Fascicle switching generates a chiasmal neuroarchitecture in the embryonic central body of the grasshopper Schistocerca gregaria

The central body is a prominent neuropilar structure in the midbrain of the grasshopper and is characterized by a fan-shaped array of fiber columns, which are part of a chiasmal system linking anterior and posterior commissures. These columns are established during embryogenesis and comprise axons from cell clusters in the pars intercerebralis, which project to the central body via the so-called w, x, y, z tracts. Up to mid-embryogenesis the primary axon scaffold in both the brain and ventral nerve cord comprises a simple orthogonal arrangement of commissural and longitudinal fiber pathways. No chiasmata are present and this pattern is maintained during subsequent development of the ventral nerve cord. In the midbrain, individual axons entering the commissural system from each of the w, x, y, z tracts after mid-embryogenesis (55%) are seen to systematically de-fasciculate from an anterior commissure and re-fasciculate with another more posterior commissure en route across the midline, a feature we call "fascicle switching". Since the w, x, y, z tracts are bilaterally symmetrical, fascicle switching generates chiasmata at stereotypic locations across the midbrain. Choice points for leaving and entering fascicles mark the anterior and posterior positions of each future column. As the midbrain neuropil expands, the anterior and posterior groups of commissures condense, so that the chiasmata spanning the widening gap between them become progressively more orthogonally oriented. A columnar neuroarchitecture resembling that of the adult central body is already apparent at 70% of embryogenesis.     

Gliogenesis in the embryonic brain of the grasshopper Schistocerca gregaria with particular focus on the protocerebrum prior to mid-embryogenesis

I investigate the pattern of gliogenesis in the brain of the grasshopper Schistocerca gregaria prior to mid-embryogenesis, with particular focus on the protocerebrum. Using the glia-specific marker Repo and the neuron-specific marker HRP, I identify three types of glia with respect to their respective positions in the brain: surface glia form the outmost cell layer ensheathing the brain; cortex glia are intermingled with neuronal somata forming the brain cortex; and neuropil glia are associated with brain neuropils. The ontogeny of each glial type has also been studied. At 24 % of embryogenesis, a few glia are observed in each hemisphere of the proto-, deuto- and tritocerebrum. In each protocerebral hemisphere, such glia form a cluster that expands rapidly during later development. Closer examination reveals proliferative glia in such clusters at ages spanning from 24 to 36 % of embryogenesis, indicating that glial proliferation may account for the expansion of the clusters. Data derived from 33–39 % of embryogenesis suggest that, in the protocerebrum, each type of glia is likely to be generated by its respective progenitor-forming clusters. Moreover, the glial cluster located at the anterior end of the brain can give rise to both surface glia and cortex glia that populate the protocerebrum via subsequent migration. Proliferation is observed for all three glial types, indicating a possible source for the glia.     

Embryonic development of the central projection of auditory afferents (Schistocerca gregaria, Orthoptera, Insecta)

The auditory system of Schistocerca gregaria is a well investigated sensory network in the adult grasshopper. Here we present a first study on the embryonic development of this neuronal network. Focussing on the auditory receptor cells we show that they differentiate axonal processes at around 45% of embryonic development. These axons fasciculate with the intersegmental nerve and enter the central nervous system by 45-50% of development. First collaterals sprout into the major arborization area, the frontal auditory projection area of the metathoracic ganglion by 60%. This projection increases in density until an adult-like morphology is established by 90% of development. Furthermore, by the end of embryogenesis all three types of receptor fiber projections can be distinguished. This development is independent of a hearing ability, which develops much later during postembryonic life. The auditory projection co-develops with the fusion of neuromeres to the metathoracic ganglion, the formation of the target neuropile areas and the expression of the synapse associated molecule synapsin. Fasciclin I and Lachesin, both potential axon-guidance molecules, are expressed strongly on both, peripheral and central auditory pathways and, although much weaker, within the synaptic target area.     

Cell interactions involved in development of the bilaterally symmetrical intestinal valve cells during embryogenesis in Caenorhabditis elegans.

