碱性成纤维细胞生长因子及其受体在神经管畸形发生中作用的研究

用免疫组织化学方法研究了碱性成纤维细胞生长因子及其受体在正常受体在正常金黄地鼠和过量维生素Ａ酸致大鼠胚胎神经管及其周围间充质中,实验组在服药２２小时后,上述各部位碱性成纤维细胞生长因子及其受体含量均明显减少。说明碱性成纤维细胞生长因子及其受体的含量均明显减少。说明碱性成纤维细胞生长因子及其受体与神经管正常发生密切相关：其含量的减少可能是过量维生素Ａ酸致神经管有机制之一。     

Aberrant differentiation of the axially condensed tail bud mesenchyme in human embryos with lumbosacral myeloschisis

Development of the posterior neural tube (PNT) in human embryos is a complicated process that involves both primary and secondary neurulation. Recently, we histologically examined 20 human embryos around the stage of posterior neuropore closure and found that the axially condensed mesenchyme (AM) intervened between the neural plate/tube and the notochord in the junctional region of the primary and secondary neural tubes. The AM appeared to be incorporated into the most ventral part of the primary neural tube, and no cavity was observed in the AM. In this study, we report three cases of human embryos with myeloschisis in which the open primary neural tube and the closed secondary neural tube overlap dorsoventrally. In all three cases, part of the closed neural tube was located ventrally to the open neural tube in the lumbosacral region. The open and closed neural tubes appeared to be part of the primary and the AM‐derived secondary neural tubes, respectively. Thus, these findings suggest that, in those embryos with myeloschisis, the AM may not be incorporated into the ventral part of the primary neural tube but aberrantly differentiate into the secondary neural tube containing cavities, leading to dorsoventral overlapping of the primary and secondary neural tubes. The aberrant differentiation of the AM in embryos with lumbosacral myeloschisis suggests that the AM plays some roles in normal as well as abnormal development of the human posterior neural tube. Anat Rec, 2007. © 2007 Wiley‐Liss, Inc.     

Differentiation of the chick embryo floor plate

Summary In a number of species, the floor plate of the developing neural tube and spinal cord has been ascribed specialized functions associated with the patterning of neuronal differentiation. The differentiation of the floor plate itself is believed to be closely related to the presence of the underlying notochord. Grafting experiments have previously shown that in the chick embryo an implanted segment of notochord is capable of inducing the adjacent host neural plate or neural tube to produce an additional floor plate, although the inductive effect diminishes with increasing age of the host. We have examined the potential of notochord to promote the appearance of floor plate-like structures from neural tube tissue in culture. To facilitate this, it was necessary initially to examine the immunoreactivity of the early neural tube and floor plate in situ and in vitro with a panel of antibodies to identify a suitable marker for floor plate differentiation in vitro. In situ, the differentiation of the floor plate was characterized by a lack of immunoperoxidase staining with antibody to neurofilaments and the monoclonal antibody HNK-1 throughout the period examined. This distinguished the floor plate from other regions of the neural tube, and was in contrast to its conspicuous affinity for antibodies to N-CAM and highly sialylated N-CAM, which also stained several closely adjacent regions of the neural tube over the period examined. We also found that oligodendrocytes occurred both in the floor plate and in the flanking ventral neural tube, and that astrocytes were too poorly represented throughout the neural tube at the stages examined to be useful markers of floor plate differentiation. We therefore concluded that only the anti-neurofilament and the HNK-1 antibodies were potentially useful markers for floor plate differentiation. When these antibodies were tested on cells in culture, neural tube tissue showed the presence of neurofilament and HNK-1-positive neurites, while floor plate cultures showed few of these. These distributions were consistent with those demonstrated in situ. However, cells staining positively for N-CAM, sialylated N-CAM and the glial cell markers were relatively sparse in floor plate cultures, suggesting that these epitopes were not retained or were masked in cultured cells. As a result of these experiments, we selected the absence of neurofilament-positive cells as a marker for floor plate differentiation in culture. Co-culture of pieces of neural tube from 3-day embryos with notochord segments resulted in the suppression of neurofilament-positive neurite outgrowth from the former, and the consequent production of tissue with floor plate-like characteristics. The absence of neurites was most marked on the side of the neural tube tissue that was apposed to the notochord. Co-culture of neural tube with other tissues did not produce this effect. We suggest that the neurite suppression by notochord in vitro is analogous to its activity in situ, and that neural tubes from 3-day embryos are still competent to respond to notochordal tissue.     

