The development of mouse spinal cord in tissue culture

Summary Whole mouse embryos were grown in vitro from Theiler stage 12 (1 to 7 somites) to Theiler stages 15 and 16 (25 to 35 somites). This procedure gives experimental access to precisely staged embryos during the early period of neurogenesis. To follow the further development of neurons in vitro, fragments of spinal primordia were set up from these cultured embryos. In such cultures, the proliferation of precursor cells, the formation of postmitotic cells and, finally, the cytodifferentiation of neurons were observed. A preliminary account of this work was given at the Tissue Culture Association Meeting in 1977, and the Canadian Federation of Biological Societies Meeting in 1977 (1,2). This work was supported by Grant MT 4235 from the Medical Research Council of Canada.     

The development of mouse spinal cord in tissue culture

Summary Neural tubes of mouse embryos at Theiler Stages 14, 15, and 16 were grown in cultures for 21 d with 0.5 μCi/ml tritiated thymidine or cold growth medium. It was found that 50 to 60% of the neurons formed in the outgrowth zone were labeled, indicating that they formed from precursor cells that proliferated in the cultures. The unlabeled neurons must have formed from cells that were already postmitotic when the cultures were started. By comparing the total number of neurons per neuromere formed in vivo and in vitro, it seems that the postmitotic precursor cells survive better in cultures and only a small percentage of proliferative precursor cells in cultures enter the postmitotic stage and form neurons. This work was supported by Grant MT4235 from the Medical Research Council of Canada.     

The allocation of cells in the presomitic mesoderm during somite segmentation in the mouse embryo

Orthotopic grafts of wheat germ agglutinin-colloidal gold conjugate (WGA-gold) labelled cells were used to demonstrate differences in the segmental fate of cells in the presomitic mesoderm of the early-somite-stage mouse embryos developing in vitro. Labelled cells in the anterior region of the presomitic mesoderm colonized the first three somites formed after grafting, while those grafted to the middle region of this tissue were found mostly in the 4th-7th newly formed somites. Labelled cells grafted to the posterior region were incorporated into somites whose somitomeres were not yet present in the presomitic mesoderm at the time of grafting. There was therefore an apparent posterior displacement of the grafted cells in the presomitic mesoderm. Colonization of somites by WGA-gold labelled cells was usually limited to two to three consecutive somites in the chimaera. The distribution of cells derived from a single graft to two somites was most likely due to the segregation of the labelled population when cells were allocated to adjacent meristic units during somite formation. Further spreading of the labelled cells to several somites in some cases was probably the result of a more extensive mixing of mesodermal cells among the somitomeres prior to somite segmentation.     

The rostral level of origin of sympathetic neurons in the chick embryo, studied in tissue culture.

Groups of three consecutive somites from the first to the eleventh somite from chick embryos of stages 17-18 were grown in tissue culture for seven days. Sympathetic neurons, identified both by phase contrast microscopy and FIF histochemistry, occurred only in cultures which included the sixth, or more caudal, somites. If it is assumed that sympathetic precursor cells (neural crest cells) have not undergone a caudal shift prior to stages 17-18, and taking into account the loss of one or two rostral somites, then the anterior sympathetic ganglia are derived from neural crest caudal to the sixth or seventh somite. Thus, the vagal zone (level with somites 1-7) contributes little to the sympathetic nervous system.     

Desmin expression during early mouse tongue morphogenesis.

