians has shown that the optic vesicle is not the primary inducer of lens formation. Rather, an earlier interaction between anterior neural plate and presumptive lens ectoderm appears to direct lens formation. One problem with many early experiments was the absence of an unambiguous assay for lens formation. Before being able to test whether the revised model of lens induction applies to chicken embryos, we examined the suitability of using delta-crystallin as a marker of lens formation. Although delta-crystallin is the major protein synthesized in the chick lens, one or both of the two delta-crystallin genes found in chickens is transcribed in many non-lens tissues as well. In studies of lens formation where appearance of the delta-crystallin protein is used as a positive assay, synthesis of delta-crystallin outside of the lens could make experiments difficult to interpret. Therefore, polyacrylamide gel electrophoresis, immunoblotting, and immunofluorescence were used to determine whether the delta-crystallin messenger RNA detected in non-lens tissues is translated into protein, as it is in the lens. On Coomassie-blue-stained gels of several tissues from stage-22 embryos, a prominent protein was observed that co-migrated with delta-crystallin. However, on immunoblots, none of the non-lens tissues tested contained detectable levels of delta-crystallin at this stage. By imunofluorescence, delta-crystallin was observed in Rathke's pouch and in a large area of oral ectoderm near Rathke's pouch, yet none of the cells in these non-lens tissues showed the typical elongated morphology of lens fiber cells. When presumptive lens ectoderm or other regions of ectoderm from stage-10 embryos were cultured and tested for lens differentiation, both cell elongation and delta-crystallin synthesis were observed, or neither were observed. The results suggest that delta-crystallin synthesis and cell elongation together serve as useful criteria for assessing a positive lens response.     

The appearance of alpha-crystallin in relation to cell cycle phase in the embryonic mouse lens

Specific protein synthesis in the embryonic mouse lens was studied by immunofluorescence with antisera to adult mouse lens or crystallin fractions. Positive reactions were first detected in a few cells of the lens cup 18-24 hr after contact between optic vesicle and presumptive lens ectoderm had been established. During formation of the lens vesicle a rapidly increasing fraction of cells produced crystallins. At the time of detachment of the vesicle from the surface all cells of its posterior wall showed immunofluorescence. After fiber elongation became distinct cells of the anterior epithelium began to fluoresce and shortly afterwards the entire rudiment produced crystallins. The early reactions were due entirely to the presence of alpha-crystallin. Reactions were restricted to the lens. Thus, in the mouse as in other species crystallins were detectable by immunofluorescence in vivo only after lens morphogenesis was well underway and only in the lens rudiment itself. Cells first synthesizing crystallins always had an elongated shape and their nuclei were in a basal position. A few hours later mitotic cells displayed fluorescence. Taking into account earlier found relations between cell morphology and cell cycle phase, this indicates that alpha-crystallin is first demonstrable in the S-or early G-2 phase of the cell cycle, and that the start of its synthesis does not preclude continued cell replication. It is interesting that the cellular location, cell cycle phase, and developmental stage, in which crystallins first appear, are comparable in mouse and chick embryo. Yet, entirely different proteins are involved: alpha-crystallin in the first, delta-crystallin in the latter. Implications of this for our understanding of lens induction are discussed.     

Prebursal stem cells in the intraembryonic mesenchyme of the chick embryo at 7 days of incubation.

Cyclophosphamide-treated 18-day-old chick embryos were transplanted with cells from 7-day intraembryonic mesenchyme; the recipients and donors were identical at the major histocompatibility locus. At the age of 35 days, the cell recipients were studied to determine the reconstitution capacity of the transplanted cells. The transplantation resulted in a complete restoration of IgM and IgG class antibody production against human gammaglobulin and Brucella abortus, and of microscopic morphology of the bursa of Fabricius and of the germinal center formation in the spleen. These findings demonstrate that 7-day intraembryonic mesenchyme of the chick embryo harbor prebursal stem cells. These findings confirm our previous observations in the yolk sac-embryo chimeras indicating that lymphoid stem cells originate in the intraembryonic hematopoietic sites.     

Proteolytic activity in the yolk sac membrane of quail eggs.

Extraembryonal degradation of yolk protein is necessary to provide the avian embryo with required free amino acids during early embryogenesis. Screening of proteolytic activity in different compartments of quail eggs revealed an increasing activity in the yolk sac membrane during the first week of embryogenesis. In this tissue, the occurrence of cathepsin B, a lysosomal cysteine proteinase, and cathepsin D, a lysosomal aspartic proteinase, has been described recently (Gerhartz et al., Comp Biochem Physiol, 118B:159-166, 1997). Determination of cathepsin B-like and cathepsin D-like proteolytic activity in the yolk sac membrane indicated a significant correlation between growth of the yolk sac membrane and proteolytic activity, shown by an almost constant specific activity. Both proteinases could be localized in the endodermal cells, which are in direct contact to the yolk. The concentration of proteinases in the endodermal cells appears to be almost unaltered in the investigated early stage of quail development, whereas the amount of endodermal cells increases rapidly, seen by a complicated folding of the yolk sac membrane. In the same cells quail cystatin, a potent inhibitor of quail cathepsin B (Ki 0.6 nM), has been localized at day 8 of embryonic development. Approximately at this stage of development, the quail embryo stops metabolizing yolk. In conclusion, it is strongly indicated that the amount of available free amino acids, produced by proteolytic degradation and supporting embryonic growth, is regulated by the growth of the yolk sac membrane.     