Homozygous Ft embryos are affected in floor plate maintenance and ventral neural tube patterning.

Sonic hedgehog (Shh), produced by the notochord and floor plate cells of the neural tube, plays a critical role in organizing dorsal-ventral patterning in the developing neural tube. We have investigated neural tube development in mouse embryos homozygous for the Fused toes (Ft) mutation, a deletion composed of genes of the Iroquois B (IrxB) cluster and of Fts, Ftm, and Fto. In Ft mutants starting from embryonic day 10.5, the floor plate appeared to degenerate and the notochord failed to undergo ventral displacement from the spinal cord. Consistent with the loss of Shh signalling from the floor plate, V3 neuron generation was reduced in Ft/Ft embryos and the domain of motor neuron generation expanded ventrally at the expense of V2 neurons. These data support the idea that Ft genes play an important role in dorsal-ventral patterning of the neural tube acting to define the extent of motor neuron generation; moreover, the data reveal a previously unanticipated function for Ft genes in the maintenance of the floor plate.     

Expression of the murine retinol dehydrogenase 10 (Rdh10) gene correlates with many sites of retinoid signalling during embryogenesis and organ differentiation.

Retinoic acid acts as a signalling molecule regulating many developmental events in vertebrates. As this molecule directly influences gene expression by activating nuclear receptors, its patterns of synthesis have to be tightly regulated, and it is well established that at least three retinaldehyde dehydrogenases (RALDHs) are involved in such tissue-specific synthesis. Whereas embryos from oviparous species can obtain retinaldehyde by metabolizing carotenoids stored in the yolk, placental embryos rely on retinol transferred from the maternal circulation. Here, we show that the gene encoding one of the murine retinol dehydrogenases, Rdh10, is expressed according to complex profiles both during early embryogenesis and organ differentiation. Many of its expression sites correlate with regions of active retinoid signalling and Raldh gene expression, especially with Raldh2 in the early presomitic and somitic mesoderm, retrocardiac and posterior branchial arch region, or later in the pleural mesothelium and kidney cortical region. Rdh10 also shows cell-type and/or regional specificity during development of the palate, teeth, and olfactory system. During limb bud development, it may participate in retinoic acid production in proximal/posterior cells, and eventually in interdigital mesenchyme. These data implicate the retinol to retinaldehyde conversion as the first step in the tissue-specific regulation of retinoic acid synthesis, at least in mammalian embryos.     

Retinoic acid-induced neural tube defects with multiple canals in the chick: immunohistochemistry with monoclonal antibodies.

When retinoic acid was injected into chicken yolks before incubation, various types of neural tube defect (NTD) were induced in 38-46% of the embryos after 48-96 h of incubation. The cranial NTD consisted of a delay in closing of the neural plate in 48-h embryos and some remained as disorganized, hyperplastic masses in older embryos. In spinal NTD of 48-h embryos the posterior neuropore remained widely open. In older embryos with a closed posterior neuropore, the neural tube appeared dissociated or disorganized locally at the trunk level. The tissue consisted of a dorsally-situated, neural-plate-like structure and a ventrally-located cell mass containing multiple canals. Although the location was different, this arrangement was similar to the overlap zone which appears between primary and secondary neurulation in normal development. Immunohistochemistry was performed using monoclonal antibodies which selectively stained various components of chick tissue. Considering the similarity in neural tube formation between chick and human, this experimental NTD may provide clues to understanding the etiology of human myelomeningocele.     

