Trypsin-induced cell surface changes in ascidian embryonic cells: regulation of differentiation of a tissue-specific protein.

Developing animal quartets removed surgically from 8-cell stage of Ascidia malaca and Phallusia mamillata have been treated for a short time with a low concentration of trypsin. The result is a differentiation of some neural structures, of pigment spots and of a tissue-specific enzyme, brain pigment cell tyrosinase. Tyrosinase activity, as detected histochemically, appeared in the pigment cells some hours before the normal time independently of any inductive interactions with related embryonic tissues. A study with the electron microscope has given evidence of the presence of presumed nervous cells and melanin granules related to them. An autoradiographic study using [³H]uridine has demonstrated presumed RNA synthesis which suggests gene activation. The results are discussed in relation to the possible role of the plasma membrane during embryonic development.     

Design Principles of Pancreatic Islets: Glucose-Dependent Coordination of Hormone Pulses

Pancreatic islets are functional units involved in glucose homeostasis. The multicellular system comprises three main cell types; β and α cells reciprocally decrease and increase blood glucose by producing insulin and glucagon pulses, while the role of δ cells is less clear. Although their spatial organization and the paracrine/autocrine interactions between them have been extensively studied, the functional implications of the design principles are still lacking. In this study, we formulated a mathematical model that integrates the pulsatility of hormone secretion and the interactions and organization of islet cells and examined the effects of different cellular compositions and organizations in mouse and human islets. A common feature of both species was that islet cells produced synchronous hormone pulses under low- and high-glucose conditions, while they produced asynchronous hormone pulses under normal glucose conditions. However, the synchronous coordination of insulin and glucagon pulses at low glucose was more pronounced in human islets that had more α cells. When β cells were selectively removed to mimic diabetic conditions, the anti-synchronicity of insulin and glucagon pulses was deteriorated at high glucose, but it could be partially recovered when the re-aggregation of remaining cells was considered. Finally, the third cell type, δ cells, which introduced additional complexity in the multicellular system, prevented the excessive synchronization of hormone pulses. Our computational study suggests that controllable synchronization is a design principle of pancreatic islets.     

Identification of four classes of cell surface antigens appearing at gastrulation in sea urchin embryos

An antiserum to isolated membranes of gastrula-stage embryos of the sea urchin Lytechinus variegatus was characterized by absorption and cell agglutination specificities. The antiserum was found to recognize four distinct classes of antigens on the embryonic cell surface: (1) an early embryonic class or “maternal” class present from the earliest stages of development, (2) an embryonic class of antigens which appeared on all cells beginning at gastrulation, (3) a class of antigens present on ectoderm cells, and (4) a class of antigens present on endoderm cells. All four classes of antigens were shown indirectly to be synthesized on embryonic mRNA since a hybrid embryo of the cross Tripneustes ♀ × Lytechinus ♂ expressed all four classes of Lytechinus-specific antigens beginning at gastrulation. Each class was Lytechinus specific in that hybrid cells were agglutinated if beyond the beginning of gastrulation, while normal Tripneustes ♀ × Tripneustes ♂ cells were not agglutinated.     

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons¹, which has been difficult with the conventional method of injection and electroporation at E1.5².     

Evidence for the presence of precursor B cells in normal and in hormonally bursectomized chick embryos

Embryonic bone marrow of normal and hormonally bursectomized chicks was examined for the presence of hematopoietic precursor cells capable of migrating to the thymus and bursa and of differentiating into functional T and B cells, respectively. Following transfer of chromosomally marked bone marrow of normal and in ovo bursectomized 14-day-old embryos to 14-day-old γ-irradiated embryonic recipients, donor cells proliferated in the marrow, thymus, and bursa of recipients, and differentiated to PHA- and Con A-responsive T cells as well as to dextran sulfate- and anti-immunoglobulin-responsive B cells. In contrast, when marrow of 2-day-old hatched normal and in ovo-bursectomized donors was transferred to 14-day-old embryonic recipients, donor cells repopulated only the marrow and thymus of recipients which was followed by differentiation to Con A- or PHA-responsive T cells, but the same donor cells failed to proliferate in the bursa and there was no differentiation to functional B cells of donor type. The data were fitted to a model of T- and B-cell differentiation from the stem cell level and they suggest the presence of separate populations of committed precursor T (PT) and precursor B (PB) cells in the marrow of normal and in ovo bursectomized embryos with a bursa-independent selective disappearance of PB cells from the marrow during the late embryonic period.     

