A kinetic study of the effects of δ-aminolevulinic acid upon the synthesis of embryonic and fetal hemoglobins in the blood islands of the developing chick blastodisc

1970 ◽  
Vol 48 (4) ◽  
pp. 400-406 ◽  
Author(s):  
S. D. Wainwright ◽  
Lillian K. Wainwright
Keyword(s):  
Aminolevulinic Acid ◽  
Fetal Hemoglobin ◽  
Primitive Streak ◽  
Stages Of Development ◽  
In Ovo ◽  
Rapid Synthesis ◽  
Somite Stage ◽  
Stage Of Development ◽  
Solid Media

Chick blastodiscs contained and synthesized a functional (embryonic) hemoglobin as early as the stage of the definitive primitive streak. The rate of hemoglobin synthesis in ovo rose dramatically at about the stage of the 3-somite embryo.Embryos explanted onto solid media at the 3-somite stage of development continued to synthesize hemoglobin in vitro for at least 12 h. The rate of synthesis rose markedly at the 6-somite stage of development, coincident with the onset of rapid synthesis of fetal hemoglobin. A supplement of exogenous aminolevulinic acid markedly stimulated the synthesis of hemoglobin before the 6-somite stage of development, but had little or no effect thereafter. These responses were obtained on both minimal and rich media. Levels of hemoglobin formed were characteristic of the final stages of development attained, regardless of initial stage of development, medium used, or time of incubation required to attain the final level of development.A new regulatory process resulting in elimination of a requirement for continuous presence of egg homogenate for maximal rates of hemoglobin was revealed. This took place between the 6- and 8-somite stages of development at the time of onset of rapid synthesis of fetal hemoglobin.

REGULATION OF THE INITIATION OF HEMOGLOBIN SYNTHESIS IN THE BLOOD ISLAND CELLS OF CHICK EMBRYOS: I. QUALITATIVE STUDIES OF THE EFFECTS OF ACTINOMYCIN AND δ-AMINOLEVULINIC ACID

1966 ◽  
Vol 44 (11) ◽  
pp. 1543-1560 ◽  
Author(s):  
S. D. Wainwright ◽  
Lillian K. Wainwright
Keyword(s):  
Aminolevulinic Acid ◽  
Basal Medium ◽  
Primitive Streak ◽  
Chick Embryos ◽  
Messenger Rnas ◽  
Stages Of Development ◽  
Linear Rate ◽  
Hemoglobin Synthesis ◽  
Naked Eye ◽  
Somite Stage

Intact and de-embryonated blastodiscs of chick embryos from all stages of development between the definitive primitive streak and the 10-somite stage were incubated on simple solid synthetic media. On the basal medium, blastodiscs at all initial stages of development synthesized hemoglobin readily visible to the naked eye within 24 hours, incorporated leucine into protein at an approximately linear rate for 24 hours, and incorporated uridine into RNA at a roughly linear rate for at least 6 hours after a short lag.Blastodiscs taken before the 1-somite stage failed to synthesize any detectable hemoglobin on medium containing 2 μg/ml of actinomycin, whereas those token at later stages synthesized hemoglobin visible to the naked eye. This concentration of actinomycin totally inhibited the incorporation of uridine into high molecular weight RNA within 2–3 hours, but the incorporation of leucine into protein was not inhibited for 6–8 hours. The residual incorporation of uridine was entirely into the soluble RNA fraction.At 10 μg/ml, actinomycin markedly inhibited the synthesis of hemoglobin by blastodiscs taken at stages earlier than the 6-somite embryo, but did not markedly affect hemoglobin synthesis by the more advanced blastodiscs. This concentration of actinomycin caused only slightly greater inhibition of the incorporation of uridine into acid-precipitable material than the smaller concentration for all blastodiscs, and was not markedly more inhibitory for the incorporation of leucine into protein.The presence of δ-aminolevulinic acid overcame the inhibitions of synthesis of hemoglobin by actinomycin but did not prevent the inhibitions of incorporation of uridine into RNA and of leucine into protein.Regulation of the onset of rapid hemoglobin synthesis appears to be at the translation level, probably through the supply of δ-aminolevulinic acid. The latter is probably regulated through synthesis of RNAs formed at the head-fold stage. Messenger RNAs for globin synthesis are present at the stage of the definitive primitive streak.

