Proliferative Divisions Research Articles

Bone morphogenetic protein regulation of enteric neuronal phenotypic diversity: Relationship to timing of cell cycle exit

The effects of bone morphogenetic protein (BMP) signaling on enteric neuron development were examined in transgenic mice overexpressing either the BMP inhibitor, noggin, or BMP4 under control of the neuron specific enolase (NSE) promoter. Noggin antagonism of BMP signaling increased total numbers of enteric neurons and those of subpopulations derived from precursors that exit the cell cycle early in neurogenesis (serotonin, calretinin, calbindin). In contrast, noggin overexpression decreased numbers of neurons derived from precursors that exit the cell cycle late (gamma-aminobutyric acid, tyrosine hydroxylase [TH], dopamine transporter, calcitonin gene-related peptide, TrkC). The numbers of TH- and TrkC-expressing neurons were increased by overexpression of BMP4. These observations are consistent with the idea that phenotypic expression in the enteric nervous system (ENS) is determined, in part, by the number of proliferative divisions neuronal precursors undergo before their terminal mitosis. BMP signaling may thus regulate enteric neuronal phenotypic diversity by promoting the exit of precursors from the cell cycle. BMP2 increased the numbers of TH- and TrkC-expressing neurons developing in vitro from immunoselected enteric crest-derived precursors; BMP signaling may thus also specify or promote the development of dopaminergic TrkC/NT-3-dependent neurons. The developmental defects in the ENS of noggin-overexpressing mice caused a relatively mild disturbance of motility (irregular rapid transit and increased stool frequency, weight, and water content). Although the function of the gut thus displays a remarkable tolerance for ENS defects, subtle functional abnormalities in motility or secretion may arise when ENS defects short of aganglionosis occur during development.

Mind Bomb 1-Expressing Intermediate Progenitors Generate Notch Signaling to Maintain Radial Glial Cells

Notch signaling is critical for the stemness of radial glial cells (RGCs) during embryonic neurogenesis. Although Notch-signal-receiving events in RGCs have been well characterized, the signal-sending mechanism by the adjacent cells is poorly understood. Here, we report that conditional inactivation of mind bomb-1 (mib1), an essential component for Notch ligand endocytosis, in mice using the nestin and hGFAP promoters resulted in complete loss of Notch activation, which leads to depletion of RGCs, and premature differentiation into intermediate progenitors (IPs) and finally neurons, which were reverted by the introduction of active Notch1. Interestingly, Mib1 expression is restricted in the migrating IPs and newborn neurons, but not in RGCs. Moreover, sorted Mib1+ IPs and neurons can send the Notch signal to neighboring cells. Our results reveal that not only newborn neurons but also IPs are essential Notch-ligand-presenting cells for maintaining RGC stemness during both symmetric and asymmetric divisions.

Neuroepithelial Stem Cell Proliferation Requires LIS1 for Precise Spindle Orientation and Symmetric Division

Mitotic spindle orientation and plane of cleavage in mammals is a determinant of whether division yields progenitor expansion and/or birth of new neurons during radial glial progenitor cell (RGPC) neurogenesis, but its role earlier in neuroepithelial stem cells is poorly understood. Here we report that Lis1 is essential for precise control of mitotic spindle orientation in both neuroepithelial stem cells and radial glial progenitor cells. Controlled gene deletion of Lis1 in vivo in neuroepithelial stem cells, where cleavage is uniformly vertical and symmetrical, provokes rapid apoptosis of those cells, while radial glial progenitors are less affected. Impaired cortical microtubule capture via loss of cortical dynein causes astral and cortical microtubules to be greatly reduced in Lis1-deficient cells. Increased expression of the LIS/dynein binding partner NDEL1 restores cortical microtubule and dynein localization in Lis1-deficient cells. Thus, control of symmetric division, essential for neuroepithelial stem cell proliferation, is mediated through spindle orientation determined via LIS1/NDEL1/dynein-mediated cortical microtubule capture.

Amplification of neural stem cell proliferation by intermediate progenitor cells in Drosophila brain development

BackgroundIn the mammalian brain, neural stem cells divide asymmetrically and often amplify the number of progeny they generate via symmetrically dividing intermediate progenitors. Here we investigate whether specific neural stem cell-like neuroblasts in the brain of Drosophila might also amplify neuronal proliferation by generating symmetrically dividing intermediate progenitors.ResultsCell lineage-tracing and genetic marker analysis show that remarkably large neuroblast lineages exist in the dorsomedial larval brain of Drosophila. These lineages are generated by brain neuroblasts that divide asymmetrically to self renew but, unlike other brain neuroblasts, do not segregate the differentiating cell fate determinant Prospero to their smaller daughter cells. These daughter cells continue to express neuroblast-specific molecular markers and divide repeatedly to produce neural progeny, demonstrating that they are proliferating intermediate progenitors. The proliferative divisions of these intermediate progenitors have novel cellular and molecular features; they are morphologically symmetrical, but molecularly asymmetrical in that key differentiating cell fate determinants are segregated into only one of the two daughter cells.ConclusionOur findings provide cellular and molecular evidence for a new mode of neurogenesis in the larval brain of Drosophila that involves the amplification of neuroblast proliferation through intermediate progenitors. This type of neurogenesis bears remarkable similarities to neurogenesis in the mammalian brain, where neural stem cells as primary progenitors amplify the number of progeny they generate through generation of secondary progenitors. This suggests that key aspects of neural stem cell biology might be conserved in brain development of insects and mammals.