We describe two different cell interactions that appear to be required for the proper development of a pair of bilaterally symmetrical cells in Caenorhabditis elegans called the intestinal valve cells. Previous experiments have shown that at the beginning of the 4-cell stage of embryogenesis, two sister blastomeres called ABa and ABp are equivalent in development potential. We show that cell interactions between ABp and a neighboring 4-cell-stage blastomere called P2 distinguish the fates of ABa and ABp by inducing descendants of ABp to produce the intestinal valve cells, a cell type not made by ABa. A second cell interaction appears to occur later in embryogenesis when two bilaterally symmetrical descendants of ABp, which both have the potential to produce valve cells, contact each other; production of the valve cells subsequently becomes limited to only one of the two descendants. This second interaction does not occur properly if the two symmetrical descendants of ABp are prevented from contacting each other. Thus the development of the intestinal valve cells appears to require both an early cell interaction that establishes a bilaterally symmetrical pattern of cell fate and a later interaction that breaks the symmetrical cell fate pattern by restricting to only one of two cells the ability to produce a pair of valve cells.     

The boundary cap: a source of neural crest stem cells that generate multiple sensory neuron subtypes

The boundary cap (BC) is a transient neural crest-derived group of cells located at the dorsal root entry zone (DREZ) that have been shown to differentiate into sensory neurons and glia in vivo. We find that when placed in culture, BC cells self-renew, show multipotency in clonal cultures and express neural crest stem cell (NCSCs) markers. Unlike sciatic nerve NCSCs, the BC-NCSC (bNCSCs) generates sensory neurons upon differentiation. The bNCSCs constitute a common source of cells for functionally diverse types of neurons, as a single bNCSC can give rise to several types of nociceptive and thermoreceptive sensory neurons. Our data suggests that BC cells comprise a source of multipotent sensory specified stem cells that persist throughout embryogenesis.     

Joint migration of gonadotropin-releasing hormone (GnRH) and neuropeptide Y (NPY) neurons from olfactory placode to central nervous system

The olfactory epithelium in vertebrates generates the olfactory sensory neurons and several migratory cell types. Prominent among the latter are the gonadotropin-releasing hormone (GnRH) neurons that differentiate within the olfactory epithelium during embryogenesis and migrate along the olfactory nerve to the central nervous system. We initiated studies to characterize additional neuronal phenotypes of olfactory epithelial derivation. Neuropeptide Y (NPY) neurons are functionally related to the reproductive axis, modulating the release of GnRH and directly enhancing GnRH-induced luteinizing hormone (LH) secretion from gonadotrophs. We demonstrate that a population of migratory NPY neurons originates within the olfactory epithelium of the chick. At stage 25, NPY-positive fibers, but not cells, were detected in the epithelium and the nerve. By stages 28–34, NPY neurons and processes were present in the olfactory epithelium, olfactory nerve, and at the junction of the olfactory nerve and forebrain. In these regions the number of NPY neurons increased until stage 30 and then declined as development progressed. Electron microscopic immunocytochemistry confirmed the neuronal phenotype of the NPY-positive cells. The origin and migratory nature of some of these NPY cells was confirmed by double-label immunocytochemical detection of NPY and GnRH. A large percentage of the NPY-cells coexpressed the GnRH peptide. Between stages 28 and 34 single- and double-labeled NPY and GnRH neurons were found side by side along the GnRH migratory route emanating from the nasal epithelium, along the olfactory nerve, and into the ventral forebrain. These data suggest that an NPY population originates in the olfactory epithelium and migrates into the central nervous system during embryogenesis. By stage 42, no NPY/GnRH double-labeled cells were detected. © 1996 John Wiley & Sons, Inc.     

Evolutionary changes in sensory precursor formation in arthropods: embryonic development of leg sensilla in the spider Cupiennius salei

We describe here for the first time the development of mechanosensory organs in a chelicerate, the spider Cupiennius salei. It has been shown previously that the number of external sense organs increases with each moult. While stage 1 larvae do not have any external sensory structures, stage 2 larvae show a stereotyped pattern of touch sensitive ‘tactile hairs’ on their legs. We show that these mechanosensory organs develop during embryogenesis. In contrast to insects, groups of sensory precursors are recruited from the leg epithelium, rather than single sensory organ progenitors. The groups increase by proliferation, and neural cells delaminate from the cluster, which migrate away to occupy a position proximal to the accessory cells of the sense organ. In addition, we describe the development of putative internal sense organs, which do not differentiate until larval stage 2. We show by RNA interference that, similar to Drosophila, proneural genes are responsible for the formation and subtype identity of sensory organs. Furthermore, we demonstrate an additional function for proneural genes in the coordinated invagination and migration of neural cells during sensory organ formation in the spider.     