Occipital somites provide progenitor cells for craniofacial muscle development including the tongue musculature. Serum-derived factors are assumed to be pre-requisite for myogenesis in vitro. To test these assertions, we designed experiments to determine whether early mouse tongue development in general, and desmin localization in particular, were expressed during the development of embryonic mouse first branchial arch explants cultured in serumless, chemically-defined medium. Immunohistochemical techniques determined the chronology and positions of desmin expression during early craniofacial development. Occipital somites expressed desmin at E9 (9 days +/- 2 h post-fertilization, 18-20 somites). A discrete cell migration pathway initiating in the somites and terminating in the lateral lingual processes of the tongue primordium was defined based upon desmin expression patterns in E9-E11 embryos and computer-assisted three dimensional reconstructions. The in vitro model system was permissive for tongue morphogenesis, allowing development and fusion of the lateral lingual processes with the tuberculum impar. During culture myoblasts were not observed to fuse into myotubes with sarcomeric assembly, even though explant myoblasts produced muscle-specific protein. E10 explants cultured for 9 days demonstrated a five-fold increase in cell number that expressed desmin (P less than 0.05) when compared to the E10 starting material. We interpret these results to indicate that the tongue myogenic cell lineage was determined between E8 and E11, and that this resident population expanded within explants cultured in serumless medium by several explanations: (i) cells other than progenitor myoblasts (e.g., satellite cells) were induced to become myoblasts, and/or (ii) progenitor myoblasts within the original explants expanded by cell division in the absence of serum factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differentiation capabilities of mouse optic stalk in isolation of its immediate in vivo environment

During normal in vivo development, the optic stalk gives rise only to macroglial cells. When we cultured optic stalks isolated from their immediate in situ environment, we found that optic stalks obtained from embryos at Theiler stages 16 to 19 gave rise to both neurons and glial precursor cells, whereas optic stalks obtained from embryos at stages 20 to 23 gave rise to only glial precursor cells. Between stages 19 and 20 (a period of 12 hr of development) the optic stalk changes from a pseudostratified to a simple epithelium, and concomitant with these changes is the growth of the neural retinal axons along the optic stalk. An attractive hypothesis to explain these observations is that the environmental cues that restrict the differentiation capability of the optic stalk ventricular cell population in vivo emanate from the retinal axons. Whether this is due to a restriction in the differentiation capability of a pleuripotential ventricular cell or to a selective cell death of a subpopulation of ventricular cells already committed to the neuronal lineage of differentiation is not yet resolved.     

New in vitro procedures for experimental studies on the development of 11-day mouse embryo forelimb buds

A new method has been developed for culturing 11-day mouse forelimb buds in vitro. In cultures performed with conventional procedures, skeletal pieces frequently appeared distorted and reduced in size. Moreover, forelimb buds explanted from embryos younger than a stage corresponding to 50 pairs of somites developed narrow hand plates devoid of radiated autopods. By contrast, in the new procedure using media supplemented with fetal calf serum and growth factors and enhancing distal feeding with carrier implants of catgut, enlarged pads were obtained that exhibited at least 4 digital rays in buds explanted from embryos with 40-44 pairs of somites. Compared with conventional procedures, the mean value of DNA content per limb bud was twice as great with use of our improved method. The ability of limb bud cells to proliferate and differentiate when cultured either in classical or in modified conditions, and the importance of the technical procedures, are discussed in the new prospect of in vitro developmental studies.     

Migration of zebrafish spinal motor nerves into the periphery requires multiple myotome-derived cues

In vertebrate embryos, spinal motor neurons project through segmentally reiterated nerves into the somites. Here, we report that zebrafish secondary motor neurons, which are similar to motor neurons in birds and mammals, depend on myotomal cues to navigate into the periphery. We show that the absence of myotomal adaxial cells in you-too/gli2 embryos severely impairs secondary motor axonal pathfinding, including their ability to project into the somites. Moreover, in diwanka mutant embryos, in which adaxial cells are present but fail to produce cues essential for primary motor growth cones to pioneer into the somites, secondary motor axons display similar pathfinding defects. The similarities between the axonal defects in you-too/gli2 and diwanka mutant embryos strongly suggest that pathfinding of secondary motor axons depends on myotome-derived cues, and that the diwanka gene is a likely candidate to produce or encode such a cue. Our experiments also demonstrate that diwanka plays a central role in the migration of primary and secondary motor neurons, suggesting that both neural populations share mechanisms underlying axonal pathfinding. In summary, we provide compelling evidence that myotomal cells produce multiple signals to initiate and control the migration of spinal nerve axons into the somites.     

