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At least 433 records · Page 24Linked to original sources

PubMed1985

The ontogenesis of cranial neuromeres in the rat embryo. I. A scanning electron microscope and kinetic study.

The development of the cranial neuromeres, rostral to the level of the first somite, in rat embryos during neurulation is described. Examination of scanning electron micrographs of 4-, 8-, 12- and 16-somite-stage embryos has shown that the neuromeres develop in a strictly ordered pattern but the sequence is not ordered in either a rostrocaudal or caudorostral direction. The two mesomeres are shown to develop by subdivision of a previously formed mesomere, whilst the rhombomeres develop independently of each other. At the completion of cranial neurulation two mesomeres and nine rhombomeres (including the preotic sulcus) but no prosomeres are identifiable. A direct correlation of the position of peaks in mitotic index along the embryonic axis with the position of sulci on the surface of the neural epithelium, was not possible. However, it is suggested that the mitotic index peak is the initial means of identifying a neuromere, and once the neuromere is expressed morphologically as a surface sulcus the mitotic index returns to its basal level.

Animals↗

PubMed1985

Highlights of craniofacial morphogenesis in mammalian embryos, as revealed by scanning electron microscopy.

The craniofacial region has a complex developmental history. Some of the major morphogenetic events of this region are described for the mouse embryo, as revealed by the use of scanning electron microscopy. The three germ layers, the primitive building blocks of the embryo, are delineated during gastrulation. During neurulation, the outer ectodermal layer gives rise to the nervous system. Several other events occur concomitantly with neurulation. These include regional subdivision of the mesoderm, body folding, turning, and formation of the heart and gut. The craniofacial region, as well as a number of other regions or organ systems, undergoes major developmental events during the phase of embryogenesis called organogenesis. The visceral (branchial) apparatus develops around the primitive oral cavity and pharyngeal region of the foregut, and gives rise to many of the structures of the head and neck. The face develops from the first visceral arches and derivatives of the frontonasal prominence. The tongue also originates from tissue prominences derived from the visceral arches. The oral and nasal cavities are initially continuous broadly with one another. As a result of partitioning, paired nasal cavities develop and become largely separated from the underlying oral cavity. Scanning electron microscopy reveals much about the morphological events occurring during formation of the craniofacial region. However, emphasis in future studies should be placed on determining the underlying cellular and molecular mechanisms causing these events.

Animals↗

PubMed1978

The effects of high hydrostatic pressure on microfilaments and microtubules in Xenopus laevis.

Xenopus laevis embryos of stages 14-20 were subjected, for periods of 5-330 min, to hydrostatic pressures ranging from 500 to 10 000 psi. The specimens were fixed under corresponding pressures and their neuroepithelium was studied under light and electron microscopy. A pressure of 3000 psi, maintained for as long as 180 min, did not inhibit neurulation though it induced slight deformities of the neuroepithelium. A pressure of 4000 psi, applied for 180 min, disrupted the apical ring of microfilaments and blocked neurulation. The cells lost their dissymmetry. The effect was reversible. Lengthening the duration of treatment to 330 min caused the neuroepithelial cells to loose their microtubules and to become round. This situation was not reversible. Our results indicated that microfilaments are more sensitive than microtubules, that both organelles became increasingly sensitive as the exerted pressure was increased and that microtubules of older embryos exhibited a better resistance. Finally, we showed a correlation between the presence of microfilaments and the constricted state of the cellular apices and a relationship between the presence of microtubules and cell elongation.

Animals↗

PubMed1983

Analysis of the effects of Streptomyces hyaluronidase on formation of the neural tube.