The fine structure of the newly discovered propodial ganglia of the veliger larva of the nudibranch Onchidoris bilamellata

Summary Two pairs of ganglia are found in the propodial region of the veliger of Onchidoris bilamellata: the anterolateral pair is located at the foremost corners of the propodium, and the frontal pair is located beside the propodial midline. Both sets of ganglia are positioned below the epidermis, and they are joined to the cerebral ganglia by large, common connectives. Each ganglion possesses sensory cells, nerve cells and sheath cells, and the frontal pair contains a complement of secretory cells. Externally, the propodial ganglia are manifested as sensory fields. The fields of the anterolateral pair are elliptical in shape, and each appears as a band of cilia bordering an unciliated zone. The region devoid of cilia is composed of ordinary epidermal cells, whereas the ciliated portion is comprised of dendritic endings originating from cells in the ganglion. Dendrites arise from one type of sensory cell and pass through the epidermis in bundles. Each dendrite terminates as a single cilium at the epidermal surface. Sensory fields of the frontal ganglia are key-shaped and oppose one another on the anterior end of the foot. Each field appears as a flat, circular, unciliated region which extends into a ciliated groove that runs dorsally toward the mouth. The groove contains the terminals of secretory cells, ciliated sensory cells, and the cell bodies of nonciliated sensory cells. The nonciliated sensory cells, characterized by a microvillous apex, are the dominant cells in the flattened circular zone. The space between the frontal ganglia and the epidermis is bridged by bundles of processes which are similar to those of the anterolateral ganglia. However, these tracts contain collections of the apical processes of secretory cells, the dendrites of ciliated sensory cells, and the axons of nonciliated sensory cells. Morphological and behavioral evidence indicates that the propodial ganglia serve a chemosensory function during settlement and metamorphosis.     

Immunocytological characterization of the expression of cell adhesion molecule L1 during early innervation of mouse otocysts

Summary Doubts exist as to whether afferent nerve fibers exert a neurotrophic effect on the differentiation of sensory cells in the developing vestibular neuroepithelium. To determine whether innervation of hair cells precedes their differentiation, we have used the L1 adhesion molecule as a marker for axons. The detection of L1 on afferent axons in the otic vesicle of mouse embryos on gestation day 11 shows that nerve fibers penetrate the neuroepithelium before the sensory cells differentiate. L1-immunoreactivity of nerve endings also reveals the considerable fiber ramification on gestation days 14 and 15, i.e., corresponding to the first stages of sensory cell differentiation. The expression of L1 at successive stages of nerve fiber growth in the neuroepithelium, such as fasciculation and ramification, is not consistent with the previous role proposed for L1 as a fascicule-promoting factor and raises the possibility that other mechanisms are involved in L1 mediaded adhesion.     

Bilaterally symmetrical rhopalial nervous system of the box jellyfish Tripedalia cystophora

Cubomedusae, or box jellyfish, have the most elaborate visual system of all cnidarians. They have 24 eyes of four morphological types, distributed on four sensory structures called rhopalia. Box jellyfish also display complex, probably visually guided behaviors such as obstacle avoidance and fast directional swimming. Here we describe the strikingly complex and partially bilaterally symmetrical nervous system found in each rhopalium of the box jellyfish, Tripedalia cystophora, and present the rhopalial neuroanatomy in an atlas-like series of drawings. Discrete populations of neurons and commissures connecting the left and the right side along with two populations of nonneuronal cells were visualized using several different histochemical staining techniques and electron microscopy. The number of rhopalial nerve cells and their overall arrangement indicates that visual processing and integration at least partly happen within the rhopalia. The larger of the two nonneuronal cell populations comprises approximately 2,000 likely undifferentiated cells and may support a rapid cell turnover in the rhopalial nervous system.     