Effects of fibroblast growth factor 2 and insulin-like growth factor II on the development of parthenogenetic mouse embryos in vitro

Most parthenogenetic embryos (PEs) in mammals die shortly after implantation, and this failure to develop is associated with genomic imprinting. We have examined the influence of human recombinant basic fibroblast growth factor 2 (FGF-2) and human recombinant insulin-like growth factor II (ICF-II) on the development of (CBA x C57BL/6)F1 parthenogenetic mouse embryos. Embryos were treated in vitro at the morula stage with different doses of FGF-2 and, after their development to blastocysts, transferred to pseudopregnant recipients. The optimal doses of FGF-2 did not affect the number of forming and implanting blastocysts, but increased, from 20 to 42%, the number of embryos developing to somite stages. PEs (18-21 somites) treated with an optimal dose of FGF-2 were explanted for further development in culture by treatment with the second growth factor, IGF-II. Eighty-three percent of those embryos cultured with IGF-II (2.5 microg/ml) developed to 35 or more somites, as compared with 36% of embryos cultured without any growth factors (P < 0.01). Also, a significantly higher proportion of PEs developed to 40-50 somites in this case. These results show that the in vitro treatment of PEs with FGF-2 at the morula stage increases the number of somite embryos, and the second treatment of somite PEs with IGF-II in culture medium prolongs their development significantly.     

Effects of growth factors FGF2 and IGF2 on the development of parthenogenetic mouse embryos in utero and in vitro

We studied the effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 2 (IGF2) on the development of parthenogenetic mouse embryos (CBA x C57BK/6)F1. The parthenogenetic embryos were treated in vitro during the preimplantation period and, at the blastocyst stage, transplanted into the uterus of pseudopregnant females. The addition of FGF2 at an optimal dose (2.5 ng/ml) to the culture medium increased twofold the number of embryos developed in utero to the somite stages as compared to the control: 18 and 43%, respectively. The parthenogenetic embryos (18-21 somites), treated and nontreated with FGF2 during the preimplantation period, were explanted for further development in vitro and treated with IGF2 at 2.5 micrograms/ml. As a result, many more parthenogenetic embryos (> 87%) of both groups developed in vitro to the stage of 30 or more somites as compared to the control (59%). The treatment of the parthenogenetic embryos with FGF2 alone at the preimplantation stages did not improve their development in vitro at the postimplantation stages. The results we obtained suggest that the treatment of parthenogenetic embryos in vitro with FGF2 during the preimplantation period increased twofold the number of somite embryos in utero, while their subsequent treatment in vitro with IGF2 leads to a significant prolongation of their development, as compared to the control.     

Teratogenic effects of valproic acid and diphenylhydantoin on mouse embryos in culture

Teratogenic effects of the anticonvulsant drugs valproic acid (VPA) and diphenylhydantoin (DPH) on the development of mouse embryos during early organogenesis were studied using the whole embryo culture technique. Embryos with one to seven somites were exposed in vitro to 50-375 micrograms/ml VPA or 15-135 micrograms/ml DPH for up to 42 hours and compared to control embryos cultured in 80% rat serum without either drug. For both VPA- and DPH-treated embryos, a dose-dependent increase in the frequency of abnormal embryos and a decrease in viability were found. VPA and DPH produced a similar pattern of defects. Drug-induced anomalies included open neural tubes in the cranial regions, abnormal body curvature, craniofacial deformities, and yolk sac defects. Ultrastructural changes were noted in the neuroepithelium of exencephalic VPA-treated embryos. Growth and development were retarded in embryos exposed to greater than 35 micrograms/ml DPH or greater than 50 micrograms/ml VPA as indicated by the decrease in protein and DNA content and the reduction in somite number, crown-rump length, and yolk sac diameter. On a molar basis DPH was potentially more teratogenic than VPA, which correlates with the higher lipid solubility of DPH. With VPA, susceptibility to the drug depended on the developmental stage; e.g., at 150 micrograms/ml VPA the frequency of malformations was 70% in embryos with one to four somites as compared to 35% in embryos with five to seven somites.     

