Microtubules and specification of the dorsoventral axis in frog embryos

The body plan of the frog is set-up by a rearrangement of the egg cytoplasm shortly after fertilization. Microtubules play several roles in this critical developmental event.     

Identification of PGC7, a new gene expressed specifically in preimplantation embryos and germ cells

The gene expression patterns of primordial germ cells (PGCs) and embryonic stem cells were analyzed by a modified serial analysis of gene expression. During the process, we cloned a novel gene, PGC7, which was preferentially expressed in PGCs. Immunohistochemical analysis revealed that PGC7 was specifically expressed in early pre-implantation embryos, PGCs and oocytes. These results suggest that PGC7 might play an important role in the development of PGCs and oocytes.     

An amphioxus netrin gene is expressed in midline structures during embryonic and larval development

Members of the netrin gene family have been identified in vertebrates, Drosophila and Caenorhabditis elegans and found to encode secreted molecules involved in axon guidance. Here I use the conserved function of netrins in triploblasts, coupled with the phylogenetic position of amphioxus (the closest living relative of the vertebrates), to investigate the evolution of an axon guidance cue in chordates. A single amphioxus netrin gene was isolated by PCR and cDNA library screening and named AmphiNetrin. The predicted AmphiNetrin protein showed high identity to other netrin family members but differed in that the third of three EGF repeats found in other netrins was absent. Molecular phylogene-tic analysis showed that despite the absent EGF repeat AmphiNetrin is most closely related to the vertebrate netrins. AmphiNetrin expression was identified in embryonic notochord and floor plate, a pattern similar to that of vertebrate netrin-1 expression. AmphiNetrin expression was also identified more widely in the posterior larval brain, and in the anterior extension of the notochord that underlies the anterior of the amphioxus brain. All of these areas of expression are correlated with developing axon trajectories: The floor plate with ventrally projecting somatic motor neurons and Rohde cell projections, the posterior brain with the ventral commissure and primary motor centre and the anterior extension of the notochord with ventrally projecting neurons associated with the median eye. Amphioxus is naturally cyclopaedic and also lacks the ventral brain cells that the induction of which results in the splitting of the vertebrate eye field and, when missing, result in cyclopaedia. These cells normally express netrins required for developing axon tracts in the brain, and the expression of AmphiNetrin in the anterior extension of the notochord underlying the brain may explain how amphioxus is able to maintain ventral guidance cues while lacking these cells. Received: 15 November 1999 / Accepted: 27 January 2000     

Alteration of the anterior-posterior embryonic axis: the pattern of gastrulation in macrocephalic frog embryos

The production of an enlarged head or macrocephaly in frog embryos can be achieved by interspecific hybridization or by injection of the contents of the germinal vesicle (GV), the large nucleus of immature oocytes, into the blastocoels of embryos before they gastrulate. The macrocephalic embryos have large suckers and their neural tubes are larger anteriorly but smaller posteriorly as compared to controls. This abnormal syndrome has previously been thought to arise as a result of an axial structure determinant present in the germinal vesicle. When examined during gastrulation, however, Xenopus laevis and Rana pipiens macrocephalic embryos produced by GV injection as well as macrocephalic embryos produced by the hybrid cross, Rana septentrionalis female X Rana catesbeiana male, all exhibit alterations in the pattern of gastrulation. The most striking of these alterations is the persistence throughout gastrulation of a thick blastocoel roof composed of many cell layers, suggesting that there is an inhibition of posterior spreading of the roof normally associated with epiboly. In R. pipiens, the dorsal mesodermal mantle of GV-injected gastrulae is thicker as compared to controls, accounting for a neural plate which is wide at the anterior end. Vital dye mapping experiments on Xenopus laevis embryos show that dye marks placed on regions normally fated to become trunk epidermis become localized anteriorly when the embryos are GV injected, consistent with the idea that ectodermal cells are inhibited from moving posteriorly. These results indicate that the macrocephalic syndrome can be attributed to a localized inhibition of cell rearrangements during gastrulation as opposed to the effects of altered inducers or to axial determinants.     