A cellular network of dye-coupled glia associated with the embryonic central complex in the grasshopper Schistocerca gregaria

The central complex of the grasshopper (Schistocerca gregaria) brain comprises a modular set of neuropils, which develops after mid-embryogenesis and is functional on hatching. Early in embryogenesis, Repo-positive glia cells are found intermingled among the commissures of the midbrain, but then redistribute as central complex modules become established and, by the end of embryogenesis, envelop all midbrain neuropils. The predominant glia associated with the central body during embryogenesis are glutamine synthetase-/Repo-positive astrocyte-like glia, which direct extensive processes (gliopodia) into and around midbrain neuropils. We used intracellular dye injection in brain slices to ascertain whether such glia are dye-coupled into a communicating cellular network during embryogenesis. Intracellular staining of individual cells located at any one of four sites around the central body revealed a population of dye-coupled cells whose number and spatial distribution were stereotypic for each site and comparable at both 70 and 100% of embryogenesis. Subsequent immunolabeling confirmed these dye-coupled cells to be astrocyte-like glia. The addition of n-heptanol to the bathing saline prevented all dye coupling, consistent with gap junctions linking the glia surrounding the central body. Since dye coupling also occurred in the absence of direct intersomal contacts, it might additionally involve the extensive array of gliopodia, which develop after glia are arrayed around the central body. Collating the data from all injection sites suggests that the developing central body is surrounded by a network of dye-coupled glia, which we speculate may function as a positioning system for the developing neuropils of the central complex.     

Anatomy of neurons crossing the tritocerebral commissures of the cockroach Periplaneta americana (Blattaria)

The neuronal connections of the tritocerebral commissures of Periplaneta americana were studied in the brain-suboesophageal ganglion complex and the stomatogastric nervous system by means of heavy metal iontophoresis through cut nerve ends followed by silver intensification. The tritocerebral commissure 1 (Tc1) contains mainly the processes of the subpharyngeal nerve (Spn) whose neurons are located in both tritocerebral lobes and in the frontal ganglion. Some neurons of the frontal ganglion project through the Tc1 to the contralateral tritocerebrum. A few fibers in this commissure were observed projecting to the protocerebrum and the suboesophageal ganglion. There are tritocerebral neurons which pass through the Tc1 or the tritocerebral commissure 2 (Tc2) and extend on into the stomatogastric nervous system. One axon of a descending gaint neuron appears in the Tc2. This neuron lies in the tritocerebrum and connects the brain to the contralateral side of the ventral nerve cord. In addition, sensory fibers of the labral nerve (Ln) traverse both commissures to the opposite tritocerebrum. The anatomical and physiological relevance of the identified neuronal pathways is discussed. © 1995 Wiley-Liss, Inc.     

Visualization of proprioceptors in Drosophila larvae and pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs). Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis. The development of larval ChOs during embryogenesis has been studied extensively. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system. Multiple ChOs have been described in the adult fly. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy. In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary. Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.     

Neuronal cell death during metamorphosis of Hydractina echinata (Cnidaria, Hydrozoa)

In planula larvae of the invertebrate Hydractinia echinata (Cnidaria, Hydrozoa), peptides of the GLWamide and the RFamide families are expressed in distinct subpopulations of neurons, distributed in a typical spatial pattern through the larval body. However, in the adult polyp GLWamide or RFamide-expressing cells are located at body parts that do not correspond to the prior larval regions. Since we had shown previously that during metamorphosis a large number of cells are removed by programmed cell death (PCD), we aimed to analyze whether cells of the neuropeptide-expressing larval nerve net are among those sacrificed. By immunohistochemical staining and in situ hybridization, we labeled GLWamide- and RFamide-expressing cells. Double staining of neuropeptides and degraded DNA (TUNEL analysis) identified some neurosensory cells as being apoptotic. Derangement of the cytoplasm and rapid destruction of neuropeptide precursor RNA indicated complete death of these particular sensory cells in the course of metamorphosis. Additionally, a small group of RFamide-positive sensory cells in the developing mouth region of the primary polyp could be shown to emerge by proliferation. Our results support the idea that during metamorphosis, specific parts of the larval neuronal network are subject to neurodegeneration and therefore not used for construction of the adult nerve net. Most neuronal cells of the primary polyp arise by de novo differentiation of stem cells commited to neural differentiation in embryogenesis. At least some nerve cells derive from proliferation of progenitor cells. Clarification of how the nerve net of these basal eumetazoans degenerates may add information to the understanding of neurodegeneration by apoptosis as a whole in the animal kingdom.     