Chick embryos at stages 8 to 9 were treated in ovo with Streptomyces hyaluronidase (SH) to determine whether neurulation occurs normally in embryos depleted of hyaluronic acid, a major component of the extracellular matrix. Open neural tube defects occurred in 60-94% (depending on the particular enzyme batch) of the embryos treated with SH and examined after an additional 24 h of incubation. Defects were confined mainly to the spinal cord. The neural folds underwent elevation in defective regions but failed to converge and fuse across the dorsal midline. The extracellular matrix of embryos treated with SH was depleted consistently, as determined with sections stained with Alcian blue. Control experiments were done to ensure that neural tube defects were not caused by non-specific protease contamination of SH, or by digestion products of hyaluronic acid. We propose several plausible and testable mechanisms through which the extracellular matrix might influence the complex developmental process of neurulation.

Animals↗

PubMed1984

Regional biosynthetic markers in the early amphibian embryo.

A search has been made for regional differences in macromolecular synthesis in the axolotl embryo at a stage when the principal regions have become determined but terminal differentiation has not yet begun. The epidermis of the neurula makes a number of abundant proteins which are not made elsewhere. Some of these are identified by immunoprecipitation as cytokeratins (relative molecular masses (Mr) 62,59,54,51 and 46 X 10(3)). At the same stage a network of tonofilaments becomes visible by electron microscopy and is also confined to the epidermis. There is no significant incorporation of 3H-sugars into glycoproteins before neurulation. During neurulation specific species begin to be made by the notochord (Mr 265 and 185 X 10(3)), the mesoderm (315 X 10(3)) and the epidermis (170 X 10(3)). The latter species is water soluble, has a native Mr of 470 X 10(3), is somewhat resistant to trypsin and has a high sugar content. Since these characteristics do not relate to any known glycoprotein it has been given a new name: epimucin. Several neutral glycolipids and gangliosides are present in the early embryo, the most abundant of which is identified as galactocerebroside. Synthesis occurs from fertilization onwards, however even by the neurula stage the qualitative pattern is the same in all parts except for the epidermis which shows two substantial enhancements and one reduction compared to other regions. The differences reported probably relate to physiological functions rather than to the mechanism of determination itself, so their chief importance is as markers of early embryo regions in experiments on induction.

Ambystoma↗

PubMed1981

An experimental study on the morphogenesis of spina bifida.

This investigation was performed to study the morphogenesis of caudal meningomyeloschisis or spina bifida aperta during neurulation. Pregnant rats were treated wih trypan blue to induce these defects in the closure of the neural walls. Maternal serum was studied to determine trypan blue levels. Both control and treated embryos were examined grossly, at cellular and subcellular level on days 10 through 13 of gestation. Trypan blue was distributed throughout the maternal tissues. There existed a maternal blood-brain barrier and a barrier at the level of the visceral yolk sac and embryonic gut preventing passage of the dye through these entodermal structures. The interstitial space in the paraxial mesenchyme was considerably increased in day 10 treated embryos (6--7 somites) but cellular and ultrastructural changes were not seen. This accumulation of intercellular fluid was only seen in the restricted area of the mesoderm above the invaginating intra-embryonic entoderm. On day 11 the closure of the neural walls was completed in control embryos. In the treated embryos of the similar stages a blister which contained embryonic blood cells had replaced the interstitial mesenchymal fluid. This structural disturbance in the non-segmented mesenchyme interfered primarily with the neurulation and resulted in caudal meningomyeloschisis (spina bifida aperta). Trypan blue was thought to interfere with the function of the extra-embryonic membranes and the primitive gut. The results of this study supported the hypothesis of indirect teratogenic effect upon the embryo.

Abnormalities, Drug-Induced↗

PubMedDate not supplied

[The anomalous development of the nervous system of the human embryo at 3.2 mm of length].

Serial histological preparations of a human embryo of 3.2 mm parieto-coccygeal length were studied. The morphological organization of the embryo had a combination of signs typical for the 10, 11 and 12th stages of the human development. Neurulation in the abnormal embryos was completed at the stage of 6 somites which is 2-3 days earlier than in a normally developing embryo. The embryo had no neural tube and the neuroepithelial cells represented a homogeneous cord without signs of striatal or nuclear organization. The embryo was found to have germs of the ocular vesicles, induced germs of the lens, olfactory and auditory placodes. Disturbances in the differentiation of the chordomesoderm and germs of branchial pockets took place. The changes observed are supposed to be a result of relaxation reactions of the neuroepithelium of the neural tube in response to nonspecific mechanico-chemical actions at the end of neurulation.

