Can a non-gut mesenchyme support differentiation of gut endocrine cells?

Summary This experiment was designed to find out if endoderm lacks an intrinsic ability to give rise to gut endocrine cells, and, if not, whether differentiation of endocrine cells can be supported by mesenchyme from a source outside the digestive tract. Heterospecific combinations of proventricular endoderm and flank mesenchyme from chick and quail embryos at 3.25–4 days of incubation were grown as chorio-allantoic grafts to a final incubation age of 21 days. Re-associated proventricular endoderm and mesenchyme served as controls. The proventricular endoderm induced some smooth muscle in the flank mesenchyme but the latter did not support as advanced glandular morphogenesis as did proventricular mesenchyme. Nevertheless, endocrine cells differentiated in experimental as in control grafts and at similar frequencies. The various types were distinguished immunocytochemically by their contained peptides; the range of types found was specific for the proventriculus. Hence it is concluded not only that the particular non-gut mesenchyme used does support differentiation of gut endocrine cells, but also that the determination of the progenitors of endocrine cells, and the selection of the range of types destined to differentiate in a particular part of the digestive tract under normal circumstances, occurs early in development — before 3.25 days of incubation in the case of the proventriculus.     

Differentiation of gut endoderm in dependence of the notochord

Summary Presumptive intraembryonic endoderm, either isolated or together with adhering mesoderm, from 19-h chick embryos, was grafted to the coelom of 50-h host embryos. The viability of such grafts was low and endodermal differentiation was poor. In a second series the endoderm (with or without adhering mesoderm) was combined with a fragment of notochordal tissue from 48–60-h donor embryos. Then the recovery was much higher, notably after longer periods of in vivo culture. After 10 days of cultivation well-developed entero-endocrine (argyrophilic) cells were found among the regular enterocytes in both series.     

Epithelial influences on skeletogenesis in the mandible of the embryonic chick

Intact mandibular processes and the enzymatically separated mesenchymal and epithelial components of the mandible from embryonic chicks of 2.5- to 5-day incubation (Hamburger and Hamilton, '51: stages 16-25) were grown individually, either in organ culture or as grafts to the chorioallantoic membranes of host embryos. The differentiation of cultured and grafted intact mandibular processes was histologically normal, but the time of histodifferentiation differed from that in vivo. The histodifferentiation of cultured and grafted mandibular mesenchyme grown isolated from its epithelium depended upon the age of the embryo from which the mesenchyme had been obtained. Intramembranous ossification producing membrane bones of the mandible occurred in mesenchyme isolated from 4.5- to 5-day embryos (HH 24–25), but did not occur in mesenchyme isolated from younger embryos. Cartilage (Meckel's) and subperichondrial bone in the articular process of Meckel's cartilage differentiated in mesenchyme isolated from embryos of all age groups tested (HH 16–25). Mandibular mesenchyme, therefore, requires the presence of epithelium until 4.5 days of incubation if the membrane bones of the mandible are to differentiate; if epithelial influences are required for Meckel's cartilage and subperichondrial bone formation, they are not required beyond 2.5 days of incubation. Mandibular epithelium isolated from its mesenchyme became layers of squamous cells in culture; but when grafted onto the chorioallantoic membrane, the epithelium became underlain by host fibroblasts and differentiated into a stratified squamous epithelium. Mandibular epithelium, therefore, is capable of differentiation in the presence of foreign fibroblasts derived from the chorioallantoic membrane.     

A substance secreted by rat Sertoli cells induces feminization of embryonic chick testes in vitro

Male and female gonads from 7- to 9-day-old chick embryos were cultured for 6 days in Sertoli cell-conditioned medium or in serum-free medium to investigate the possible effect of substances secreted by rat Sertoli cells on chick gonad development. Histological analysis showed that whereas all female gonads proceed through normal ovarian development in both culture media, most of male gonads showed clear feminization only when cultured in Sertoli cell-conditioned medium; male gonads cultured in serum-free medium developed as normal testes. Because the only substance detected in our conditioned medium with the potential to cause these effects was sex-specific antigen (Sxs), our results provide further evidence that Sxs antigen may play a role in sexual differentiation in birds, and probably in mammals.     