The development of diploid parthenogenetic mouse embryos of the inbred C57BL and CBA strains]

We studied preimplantation development in vitro and postimplantation development in vivo of diploid parthenogenetic mouse embryos of C57BL/6 and CBA strains, as well as of (CBA x C57BL/6)F1 hybrids. Development to blastocyst stage of diploid eggs obtained from C57BL/6, CBA, and hybrid mice was observed in 90, 15, and 73% cases, respectively. After implantation, C57BL/6 embryos did not develop to somite stages, while CBA and hybrid embryos reached various stages of somite formation in 45 and 30% cases, respectively. Cultivation of embryos beginning from one-cell stage in the medium containing 2% newborn calf serum increased the yield of blastocysts from 15 to 59% in CBA embryos and from 73 to 90% in hybrids; However, such effect was not observed with C57BL/6 embryos. The latest stages of development observed in CBA and hybrid diploid parthenogenetic embryos were 33-35 somites and 25-30 somites, respectively. Imprinting patterns in chromosomes of CBA and C57BL/6 gametes are discussed.     

The distribution of tenascin coincides with pathways of neural crest cell migration

The distribution of the extracellular matrix (ECM) glycoprotein, tenascin, has been compared with that of fibronectin in neural crest migration pathways of Xenopus laevis, quail and rat embryos. In all species studied, the distribution of tenascin, examined by immunohistochemistry, was more closely correlated with pathways of migration than that of fibronectin, which is known to be important for neural crest migration. In Xenopus laevis embryos, anti-tenascin stained the dorsal fin matrix and ECM along the ventral route of migration, but not the ECM found laterally between the ectoderma and somites where neural crest cells do not migrate. In quail embryos, the appearance of tenascin in neural crest pathways was well correlated with the anterior-to-posterior wave of migration. The distribution of tenascin within somites was compared with that of the neural crest marker, HNK-1, in quail embryos. In the dorsal halves of quail somites which contained migrating neural crest cells, the predominant tenascin staining was in the anterior halves of the somites, codistributed with the migrating cells. In rat embryos, tenascin was detectable in the somites only in the anterior halves. Tenascin was not detectable in the matrix of cultured quail neural crest cells, but was in the matrix surrounding somite and notochord cells in vitro. Neural crest cells cultured on a substratum of tenascin did not spread and were rounded. We propose that tenascin is an important factor controlling neural crest morphogenesis, perhaps by modifying the interaction of neural crest cells with fibronectin.     

Quantitative distribution of chick neural crest cells during gangliogenesis

Summary We have quantitated the distribution of chick neural crest cells after they have completed early migration and are aggregating into ganglia. Variables tested for an influence on the distribution of cells include stage, level of somites, position in each of the primary body axes, and individual embryo. The 11th–15th cervical somites of embryos at stages 30, 35, and 40 somites (s) incubated for 2.5, 3.0, and 3.5 days were labeled with antibody to HNK-1 to detect neural crest cells, and doubly labeled with antibody to HNK-1 and to the 150 kD neurofilament subunit to detect neural crest-derived neurons. Significantly more neural crest cells appear at older stages, but cells are uniformly distributed among the 11th–15th somites at any given stage. Significant differences in the total number of neural crest cells among three embryos sampled at the same stage indicate that the number of cells is independent of the staging series used. As early as the 35 s stage about one-third of the neural crest cells throughout the somite exhibit NF staining. At the 40 s stage, doubly labeled NF cells, as well as HNK-1 labeled cells, aggregate in a circumscribed portion of the mediolateral axis to form presumptive sensory ganglia in the dorsal region of the somites. Also at 40 s a wave of cell aggregation into sympathetic ganglia proceeds anteroposteriorly along the ventral border of the somitic mesenchyme. The results show a sequence of phenotypic expression beginning with neurofilament antigen, then ganglionic aggregation, and finally, in the case of sympathetic neurons, catecholamine transmitter.     