Humans↗

PubMed1998

Embryonic and fetal development of structures associated with the cerebro-spinal fluid in man and other species. Part I: The ventricular system, meninges and choroid plexuses.

Little is known about the development of the central nervous system (CNS) in humans. Ethical considerations preclude experimental studies in this field, and as a result most available data on human ontogenesis are descriptive. Comparative anatomic and embryologic studies have demonstrated that the main developmental milestones are conserved across species, and their results can be used to suggest a likely scenario for human development. The development of the ventricles, meninges, and choroid plexuses are discussed in this article. The central cavity of the neural tube is formed during neurulation, which occurs during the fourth gestational week. The first milestone is occlusion of the spinal neurocele (the central canal in the neural tube) shortly after neurulation. This prevents free communication between the ventricular system and the amniotic cavity. The second milestone is development of the meninges, which separate the central nervous system from the rest of the body. The embryonic origin of the meninges varies across species. In birds (and probably in mammals), the spinal meninges are derived from the somitic mesoderm, the brainstem meninges from the cephalic mesoderm, and the telencephalic meninges from the neural crest. Differentiation of the meninges, which involves formation of the subarachnoid space, occurs early, before the cerebrospinal fluid (CSF) begins to flow around the CNS. During ontogenesis, the meninges play a key role in regulating the growth of underlying nervous structures. They induce the formation of the superficial glial limiting layer and stimulate the growth of precursors located in the superficial blastemas of the cerebellum and hippocampus. The choroid plexuses are complex specialized structures that produce most of the CSF. Their epithelium derives from the neural tube epithelium and their mesenchyma from the meninges. Of the many enzymes produced in the choroid plexuses, some reflect the pivotal metabolic role of these structures (alkaline and acid phosphatases, magnesium-dependent ATPase, glucose-6-phosphatase, thiamine pyrophosphatase, adenylate cyclase, oxidoreductase, esterases, hydrolases, cathepsin D, and glutathion S-transferase). The two enzymes that are crucial to the production of CSF are Na+/K+ ATPase and carbonic anhydrase. Inactivation of catecholamines is mediated by catechol-O-methyltransferase and by the monoamine oxidases A and B. The morphology and synthesis profile of the choroid plexuses changes during development, although little is known about these changes in humans.

Animals↗

PubMed2024

A time-resolved single-cell roadmap of the logic driving anterior neural crest diversification from neural border to migration stages.

Neural crest cells exemplify cellular diversification from a multipotent progenitor population. However, the full sequence of early molecular choices orchestrating the emergence of neural crest heterogeneity from the embryonic ectoderm remains elusive. Gene-regulatory-networks (GRN) govern early development and cell specification toward definitive neural crest. Here, we combine ultradense single-cell transcriptomes with machine-learning and large-scale transcriptomic and epigenomic experimental validation of selected trajectories, to provide the general principles and highlight specific features of the GRN underlying neural crest fate diversification from induction to early migration stages using Xenopus frog embryos as a model. During gastrulation, a transient neural border zone state precedes the choice between neural crest and placodes which includes multiple converging gene programs. During neurulation, transcription factor connectome, and bifurcation analyses demonstrate the early emergence of neural crest fates at the neural plate stage, alongside an unbiased multipotent-like lineage persisting until epithelial-mesenchymal transition stage. We also decipher circuits driving cranial and vagal neural crest formation and provide a broadly applicable high-throughput validation strategy for investigating single-cell transcriptomes in vertebrate GRNs in development, evolution, and disease.

Animals↗

PubMed1996

Embryological origin for autism: developmental anomalies of the cranial nerve motor nuclei.

