Growth Based Morphogenesis of Vertebrate Limb Bud

Many genes and their regulatory relationships are involved in developmental phenomena. However, by chemical information alone, we cannot fully understand changing organ morphologies through tissue growth because deformation and growth of the organ are essentially mechanical processes. Here, we develop a mathematical model to describe the change of organ morphologies through cell proliferation. Our basic idea is that the proper specification of localized volume source (e.g., cell proliferation) is able to guide organ morphogenesis, and that the specification is given by chemical gradients. We call this idea “growth-based morphogenesis.” We find that this morphogenetic mechanism works if the tissue is elastic for small deformation and plastic for large deformation. To illustrate our concept, we study the development of vertebrate limb buds, in which a limb bud protrudes from a flat lateral plate and extends distally in a self-organized manner. We show how the proportion of limb bud shape depends on different parameters and also show the conditions needed for normal morphogenesis, which can explain abnormal morphology of some mutants. We believe that the ideas shown in the present paper are useful for the morphogenesis of other organs.     

Cell Proliferation during Bud Formation of the Quail Uropygial Gland

A localized increase of labeling index (L.I.) was observed in bud-forming epithelium of the uropygial gland of the Japanese quail embryo through computer-aided analysis. At day 10 glandular buds are not yet formed anywhere in the epithelium and the distribution of proliferative activity in the epithelium is not differential. On the following day the peripheral buds are being formed at the externo-lateral end of the basal luminal epithelium, L.I. increases locally in these buds. At day 12 a localized increase of L.I. takes place also at the median end; extremely high labeling indices are seen at the tip of the peripheral buds and developing central buds.     

Cell Sorting and Chondrogenic Aggregate Formation in Limb Bud Recombinants and in Culture

The cartilage pattern of the developing chick limb changes along the proximal-distal (PD) axis. It is assumed that these spatial changes are brought about by differences in the cellular properties of distal mesoderm, the progress zone (PZ). To examine whether these differences are actually maintained in the individual cells composing the PZ, we dissociated early (stage 20) and late (stage 25) PZ tissues into single cells, then mixed and recombined them with ectodermal jackets. The recombinants were grafted to limb bud stumps and allowed to develop into limb-like structures. Early PZ cells were distributed within whole cartilage elements along the PD axis of the limb-like structures, while cells from late PZ participated only in the formation of distal cartilage elements.
A difference in distribution pattern between the cells of early and late PZ in mixed culture was also observed. Cells of early PZ aggregated rapidly in patches and formed cartilage nodules, while the cells of late PZ distributed in regions surrounding these cell aggregates and gradually differentiated to cartilage cells. These results suggest that the cellular properties in the PZ concerning the rate of chondrogenic aggregate formation change during limb bud development, and that this change may relate to the cartilage pattern formation along the PD axis.     

The Role of Cell Proliferation in Tubulogenesis of Human Keratinocytes

We studied kinetics and role of keratinocyte proliferation in the process of cysto- and tubulogenesis of human postnatal epidermal keratinocytes in collagen gel. The influence of media conditioned by embryonic and postnatal fibroblasts on morphogenesis has been comparatively analyzed. We studied the influence of proliferation inhibitors and inductors on tubulogenesis. The obtained data indicate partial dissociation of migration, proliferation, and differentiation of keratinocytes during morphogenetic processes in culture. We propose a new model considering proliferation of epithelial cells during cysto- and tubulogenesis.     

The Embryonic Limb Bud of the Urodele:

Scanning electron microscopy and histological investigation of the embryonic fore-limb bud of Notophthalamus (Triturus) cristatus and N. vulgaris has revealed that an apical ectodermal ridge (AER) is not detectable in these species of newt (Urodela). The limb buds examined ranged from those just visible as a slight projection from the body surface through to buds possessing two prominent digits. These stages correspond to the stages in Anurans during which the AER first appears, reaches its maximum size and then regresses. The significance of this observation is discussed.     