Low expression of activin a in mouse and human embryonic teratocarcinoma cells

TGFβ family factors play an important role in regulating the balance of self-renewal and differentiation of mouse and human pluripotent stem and embryonic teratocarcinoma cells. The expression patterns of TGFβ family signaling ligands and functional roles of these signaling pathways differ significantly in mouse and human embryonic stem cells, but the activity and functional role of these factors in mouse and human embryonic teratocarcinoma cells were not sufficiently investigated. Comparative quantitative real-time PCR analysis of the expression of TGFβ family factors in mouse embryonic stem, embryonic germ, and embryonic teratocarcinoma cells showed that embryonic teratocarcinoma cells express lower ActivinA than pluripotent stem cells but similar levels of factors Nodal, Lefty1, TGFβ1, BMP4, and GDF3. In human nullipotent embryonic teratocarcinoma PA-1 cells, most factors of the TGFβ family (ACTIVINA, NODAL, LEFTY1, BMP4, and GDF3) are expressed at lower levels than in human embryonic stem cells. Thus, in mouse and human nullipotent teratocarcinoma cells, the expression of ActivinA is significantly reduced compared with embryonic stem cells. Presumably, these differences may be associated with changes in the functional activity of the respective signaling pathways and deregulation of proliferative and antiproliferative mechanisms in embryonic teratocarcinoma cells.     

Isolation and identification of a specific and reversible inhibitor of starfish development

An inhibitor of development of the starfish Asterina pectinifera was purified to homogeneity from a culture of the bacterium Bacillus subtilis, and was identified as adenosine. Adenosine at 6 μg/ml was shown to halt embryonic development specifically at the 256-cell stage when all the embryonic cells differentiate into epithelial cells. By returning treated embryos to normal seawater, they developed normally to the bipinnaria stage.     

Immunohistochemical Study of Expression of Sohlh1 and Sohlh2 in Normal Adult Human Tissues

The expression pattern of Sohlh1 (spermatogenesis and oogenesis specific basic helix-loop-helix 1) and Sohlh2 in mice has been reported in previous studies. Sohlh1 and Sohlh2 are specifically expressed in spermatogonia, prespermatogonia in male mice and oocytes of primordial and primary follicles in female mice. In this report, we studied the expression pattern of Sohlh1 and Sohlh2 in human adult tissues. Immunohistochemical staining of Sohlh1 and Sohlh2 was performed in 5 samples of normal ovaries and testes, respectively. The results revealed that Sohlh genes are not only expressed in oocytes and spermatogonia, but also in granular cells, theca cells, Sertoli cells and Leydig cells, and in smooth muscles of blood vessel walls. To further investigate the expression of Sohlh genes in other adult human tissues, we collected representative normal adult tissues developed from three embryonic germ layers. Compared with the expression in mice, Sohlhs exhibited a much more extensive expression pattern in human tissues. Sohlhs were detected in testis, ovary and epithelia developed from embryonic endoderm, ectoderm and tissues developed from embryonic mesoderm. Sohlh signals were found in spermatogonia, Sertoli cells and also Leydig cells in testis, while in ovary, the expression was mainly in oocytes of primordial and primary follicles, granular cells and theca cells of secondary follicles. Compared with Sohlh2, the expression of Sohlh1 was stronger and more extensive. Our study explored the expression of Sohlh genes in human tissues and might provide insights for functional studies of Sohlh genes.     

The ferritin content of human red blood cells during the replacement of embryonic cells by fetal cells

Mature human embryonic erythrocytes (hemoglobin is ≥ 90% of the cellular protein) contained at least 20 times as much ferritin as human adult erythrocytes, suggesting the possibility that the embryonic red cells participate in iron storage as they do in other embryonic or larval vertebrates. The ferritin content of mature red cells in the circulation declined when fetal red cells replaced embryonic red cells; the cell replacement was monitored by the disappearance of embryonic ε-chains and the appearance of the fetal globin chains, γ^A and γ^G. A constant ratio of 0.67 was obtained for $\text{γ}{}^{\mathrm{<mtext>G</mtext>}}\text{γ}{}^{\mathrm{<mtext>A</mtext>}}\text{+ γ}{}^{\mathrm{<mtext>G</mtext>}}$ from the first detectable appearance (4 weeks after conception) until 13 weeks, a value which is similar to the value previously obtained at 20 weeks gestation and birth but higher than that observable in adults; thus, both γ^G and γ^A chains are produced in similar amounts throughout gestation. The high levels of ferritin in normal human embryonic erythrocytes emphasize the similarity of erythropoiesis in human embryos and other vertebrates. In addition, the results show that red cell ferritin can be used as a marker for studying the mechanism of induction of embryonic erythropoiesis in cultured cell lines, such as K562 from human chronic myelocytic leukemia, and that ferritin content may also serve as a marker for cellular transformations involving reversions to embryonic erythropoiesis.     

