John Gerhart - Academia.edu (original) (raw)

Papers by John Gerhart

Science, 1994

Transgenic Xenopus laevis embryos were produced by transplantation of transfected cultured cell n... more Transgenic Xenopus laevis embryos were produced by transplantation of transfected cultured cell nuclei into unfertilized eggs. A Xenopus cell line, X-C, was stably transfected with plasmids containing a hygromycin-resistance gene and genes for either beta-galactosidase with a heat shock promoter or chloramphenicol acetyltransferase (CAT) with a muscle-specific actin promoter. Nuclei transplanted from these cells into unfertilized eggs directed development of embryos containing stably integrated copies of the plasmids in each cell. Transgenic embryos showed somite-specific expression of CAT and uniform expression of beta-galactosidase. Transgenic embryos produced by nuclear transplantation should be useful for testing the function of cloned genes in amphibian development.

Molecular Biology of the Cell, 2001

The organization of the endoplasmic reticulum (ER) in the cortex ofXenopus oocytes was investigat... more The organization of the endoplasmic reticulum (ER) in the cortex ofXenopus oocytes was investigated during maturation and activation using a green fluorescent protein chimera, immunofluorescence, and electron microscopy. Dense clusters of ER developed on the vegetal side (the side opposite the meiotic spindle) during maturation. Small clusters appeared transiently at the time of nuclear envelope breakdown, disappeared at the time of first polar body formation, and then reappeared as larger clusters in mature eggs. The appearance of the large ER clusters was correlated with an increase in releaseability of Ca2+by IP3. The clusters dispersed during the Ca2+wave at activation. Possible relationships of ER structure and Ca2+regulation are discussed.

Development, 1996

The dorsoventral body axis in amphibian embryos is established by a rotation of the outer cortex ... more The dorsoventral body axis in amphibian embryos is established by a rotation of the outer cortex relative to the inner cytoplasmic core. This cortical rotation depends on microtubules and is correlated with a parallel array of microtubules just inside the vegetal cortex. Since the parallel array moves with the inner cytoplasm and most of its microtubules are oriented with their plus ends facing the direction of cortical movement, it has been suggested that plus end-directed motor molecules attached to the cortex drive the rotation by moving along microtubules of the parallel array. Using an inverted confocal microscope to examine living eggs, however, we found that rotation movements precede the formation of a detectable parallel array at the vegetal pole, that the parallel array consists of multiple layers of microtubules at depths ranging from 4 to 8 μm inside the plasma membrane and that the velocity of rotation in immobilized eggs increases with depth in this region. These findi...

Developmental Biology, 1985

In Xenopus laevis, the dorsal structures normally develop from regions of the egg opposite the si... more In Xenopus laevis, the dorsal structures normally develop from regions of the egg opposite the side of sperm entry. Gravity is known to affect this topographic relationship in eggs inclined obliquely from their normal vertical orientation in the period before first cleavage. This effect has been explored in detail, making use of low-speed centrifugation (10-50 g) for short durations (4 min). Eggs were immobilized in gelatin and oriented with their animal-vegetal axes 90 degrees to the force vector, with the sperm entry point (SEP) side of the egg either toward or away from the center of the rotor. It has been found that the egg shows three distinct periods of response to centrifugal force in the interval from fertilization to first cleavage: Prior to 0.4 (40% of the first cleavage interval), the egg is very sensitive to centrifugal force and develops dorsal structures from its centrifugal side, regardless of the position of the SEP in the centrifugal field. Thus, the dorsal structures of the embryo are reversed from normal in eggs centrifuged with the SEP away from the center of the rotor. In the period 0.4 to 0.7, the egg is still very sensitive to centrifugal force and develops dorsal structures from its centripetal side, regardless of the position of the SEP in the centrifugal field. Thus, the dorsal structures of the embryo are reversed from normal in eggs centrifuged with the SEP toward the center of the rotor. In the period 0.7-1.0, the egg becomes increasingly resistant to centrifugal force and forms dorsal structures at the normal position opposite the SEP side. This resistance can be overcome in some egg clutches by 50 g centrifugation followed by prolonged 90 degrees off-axis inclination at 1g. Midway in the second cell cycle, there is a brief period of sensitivity to centrifugal force. These These results are discussed in terms of the types of cytoplasmic rearrangements occurring in the egg at different times of the cell cycle, and in terms of the process of cytoplasmic localization of determinants of dorsal axial development.