Knock-in Mutation Reveals an Essential Role for Focal Adhesion Kinase Activity in Blood Vessel Morphogenesis and Cell Motility-Polarity but Not Cell Proliferation

Focal adhesion kinase (FAK) associates with both integrins and growth factor receptors in the control of cell motility and survival. Loss of FAK during mouse development results in lethality at embryonic day 8.5 (E8.5) and a block in cell proliferation. Because FAK serves as both a scaffold and signaling protein, gene knock-outs do not provide mechanistic insights in distinguishing between these modes of FAK function. To determine the role of FAK activity during development, a knock-in point mutation (lysine 454 to arginine (R454)) within the catalytic domain was introduced by homologous recombination. Homozygous FAK^R454/R454 mutation was lethal at E9.5 with defects in blood vessel formation as determined by lack of yolk sac primary capillary plexus formation and disorganized endothelial cell patterning in FAK^R454/R454 embryos. In contrast to the inability of embryonic FAK^−/− cells to proliferate ex vivo, primary FAK^R454/R454 mouse embryo fibroblasts (MEFs) were established from E8.5 embryos. R454 MEFs exhibited no difference in cell growth compared with normal MEFs, and R454 FAK localized to focal adhesions but was not phosphorylated at Tyr-397. In E8.5 embryos and primary MEFs, FAK R454 mutation resulted in decreased c-Src Tyr-416 phosphorylation. R454 MEFs exhibited enhanced focal adhesion formation, decreased migration, and defects in cell polarity. Within immortalized MEFs, FAK activity was required for fibronectin-stimulated FAK-p190RhoGAP association and p190RhoGAP tyrosine phosphorylation linked to decreased RhoA GTPase activity, focal adhesion turnover, and directional motility. Our results establish that intrinsic FAK activity is essential for developmental processes controlling blood vessel formation and cell motility-polarity but not cell proliferation. This work supports the use of FAK inhibitors to disrupt neovascularization.     

Directional Cell Migration,but Not Proliferation,Drives Hair Placode Morphogenesis

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The Mechanisms of Apple Bud Morphogenesis: Analysis and a Model

Information in the literature suggests that the process of applebud morphogenesis is controlled by the level of growth substances,notably gibberellins in the bud, and by the availability ofgrowth substrate (S). The spur buds of apples must contain acertain minimum number of nodes (N_m) before flower primordiacan be formed, and the process of bud morphogenesis can be describedby a rate of node production with time, t by the equation It is postulated that the parameter k_G is a function of thebalance between the enzymes systems in the bud, the rate ofsynthesis of the enzymes associated with the development offloral bud parts (floral enzymes) being inversely related tothe level of gibberellins (G) in the bud. The parameter k_G isa function of the nutritional state of the bud; a is a rateparameter, and i is a conversion factor. Node production rateis asymptotic with increase in S, but switches from high tolow values as G varies about a critical concentration. The implicationsof the model, which appears to describe most observations, arediscussed, and some suggestions for testing it are put forward.     

The Morphogenesis of Apple Buds: II. The Development of the Bud

The formation of the terminal resting bud of apple spurs wasstudied on untreated and defoliated spurs by dissection of budscollected at intervals during the season. A distinctive patternof bud development was found in all treatments, the principalfeatures being firstly the steady rate over long periods ofthe season at which successive primordia began or completedbud-scale development, and secondly a change in rate of initiationof bud-scales several weeks after the resting bud had begunto form. Bud-scale initiation was invariably delayed in primordia inthe early period of bud development, when primordia adjacentto the apical meristem were affected directly by the foliage.The rate at which the number of mature bud-scales in the budincreased during the season was not related to the extent offoliar development, but appeared to depend upon the degree towhich the bud, during its early development, was affected bythe foliage. Whilst defoliation of spurs showed that the formation of bud-scaleswas dependent upon an effect of the foliage, no simple relationshipwas found. The number and size of leaves on defoliated spurswas much less than on untreated spurs, but the pattern of buddevelopment was very similar. Since the morphogenetic effectof foliage is known to vary with its age, the constant rateof formation of bud-scales during the season was consideredto be evidence of a second factor in the bud which counteractedvariations in effect of the foliage. A change in rate of increasein number of bud-scales could only occur therefore if the effectivelevel of one of these factors was altered independently of theother. This, it is suggested, is brought about by the accumulationof a further factor in the bud-scales, at a rate determinedduring the early formation of each primordium. From the similaritybetween the effects of these hypothetical factors and thoseof growth-regulating substances extracted from or applied tobuds of fruit trees it is thought that the second factor maybe a gibberellin-like substance, and the bud-scale factor agrowth inhibitor.     