The underlying brain injury that leads to autism has been difficult to identify. The diagnostic criteria of the disease are not readily associated with any brain region or system, nor are they mimicked by vascular accidents, tumors, or degenerative neurological diseases occurring in adults. Fortuitously, a recent report of autism induced by thalidomide exposure provides evidence that the disease originates by an injury at the time of closure of the neural tube. The human data suggest that the initiating lesion includes the motor cranial nerve nuclei. To test this hypothesis, we first examined motor nuclei in the brainstem of a human autistic case. The autopsy brain exhibited near-complete absence of the facial nucleus and superior olive along with shortening of the brainstem between the trapezoid body and the inferior olive. A similar deficit has been reported in Hoxa-1 gene knockout mice in which pattern formation of the hindbrain is disrupted during neurulation. Alternatively, exposure to antimitotic agents just after neural tube closure could produce the observed pattern of deficits. Thus, the lesions observed in the autopsy case appear to match those predicted by the thalidomide cases in both time of origin and central nervous system (CNS) location. To produce similar brain lesions experimentally, we exposed rat embryos to valproic acid, a second teratogen newly linked to autism. Dams received 350 mg/kg of valproic acid (VPA) on day 11.5 (the day of neural tube closure), day 12, or day 12.5 gestation. Each treatment significantly reduced the number of motor neurons counted in matched sections of the earliest-forming motor nuclei (V, XII), and progressively later exposures affected the VIth and IIIrd cranial nerve nuclei. All treatments spared the facial nucleus, which forms still later. Counts from the mesencephalic nucleus of trigeminal, the dorsal motor nucleus of the vagus, and the locus ceruleus were not affected by exposure to VPA, even though these nuclei form during the period when exposure occurred. Despite its effects on the motor nuclei, valproic acid exposure did not alter the further development of the brain in any obvious way. Treated animals were robust and had no external malformations. The autopsy data and experimental data from rats confirm that CNS injuries occurring during or just after neural tube closure can lead to a selective loss of neurons derived from the basal plate of the rhombencephalon. The results add two new lines of evidence that place the initiating injury for autism around the time of neural tube closure.

Aged↗

PubMed1997

Induction of thermotolerance in early postimplantation rat embryos is associated with increased resistance to hyperthermia-induced apoptosis.

Previously we reported that hyperthermia (43 degrees C) induces cell death in neurulation stage rat embryos as part of the pathogenesis culminating in abnormal growth and development. We now show that hyperthermia-induced cell death occurs by a process termed apoptosis. DNA fragmentation, a hallmark of apoptosis, was noted as early as 2.5 hr after embryos were exposed to 43 degrees C. A smaller but significant increase in DNA fragmentation was also observed in embryos exposed to 42 degrees C, but only at the 5 hr time point. In control embryos, TUNEL-positive apoptotic bodies were consistently observed in the neuroepithelium at the point of neural tube closure and in the optic stalk. In embryos exposed to 43 degrees C, the number of TUNEL-positive apoptotic bodies was significantly increased. Using both gel electrophoresis and TUNEL, we also show that the induction of thermotolerance is associated with a significant reduction in DNA fragmentation. Together our results show that specific programmed cell death and hyperthermia-induced cell death correlate with internucleosomal DNA fragmentation characteristic of apoptosis. Finally, we show that the induction of thermotolerance in rat embryos is associated with a significant reduction in internucleosomal DNA fragmentation and associated apoptosis.

Animals↗

PubMed1998

Characterization of cell death induced by 2-methoxyethanol in CD-1 mouse embryos on gestation day 8.