Time disparity sensitive behavior and its neural substrates of a pulse-type gymnotiform electric fish, Brachyhypopomus gauderio

Roles of the time coding electrosensory system in the novelty responses of a pulse-type gymnotiform electric fish, Brachyhypopomus, were examined behaviorally, physiologically, and anatomically. Brachyhypopomus responded with the novelty responses to small changes (100 μs) in time difference between electrosensory stimulus pulses applied to different parts of the body, as long as these pulses were given within a time period of ~500 μs. Physiological recording revealed neurons in the hindbrain and midbrain that fire action potentials time-locked to stimulus pulses with short latency (500–900 μs). These time-locked neurons, along with other types of neurons, were labeled with intracellular and extracellular marker injection techniques. Light and electron microscopy of the labeled materials revealed neural connectivity within the time coding system. Two types of time-locked neurons, the pear-shaped cells and the large cells converge onto the small cells in a hypertrophied structure, the mesencephalic magnocellular nucleus. The small cells receive a calyx synapse from a large cell at their somata and an input from a pear-shaped cell at the tip of their dendrites via synaptic islands. The small cells project to the torus semicircularis. We hypothesized that the time-locked neural signals conveyed by the pear-shaped cells and the large cells are decoded by the small cells for detection of time shifts occurring across body areas.     

Pole cells of Drosophila melanogaster in culture. Normal metabolism, ultrastructure, and functional capabilities.

The metabolism, ultrastructure, and function of mass-isolated pole cells were examined during short-term culture in vitro. In addition to demonstrating that these cells functioned normally in culture, a number of new features of embryonic pole cells were discovered. Cell populations isolated from Renografin density gradients were incubated in medium containing tritiated valine, uridine, or thymidine. Although pole cells incorporated similar amounts of valine into protein as other embryonic cells throughout the first 6 hr in culture, they began to synthesize RNA only after 2 hr in culture. Approximately 30% of the pole cells synthesized DNA in vitro and this synthetic activity occurred largely during the first hour of culture. An ultrastructural analysis of colcemid-treated cells showed that 10% of the pole cells divide shortly after placement in culture. During pole cell culture in vitro, polar granules and nuclear bodies fragment and disperse so that they are eventually not detected in these cells. These changes also occur during pole cell development in vivo. Finally, we have obtained 25 to 33% germ line mosaicism among the fertile adults which were derived from embryos receiving transplantation of isolated pole cells before and after culture in vitro. These results demonstrate that these cells are able to follow their normal developmental program in vitro and are able to give rise to functional germ cells in vivo.     

Mammalian COPII Coat Component SEC24C Is Required for Embryonic Development in Mice

COPII-coated vesicles mediate the transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi. SEC24 is the COPII component primarily responsible for recruitment of protein cargoes into nascent vesicles. There are four Sec24 paralogs in mammals, with mice deficient in SEC24A, -B, and -D exhibiting a wide range of phenotypes. We now report the characterization of mice with deficiency in the fourth Sec24 paralog, SEC24C. Although mice haploinsufficient for Sec24c exhibit no apparent abnormalities, homozygous deficiency results in embryonic lethality at approximately embryonic day 7. Tissue-specific deletion of Sec24c in hepatocytes, pancreatic cells, smooth muscle cells, and intestinal epithelial cells results in phenotypically normal mice. Thus, SEC24C is required in early mammalian development but is dispensable in a number of tissues, likely as a result of compensation by other Sec24 paralogs. The embryonic lethality resulting from loss of SEC24C occurs considerably later than the lethality previously observed in SEC24D deficiency; it is clearly distinct from the restricted neural tube phenotype of Sec24b null embryos and the mild hypocholesterolemic phenotype of adult Sec24a null mice. Taken together, these results demonstrate that the four Sec24 paralogs have developed unique functions over the course of vertebrate evolution.     