Shoot Axillary Bud Morphogenesis in Kiwifruit (Actinidia deliciosa)

The annual cycle of kiwifruit [Actinidia deliciosa(A. Chev.)C. F. Liang et A. R. Ferguson var.deliciosacv. Hayward] shootaxillary bud (first-order axillary bud, FOAB) morphogenesisis described. FOABs developed quickly with the majority of budscales and leaf primordia present approx. 125 d after budbreak(dab). Mature FOABs had, on average, 23.2 bud scales and leafprimordia. Most second-order axillary structures were also presentapprox. 125 dab. During the growing season, the second-orderstructures developed into second-order axillary buds (SOABs)or remained as simple, dome-shaped meristems (SDSMs). At maturity,nearly all FOABs had four SOABs and, on average, 12.4 SDSMs.Most SDSMs were fused to the subtending leaf primordia, butsome SDSMs developed so that they were ‘free’ fromthe subtending leaf primordia. Third-order axillary meristems(third-order SDSMs) were observed in the axils of most SOABs,and, on average, there were 20.6 per FOAB. Our observationson the development of second-order axillary structures are consistentwith evocation in kiwifruit occurring earlier than the generally-acceptedtime of late summer. Actinidia deliciosa; bud morphogenesis; development; flowering; evocation     

Glioma Cell Proliferation Controlled by ERK Activity-Dependent Surface Expression of PDGFRA

Increased PDGFRA signaling is an essential pathogenic factor in many subtypes of gliomas. In this context the cell surface expression of PDGFRA is an important determinant of ligand sensing in the glioma microenvironment. However, the regulation of spatial distribution of PDGFRA in glioma cells remains poorly characterized. Here, we report that cell surface PDGFRA expression in gliomas is negatively regulated by an ERK-dependent mechanism, resulting in reduced proliferation of glioma cells. Glioma tumor tissues and their corresponding cell lines were isolated from 14 patients and analyzed by single-cell imaging and flow cytometry. In both cell lines and their corresponding tumor samples, glioma cell proliferation correlated with the extent of surface expression of PDGFRA. High levels of surface PDGFRA also correlated to high tubulin expression in glioma tumor tissue in vivo. In glioma cell lines, surface PDGFRA declined following treatment with inhibitors of tubulin, actin and dynamin. Screening of a panel of small molecule compounds identified the MEK inhibitor U0126 as a potent inhibitor of surface PDGFRA expression. Importantly, U0126 inhibited surface expression in a reversible, dose- and time-dependent manner, without affecting general PDGFRA expression. Treatment with U0126 resulted in reduced co-localization between PDGFRA and intracellular trafficking molecules e.g. clathrin, RAB11 and early endosomal antigen-1, in parallel with enhanced co-localization between PDGFRA and the Golgi cisternae maker, Giantin, suggesting a deviation of PDGFRA from the endosomal trafficking and recycling compartment, to the Golgi network. Furthermore, U0126 treatment in glioma cells induced an initial inhibition of ERK1/2 phosphorylation, followed by up-regulated ERK1/2 phosphorylation concomitant with diminished surface expression of PDGFRA. Finally, down-regulation of surface PDGFRA expression by U0126 is concordant with reduced glioma cell proliferation. These findings suggest that manipulation of spatial expression of PDGFRA can potentially be used to combat gliomas.     

The PICALM Protein Plays a Key Role in Iron Homeostasis and Cell Proliferation

The ubiquitously expressed phosphatidylinositol binding clathrin assembly (PICALM) protein associates with the plasma membrane, binds clathrin, and plays a role in clathrin-mediated endocytosis. Alterations of the human PICALM gene are present in aggressive hematopoietic malignancies, and genome-wide association studies have recently linked the PICALM locus to late-onset Alzheimer's disease. Inactivating and hypomorphic Picalm mutations in mice cause different degrees of severity of anemia, abnormal iron metabolism, growth retardation and shortened lifespan. To understand PICALM's function, we studied the consequences of PICALM overexpression and characterized PICALM-deficient cells derived from mutant fit1 mice. Our results identify a role for PICALM in transferrin receptor (TfR) internalization and demonstrate that the C-terminal PICALM residues are critical for its association with clathrin and for the inhibitory effect of PICALM overexpression on TfR internalization. Murine embryonic fibroblasts (MEFs) that are deficient in PICALM display several characteristics of iron deficiency (increased surface TfR expression, decreased intracellular iron levels, and reduced cellular proliferation), all of which are rescued by retroviral PICALM expression. The proliferation defect of cells that lack PICALM results, at least in part, from insufficient iron uptake, since it can be corrected by iron supplementation. Moreover, PICALM-deficient cells are particularly sensitive to iron chelation. Taken together, these data reveal that PICALM plays a critical role in iron homeostasis, and offer new perspectives into the pathogenesis of PICALM-associated diseases.     