Special Problems of Experimenting in ovo on the Early Chick Embryo, and a Solution

Development ◽  
1960 ◽  
Vol 8 (4) ◽  
pp. 369-375
Author(s):  
P. H. S. Silver
Keyword(s):  
Chick Embryo ◽  
Short Term ◽  
Stages Of Development ◽  
In Ovo ◽  
Technical Problems ◽  
Early Chick Embryo ◽  
In Vitro Methods ◽  
Early Stages

It seems to be generally accepted that experimenting in ovo on the chick during the early stages of development (up to about 48 hours) is fraught with the greatest difficulty. After about this time no serious technical problems arise and a high proportion of successful results can be expected. It is natural to ask why there should be this change-over from extreme difficulty to reasonable simplicity. New (1955) attributed to this ‘inaccessibility of the chick embryo in the egg’ the invention of his own and many other in vitro methods during the last 30 years. There is no doubt that, when short-term experiments only are required, in vitro methods will probably always be preferred. But all in vitro methods suffer from the disadvantage that the embryo cannot be expected to survive for more than 48 hours or so after explantation.

A spinal cord fate map in the avian embryo: while regressing, Hensen's node lays down the notochord and floor plate thus joining the spinal cord lateral walls

Development ◽  
1996 ◽  
Vol 122 (9) ◽  
pp. 2599-2610 ◽  
Author(s):  
M. Catala ◽  
M.A. Teillet ◽  
E.M. De Robertis ◽  
M.L. Le Douarin
Keyword(s):  
Spinal Cord ◽  
Primitive Streak ◽  
Floor Plate ◽  
Dorsal Side ◽  
Avian Embryo ◽  
Tail Bud ◽  
Fate Map ◽  
In Ovo ◽  
Somite Stage ◽  
Lateral Walls

The spinal cord of thoracic, lumbar and caudal levels is derived from a region designated as the sinus rhomboidalis in the 6-somite-stage embryo. Using quail/chick grafts performed in ovo, we show the following. (1) The floor plate and notochord derive from a common population of cells, located in Hensen's node, which is equivalent to the chordoneural hinge (CNH) as it was defined at the tail bud stage. (2) The lateral walls and the roof of the neural tube originate caudally and laterally to Hensen's node, during the regression of which the basal plate anlage is bisected by floor plate tissue. (3) Primary and secondary neurulations involve similar morphogenetic movements but, in contrast to primary neurulation, extensive bilateral cell mixing is observed on the dorsal side of the region of secondary neurulation. (4) The posterior midline of the sinus rhomboidalis gives rise to somitic mesoderm and not to spinal cord. Moreover, mesodermal progenitors are spatially arranged along the rest of the primitive streak, more caudal cells giving rise to more lateral embryonic structures. Together with the results reported in our study of tail bud development (Catala, M., Teillet, M.-A. and Le Douarin, N.M. (1995). Mech. Dev. 51, 51–65), these results show that the mechanisms that preside at axial elongation from the 6-somite stage onwards are fundamentally similar during the complete process of neurulation.

scholarly journals Fetal hemoglobin and potassium in isolated transferrin receptor- positive dense sickle reticulocytes

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 2013-2020 ◽  
Author(s):  
RS Franco ◽  
R Barker-Gear ◽  
MA Miller ◽  
SM Williams ◽  
CH Joiner ◽  
...  
Keyword(s):  
Cell Formation ◽  
Fetal Hemoglobin ◽  
Density Fractions ◽  
Sickle Cells ◽  
Stage Of Development ◽  
Short Lifespan ◽  
F Cells ◽  
Low Levels

A subset of sickle cells have an increased density at the reticulocyte stage of development, indicating that they are either abnormally dense upon release from the bone marrow or become dense quickly in the circulation. These cells are of interest because they most likely have severely disrupted cation regulation and a short lifespan. Based on the distribution of fetal hemoglobin (HbF) in the density fractions of sickle red blood cells (RBCs) and in vitro studies of cellular K+ loss, it seems likely that HbF content is an important in vivo determinant of dense cell formation. In this study, we tested the hypothesis that young, dense cells have low HbF content. Sickle RBCs were first separated into light and dense fractions. Reticulocytes were isolated from unfractionated cells and from each density fraction with an immunomagnetic technique directed against transferrin receptors (TfR) and assayed for the percentage of HbF and K+/Hb ratio. TfR+ reticulocytes isolated from unfractionated cells had a much lower HbF content when compared with all the unfractionated RBCs. This is most likely caused by enrichment of F cells because of a longer circulation life span. Heavy TfR+ reticulocytes had a K+/Hb ratio similar to that measured in the entire dense population and contained very low levels of HbF, averaging 2.5% of the level in all RBCs, 11.7% of the level in all TfR+ reticulocytes, and 4.0% of the level in all dense RBCs. These findings suggest that TfR+ dense cells derive predominantly from non-F cells. Furthermore, the amount of HbF in the circulating dense cells suggests that many of these cells do not derive from the TfR+ dense cells.