Par-complex proteins promote proliferative progenitor divisions in the developing mouse cerebral cortex

The size of brain regions depends on the balance between proliferation and differentiation. During development of the mouse cerebral cortex, ventricular zone (VZ) progenitors, neuroepithelial and radial glial cells, enlarge the progenitor pool by proliferative divisions, while basal progenitors located in the subventricular zone (SVZ) mostly divide in a differentiative mode generating two neurons. These differences correlate to the existence of an apico-basal polarity in VZ, but not SVZ, progenitors. Only VZ progenitors possess an apical membrane domain at which proteins of the Par complex are strongly enriched. We describe a prominent decrease in the amount of Par-complex proteins at the apical surface during cortical development and examine the role of these proteins by gain- and loss-of-function experiments. Par3 (Pard3) loss-of-function led to premature cell cycle exit, reflected in reduced clone size in vitro and the restriction of the progeny to the lower cortical layers in vivo. By contrast, Par3 or Par6 (Pard6alpha) overexpression promoted the generation of Pax6+ self-renewing progenitors in vitro and in vivo and increased the clonal progeny of single progenitors in vitro. Time-lapse video microscopy revealed that a change in the mode of cell division, rather than an alteration of the cell cycle length, causes the Par-complex-mediated increase in progenitors. Taken together, our data demonstrate a key role for the apically located Par-complex proteins in promoting self-renewing progenitor cell divisions at the expense of neurogenic differentiation in the developing cerebral cortex.

Control of planar divisions by the G-protein regulator LGN maintains progenitors in the chick neuroepithelium

The spatio-temporal regulation of symmetrical as opposed to asymmetric cell divisions directs the fate and location of cells in the developing CNS. In invertebrates, G-protein regulators control spindle orientation in asymmetric divisions, which generate progeny with different identities. We investigated the role of the G-protein regulator LGN (also called Gpsm2) in spindle orientation and cell-fate determination in the spinal cord neuroepithelium of the developing chick embryo. We show that LGN is located at the cell cortex and spindle poles of neural progenitors, and that it regulates spindle movements and orientation. LGN promotes planar divisions in the early spinal cord. Interfering with LGN function randomizes the plane of division. Notably, this does not affect cell fate, but frequently leads one daughter of proliferative symmetric divisions to exit the neuroepithelium prematurely and to proliferate aberrantly in the mantle zone. Hence, tight control of planar spindle orientation maintains neural progenitors in the neuroepithelium, and regulates the proper development of the nervous system.

Interkinetic Nuclear Migration and the Selection of Neurogenic Cell Divisions during Vertebrate Retinogenesis

During retinal development, neuroepithelial progenitor cells divide in either a symmetric proliferative mode, in which both daughter cells remain mitotic, or in a neurogenic mode, in which at least one daughter cell exits the cell cycle and differentiates as a neuron. Although the cellular mechanisms of neurogenesis remain unknown, heterogeneity in cell behaviors has been postulated to influence this cell fate. In this study, we analyze interkinetic nuclear migration, the apical-basal movement of nuclei in phase with the cell cycle, and the relationship of this cell behavior to neurogenesis. Using time-lapse imaging in zebrafish, we show that various parameters of interkinetic nuclear migration are significantly heterogeneous among retinal neuroepithelial cells. We provide direct evidence that neurogenic progenitors have greater basal nuclei migrations during the last cell cycle preceding a terminal mitosis. In addition, we show that atypical protein kinase C (aPKC)-mediated cell polarity is essential for the relationship between nuclear position and neurogenesis. Loss of aPKC also resulted in increased proliferative cell divisions and reduced retinal neurogenesis. Our data support a novel model for neurogenesis, in which interkinetic nuclear migration differentially positions nuclei in neuroepithelial cells and therefore influences selection of progenitors for cell cycle exit based on apical-basal polarized signals.

ASPM and Citron Kinase Co-Localize to the Midbody Ring during Cytokinesis

Mutations in ASPM (abnormal spindle-like microcephaly associated) and Citron Kinase (CITK) cause primary microcephaly in humans and rodents, respectively. Both proteins are expressed during neurogenesis and play important roles in neuronal progenitor cell division. ASPM is localized to the spindle pole, and is essential for maintaining proliferative cell division. CITK is present at the cytokinesis furrow and midbody ring, and it is essential for cellular abscission. We report here that ASPM also localizes to the midbody ring in mammalian cells. ASPM co-localizes with CITK at the midbody ring and co-immunoprecipitates with CITK in lysates prepared from HeLa cells and embryonic neuroepithelium. Furthermore, a GFP-tagged fragment of the N-terminus of ASPM localizes to centrosomes and spindle poles, while a GFP-tagged fragment of the C-terminus localizes to midbodies. All reported ASPM mutations that cause microcephaly involve a truncation or mutation of the C-terminus. In addition, at least two other microcephaly-related proteins, CENPJ and CDK5RAP2, previously localized to spindle poles, also localize to midbodies. Together our observations support a model of neurogenesis in which spindle dynamics and cellular abscission are coordinated.