N-CAM, polysialic acid and chick tail bud development

Summary We have previously shown that the binding of the lectin wheat germ agglutinin (WGA) to developing tail buds results in a range of caudal axial defects, which were most likely due to the affinity of the lectin for sialic acid residues. In the present study, we examined the distribution and role of a sialic acid-containing glycoprotein, N-CAM, in chick tail bud development. In the early tail bud, anti N-CAM, staining was found in the medullary cord. However, there was no uptake of an antibody specific to N-CAM containing moderate to long chains of polysialic acid (5A5 monoclonal antibody). At later stages, while N-CAM localized throughout the neural tube, staining with the 5A5 antibody was restricted to the floor plate. Sub-blastodermal injection of the anti N-CAM antibody beneath the tail bud region of HH stages 13–14 embryos produced caudal axial malformations. These malformations included the presence of accessory segments of neural tube and/or notochord, and fusion between the neural tube and underlying segment of notochord. Our results suggest that N-CAM is present during the development of the secondary neuraxis from the tail bud, although the highly sialylated form of this molecule could not be visualized until relatively late stages. N-CAM probably plays a role in the normal course of tail bud development, since perturbation of the molecule with an antibody resulted in malformations. Since these malformations were similar to those we have previously reported when we treated similarly staged chick embryos with WGA, there is a possibility that the sialic acid residues recognized and bound by the lectin are those associated with the N-CAM molecule.     

Cephalic neurulation in the mouse embryo analyzed by SEM and morphometry

A detailed account of mouse neurulation is given based mostly on SEM analysis over 20 hr of development. Many observations and measurements were made on staged living embryos and on embryos prepared for scanning and light microscopy to help deduce what mechanisms may contribute to neural tube formation. Each-lateral half of the early cephalic neural plate makes a convex bulge, opposite to the way it must fold to form a tube. Underlying mesenchyme and matrix are reported to have a role in forming these bulges. Processes that form the tube must overcome this opposed folding and the forces that produce it. Cranial flexure begins long before tube formation. The flexure commences at the rostral tip of the cephalic neural plate, then the apex of the flexure migrates caudally to the mesencephalic region. Early appearance of this flexure imposes a mechanical impediment to tube closure in forebrain and midbrain regions. Tube closure begins in the cervical region exactly where the neural plate is reflected dorsally by a bend in the embryo. This bend may mechanically assist closure in this region. Cells of the mouse neural plate are reported to contain organized microfilaments and microtubules, and the plate cells appear to change shape (reduce apical area and increase cell height) in the same manner as that suggested in embryos of some other species to contribute to neural tube formation. Measurements show that the lateral edges of the cephalic neural plate elongate craniocaudally more than the midline of the plate through each period. This elongation could contribute to the folding of the plate into a tube. The progress of cranial ventral flexure pauses while tube formation occurs, but edge elongation continues, presumably contributing to tube formation. There is considerable increase in volume of the neural plate during tube closure, and cell proliferation and enlargement of daughter cells seem sufficient to account for this growth. Mitotic spindles are positioned to place the majority of the daughter cells into the long axis of the neural plate, so ordered growth may be the main mechanism of elongation of the plate in the craniocaudal direction, which in turn may assist in tube formation. Mouse cephalic neural plates appear overlying already segmented cranial mesenchyme according to previous reports, and neuromeres develop precociously in the open plates, where their positions correlate exactly with the underlying segmented mesenchyme.     

灵芝孢子降低维甲酸诱导的孕鼠胚胎神经管畸形的发生

目的 探讨灵芝孢子能否促进受维甲酸诱导,而停滞在G0/G1期的胚胎神经管神经上皮细胞重新进入细胞周期循环,继续增殖和分化,减少神经管畸形的发生.方法 于实验对照组和灵芝孢子组小鼠孕期胚胎E17.75d时一次性胃饲全反式维甲酸,造成这两组孕鼠的胚胎出现神经管畸形.然后,给予灵芝孢子组孕鼠胃饲灵芝孢子溶液.在孕期E10.5d时取出两组孕鼠的胚胎,分别计数胚胎的神经管畸形发生率.应用免疫荧光组织化学染色和流式细胞仪检测胚胎神经管神经上皮的巢蛋白(nestin)表达情况.应用DNA定量荧光染色和RT-PCR技术检测胚胎神经管依赖细胞周期蛋白激酶2(Cdk2) mRNA和Cdk4 mRNA的转录.结果 灵芝孢子组孕鼠胚胎的神经管畸形发生率显著低于实验对照组.神经管神经上皮细胞表达nestin的百分率也明显大于实验对照组.实验对照组孕鼠胚胎的神经管细胞在G0/G1期的比例则显著高于灵芝孢子组,但是实验对照组神经管细胞S期的比例低,低于灵芝孢子组形态正常的胚胎神经管细胞.RT-PCR检测发现,灵芝孢子组孕鼠胚胎神经管细胞能够正常转录Cdk4 mRNA,而实验对照组则低转录Cdk4 mRNA.结论 灵芝孢子能够减少维甲酸诱导的妊娠小鼠孕期E10.5d胚胎神经管畸形的发生.     