Can gastric endoderm change the regionally specific inducing ability of presumptive small intestinal mesoderm?

This study was designed to establish the source of gut mesoderm's ability to induce regional pattern in the endoderm. The most obvious possibility is induction by the endoderm through epithelial-mesenchymal interaction. To test this experimentally, reciprocal quail/chick combinations were prepared of early proventricular endoderm (that is already known to be regionally determined) and presumptive small intestinal mesoderm. The combinations were cultured for 7 days to allow for 'programming' of the mesoderm by the endoderm. After removal of the proventricular endoderm the mesoderm was combined with young gizzard endoderm. It is known that gizzard endoderm can be provoked to develop in either a proventricular or a small intestinal direction by association with the appropriate mesoderm. Thus, by combining intestinal mesoderm 'programmed' by association with proventricular endoderm with gizzard endoderm, the subsequent differentiation of the gizzard endoderm would indicate whether or not the inducing ability of the intestinal mesenchyme had been altered. In addition to such experimental grafts, three types of control graft were prepared. The results of the experiment, based on the morphology of the grafts and the immunocytochemical analysis of selected endocrine cell types, showed that in the majority of cases the gizzard endoderm developed the features of small intestine, not those of proventriculus. This indicates that at the stages studied, endoderm does not act to program mesoderm with which it is associated. If this does occur, it must take place at an earlier stage, i.e., before the time of explantation of the presumptive small intestinal mesoderm (1.25 days of incubation).     

Normal transitions in synthesis of replacement histones H2A.Z and H3.3 during differentiation of dystrophic myotube cells. A brief note

We previously reported that differentiating G0 myotube cells cultured from normal chicken embryos exhibit a histone synthesis pattern that is highlighted by transitions in the expression of the minor replacement variants H3.3 and perhaps H2A.Z (Wunsch and Lough, Dev. Biol. 119 (1987) 94-99). Because these proteins may be synthesized to maintain chromatin structure during the differentiation and maturation of the skeletal muscle fiber, it was of interest to determine whether they are made at normal levels during the differentiation of dystrophic muscle. To this end, the synthesis of histone proteins in cultured myoblasts and myotubes from normal and dystrophic avian embryos has been characterized by two-dimensional polyacrylamide gel electrophoresis and fluorography. Proliferating myoblasts (day 1) as well as two stages of differentiating myotubes (days 3, 4) exhibited histone synthesis patterns that were indistinguishable when comparing normal and dystrophic cells. It is noteworthy that this study also revealed that, in both cell types, the change in H2A.Z synthesis during the myoblast/myotube transition was remarkable, increasing from approximately 20% of the non-ubiquitinated H2As in myoblasts to 80% in myotubes. Also, gel staining patterns and immunoblotting detected no differences in the degree of histone ubiquitination between normal and dystrophic cells. These findings indicate that, up to this point in dystrophic differentiation, neither the synthesis nor ubiquitination of histones are perturbed.     

Is chemotaxis a factor in the migration of precardiac mesoderm in the chick?

Summary The chick heart is formed from bilateral patches of presumptive cardiac mesoderm cells which migrate over the endoderm and fuse in the midline. We have tested the possibility that this migration is controlled, at least in part, by a chemotactic substance exuded by the anterior end of the endoderm. We have used chick/quail combinations to follow naturally marked cells during the course of their migration. Chimaeric embryos were formed by fusing together parts of chick and quail embryos of stage 5–6. Each embryo possessed two pairs of precardiac regions, the quail pair lying immediately anterior to that of the chick. These chimaeras were then explanted in embryo culture. In the event of chemotaxis, cells from the posterior end of the quail precardiac mesoderm might be expected to invade the chick area. Samples of explants and chimaeras were examined at intervals from 2 to 24 h, but in no case were cells found to have changed their direction of migration as a result of the proximity of anterior endoderm. It is concluded that this work does not provide evidence for a chemotactic attraction by the anterior end of the endoderm. Supported by the following grants: NIH HD 21048, HD 06819, and AHA 880696 (JWL); the British Heart Foundation, and Action Research (R.B.); and an SERC postgraduate studentship (HSE).     