Cell death was analyzed in neurulating mouse embryos after in vivo doses of 2-methoxyethanol (2-ME) that produce anterior neural tube defects. Characterization of 2-ME-induced cell death was performed by evaluating: (1) vital fluorochrome staining in whole embryos applying confocal laser scanning microscopy; (2) characteristics of cell debris in conventional histological sections revealed by light microscopy; and (3) Apoptag in situ immunohistochemical staining for apoptosis using light microscopy. Methods for quantification of cell death identified by these three techniques were explored using computerized image analysis. Physiological cell death in control embryos primarily occurred in the neural crest region during neural fold elevation. Embryos exposed to 2-ME had expanded areas of cell death in the neural crest and also new areas of cell death in medial regions of the anterior neural tube. Both physiological and 2-ME-induced embryonic cell death had morphological, immunohistochemical, and fluorochrome staining characteristics of apoptosis. When fluorescence data from confocal microscopic analysis of vital fluorochrome-stained embryos were analyzed, a dose-dependent increase was found in embryos exposed to 2-ME. Similar results were obtained when cell death was analyzed in either conventional histological sections or sections prepared for immunohistochemical detection of apoptosis. The cell death data obtained in this study correlate with previously observed near-term malformation rates, suggesting that a quantitative relationship exists between 2-ME-induced embryonic cell death and neural tube defects.

Acridine Orange↗

PubMed1996

Apoptosis removes chick embryo tail gut and remnant of the primitive streak.

Removal of transient features in morphogenesis of chick embryo tail is by programmed cell death. We used ApopTagTM (Oncor, Gaithersburg, MD) with the peroxidase/diaminobenzidine (DAB) procedure to correlate apoptosis with earlier reports of patterns of cell death in stage HH17-25 embryos, and our results suggest that the cell death inferred with supravital staining and appearance of cells in morphogenesis of the tail bud is programmed cell death called apoptosis. Apoptosis markers in tail bud are most abundant in the median cell cord of occluded degenerating tail gut. Tail bud mesenchyme marks for apoptosis most frequently in the ventrum of older stages, where cell death has been reported. Cells of the remnant of the primitive streak (Hensen's node) mark for apoptosis, suggesting that programmed cell death is a stop signal for axial organization at the caudal terminus. Apoptosis markers in postmembrane cloacal endoderm anticipate the transient cloacal fenestra. Lack of apoptosis markers in neural tube, notochord, and somites supports the suggestion of Schoenwolf ([1981] Anat, Embryol. (Beri.) 162:183-197) that cells of those areas in the tail bud are assimilated into the growing rump of the chick embryo. Lack of markers in neural tube of tail bud formed by secondary neurulation suggests that apoptosis is not involved in cavitation of medullary cord, but further investigation is necessary. A limited investigation of pharyngeal membranes and midgut, where cell death has not been reported to be as important in morphogenesis, did not show apoptosis markers in those tissues (Miller and Briglin [1994] "Cell Death in Development and Cancer," Houston: University of Texas MD Anderson Cancer Center, pp, 82-83). Absence of apoptosis markers in roof of gut tube suggests that the lower frequency of thymidine labeling reported for those cells (Miller [1986] Anat. Rec. 214: 87A) is not a result of apoptosis. Clearly marked cells correlated with expected locations of migrating neural crest and primordial germ cells in these stages, but distribution of apoptosis markers was not abundant or general for either cell type.

Animals↗

PubMed1997

Disruption of Msx-1 and Msx-2 reveals roles for these genes in craniofacial, eye, and axial development.

In mouse embryos, the muscle segment homeobox genes, Msx-1 and Msx-2 are expressed during critical stages of neural tube, neural crest, and craniofacial development, suggesting that these genes play important roles in organogenesis and cell differentiation. Although the patterns of expression are intriguing, little is known about the function of these genes in vertebrate embryonic development. Therefore, the expression of both genes, separately and together, was disrupted using antisense oligodeoxynucleotides and whole embryo culture techniques. Antisense attenuation of Msx-1 during early stages of neurulation produced hypoplasia of the maxillary, mandibular, and frontonasal prominences, eye anomalies, and somite and neural tube abnormalities. Eye defects consisted of enlarged optic vesicles, which may ultimately result in micropthalmia similar to that observed in Small eye mice homozygous for mutations in the Pax-6 gene. Histological sections and SEM analysis revealed a thinning of the neuroepithelium in the diencephalon and optic vesicle and mesenchymal deficiencies in the craniofacial region. Injections of Msx-2 antisense oligodeoxynucleotides produced similar malformations as those targeting Msx-1, with the exception that there was an increase in number and severity of neural tube and somite defects. Embryos injected with the combination of Msx-1 + Msx-2 antisense oligodeoxynucleotides showed no novel abnormalities, suggesting that the genes do not operate in a redundant manner.