Growth of the clavicle and development of clavicular secondary cartilage in the embryonic mouse

Whether secondary cartilage develops in the mammalian clavicle has been a matter of controversy. This study documents, in the embryonic mouse: (a) the onset of clavicular osteogenesis at 14 days of gestation (Theiler stage 22); (b) the appearance of secondary cartilage at 16 days of gestation (Theiler stage 24) and its persistence as a prominent cartilage until 18 days of gestation; (c) that the relative growth rate of the clavicle is much higher (0.097 mg/g body weight/day) between 16 and 17 days of gestation than at later ages (mean of 0.005 mg/g/day between 17 days of gestation and 4 days postnatally), and (d) that secondary cartilage failed to form in clavicles from 15-day-old embryos maintained in vitro. We conclude that secondary cartilage is a feature of the developing mouse clavicle, that it arises when the relative growth rate of the clavicle is highest, and that the most likely stimulus for differentiation of this cartilage is mechanical, muscle-based and associated with rapid relative clavicular growth.     

Difference in the expression of asymmetric acetylcholinesterase molecular forms during myogenesis in early avian dermomyotomes and limb buds in ovo and in vitro

The A12 (asymmetric) form of acetylcholinesterase (AChE) is generally considered to be synthesized in leg muscle tissues by myotubes under neural influence, but not by myoblasts. We have examined the expression of the different molecular forms of AChE in explants of developing limb buds and dermomyotomes (the myogenic part of the somites) obtained from 3-day-old chick and quail embryos, either directly after removal or during in vitro culture. We describe a muscular differentiation of both territories in vitro, leading to the formation of myotubes which are morphologically similar to the class of early muscle cells described by Bonner and Hauschka (1974). In vivo the A12 form is present in quail dermomyotomes which are almost entirely composed of mononucleated poorly differentiated cells; in contrast, it is absent from similar cells in chick dermomyotomes and from limb buds in both species. This shows that in the case of quail embryos the appearance of the A12 form precedes the fusion of myoblasts into myotubes. In both species, dermomyotome explants express asymmetric and globular forms of the enzyme during muscular differentiation in vitro, whereas limb buds synthesize only globular forms. After surgical removal of neural tube and/or neural crest at 2 days in ovo, the biosynthesis of the A forms in quail dermomyotomes is not suppressed and is consequently not dependent upon prior connection of the dermomyotomes to central neurons or upon the presence of autonomic precursors. Since limb bud muscle cells derive from somites our results raise questions concerning the differentiation of migrating somitic cells in this territory where a neural influence appears necessary to induce the biosynthesis of asymmetric AChE forms.     

A traction-based mechanism for somitogenesis in the chick

Summary This paper suggests that chick somites form because presomitic cells exert tractional forces on one another. These forces derive from the increase in cell adhesion and density that occurs as N-CAM and N-cadherin are laid down by the motile cells of the presomitic mesoderm, well before the somites form. Harris et al. (1984) have shown that adhesive and motile cells in an appropriate environment in vitro can spontaneously form aggregates under the influence of the tractional forces that they exert. Presomitic mesodermal cells may behave similarly: as CAM production increases local adhesivity, the tractional forces between the cells should become sufficiently strong for groups of cells to segment off the mesenchyme as somites. The successive expression of CAMs down the presomitic mesoderm will thus lead to the formation of an anterior-posterior sequence of somites. This mechanism can explain several aspects of somitogenesis that models generating a repetitive pre-pattern through gating cohorts of cells find hard to explain: first, mesodermal segregation occurs among highly adherent cells; second, that multiple rows of somites can form in embryos cultured on highly adherent substrata; third, that stirred mesoderm will still form normal somites; and, fourth, how somite size can be altered in heat-shocked embryos and elsewhere. Suggestions are given as to how the mechanism may be tested and where else in the embryo it could apply.     

In vitro development of the postimplantation embryos of laboratory rodents in human blood serum

Rat and mouse embryos at the stage of the first somites formation (1-5 pairs) cultivated in human blood serum demonstrated its embryolethal and teratogenic effect. The embryos taken at a later stage (11-18 pairs of somites) developed normally and could be compared with the development of the rat embryos in homologous blood serum. There was no difference in the development when the embryos were cultivated either in male or female blood serum. The stage of embryogenesis 11-18 pairs of somites is recommended for in vitro experimental revealing in the human serum of embryotoxic factors induced by certain external influences.