Direct exposure of mouse embryonic limb-buds to 5-bromodeoxyuridine in vitro and its effect on chondrogenesis: increasing resistance to the analog at successive stages of development

Development ◽  
1976 ◽  
Vol 36 (3) ◽  
pp. 639-652
Author(s):  
Narsingh D. Agnish ◽  
Devendra M. Kochhar
Keyword(s):  
Developmental Stage ◽  
Prolonged Exposure ◽  
Inhibitory Effect ◽  
Limb Buds ◽  
Embryonic Mouse ◽  
Continuous Growth ◽  
Somite Stage ◽  
Stage Of Development ◽  
Short Period

The inhibitory effect of 5-bromodeoxyuridine (BudR) — an analog of thymidine — on embryonic mouse limb-buds was studied in vitro employing an organ-culture system. The effect was found to be dose-related and also depended on the developmental stage of the donor embryos. Limbs at an early stage of development (early llth-day embryos, somite stage 26–29) were extremely sensitive to the analog. Treatment with low levels (2–4µml) and for a relatively short period of time in culture (2–3 days) completely and irreversibly suppressed chondrogenesis in these explants. Limbs from older embryos (somite stage 40 and up) were found to be much less sensitive to the inhibitory effect of the drug; a prolonged exposure to a much higher dose (100–150 µml) resulted in an incomplete suppression of chondrogenesis. Only a 20% inhibition was observed in the cultures of limbs from mid-13th-day mouse embryos. After continuous growth in vitro, the limbs became progressively resistant to the analog and towards the end of the culture period had become refractory to the drug. The time of complete insensitivity appeared earlier in the cultures of the limbs taken from older embryos than in the explants of younger limbs. These studies show that as limbs continue to differentiate in vivo or in vitro, they become increasingly resistant to the inhibitory effect of BudR in at least as far as the effect on the process of chondrogenesis is concerned. It is suggested that the relative sensitivity or insensitivity to the inhibitory effect of BudR may prove to be a useful parameter in evaluating the developmental stage of an organ.

Influence of diandric and digynic triploid genotypes on early mouse embryogenesis

Development ◽  
1989 ◽  
Vol 105 (1) ◽  
pp. 137-145
Author(s):  
M.H. Kaufman ◽  
K.K. Lee ◽  
S. Speirs
Keyword(s):  
Embryonic Development ◽  
Primitive Streak ◽  
Stages Of Development ◽  
Mouse Embryogenesis ◽  
Somite Stage ◽  
Wide Range ◽  
Extraembryonic Membranes ◽  
Early Mouse ◽  
Hydatidiform Moles

Standard micromanipulatory techniques were used to produce tripronucleate diandric and digynic triploid mouse conceptuses. When these were transferred to suitable recipients, most implanted. A wide range of embryonic stages from the primitive streak to the 15- to 25-somite stage were isolated in both triploid series in otherwise identical recipients. In the diandric triploid series, all of the embryos recovered appeared to be morphologically normal, but considerably smaller than fertilized embryos analysed at similar stages of development. This contrasts with the digynic triploid conceptuses which, though also ranging from the primitive-streak stage to about the 10- to 15-somite stage at the time of their isolation, generally showed poorer embryonic development than the diandric triploids, and were invariably morphologically abnormal. Unlike the situation observed in man, where the placentas of diandric triploid conceptuses commonly display widespread trophoblastic hyperplasia and form the characteristic ‘partial’ or ‘incomplete’ type of hydatidiform moles, the extraembryonic membranes of the diandric triploid mouse conceptuses (as well as the digynic triploids) did not appear to be grossly abnormal).

scholarly journals Turnover of Carbon Pools Labelled with [14C]Glucose during in vitro Culture of Preimplantation Mouse Embryos