The Role of Phosphometabolites in Cell Proliferation, Energy Metabolism, and Tumor Therapy

A common characteristic of tumor cells is the constant overexpression of glycolytic and glutaminolytic enzymes. In tumor cells the hyperactive hexokinase and the partly inactive pyruvate kinase lead to an expansion of all phosphometabolites from glucose 6-phosphate to phosphoenolpyruvate. In addition to the glycolytic phosphometabolites, synthesis of their metabolic derivatives such as P-ribose-PP, NADH, NADPH, UTP, CTP, and UDP-N-acetyl glucosamine is also enhanced during cell proliferation. Another phosphometabolite derived from P-ribose-PP, AMP, inhibits cell proliferation. The accumulation of AMP inhibits both P-ribose-PP-synthetase and the increase in concentration of phosphometabolites derived from P-ribose-PP. In cells with low glycerol 3-phosphate and malate-aspartate shuttle capacities the inhibition of the lactate dehydrogenase by low NADH levels leads to an inhibition of glycolytic ATP production. Several tumor-therapeutic drugs reduce NAD and NADH levels, thereby inhibiting glycolytic energy production. The role of AMP, NADH, and NADPH levels in the success of chemotherapeutic treatment is discussed.     

An in vitro Analogue of Early Chick Limb Bud Outgrowth

Our culture system appears to represent an in vitro analogue of early chick limb morphogenesis. Organized mesodermal cell accumulations resembling limb buds were derived from a monolayer of limb mesoderm cells when covered by limb ectoderm which included the apical ectodermal ridge (AER). The ridge retained its normal configuration when grown over a limb mesoderm monolayer and the mesoderm cells accumulated under the ridge to form a multilayered structure (10–25 cells in thickness) with the characteristic shape of a limb bud. Ectoderm which did not include the ridge failed to promote the formation of limb-like mesodermal accumulations thus the action of the ridge appears to be specific. The AER-elicited expression of mesodermal cell behaviour leading to early limb outgrowth is discussed in terms of possible morphogenetic mechanisms involved i.e. differential mitosis, cell migration, changes in cell shape and especially the adhesive properties of the cells.     

Biochemical Inhibition of Positional Signalling in the Avian Limb Bud

A region at the posterior margin of the developing avian limb bud, the zone of polarizing activity, appears to be responsible for signalling positional information along the limb antero-posterior axis. The mechanism of signalling is unknown and, unfortunately, no subcellular preparation from the polarizing region has shown polarizing activity in vivo. We have performed a series of experiments in which isolated polarizing regions were. treated with chemical agents or inhibitors prior to being grafted into anterior sites on host limb buds. A previous paper described the effects of some metabolic and biosynthetic inhibitors [5]. This paper describes the results of treatments with agents that primarily affect cell or cell surface integrity, or intracellular small molecules such as those involved in sulphydryl or cation balance. Chick and quail polarizing regions are compared, and a disaggregated cell assay is used to analyze inhibition. Drugs affecting cytostructure (colchicine, vinblastine, and cytochalasin B) inhibited the activity of polarizing regions, but did not affect the activity of treated cell suspensions, thus their action seemed dependent on retention of the agent by tissue. Inhibition observed with metabolic and biosynthetic inhibitors did not appear to involve drug retention. Agents interfering with cell surface integrity (meta-periodate, endoglycosidases, and concanavalin A) did not greatly interfere with polarizing activity at concentrations where they effectively eliminated cell spreading. Aldehyde fixatives or Triton X-100 abolished polarizing activity. Ouabain had little effect on positional signalling, but valinomycin abolished activity.     

Evidence For Cell Generation Controlled Proliferation In the Small Intestinal Crypt

Abstract. In this paper we discuss the hypothesis that cell proliferation is controlled by the number of generations after leaving an 'eternal' stem cell. the theory is based on a simulation of the kinetic behaviour of cells in the intestinal crypts. There is evidence of three, four and five generations of cells which are allowed to enter mitosis in the lower and upper part of the normal intestinal tract, and in some disease states, respectively. We suggest an internal proliferation control: some kind of knowledge that cells carry from generation to generation. It is an open question what sets and changes the generation counter: internal genetic information or external influences such as growth factors or chalones.
The geometric shape of the epithelial tissue in the intestinal tract can be understood as the steady state of a highly dynamic process. Age and death are determined from the beginning; cell-cell interaction or communication is not necessary and can be neglected. Our theory will be illustrated using the intestinal crypts as they are easily accessible, of a simple structure and completely described in the literature.