Ascidian Wnt-7 gene is expressed exclusively in the tail neural tube of tailbud embryos

In vertebrate embryogenesis, many Wnt genes are expressed in the neural tube and play important roles in regional specifications. There are many subfamilies of Wnt, and each subfamily shows distinct expression patterns in the neural tube. Ascidian larvae have a dorsal hollow neural tube similar to that of vertebrates. To date, the degree of correspondence between regionality of the neural tubes of ascidians and vertebrates remains unclear. To compare cellular differences in neural tubes, Wnt genes can be used as molecular probes. We report here that a new member of the ascidian Wnt gene family, HrWnt-7, was expressed in the tail neural tube at the early tailbud stage. Moreover, in cross-section, HrWnt-7 was expressed in the dorsal and ventral ependymal cells. Received: 14 July 2000 / Accepted: 1 August 2000     

DBHR, a gene with homology to dopamine beta-hydroxylase, is expressed in the neural crest throughout early development

In a screen for genes involved in neural crest development, we identified DBHR (DBH-Related), a putative monooxygenase with low homology to dopamine beta-hydroxylase (DBH). Here, we describe novel expression patterns for DBHR in the developing embryo and particularly the neural crest. DBHR is an early marker for prospective neural crest, with earliest expression at the neural plate border where neural crest is induced. Furthermore, DBHR expression persists in migrating neural crest and in many, though not all, crest derivatives. DBHR is also expressed in the myotome, from the earliest stages of its formation, and in distinct regions of the neural tube, including even-numbered rhombomeres of the hindbrain. In order to investigate the signals that regulate its segmented pattern in the hindbrain, we microsurgically rotated the rostrocaudal positions of rhombomeres 3/4. Despite their ectopic position, both rhombomeres continued to express DBHR at the level appropriate for their original location, indicating that DBHR is regulated autonomously within rhombomeres. We conclude that DBHR is a divergent member of a growing family of DBH-related genes; thus, DBHR represents a completely new type of neural crest marker, expressed throughout the development of the neural crest, with possible functions in cell-cell signaling.     

Serotonin signaling is a very early step in patterning of the left-right axis in chick and frog embryos

BACKGROUND: Consistent left-right (LR) asymmetry is a fascinating problem in developmental and evolutionary biology. Conservation of early LR patterning steps among vertebrates as well as involvement of nonprotein small-molecule messengers are very poorly understood. Serotonin (5-HT) is a key neurotransmitter with crucial roles in physiology and cognition. We tested the hypothesis that LR patterning required prenervous serotonin signaling and characterized the 5-HT pathway in chick and frog embryos. RESULTS: A pharmacological screen implicated endogenous signaling through receptors R3 and R4 and the activity of monoamine oxidase (MAO) in the establishment of correct sidedness of asymmetric gene expression and of the viscera in Xenopus embryos. HPLC and immunohistochemistry analysis indicates that Xenopus eggs contain a maternal supply of serotonin that is progressively degraded during cleavage stages. Serotonin's dynamic localization in frog embryos requires gap junctional communication and H,K-ATPase function. Microinjection of loss- and gain-of-function constructs into the right ventral blastomere randomizes asymmetry. In chick embryos, R3 and R4 activity is upstream of the asymmetry of Sonic hedgehog expression. MAO is asymmetrically expressed in the node. CONCLUSIONS: Serotonin is present in very early chick and frog embryos. 5-HT pathway function is required for normal asymmetry and is upstream of asymmetric gene expression. The microinjection data reveal asymmetry existing in frog embryos by the 4-cell stage and suggest novel intracellular 5-HT mechanisms. These functional and localization data identify a novel role for the neurotransmitter serotonin and implicate prenervous serotonergic signaling as an obligate aspect of very early left-right patterning conserved to two vertebrate species.     

Timekeeping by frog embryos, in normal development and after heat shock

(1) Timekeeping refers to the uniformity of development in time. The precision of timekeeping is measured by the extent to which embryos, within an initially synchronous population, come to diverge in the course of their development. (2) Divergence is measured as the variation in the stage of development reached between embryos allowed to develop for a fixed period of time. The lower the variation the better the timekeeping. (3) Divergence among frog embryos that started development at the same time is hardly measurable after approx. 100 h of development. This striking uniformity indicates good timekeeping. (4) Timekeeping is not impaired among the survivors following heat shocks that retard development and disturb and curtail morphogenesis. (5) The immediate effect of heat shock is a stoppage of development, the duration of which is the same for all embryos in the same treatment batch. The embryos react to heat shock by rescheduling their development with the interpolation of a rest, the duration of which is controlled to the same precision as normal development. The postponement of development, without impairment of timekeeping, implies dis-engagement of the processes of morphogenesis from, and their subsequent re-engagement with, an enduring rate-determining activity unaffected by heat shock. (6) We have searched for embryos whose rate of development was disturbed by heat shock to run slower or faster than the norm. We have found none. It seems that the (temperature-compensated) rate of development is invariant up to the moment of failure, or a change is immediately lethal.     