Development, 1989

We first review cortical-cytoplasmic rotation, a microtubule-mediated process by which the Xenopu... more We first review cortical-cytoplasmic rotation, a microtubule-mediated process by which the Xenopus egg, like other amphibian eggs, transforms its polarized cylindrical symmetry into bilateral symmetry within the first cell cycle after fertilization. This transformation, the earliest of many steps leading to dorsal development, involves the displacement of the egg’s cortex relative to its cytoplasmic core by 30° in an animal-vegetal direction. As rotation is progressively reduced by microtubuledepolymerizing agents, embryos develop with body axes progressively deleted for dorsal structures at the anterior end. With no rotation, ventralized embryos are formed. In an effort to comprehend this progressive effect on embryonic organization, we go on to review subsequent developmental processes depending on rotation, and we propose, with evidence, that reduced rotation leads to a reduced number of vegetal dorsalizing cells, which induce during the blastula stage a Spemann organizer region ...

Boston Studies in the Philosophy and History of Science

The 1981 session on the Cellular Basis of Morphogenetic Change included discussions of pattern fo... more The 1981 session on the Cellular Basis of Morphogenetic Change included discussions of pattern formation by positional information and cellular interpretation, polyclonal compartments as developmental units of selector gene expression, morphogenetic activities of cells, conserved cell types, and the developmental pathways revealed by newly collected Drosophila mutants. All of this was contributory to and preparatory for the great advances of the next decades in the understanding of pattern formation in terms of cell-cell signaling and transcriptional regulation, and in the evolution of development. The present article is a summary of some of these advances, taking the Bmp and Wnt signaling pathways in vertebrate embryos, which mediate the large scale patterning of the dorsoventral and anteroposterior axes of the embryo. The summary covers the different transduction components and transcription factors activated within the two pathways when signals are received, and the complex yet robust and adaptable means by which cells generate graded distributions of signals over specific cell groups. Key to these distributions at the gastrula-neurula stages is Spemann’s organizer, a signaling and morphogenetic center of the early embryo that produces Bmp and Wnt antagonists. The pathways themselves date back to the earliest metazoa, and their use in patterning the embryonic axes dates back at least to the bilaterian common ancestor. The evolution of development has involved changes in the times and places of repeated usage of these pathways, in the genes targeted by them (changing cell competence), and in the means and location of signal distribution.

Current Topics in Cellular Regulation, 1970

Publisher Summary This chapter discusses the regulatory properties of aspartate transcarbamylase ... more Publisher Summary This chapter discusses the regulatory properties of aspartate transcarbamylase (ATCase) from Esterichia coli . Early circumstantial evidence for regulation in the pyrimidine biosynthetic pathway at the level of enzyme activity came in 1952 from the isotope competition studies of Bolton et al. , in which they found that nonradioactive uracil or uridine added in small amounts to the nutrient medium of growing Escherichia coli immediately and extensively suppresses the endogenous bacterial synthesis of pyrimidines from 14 C-labeled CO 2 . When uracil becomes exhausted, the mutant begins producing carbamyl aspartate in large quantities, up to one-half its dry weight in 4 hours, reflecting the derepression and uncontrolled activity of ATCase. When uracil is again provided to the mutant, the formation of carbamyl aspartate ceases within minutes, indicating the inhibition of ATCase activity.