脑源性神经营养因子在早期人胚神经管发育中的定位表达

目的研究脑源性神经营养因子(BDNF)在早期人胚神经管发育过程中的定位表达。方法采用免疫细胞化学ABC法染色,研究35天人胚的发育情况。结果在人胚神经管的室带中,神经元的细胞质BDNF免疫反应阳性;在中间带,神经元的突起BDNF免疫反应阳性,一部分神经元的细胞核BDNF免疫反应阳性,另外一部分神经元的细胞核BDNF免疫反应阴性;在缘带BDNF的分布与中间带相似。神经管的头侧较尾侧BDNF阳性反应较强,神经管的腹侧BDNF阳性反应较背侧强。结论BDNF在人胚神经管免疫反应阳性,表明BDNF是诱导神经管分化发育的重要信号分子,提示BDNF在人胚神经管的发育中具有十分重要的作用。     

Conserved RARE localization in amphioxus Hox clusters and implications for Hox code evolution in the vertebrate neural crest.

The Hox code in the neural crest cells plays an important role in the development of the complex craniofacial structures that are characteristic of vertebrates. Previously, 3' AmphiHox1 flanking region has been shown to drive gene expression in neural tubes and neural crest cells in a retinoic acid (RA)-dependent manner. In the present study, we found that the DR5-type RA response elements located at the 3' AmphiHox1 flanking region of Branchiostoma floridae are necessary and sufficient to express reporter genes in both the neural tube and neural crest cells of chick embryos, specifically at the post-otic level. The DR5 at the 3' flanking region of chick Hoxb1 is also capable of driving the same expression in chick embryos. We found that AmphiHox3 possesses a DR5-type RARE in its 5' flanking region, and this drives an expression pattern similar to the RARE element found in the 3' flanking region of AmphiHox1. Therefore, the location of these DR5-type RAREs is conserved in amphioxus and vertebrate Hox clusters. Our findings demonstrate that conserved RAREs mediate RA-dependent regulation of Hox genes in amphioxus and vertebrates, and in vertebrates this drives expression of Hox genes in both neural crest and neural tube. This suggests that Hox expression in vertebrate neural crest cells has evolved via the co-option of a pre-existing regulatory pathway that primitively regulated neural tube (and possibly epidermal) Hox expression.     

Female predisposition to cranial neural tube defects is not because of a difference between the sexes in the rate of embryonic growth or development during neurulation.

The susceptibility of females to anencephaly is well established and has been suggested to result from a slower rate of growth and development of female embryos during cranial neurulation. We have tested this hypothesis by measuring the rates of growth and development, both in utero and in vitro, of male and female embryos of the curly tail (ct) mutant mouse strain, in which cranial neural tube defects occur primarily in females. Embryonic growth was assessed by increase in protein content, while development progression was judged from increase in somite number and morphological score. Embryos were sexed by use of the polymerase chain reaction to amplify a DNA sequence specific to the Y chromosome, and by sex chromatin analysis. We find that, during neurulation (between 8.5 and 10.5 days of gestation), males are advanced in growth and development relative to their female litter mates, but that the rates of growth and development do not differ between the sexes during this period. We conclude that rate of embryonic growth and development is unlikely to determine susceptibility to cranial neural tube defects. It seems more likely that male and female embryos differ in some specific aspect(s) of the neurulation process that increases the susceptibility of females to development of anencephaly.     

Enforced expression of the transcription factor HOXD3 under the control of the Wnt1 regulatory element modulates cell adhesion properties in the developing mouse neural tube