The role of muscle activity in the differentiation of neuromuscular junctions in slow and fast chick muscles

Summary The differentiation of neuromuscular junctions of multiply innervated, slow, anterior latissimus dorsi (ALD) and focally innervated, fast, posterior latissimus dorsi (PLD) muscles was studied in normal and curarized chick embryos. At 16 days of incubation, fibres of both muscles are contacted by several axon profiles, the number of which falls with age. In 18-day-old embryos individual endplates in ALD are usually contacted by three axon profiles, whereas in PLD, endplates are contacted only by a single large terminal profile. At this time, there is already a significant accumulation of cell organelles in the postsynaptic area.Treatment of embryos with curare during the 7th and 12th day of incubation delays the differentiation of the neuromuscular junction in both muscles. The paralysis dramatically affects the decrease of the number of axon profiles at individual endplates in both muscles. At 16 days the number of axon profiles was greater in embryos treated with curare than in the untreated controls. At 18 days when the number of axon profiles normally decreases, the endplates of both types of curarized muscles have an even greater number of axon profiles than at 16 days. Endplates in curarized PLD had up to 13 and in curarized ALD up to 12 axon profiles. The effects of curare gradually wore off and when the movements of the embryos again became more vigorous, the normal differentiation of neuromuscular junctions continued. At 21 days of incubation many embryos recover from curare and show endplates of normal appearance in both muscles. These results suggest that activity of the muscle is essential for the maturation of the neuromuscular junctions.     

Notch signaling can regulate endoderm formation in zebrafish.

Early in vertebrate development, the processes of gastrulation lead to the formation of the three germ layers: ectoderm, mesoderm, and endoderm. The mechanisms leading to the segregation of the endoderm and mesoderm are not well understood. In mid-blastula stage zebrafish embryos, single marginal cells can give rise to both endoderm and mesoderm (reviewed by Warga and Stainier [2002] The guts of endoderm formation. In: Solnica-Krezel L, editor. Pattern formation in zebrafish. Berlin: Springer-Verlag. p 28-47). By the late blastula stage, however, single marginal cells generally give rise to either endoderm or mesoderm. To investigate this segregation of the blastoderm into cells with either endodermal or mesodermal fates, we analyzed the role of Notch signaling in this process. We show that deltaC, deltaD, and notch1 are expressed in the marginal domain of blastula stage embryos and that this expression is dependent on Nodal signaling. Activation of Notch signaling from an early stage leads to a reduction of endodermal cells, as assessed by sox17 and foxA2 expression. We further find that this reduction in endoderm formation by the activation of Notch signaling is preceded by a reduction in the expression of bonnie and clyde (bon) and faust/gata5, two genes necessary for endoderm formation (Reiter et al. [1999] Genes Dev 13:2983-2995; Reiter et al. [2001] Development 128:125-135; Kikuchi et al. [2001] Genes Dev 14:1279-1289). However, activation of Notch signaling in bon mutant embryos leads to a further reduction in endodermal cells, also arguing for a bon-independent role for Notch signaling in endoderm formation. Altogether, these results suggest that Notch signaling plays a role in the formation of the endoderm, possibly in its segregation from the mesoderm.     

Early mesoderm differentiation in the chick embryo

Summary Two groups of experiments were carried out. In the first group, grafts of quail mesoderm whose presumptive fate was to form somites or heart tissues, were taken from quail embryos (stage 4–5 of Hamburger and Hamilton 1951) and inserted beneath the ectoderm of chick embryos of stage 3–4 immediately lateral to the primitive streak. Whilst most grafts contributed to the somites and/or the heart, 22 out of a total of 46 were found to have contributed also to the pharyngeal endoderm. Although three of these grafts were known to have included some quail endoderm cells, the remainder were considered to consist of mesoderm alone. It is concluded that mesoderm at the primitive streak stages is still capable of forming endoderm.In the second group of experiments, grafts of quail somites (stage 10–14) were inserted beneath the ectoderm of chick embryos of stage 3–4. In 18 out of 23 cases the graft cells were found in somitic tissue, but they were also found in the endoderm (4 specimens), lateral plate (3 specimens) and endothelium (4 specimens). It is concluded that even at stages 10–14, the somite-derived cells are still not completely determined to form somite derivatives. In those cases where the grafted somites differentiated further, sclerotome cells which migrated from them did not necessarily move towards the host notochord.     