Brookhaven symposia in biology, 1964

Cytobios, 1985

When an M-phase promoting factor (MPF) is injected into Xenopus oocytes, which are naturally arre... more When an M-phase promoting factor (MPF) is injected into Xenopus oocytes, which are naturally arrested at the G2/prophase boundary, it induces rapid entry of the cells into M-phase. MPF is present in late G2 and in M-phase of a variety of cell types, such as Xenopus eggs (naturally arrested in M), cleaving embryos, yeast, HeLa, and CHO cultures. MPF has been purified approximately 50-fold from eggs. It is stabilized by gamma-thio-ATP and by phosphoprotein phosphatase inhibitors. It runs as a protein of approximately 100 kd size on gel filtration. Oocytes contain a precursor of MPF, which is activated by post-translational means when a small amount of purified MPF is injected into the cell. Thus, MPF appears to be an auto-activating cytoplasmic trigger of M-phase. At anaphase of the cell cycle, MPF is inactivated due to the appearance of an 'anti-MPF' activity. Monoclonal antibodies have been prepared to partially purified MPF stabilized by gamma-thio-ATP, and several preparat...

Evolution—the Extended Synthesis, 2010

Developmental Biology, 1989

Excessively dorsalized embryos of Xenopus laevis develop from eggs treated with 30-70% D2O for a ... more Excessively dorsalized embryos of Xenopus laevis develop from eggs treated with 30-70% D2O for a few minutes within the first third of the cell cycle following fertilization. As the concentration of D2O and the duration of exposure are increased, the anatomy of these embryos shifts in the direction of enlarged dorsal and anterior structures and reduced ventral and posterior ones. Twinning of dorsoanterior structures is frequent. Intermediate forms include embryos with large heads but no trunks or tails. The limit form of the series has cylindrical symmetry, with circumferential bands of eye pigment and cement gland, a core of notochord-like tissue, and a centrally located beating heart. D2O treatment seems to increase the egg's sensitivity to the dorsalizing effects of cortical rotation and to stimulate the egg to initiate two or more directions of rotation. Such eggs probably establish thereafter a widened and/or duplicated Nieuwkoop center in the vegetal hemisphere, with the subsequent induction of a widened and/or duplicated Spemann organizer region in the marginal zone, which leads to excessive dorsal development. The existence of these anatomical forms indicates the potential of the egg to undertake dorsal development at all positions of its circumference and suggests that normal patterning depends on the limited and localized activation or disinhibition of this widespread potential.

Developmental Biology, 1990

Protein secretion is blocked in Xenopus oocytes arrested at second meiotic metaphase. In this rep... more Protein secretion is blocked in Xenopus oocytes arrested at second meiotic metaphase. In this report, we show that secretion becomes blocked coincident with germinal vesicle breakdown (GVBD). Transport through the metaphase-arrested oocyte's secretory pathway continues unimpeded until proteins reach the trans-Golgi. These conclusions are drawn from experiments using exogenous prolactin and vesicular stomatitis virus G protein (VSV G) encoded by SP6 transcripts and endogenous glycosaminoglycan (GAG) chains initiated on beta-D-4-methylumbelliferyl-xyloside. From the initiation of maturation with progesterone until GVBD, secretion of prolactin synthesized before the start of maturation is comparable to secretion in immature oocytes, but after GVBD secretion of prolactin declines approximately 63% in the first hour. Not all steps in the secretory pathway are blocked when oocytes mature. Since VSV G protein acquires resistance to endo H digestion with equal efficiency in immature oocytes (arrested in first meiotic prophase) and matured oocytes (arrested in second meiotic metaphase), we conclude that transport of this protein from the ER to the Golgi is not inhibited at meiotic metaphase. Using [35S]sulfate to label xyloside-initiated GAG chains we find that transport of GAG chains from the trans-Golgi to the cell surface is 15-fold lower in matured oocytes than in immature oocytes. Examination of the size of GAG chains by SDS-PAGE and HPLC indicates that matured oocytes produce GAG chains significantly larger than GAG chains from immature oocytes. This increase in size suggests that GAG chains from matured oocytes have a longer residence time in the trans-Golgi than GAG chains from immature oocytes. Hence, part of the block to secretion in metaphase-arrested oocytes could be an inhibition of vesicle budding from the trans-Golgi.