HOX genes expressed in a specific spatial and temporal manner play a crucial role in determining the body plan during the early development of vertebrates. In adult tissues, many HOX genes participate in normal hematopoiesis and carcinogenesis. We previously found that overexpression of the homeobox gene HOXD3 alters expression levels of cell adhesion molecules in human cancer cell lines. Here, we have investigated whether HOXD3 expression is related to the cell adhesion processes during mouse development focusing on dorsal midline cells or roof-plate cells of the neural tube and neural crest cells. We created transgenic mouse embryos, in which HOXD3 is expressed in the dorsal midline under the control of the Wnt1 regulatory element, and analyzed these embryos at embryonic day 10.5-13.5. In HOXD3-expressing transgenic embryos, although neural crest-derived structures in the trunk region appeared to be normal, striking abnormalities were found in the neural tube. In transgenic embryos expressing the lacZ gene under the control of the Wnt1 regulatory element, expression of lacZ was restricted to roof-plate cells within the neural tube. By contrast, in HOXD3-expressing transgenic embryos, expression of HOXD3 was not only located in the dorsal neural tube, but also had spread inside the ventricular zone in more ventral regions of the neural tube. These findings show that the HOXD3 transgene is expressed more broadly than the Wnt1 gene is normally expressed. Expression of both Wnt1 and Msx1, marker genes in the roof plate, was further extended ventrally in HOXD3-expressing embryos than in normal embryos, suggesting that expression of the HOXD3 transgene expands the roof plate ventrally within the neural tube. In the ventricular zone of HOXD3-expressing embryos at embryonic day 10.5, we observed an increase in the number of mitotic cells and failure of interkinetic nuclear migration of progenitor cells. Furthermore, in HOXD3-expressing embryos at embryonic day 12.5, the ventricular zone, in which progenitor cells became more loosely connected to each other, was composed of a large number of cells that did not express N-cadherin. Our results indicate that expression of HOXD3 is closely associated with modulation of cell-adhesive properties during embryonic development.     

Histological and ultrastructural studies of secondary neurulation in mouse embryos

The histological and ultrastructural features of secondary neurulation in C57BL/6 mouse embryos were examined as a first step in the analysis of how this process occurs in mammalian embryos. Secondary neurulation involves two major events in mouse embryos: (1) formation of the medullary rosette (9.5- to 10-day embryos) or plate (11- to 12-day embryos), and (2) cavitation. These two events occur simultaneously. The medullary rosette consists of elongated tail bud cells, radially arranged around a central lumen formed by cavitation. The secondary portion of the neural tube forms in 9.5- to 10-day embryos by progressive enlargement of the central lumen and addition (by cell recruitment or mitosis) of tail bud cells to the rosette. The medullary plate likewise consists of elongated tail bud cells, but these cells do not surround a central cavity. Instead, cells of the medullary plate extend ventrad from the basal aspect of the dorsal surface ectoderm to a slit-like cavity formed by cavitation. Formation of the secondary neural tube occurs in 11- to 12-day embryos, principally by the recruitment of more lateral and ventral tail bud cells into the medullary plate. Free cells and cellular debris are frequently encountered in the forming lumen of the secondary neural tube, but cells exhibiting signs of necrosis were absent in cavitating regions. Numerous small intercellular junctions form at the inner (juxtaluminal) ends of tail bud cells as the medullary rosette or plate is forming and cavitation is occurring. These observations suggest that cavitation per se (i.e., formation of a lumen) during secondary neurulation is a relatively passive phenomenon, which results principally from neighboring cells becoming polarized apicobasally and incorporated into a primitive neuroepithelium. The latter constitutes the walls of the forming secondary neural tube.     

Cyclops-independent floor plate differentiation in zebrafish embryos.

In zebrafish, development of the ventral neural tube depends on the Nodal-related signal Cyclops (Cyc). One-day-old cyc mutant embryos lack the medial floor plate (MFP). We show here that cells expressing MFP marker genes differentiate gradually in cyc mutant embryos in a delayed manner during the second day of development. This late differentiation is restricted to the hindbrain and spinal cord and depends on an intact Hedgehog (Hh) signalling pathway. Cells expressing MFP marker genes in cyc mutant embryos appear to be derived from lateral floor plate (LFP) cells as they coexpress LFP and MFP marker genes. This finding suggests that the correct temporal development of the MFP is required for the distinction of LFP and MFP cells in wild-type embryos.     