Animals↗

PubMed1997

Initial closure of the mesencephalic neural groove in the chick embryo involves a releasing zipping-up mechanism.

According to a traditional viewpoint, initial closure of the anterior neural groove involves bilateral elevation of the edges of the neural plate, flattening of the midline area, subsequent convergence of the dorsal neural folds, and finally adhesion and fusion of the medial fold edges. In a transverse view, the shape of the neural groove thereby changes from V > U > toppled C > O. This sequence implicates that the neural groove is wide almost from its inception. In the present study, a new mechanism of initial closure is proposed, based on observations in living chick embryos and on light and scanning electron microscopic observations during neurulation in the presumptive mesencephalic region. The medial part of the neural plate invaginates in ventral direction. The walls of the arising neural groove appose, beginning in the depth, and make subsequent contact. During continued invagination the neural walls extend in ventral direction, the apposition/contact zone shifts in dorsal direction up to the neural folds and the neural walls separate ventrally, resulting in the incipient neural tube lumen. The mechanism is best compared with a zipping-up releasing model. In a transverse view, the shape of the neural groove changes from V > Y > I > O. While, according to the traditional view, the neural folds have to converge from a distance in order to contact each other, in the present mechanism the walls and folds are sequentially in contact by the ventro-dorsal zipping-up mechanism, thereby avoiding the possibility of mismatch of the neural folds. The above process is initiated over a considerable longitudinal distance along the neural plate, but only at the mesencephalic level does the dorsal shift of the contact zone become complete. At other levels of the neuraxis, the contact zone releases prematurely and the neural walls become widely separated well before their dorsal neural folds are in contact. These folds have to converge, therefore, in order to close, but their matching is facilitated by the alignment of the previously contacted neural folds at the mesencephalic level as well as by guidance underneath the vitelline membrane.

Animals↗

PubMed1997

Murine cardiac progenitor cells require visceral embryonic endoderm and primitive streak for terminal differentiation.

Cardiac progenitor cells in avian and amphibian embryos are known to commit to cardiac lineage during gastrulation or early neurulation. These cells require cell interaction with anterior endoderm for their differentiation into cardiomyocytes. However, little is known about cell interaction in mammalian cardiogenesis. We investigated the staging of murine cardiomyocyte commitment and the role of cell interaction in differentiation of cardiac progenitor cells into cardiomyocytes, using cultures of various embryonic regions at 7.25 and 7.5 days post coitum (p.c.), respectively. To evaluate the terminal differentiation of cardiac progenitor cells, we employed three parameters; expression of spontaneous beating, myosin heavy chain (MHC) protein, and cardiac-specific genes (alpha myosin heavy chain, Csx/Nkx2.5 and myosin light chain 2V genes). mRNAs of cardiac-specific genes were detected in 7.25-day p.c. mesoderm by RT-PCR, suggesting that the genetic specification to cardiac lineage initiated in the mesoderm by 7.25 days p.c. The 7.25-day p.c. isolated mesoderm in 48 hr culture, however, failed to differentiate into spontaneous beating cardiomyocytes and exhibited non-organized MHC protein in 19% of these culture. In contrast, all of the 7.5-day p.c. isolated mesoderm differentiated into beating cardiomyocytes even in 24 hr culture. The 7.25-day p.c. mesoderm associated with primitive streak increased MHC protein expression in 93% of these cultures, although they formed beating foci in 3%. The 7.25-day p.c. explants containing both visceral embryonic endoderm and primitive streak succeeded in terminal differentiation into spontaneous beating cardiomyocytes. Our study suggests that cardiac progenitor cells obtain the potency to complete terminal differentiation autonomously at 7.5 days p.c., as a consequence of the multistep induction by cell interactions with both the primitive streak and visceral embryonic endoderm, following the genetic specification to cardiac lineage in the early gastrula stage.