Myotome and early neurogenesis in chick embryos

The present study was undertaken in order to verify the identification of profiles of presumptive growth cones in vivo. The developing spinal nerves of chick embryos were studied by light and electron microscopy. We traced the onset of efferent and afferent innervation of the myotome in 2- to 4-day-old chick embryos in order to be sure that we were examining the growing tips of axons. In the process of studying these growing axons, we were able to observe some unique relationships of neural tube, myotome, and differentiating spinal nerves. The neural tube tightly abuts the myotome in Hamburger and Hamilton's (HH) stage 14 chick embryos and cytoplasmic projections from the myotome directly abut the neural tube. The first ventral roots could be identified in HH stage 15 embryos and dorsal roots in HH stage 16 embryos, both under 2 1/2 days of age. The advancing spinal nerve courses toward the anterior or cranial half of the myotome, and growth cones directly contact the medial wall of the myotome. The spinal nerves continue to abut tightly the myotome during the succeeding day of embryonic life, and growth cones enter the substance of the myotome by 3 days, or HH stage 19 embryos. These dorsolaterally directed axons will form the dorsal ramus of the spinal nerves and the ventral ramus continues to be contiguous with the myotome. Invasion of the myotome by axons (putative innervation), and thus innervation of myotomal cells in the 3-day chick embryos, was a totally unexpected finding. The myotome and its potential derivatives thus have extensive neural contact by 3 days of embryonic life in the chick. These findings document a parallel differentiation of afferent and efferent elements of the nervous system and confirm previous accounts identifying growth cones in an intact organism. These findings suggest that afferent as well as efferent nerves may have critical roles in the differentiation of the mesodermal as well as ectodermal derivatives.     

Determination of anterior endoderm in Xenopus embryos.

Grafts of anterior endoderm from embryos at stage 28 or later developed according to their fate (i.e., anterior) when transplanted posteriorly. Conversely, grafts from earlier embryos developed according to their new location (i.e., posterior). However, endoderm grafted along with its mesodermal and ectodermal sheath retained its fate regardless of the stage of the donor. We conclude that anterior endoderm in Xenopus embryos is determined at about stage 25 under the influence of mesoderm.     

Isotransplantation of fetal mouse pancreas in experimental diabetes. Effect of gestational age and organ culture

Fetal mouse pancreatic isotransplantation was performed in CBA mice, previously made diabetic with streptozotocin. Either 17- or 12-day-old fetal tissue was used, either uncultured or after 14 days in organ culture. Grafts were placed under the renal capsule, and their effect on fasting blood glucose levels was assessed over a 10-week postgraft period. The grafts and the host pancreas were then examined by light microscopy. Uncultured 12- and 17-day fetal pancreases produced poorly developed grafts with small islets, numerous ducts, and copious adipose tissue. By contrast, if organ cultured for 14 days before transplantation, both 12- and 17-day tissue resulted in large grafts composed predominantly of well-stained islet cells, few ducts, and little connective and adipose tissue. The most functionally effective grafts were derived from cultured 17-day pancreas, and a single graft per recipient always ameliorated diabetes. By contrast, grafts from single or multiple uncultured 12-day pancreases did not function, but, after culture, grafts derived from three (but not from one) 12-day pancreas lowered blood glucose levels to almost normal levels. It is concluded that grafts of fetal mouse pancreas can function in experimental diabetes provided they are first cultured and are of adequate gestational age.     