Dmbx1, a novel evolutionarily conserved paired-like homeobox gene expressed in the brain of mouse embryos.

To identify novel homeobox genes expressed during mouse embryogenesis, we searched the databases and found a novel mouse paired-like homeobox gene, Dmbx1(diencephalon/mesencephalon-expressed brain homeobox gene 1), that is also conserved in zebrafish and human. Linkage analysis mapped mouse Dmbx1 to the mid-portion of chromosome 4 that is the homologous gene cluster region of human chromosome 1, where human DMBX1 is located. Both mouse and human Dmbx1/DMBX1 have four coding exons and their gene structures are conserved. Whole-mount in situ hybridization revealed that Dmbx1 expression is detected in 7.5-9.5 dpc mouse embryos. At 7.5 and 8.5 dpc, Dmbx1 is expressed in a sub-region of the anterior head folds. At 9.5 dpc, expression is observed in the caudal diencephalon as well as in the mesencephalon and is restricted to the neuroepithelium. Expression in adult tissues was detected in brain, stomach, and testis. Dmbx1 provides a unique marker of the developing anterior nervous system and should provide a useful molecular resource to elucidate the mechanisms that pattern the vertebrate brain.     

Proliferating ability,morphological development and acetylcholinesterase activity of the neural tube cells in early chick embryos

Summary Using ³H-thymidine autoradiography and AChE histochemistry at the electron microscopic level on the same sections, the interrelationships between loss of proliferating ability, morphological development and increase of AChE activity during the course of differentiation of the neural tube cells were investigated in early chick embryos. The neural tube wall consisted of spindle-shaped cells with no AChE activity, weakly positive spindle-shaped cells showing AChE activity in the cisternae of the nuclear envelope and in a few short profiles of r-ER, moderately positive spindle-shaped cells showing AChE activity in the nuclear envelope and in a moderate number of r-ER profiles and intensely positive large round cells showing AChE activity in the nuclear envelope and in a large number of r-ER profiles. Nuclei of the AChE-negative, weakly positive and moderately positive cells were located in the ependymal layer (matrix). The AChE-intensely positive cells were in the mantle layer. The AChE-negative and weakly positive cells were capable of proliferation and were regarded as undifferentiated neuroepithelial cells. In contrast, the moderately positive and intensely positive cells were no longer capable of proliferation and were considered to be neurons. These findings indicate that the r-ER increases rapidly in amount and volume in newly formed neurons soon after their final cell division, and that AChE increases in the neurons in parallel to the development of the r-ER.     

Drosophila single-minded gene and the molecular genetics of CNS midline development.

Our goal is to understand the molecular mechanisms that govern the formation of the central nervous system. In particular, we have focused on the development of a small group of neurons and glia that lie along the midline of the Drosophila CNS. These midline cells possess a number of unique attributes which make them particularly amenable to molecular, cellular, and genetic examinations of nervous system formation and function. In addition, the midline cells exhibit distinctive ontogeny, morphology, anatomical position, and patterns of gene expression which suggest that they may provide unique functions to the developing CNS. The single-minded gene encodes a nuclear protein which is specifically expressed in the midline cells and has been shown to play a crucial role in midline cell development and CNS formation. Genetic experiments reveal that sim is required for the expression of many CNS midline genes which are thought to be involved in the proper differentiation of these cells. In order to identify additional genes which are expressed in some or all of the midline cells at different developmental stages, a technique known as enhancer trap screening was employed. This screen led to the identification of a large number of potential genes which exhibit various midline expression patterns and may be involved in discrete aspects of midline cell development. Further molecular, genetic, and biochemical analyses of sim and several of the enhancer trap lines are being pursued. This should permit elucidation of the genetic hierarchy which acts in the specification, differentiation, and function of these CNS midline cells.     

A cell surface antigen, TER, expressed by embryos and germ cells.

An antiserum prepared in rabbits against the C3HeB/FeJ mouse ovarian teratocarcinoma E6496 was absorbed in vivo in C3HeB/FeJ mice. This absored antiserum identified an antigen, denoted TER, that is present on sperm, ova, embryonic germ cells, and cells of the early mouse embryo. TER was absent from all adult somatic cells tested, but found on several murine tumors.