Animals↗

PubMed1999

Retinoic acid biosynthetic enzyme ALDH1 localizes in a subset of retinoid-dependent tissues during xenopus development.

Control of retinoic acid synthesis in vertebrate organisms is undoubtedly important for regulating the numerous retinoid signaling events which occur during development. The mechanisms which accomplish this task involve enzymes such as class I aldehyde dehydrogenase (ALDH1), which has recently been found to be conserved from amphibians to mammals and which functions as a retinoic acid biosynthetic enzyme in vivo. Here we have found that Xenopus ALDH1 mRNA and protein is expressed in a subset of retinoid-dependent tissues which develop shortly after neurulation during the tail bud stages. ALDH1 mRNA was first clearly detectable by in situ hybridization in stage 28 tail bud embryos localized in the olfactory placode and pronephros, and at stage 35 mRNA was also detected in the pronephric duct. Antibodies were generated against Xenopus ALDH1, and immunohistochemistry was used to demonstrate that ALDH1 protein accumulates in the olfactory placode, pronephros, and dorsal retina at stage 28, and additionally in the lens placode and pronephric duct at stage 35. Neither ALDH1 mRNA nor protein was detected in the posterior region of Xenopus embryos during the tail bud stages. In contrast to neurula stage embryos in which retinoic acid is distributed in an anteroposterior gradient with the high end posteriorly, we found that tail bud stage embryos have retinoic acid present in significant levels in both the head and trunk regions, but with no detection in the posterior region. These findings are consistent with ALDH1 contributing to retinoic acid synthesis needed for development of certain head structures (olfactory placodes, dorsal retina, lens placode) and certain trunk structures (pronephros and pronephric duct). Dev Dyn 1999;215:264-272.

Aldehyde Dehydrogenase↗

PubMed1996

Improving the efficacy of fluorescent labeling for histological tracking of cells in early mammalian and avian embryos.

BACKGROUND: Previously it has been difficult to localize in histological sections fluorescent dyes used to label living cells in early embryos. Fluorescent dyes typically are readily soluble in alcohol, xylene, and other common solvents used for conventional paraffin processing. Consequently, they are lost during paraffin embedment. Loss of label can be circumvented with the use of frozen sections, but this technique is laborious to use with young gastrulating and neurulating embryos and it is difficult to obtain consistently high-quality serial sections. Alternative methods such as photoconversion have been used with the fluorescent carbocyanine dye DiI. In this procedure, a diaminobenzidine (DAB)-insoluble reaction product can be deposited in the tissues of whole embryos (using UV photoconversion), which can later be viewed in conventional paraffin sections, but this method is time intensive, technically demanding, and allows for processing of only a single embryo at a time. Moreover, in our hands photoconversion produces inconsistent results and frequently yields significant nonspecific staining. METHODS: We have developed an immunohistochemically based process for demonstrating cells labeled with the fluorescent dye [5- (and -6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE)]. With this new technique, cells labeled in living embryos with CFSE can be viewed after later development, first in whole mounts and subsequently in conventional paraffin serial sections. Typically, selected regions of mouse and chick embryos are injected with dye, cultured for periods of about 24 h, viewed with fluorescence microscopy (and recorded on videotape) using an appropriate filter set, and fixed with a formaldehyde-based fixative. An antifluorescein antibody is next bound to the fluorescein groups of CFSE, and an insoluble reaction product is then generated using a horseradish peroxidase (HRP)-conjugated secondary antibody and DAB. RESULTS: The DAB reaction product deposited with our novel technique can be seen readily in whole mounts using standard stereo light microscopy, providing a permanent label. Moreover, after routine processing for paraffin embedment and histological serial sectioning, the reaction product persists, allowing detailed analyses of the positions and fates of labeled cells. CONCLUSIONS: This simple, immunocytochemical technique, tested in both mouse and chick embryos, provides a highly specific, permanent, and reproducible method for localizing descendants of fluorescently labeled living cells in conventional paraffin sections.

Animals↗