Behavioral analysis of opiate-mediated inhibition in the early chick embryo

Morphine (opiate agonist) produced a dose-dependent decrease in the spontaneous motility of 5- and 9-day chick embryos. Naloxone (opiate antagonist) appeared to reverse competitively the inhibition of motility caused by morphine. The effects of morphine on spontaneous motility in 5-day embryos were also reversed stereospecifically by the opiate antagonist pairs WIN 44441-3/WIN 44441-2 and levallorphan/dextrallorphan. Levorphanol (opiate agonist) also produced a dose-dependent decrease in the motility of 5-day embryos while its inactive (+)-isomer, dextrophan, was not effective. Etorphine (opiate agonist) was more than 1000-fold more effective than morphine in inhibiting the motility of 5-day embryos. The effectiveness of several opiate agonists and antagonists on the spontaneous motility of 5-day embryos was similar to their effectiveness in radioligand-binding studies on isolated membrane receptors from either adult mammalian brain or ileum. Levorphanol was more effective than dextrophan and etorphine was substantially more effective than morphine in decreasing the spontaneous motility of 4-day embryos. WIN 44441-3 was more effective than WIN 44441-2 in reversing the inhibition of motility in 4-day embryos caused by morphine. Morphine inhibited spontaneous hind-limb motility in both thoracic spinal and sham-operated 7-day embryos; the inhibition of motility caused by morphine was reversed by WIN 44441-3 in both thoracic spinal and sham-operated 7-day embryos. [Leu5]enkephalin-like immunoreactivity in the lumbar spinal cord was concentrated in the superficial laminae of the dorsal horn and along the midline rostral to the central canal. A lesser concentration of immunoreactive processes occurred in the medial and lateral motor columns where labelled varicosities appeared to contact motoneurons. Opiate receptors appear to be present at least as early as day 5 (and perhaps as early as day 4) in the chick embryo. Opiate receptors appear to be present in the lumbar spinal cord of the chick embryo at least as early as day 7. The structural requirements for ligand binding to opiate receptors in the 5-day chick embryo are similar to the requirements for ligand binding to opiate receptors in the adult.     

Glucocorticoid receptor-mediated teratogenesis and cell proliferation in the limbs and face of the chick embryo

The susceptibility of the face region of the chick embryo to the teratogenic action of intraamniotically injected hydrocortisone contrasts with the resistance of the limbs to its action while at the same time their dysmorphogenesis may be induced by other agents. Since glucocorticoid receptors were shown to mediate face teratogenesis, their development was investigated in freshly dissected limb buds of 3-, 3.5-, and 4-day-old chick embryos in comparison with the face region. The specific binding of 3H-dexamethasone to molybdate-stabilized glucocorticoid receptors was estimated by the dextran-coated charcoal method and complemented by cytologic analysis of mitotic activity in control and hydrocortisone-treated embryos. The glucocorticoid receptors were found in both organ anlagen already on day 3 when their concentration in femtomoles per microgram DNA was significantly higher in the face region. Accordingly, on day 3 intraamniotic hydrocortisone inhibited the mitotic activity in the face without affecting the developing limbs. On days 3.5 and 4 the concentration of glucocorticoid receptors was similar in both organ anlagen. Administration of hydrocortisone on day 4 induced mitotic depression in the face as well as in the limbs. However, the degree of inhibition appeared to be dependent upon the actual mitotic rate. In the face region where the mitotic activity culminated at that time, the inhibition was much deeper and longer-lasting than in the developing limbs characterized by continuous decrease of proliferation rate in controls. These findings are consistent with a view that glucocorticoid receptors are a prerequisite, but not the only factor in receptor-mediated teratogenesis.     

与大鼠胚胎卵黄囊内胚层相关的巨噬细胞分化

为阐明胚胎巨噬细胞（ＭΦ）的分化和卵黄囊内胚层（ＹＳ－Ｅ）之间的关系，用近交ＤＡ大鼠胚胎的卵黄囊内胚层细胞体外培养，观察了ＹＳ－Ｅ细胞的膜抗原表型、贴壁能力及吞噬功能的变化；用免疫组织化学方法观察了ＹＳ－Ｅ样细胞在鼠胚内的发生和分布，证实了从胚胎第１０．５ｄ开始，即卵黄囊和胚体之间的血液循环相通之后，在鼠胚体内的疏松结缔组织、肝原基和脑等处，可以观察到和ＭΦ有相同抗原表型的ＹＳ－Ｅ样细胞。以上结果