1982 ◽  
Vol 35 (6) ◽  
pp. 637
Author(s):  
I L Pike ◽  
RG Wales
Keyword(s):  
In Vitro Culture ◽  
Mouse Embryos ◽  
Carbon Pools ◽  
Stages Of Development ◽  
Glucose Carbon ◽  
Stage Of Development ◽  
Preimplantation Mouse Embryos ◽  
Preimplantation Mouse

The pulse-chase technique was used to study the uptake and turnover of glucose carbon by mouse embryos in vitro. During a 1 h pulse the uptake of glucose into all embryonic fractions increased between the eight-celled and the morula-early blastocyst stages of development. Whilst most of the glucose carbon entered the non-glycogen, acid-soluble pool, significant amounts were isolated in acid-insoluble macromolecules and, at the later stage of development, in acid-soluble glycogen.

scholarly journals Fetal hemoglobin and potassium in isolated transferrin receptor- positive dense sickle reticulocytes

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 2013-2020 ◽  
Author(s):  
RS Franco ◽  
R Barker-Gear ◽  
MA Miller ◽  
SM Williams ◽  
CH Joiner ◽  
...  
Keyword(s):  
Cell Formation ◽  
Fetal Hemoglobin ◽  
Density Fractions ◽  
Sickle Cells ◽  
Stage Of Development ◽  
Short Lifespan ◽  
F Cells ◽  
Low Levels

Abstract A subset of sickle cells have an increased density at the reticulocyte stage of development, indicating that they are either abnormally dense upon release from the bone marrow or become dense quickly in the circulation. These cells are of interest because they most likely have severely disrupted cation regulation and a short lifespan. Based on the distribution of fetal hemoglobin (HbF) in the density fractions of sickle red blood cells (RBCs) and in vitro studies of cellular K+ loss, it seems likely that HbF content is an important in vivo determinant of dense cell formation. In this study, we tested the hypothesis that young, dense cells have low HbF content. Sickle RBCs were first separated into light and dense fractions. Reticulocytes were isolated from unfractionated cells and from each density fraction with an immunomagnetic technique directed against transferrin receptors (TfR) and assayed for the percentage of HbF and K+/Hb ratio. TfR+ reticulocytes isolated from unfractionated cells had a much lower HbF content when compared with all the unfractionated RBCs. This is most likely caused by enrichment of F cells because of a longer circulation life span. Heavy TfR+ reticulocytes had a K+/Hb ratio similar to that measured in the entire dense population and contained very low levels of HbF, averaging 2.5% of the level in all RBCs, 11.7% of the level in all TfR+ reticulocytes, and 4.0% of the level in all dense RBCs. These findings suggest that TfR+ dense cells derive predominantly from non-F cells. Furthermore, the amount of HbF in the circulating dense cells suggests that many of these cells do not derive from the TfR+ dense cells.

Positioning of inner cell mass determines the development of mouse blastocysts in vitro

Development ◽  
1981 ◽  
Vol 65 (1) ◽  
pp. 105-117
Author(s):  
Tsung-Chieh Wu ◽  
Yu-Jui Wan ◽  
Ivan Damjanov
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Subsequent Development ◽  
Stages Of Development ◽  
Somite Stage ◽  
Plastic Dish ◽  
Advanced Stages ◽  
Further Development

We have explanted mouse blastocysts into culture dishes and studied the positioning of the inner cell mass (ICM) at the time of attachment in order to establish what effect the location of the inner cell mass has on the subsequent development of embryos in vitro. We show that blastocysts may attach to the substrate with any portion of the trophectoderm. The location of ICM within the attaching blastocysts is unpredictable. The directions of egg-cylinder development may be either upward or downward. The axis assumed by a developing egg cylinder in vitro depends to a large extent on the initial positioning of the ICM at the time of attachment. Egg cylinders grown from blastocysts that have the ICM in the lower position (close to the plastic dish) reach advanced stages of development in greater numbers than egg cylinders grown from blastocyst with their ICM's in the upper lateral or upper position. Only upward-growing egg cylinders can develop further into somitic stage (which is equivalent to 8½- or 9-day in vivo embryos). In contrast, downward growing egg cylinders become thwarted in their further development by the surrounding trophectoderm. If the downward growing egg cylinders are freed of trophectoderm and repositioned to face upwards they will develop in a manner similar to the originally upward growing egg cylinders. These findings indicate that the initial positioning of the ICM at the time of attachment is an important factor in governing the development of blastocysts to somite stage in vitro.

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