The influence of epithelia on cartilage and loose connective tissue formation by limb mesenchyme cultures

In order to explain the observation that normally nonchondrogenic limb mesenchyme forms extensive cartilage in culture, the possibility that limb ectoderm inhibits chondrogenesis is examined. Small pieces of quail or chick ectoderm are grafted onto micromass cultures of wing mesenchyme from stage 23–34 chick embryos. The presence of nonridge wing or several other epithelia results in increased collagen accumulation in the underlying mesenchyme, a delay in cell differentiation, and the eventual formation of loose connective tissue, as determined by transmission electron microscopy. The influence can occur across Nuclepore filters having 0.1-μm-diameter pores and ultrathin Millipore filters, but not across Millipore filters of standard thickness. The influence is not contact dependent since cell processes do not cross these filters. The apical ectodermal ridge has the additional effect of stimulating mesenchymal outgrowth. These results support the idea that the ectoderm plays a direct but negative role in the formation of a chondrogenic core within the developing limb.     

Growth factor regulation of lens development

Lens arises from ectoderm situated next to the optic vesicles. By thickening and invaginating, the ectoderm forms the lens vesicle. Growth factors are key regulators of cell fate and behavior. Current evidence indicates that FGFs and BMPs are required to induce lens differentiation from ectoderm. In the lens vesicle, posterior cells elongate to form the primary fibers whereas anterior cells differentiate into epithelial cells. The divergent fates of these embryonic cells give the lens its distinctive polarity. There is now compelling evidence that, at least in mammals, FGF is required to initiate fiber differentiation and that progression of this complex process depends on the synchronized and integrated action of a number of distinct growth factor-induced signaling pathways. It is also proposed that an antero-posterior gradient of FGF stimulation in the mammalian eye ensures that the lens attains and maintains its polarity and growth patterns. Less is known about differentiation of the lens epithelium; however, recent studies point to a role for Wnt signaling. Multiple Wnts and their receptors are expressed in the lens epithelium, and mice with impaired Wnt signaling have a deficient epithelium. Recent studies also indicate that other families of molecules, that can modulate growth factor signaling, have a role in regulating the ordered growth and differentiation of the lens.     

Reinvestigation of the role of the optic vesicle in embryonic lens induction

The induction of the lens by the optic vesicle in amphibians is often cited as support for the view that a single inductive event can lead to determination in a multipotent tissue. This conclusion is based on transplantation experiments whose results indicate that many regions of embryonic ectoderm which would normally form epidermis can form a lens if brought into contact with the optic vesicle. Although additional evidence argues that during normal development other tissues, acting before the optic vesicle, also contribute to lens induction, it is still widely held, on the basis of these transplantation experiments, that the optic vesicle alone can elicit lens formation in ectoderm. While testing this conclusion by transplanting optic vesicles beneath ventral ectoderm in Xenopus laevis embryos, it became apparent that contamination of optic vesicles by presumptive lens ectoderm cells can generate lenses in these experiments, illustrating the need for adequate host and donor marking procedures. Since previous studies rarely used host and donor marking, it was not clear whether they actually demonstrated that the optic vesicle can induce lenses. Using careful host and donor marking procedures with horseradish peroxidase as a lineage tracer, we show that the optic vesicle cannot stimulate lens formation in neurula- or gastrula-stage ectoderm of Xenopus laevis. Since the general conclusion that the optic vesicle is sufficient for lens induction rests on studies in many organisms, we felt it was important to begin to test this conclusion in other amphibians as well. Similar experiments were therefore performed with Rana Palustris embryos, since it was in this organism that optic vesicle transplant studies had originally argued that this tissue alone can cause lens induction. Under conditions similar to those used in the original report, but with careful controls to assess the origin of lenses in transplants, we found that the optic vesicle alone cannot elicit lens formation. Our data lead us to propose that the optic vesicle in amphibians is not generally sufficient for lens induction. Instead, we argue that lens induction occurs by a multistep process in which an essential phase in lens determination occurs as a result of inductive interactions preceding contact of ectoderm with the optic vesicle.     

The pattern of protein and glycoprotein synthesis in presumptive lens and non-lens ectoderm of the chicken embryo

Summary Lens induction is a classic example of the tissue interactions that lead to cell specialization during early vertebrate development. Previous studies have shown that a large region of head ectoderm, but not trunk ectoderm, of 36 h (stage 10) chicken embryos retains the potential to form lenses and synthesize the protein δ-crystallin under some conditions. We have used polyacrylamide gel electrophoresis and fluorography to examine protein and glycoprotein synthesis in presumptive lens ectoderm and presumptive dorsal (trunk) epidermis to look for differentiation markers for these two regions prior to the appearance of δ-crystallin at 50 h. Although nearly all of the proteins incorporating³H-leucine were shared by presumptive lens ectoderm and trunk ectoderm, these two regions showed more dramatic differences in the incorporation of³H-sugars into glycoproteins. when non-lens head ectoderm that has a capacity for lens formation in vitro was labeled, a hybrid pattern of glycoprotein synthesis was discovered: glycoproteins found in either presumptive lens ectoderm or trunk ectoderm were oftentimes also found in other head ectoderm. Therefore, molecular markers have been identified for three regions of ectoderm committed to different fates (lens and skin), well before features of terminal differentiation begin to appear in the lens.