Temperature sensitivity of muscle and neuron differentiation in embryonic cell cultures from the Drosophila mutant, shibire.

The sex-linked temperature-sensitive mutation, shibire^ts1, which causes, at the restrictive temperature, adult paralysis and pleiotropic morphological defects in embryonic, larval, and pupal development, has been shown to exhibit temperature-sensitive inhibition of differentiation in embryonic cultures in vitro. When shi cultures were incubated at 30°C for 24 hr, both muscle and neuron differentiation were inhibited more than 90% compared to control shi cultures incubated at 20°C. Heat shift experiments showed that the temperature-sensitive periods for neuron and muscle differentiation occurred at 11 to 18 and 14 to 16 hr, respectively, where zero time was the initiation of gastrulation in donor embryos. Short heat pulses (4 and 8 hr) which extended into the temperature-sensitive period resulted in moderate inhibition of differentiation; greater inhibition occurred as the duration of the pulses increased. In contrast, heating wild-type Oregon-R cultures at 30°C for 24 hr did not inhibit muscle cell differentiation and inhibited neuron differentiation relatively little. The temperature-sensitive period in shibire for muscle differentiation occurred well after myoblast division, during the period of myocyte elongation, aggregation, and fusion, whereas that for neuron differentiation took place during a period of enzyme synthesis (acetylcholinesterase and choline acetyltransferase) and axon elongation. Thus, the shi temperature-sensitive gene product affects at least two different cell types, in vitro, at different times during differentiation.     

Egr2::Cre Mediated Conditional Ablation of Dicer Disrupts Histogenesis of Mammalian Central Auditory Nuclei

Histogenesis of the auditory system requires extensive molecular orchestration. Recently, Dicer1, an essential gene for generation of microRNAs, and miR-96 were shown to be important for development of the peripheral auditory system. Here, we investigated their role for the formation of the auditory brainstem. Egr2::Cre-mediated early embryonic ablation of Dicer1 caused severe disruption of auditory brainstem structures. In adult animals, the volume of the cochlear nucleus complex (CNC) was reduced by 73.5%. This decrease is in part attributed to the lack of the microneuronal shell. In contrast, fusiform cells, which similar to the granular cells of the microneural shell are derived from Egr2 positive cells, were still present. The volume reduction of the CNC was already present at birth (67.2% decrease). The superior olivary complex was also drastically affected in these mice. Nissl staining as well as Vglut1 and Calbindin 1 immunolabeling revealed that principal SOC nuclei such as the medial nucleus of the trapezoid body and the lateral superior olive were absent. Only choline acetyltransferase positive neurons of the olivocochlear bundle were observed as a densely packed cell group in the ventrolateral area of the SOC. Mid-embryonic ablation of Dicer1 in the ventral cochlear nucleus by Atoh7::Cre-mediated recombination resulted in normal formation of the cochlear nucleus complex, indicating an early embryonic requirement of Dicer1. Quantitative RT-PCR analysis of miR-96 demonstrated low expression in the embryonic brainstem and up-regulation thereafter, suggesting that other microRNAs are required for proper histogenesis of the auditory brainstem. Together our data identify a critical role of Dicer activity during embryonic development of the auditory brainstem.     

misshapen encodes a protein kinase involved in cell shape control in Drosophila

We have identified a novel protein kinase encoded by the misshapen gene, which is required for the normal shape and orientation of Drosophila photoreceptor cells. misshapen is also expressed in the embryonic mesoderm, pole plasm and other sites of cell shape change or movement. We propose that msn may act in a signal transduction pathway leading to cytoskeletal re-arrangements.     

Incorporation of teratocarcinoma stem cells into blastocysts by aggregation with cleavage-stage embryos

Embryonal carcinoma cells from the in vitro teratocarcinoma cell line PSA-1 were combined with normal, eight-cell stage, embryonic cells of the strain $\text{SWR}\text{J}$. The aggregates compacted and formed apparently normal blastocysts within 48 hr. Glucose phosphate isomerase (GPI) assays of the blastocysts revealed the presence of both PSA-1 and $\text{SWR}\text{J}$ GPI isozymes. Inner cell masses isolated from the blastocysts by immunosurgery expressed predominantly the PSA-1 GPI type.