Regulation of proneural gene expression and cell fate during neuroblast segregation in the Drosophila embryo.

The Drosophila embryonic central nervous system develops from sets of progenitor neuroblasts which segregate from the neuroectoderm during early embryogenesis. Cells within this region can follow either the neural or epidermal developmental pathway, a decision guided by two opposing classes of genes. The proneural genes, including the members of the achaete-scute complex (AS-C), promote neurogenesis, while the neurogenic genes prevent neurogenesis and facilitate epidermal development. To understand the role that proneural gene expression and regulation play in the choice between neurogenesis and epidermogenesis, we examined the temporal and spatial expression pattern of the achaete (ac) regulatory protein in normal and neurogenic mutant embryos. The ac protein is first expressed in a repeating pattern of four ectodermal cell clusters per hemisegment. Even though 5-7 cells initially express ac in each cluster, only one, the neuroblast, continues to express ac. The repression of ac in the remaining cells of the cluster requires zygotic neurogenic gene function. In embryos lacking any one of five genes, the restriction of ac expression to single cells does not occur; instead, all cells of each cluster continue to express ac, enlarge, delaminate and become neuroblasts. It appears that one key function of the neurogenic genes is to silence proneural gene expression within the nonsegregating cells of the initial ectodermal clusters, thereby permitting epidermal development.     

Lateral inhibition and the development of the sensory bristles of the adult peripheral nervous system of Drosophila

Cells in the neurectoderm of Drosophila face a choice between neural and epidermal fates. On the notum of the adult fly, neural cells differentiate sensory bristles in a precise pattern. Evidence has accumulated that the bristle pattern arises from the spatial distribution of small groups of cells, proneural clusters, from each of which a single bristle will result. One class of genes, which includes the genes of the achaete-scute complex, is responsible for the correct positioning of the proneural clusters. The cells of a proneural cluster constitute an equivalence group, each of them having the potential to become a neural cell. Only one cell, however, will adopt the primary, dominant, neural fate. This cell is selected by means of cellular interactions between the members of the group, since if the dominant cell is removed, one of the remaining, epidermal, cells will switch fates and become neural. The dominant cell therefore prevents the other cells of the group from becoming neural by a phenomenon known as lateral inhibiton. They, then, adopt the secondary, epidermal, fate. A second class of genes, including the gene shaggy and the neurogenic genes mediate this process. There is some evidence that a proneural cluster is composed of a small number of cells, suggesting a contact-based mechanism of communication. The molecular nature of the protein products of the neurogenic genes is consistent with this idea.     

Regulation of latent sensory hair cell precursors by glia in the zebrafish lateral line

The lateral line is a placodally derived mechanosensory organ in anamniotes that detects the movement of water. In zebrafish embryos, a migrating primordium deposits seven to nine clusters of sensory hair cells, or neuromasts, at intervals along the trunk. Postembryonically, neuromasts continue to be added. We show that some secondary neuromasts arise from a pool of latent precursors that are deposited by the primordium between primary neuromasts. Interneuromast cells lie adjacent to the lateral line nerve and associated glia. These cells remain quiescent while they are juxtaposed with the glia; however, when they move away from the nerve they increase proliferation and form neuromasts. If glia are manually removed or genetically ablated by mutations in cls/sox10, hypersensitive (hps), or rowgain (rog), neuromasts precociously differentiate. Transplantation of wt glia into mutants rescues the appropriate temporal differentiation of interneuromast cells. Our studies reveal a role for glia in regulating sensory hair cell precursors.     