Developmental Biology, 1987

The amphibian egg undergoes a rotation of its subcortical cytoplasm relative to its surface durin... more The amphibian egg undergoes a rotation of its subcortical cytoplasm relative to its surface during the first cell cycle. Nile blue spots applied to the egg periphery move with the subcortical cytoplasm and make rotation directly observable (J.-P. Vincent, G.F. Oster, and J. C. Gerhart (1986). Dev. Biol. 113, 484). We have previously shown that the direction of rotation accurately predicts the orientation of the embryonic axis developed by the egg. This suggests an important role for subcortical rotation in axis specification. In this report, we provide two kinds of experimental evidence for the essential role of rotation, and against a role for other concurrent cytoplasmic movements such as the convergence of subcortical cytoplasm toward the sperm entry point in the animal hemisphere. First, dispermic eggs develop only one embryonic axis, which is oriented accurately in line with the direction of the single rotation movement and not with the two convergence foci that form in the animal hemisphere. Rotation probably modifies the vegetal, not animal, hemisphere since axial development is normal in dispermic eggs despite highly altered animal subcortical movement. Second, we show that the amount of rotation correlates with the extent of dorsal development. UV irradiation of the vegetal hemisphere, or cold shock of the egg, inhibits rotation effectively. When there is no rotation, there is no dorsal development. On average within the egg population, increasing amounts of rotation correlate with the increasingly anterior limit of the dorsal structures of the embryonic body axis. However, individual partially inhibited eggs vary greatly in the amount of axis formed following a given amount of movement. Furthermore, the egg normally rotates more than is necessary for the development of a complete axis. These findings suggest that rotation, although essential, does not directly pattern the antero-posterior dimension of the body axis, but triggers a response system which varies from egg to egg in its sensitivity to rotation. This system is artificially sensitized by exposure of the egg to D2O shortly before rotation. We show that D2O-treated eggs produce extensive axes despite very limited rotation, often developing into hyperdorsal embryos. However, like normal eggs, they depend on rotation and cannot form dorsal structures if it is eliminated.

Developmental Biology, 1994

We have examined the involvement of proteoglycan molecules in the induction of mesodermal tissue ... more We have examined the involvement of proteoglycan molecules in the induction of mesodermal tissue in Xenopus laevis embryos. Blastocoelic injections of the enzyme heparitinase at early blastula stages lead to gastrulation defects and to failures in the development of anterior embryonic structures. The period of sensitivity of embryos to this treatment suggests a possible role for these molecules during mesoderm induction. We show that heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans are the predominant sulfated glycoconjugates synthesized in early Xenopus embryos and that HSPGs are degraded by blastocoelic injections of heparitinase. Further, bFGF induction of mesoderm in explants of Xenopus stage 8 embryonic animal cap tissue is blocked by heparitinase but not by Chondroitinase ABC, using three separate criteria of mesoderm induction. Since HSPGs present in blastula animal cap cells are digested by heparitinase under the culture conditions used in the mesoderm-induction assay, we suggest that cell-surface heparan sulfate proteoglycans are required for basic fibroblast growth factor-mediated mesoderm induction.

Roux's Archives of Developmental Biology, 1991

One important step in understanding early development is to define the cell interactions involved... more One important step in understanding early development is to define the cell interactions involved in establishing tissue types. In amphibian embryos, one such interaction is the induction by the organizer region after the late blastula stage of lateral and ventral regions of the marginal zone (MZ) to form dorsal tissue types such as muscle. It is not known whether the organizer can also induce lateral MZ to form notochord after the late blastula stage. We find that this induction occurs under experimental conditions and plays a role in normalXenopus development. The ability to induce notochord is strongest at the center of the organizer along the dorsal midline and weaker at the lateral edges of the organizer. Organizer tissue along the dorsal midline, which would differentiate as notochord in normal development, can exhibit organizer functions such as the induction of the dorsolateral MZ to form notochord without later differentiating as notochord itself. Thus organizer activity can be dissociated from subsequent notochord formation.

Current Opinion in Genetics & Development, 2008

Cold Spring Harbor Symposia on Quantitative Biology, 1963