NGF及其受体在人胚神经管早期发育中的表达

应用免疫组织化学方法研究NGF及其受体在人胚神经管早期发育中的表达。结果显示,NGF在第3周未检测到,第4周出现阳性着色,随着胚龄增加,免疫阳笥着色逐渐增强。第3周,可检测到少数散在TrkA免疫阳性细胞,随着胚胎发育,阳性细胞逐渐增多,色强度逐渐增强,TrkA、表达时序和分布与NGF基本一致。结果表明,NGF通过其特异性受体介导,在胚胎神经管形成和分化的不同阶段发挥着重要作用。     

Folic acid and methionine in the prevention of teratogen-induced congenital defects in mice

IntroductionPericonceptional supplementation with multivitamins containing folic acid reduces the risk of congenital malformations. We have previously investigated the effect on the murine development of a multiple retinoic acid competitive antagonist, Bristol-Myers-Squibb 189453, showing that treated fetuses were affected with heart defects, thymus aplasia or hypoplasia, and severe anomalies of the central nervous system. Hereby, we analyzed the effects of nutritive therapy involving folic acid and methionine on teratogen-induced congenital defects in mice.Materials and methodsA total of 132 outbred CD1 litters were studied. Pregnant mice were divided into four experimental groups, and an oral supplementation of H₂O or folic acid, or methionine, or folic acid+methionine was administered from 0.5 days postcoitum until the end of pregnancy. At 7.5 days postcoitum, mice from all these groups were administered Bristol-Myers-Squibb 189453 to induce the teratogenic effect. At the end of pregnancy, fetuses were dissected and tissues were analyzed by histology and flow cytometric assays.ResultsFolic acid reduces congenital heart diseases from 81.3% to 64.8%, neural tube defects from 20.3% to 3.7%, and thymus abnormalities from 98.4% to 27.8%, restoring a normal number of differentiated thymus cells. Methionine is less effective in contrasting congenital heart diseases and neural tube defects, and induces thymus cell proliferation but not differentiation. Folic acid+methionine weakly reduce congenital heart diseases and neural tube defects, but consistently reduce the incidence of fetuses affected with thymus pathologies from 98.4% to 67.7%.ConclusionsOur results suggest that folic acid and methionine periconceptional supplementations may influence the incidence of congenital defects and may probably induce negative selection of embryos presenting developmental anomalies.     

Development of the posterior neural tube in human embryos

Development of the posterior neural tube (PNT) in human embryos is a complicated process that involves both primary and secondary neurulation. Because normal development of the PNT is not fully understood, pathogenesis of spinal neural tube defects remains elusive. To clarify the mechanism of PNT development, we histologically examined 20 human embryos around the stage of posterior neuropore closure and found that the developing PNT can be divided into three parts: 1) the most rostral region, which corresponds to the posterior part of the primary neural tube, 2) the junctional region of the primary and secondary neural tubes, and 3) the caudal region, which emerges from the neural cord. In the junctional region, the axially-condensed mesenchyme (AM) intervened between the neural plate/tube and the notochord at the stage of posterior neuropore closure, while the notochord was directly attached to the neural plate/tube in the most rostral region. A single cavity was found to be formed in the AM as the presumptive luminal surface cells were radially aligned in the junctional region prior to the formation of the neural cord. The single cavity was continuous with the central cavity of the primary neural tube. In contrast, multiple or isolated cavities were frequently observed in the caudal region of the PNT. Our observation suggests that the junctional region of the PNT is distinct from other regions in terms of the relationship with the notochord and the mode of cavitation during secondary neurulation.     

Differentiation of the neural plate and neural tube in the young chick embryo. A study by scanning and transmission electron microscopy.

The differentiation of the presumptive neural plate, the neural plate and the neural tube have been investigated in the chick embryo by SEM, TEM and histochemical techniques. The relationship of these tissues to neighbouring structures, including extracellular materials, has also been studied. When SEM micrographs of primitive streak stage embryos were examined in stereo, it was found that cells which had been invaginating at the time of fixation were similar in shape to fibroblasts migrating in vitro. It was concluded that SEM stereo pairs could provide evidence about the mode and direction of cell migration. Many more mid-bodies have been found associated with the developing neural tissue than with the lateral ectoderm. It was found possible to recognise mid-bodies not only by TEM but also by SEM. It is therefore proposed that SEM montages may be used for assessing which regions of a tissue have recently undergone extensive mitosis. The beads on the specialised threads seen in the early stages of development are now considered to be formed from mid-bodies. Similar, but unbeaded threads have been described which span the gap between the neural folds just prior to the dorsal closure of the neural tube and it seems probably that these threads help to close the neural tube. It is suggested that the beaded threads arise by incomplete separation of two daughter cells at mitosis, whereas the unbeaded threads form by outgrowth of cell processes.