Two discrete modes of histone gene expression during oogenesis in Drosophila melanogaster

We have used in situ hybridization to ovarian tissue sections to study the pattern of histone gene expression during oogenesis in Drosophila melanogaster. Our studies suggest that there are two distinct phases of histone gene expression during oogenesis. In the first phase, which occurs during early to middle oogenesis (stages 5-10A), we observe a mosaic pattern of histone mRNA in the 15 nurse cells of the egg chamber: some cells have very high levels of mRNA, while others have little or no mRNA. Our analysis suggests that there is a cyclic accumulation and subsequent degradation of histone mRNA in the egg chamber and that very little histone mRNA is transported into the growing oocyte. Moreover, since the endomitotic replication cycles of the nurse cells are asynchronous during this period, the mosaic distribution of histone message would suggest that the expression of the histone genes in each nurse cell nucleus is probably coupled to DNA replication as in most somatic cells. The second phase begins at stage 10B. During this period, histone gene expression appears to be "induced" in all 15 nurse cells of the egg chamber, and instead of a mosaic pattern, high levels of histone mRNA are found in all cells. Unlike the earlier phase, this expression is apparently uncoupled from the endomitotic replication of the nurse cells (which are completed by the end of stage 10A). Moreover, much of the newly synthesized histone mRNA is transported from the nurse cells into the oocyte where it accumulates and is stored for use during early embryogenesis. Finally, we have also observed tightly clustered grains within nurse cell nuclei in non-denatured tissue sections. As was the case with cytoplasmic histone mRNA, there is a mosaic distribution of nuclear grains from stages 5 to 10A, while at stage 10B, virtually all nurse cell nuclei have grain clusters. These grain clusters appear to be due to the hybridization of nurse cell histone gene DNA to our probe, and are localized in specific regions of the nucleus.     

Homologous patterns in the embryonic development of the peripheral nervous system in the grasshopper Schistocerca gregaria and the fly Drosophila melanogaster.

To determine the generality of developmental mechanisms involved in the construction of the insect nervous system, the embryonic development of the peripheral nervous system in the grasshopper Schistocerca gregaria was characterized at the level of identified neurons and nerve branches and then compared to that previously described from the fly Drosophila melanogaster. For this, immunocytochemistry using a neuron-specific antibody was carried out on staged grasshopper embryos. Our results show that initially a simple peripheral nerve scaffolding is established in each segment of the animal. This scaffolding consists of a pair of intersegmental nerves that are formed by identified afferent and efferent pioneer neurons and a pair of segmental nerves that are formed by afferent pioneers situated in limb buds. Subsequently, identified sets of sensory neurons differentiate in a stereotyped spatiotemporal pattern in dorsal, lateral and ventral clusters in each segment and project their axons onto these nerves. Although segment-specific differences exist, serial homologs of the developing nerves and sensory neurons can be identified. A comparison of these results with those obtained from Drosophila shows that virtually the same pattern of peripheral nerves and sensory structures is formed in both species. This indicates that the construction of the peripheral nervous system in extremely divergent modern insects relies on conserved developmental mechanisms that evolved in ancestral insects over 300 million years ago.     

Ultrastructure of Early Secondary Embryogenesis by Multicellular and Unicellular Pathways in Cork Oak (Quercus suber L.)

Early cellular events during secondary embryogenesis were studiedin a cork oak recurrent embryogenic system in which embryosarise either in a multicellular budding pathway from a compactmass of proliferation or from isolated single cells in friablecallus. The compact mass of proliferation originated from theepidermal cells at the hypocotyl whose growth and convolutionwas characterized by a decrease in the nucleus/cytoplasm ratioand a marked increase in storage products. The transition fromthe compact mass to meristematic primordia occurred at the peripheryand was accompanied by cell dedifferentiation and a drasticreduction of storage products. Meristematic primordia evolvedto globular embryos by the organization of a protodermis andtwo internal centres. Microscope analysis of friable callusshowed an hypothetical sequence from single cells to aggregatesof a few cells, meristematic cell clusters and globular embryos.Single cells showed typical features of embryogenic cells suchas rich cytoplasm and a large number of starch grains and lipidbodies. A progressive cell dedifferentiation and a drastic reductionof storage products was observed when aggregates of a few cellsand meristematic cell clusters were compared. Progressive bipolarizationin large meristematic cell clusters initiated globular embryoformation. The comparison of both embryogenic pathways at theultrastructural level showed that subcellular changes followa similar sequential pattern, especially with regard to thestorage products. The possible role of plastid extrusions andmultivesicular bodies in the changing pattern of starch metabolismduring embryogenesis is discussed. Copyright 2001 Annals ofBotany Company Quercus suber L, cork oak, somatic embryogenesis, multicellular budding, friable callus, ultrastructural studies