Pinched off membrane

Cells slough off extra membrane by pinching it off into the extracellular space, say Dubreuil et al. on page 483. The discarded membrane can be released by either cilia or midbodies, with potentially distinct outcomes. Figure 1 Prominin-1 (red) and a midbody marker (green) are found on midbodies (arrowheads) and in lumenal particles (inset). Membrane particles can be seen floating in the extracellular space of the developing neural tube. These particles are distinct from signaling exosomes, leaving their origin in question. The authors now show that one origin of these particles is the midbody—the membrane structure that transiently connects two daughter cells and contains the contractile ring. The group identified the midbody as the source by examining EMs of neural tissue serial sections. They showed that particles emerge from midbodies of dividing neuroepithelial cells. The midbody particles are released at a developmental stage when these cells switch from symmetric, proliferative divisions to the asymmetric, differentiation-inducing divisions that produce neurons. Differentiation might be a consequence of particle release, as the discarded membrane is enriched in a stem cell marker called Prominin-1. If this protein confers a stem cell phenotype, its irreversible expulsion might drive differentiation. The authors are now testing whether cancer cells have lost the ability to discard the Prominin-1 particles, thus preserving the stem cell-like state. At the onset of neurogenesis, neuroepithelial cells released Prominin-1 particles that are smaller than midbodies and appear to originate instead from cilia. Their release might help to shrink the membrane of the cilium, which disappears during mitosis. The authors speculate that free-floating Prominin-1 bodies might also relay signaling information. Perhaps, for instance, the particles notify neighbors that a division has taken place as part of a size control mechanism. The components that make up this new membrane disposal pathway are still unknown, but they should be widely expressed; preliminary results indicate that epidermal and mesenchymal tissues also release midbody particles.

Arabidopsis FAMA controls the final proliferation/differentiation switch during stomatal development.

Coordination between cell proliferation and differentiation is essential to create organized and functional tissues. Arabidopsis thaliana stomata are created through a stereotyped series of symmetric and asymmetric cell divisions whose frequency and orientation are informed by cell-cell interactions. Receptor-like proteins and a mitogen-activated protein kinase kinase kinase were previously identified as negative regulators of stomatal development; here, we present the characterization of a bona fide positive regulator. FAMA is a putative basic helix-loop-helix transcription factor whose activity is required to promote differentiation of stomatal guard cells and to halt proliferative divisions in their immediate precursors. Ectopic FAMA expression is also sufficient to confer stomatal character. Physical and genetic interaction studies combined with functional characterization of FAMA domains suggest that stomatal development relies on regulatory complexes distinct from those used to specify other plant epidermal cells. FAMA behavior provides insights into the control of differentiation in cells produced through the activity of self-renewing populations.

Aspm specifically maintains symmetric proliferative divisions of neuroepithelial cells.

The ASPM (abnormal spindle-like microcephaly-associated) protein has previously been implicated in the determination of human cerebral cortical size, but the cell biological basis of this regulation has not been studied. Here we investigate the role of Aspm in mouse embryonic neuroepithelial (NE) cells, the primary stem and progenitor cells of the mammalian brain. Aspm was found to be concentrated at mitotic spindle poles of NE cells and to be down-regulated with their switch from proliferative to neurogenic divisions. Upon RNA interference in telencephalic NE cells, Aspm mRNA is reduced, mitotic spindle poles lack Aspm protein, and the cleavage plane of NE cells is less frequently oriented perpendicular to the ventricular surface of the neuroepithelium. The alteration in the cleavage plane orientation of NE cells increases the probability that these highly polarized cells undergo asymmetric division, i.e., that apical plasma membrane is inherited by only one of the daughter cells. Concomitant with the resulting increase in abventricular cells in the ventricular zone, a larger proportion of NE cell progeny is found in the neuronal layer, implying a reduction in the number of NE progenitor cells upon Aspm knock-down relative to control. Our results demonstrate that Aspm is crucial for maintaining a cleavage plane orientation that allows symmetric, proliferative divisions of NE cells during brain development. These data provide a cell biological explanation of the primary microcephaly observed in humans with mutations in ASPM, which also has implications for the evolution of mammalian brains.

Mutations in the Diageotropica (Dgt) gene uncouple patterned cell division during lateral root initiation from proliferative cell division in the pericycle

In angiosperms, root branching requires a continuous re-initiation of new root meristems. Through some unknown mechanism, in most eudicots pericycle cells positioned against the protoxylem change identity and initiate patterned division, leading to formation of lateral root primordia that further develop into lateral roots. This process is auxin-regulated. We have observed that three mutations in the Diageotropica (Dgt) gene in tomato prevent primordium formation. Detailed analysis of one of these mutants, dgt1-1, demonstrated that the mutation does not abolish the proliferative capacity of the xylem-adjacent pericycle in the differentiated root portion. Files of shortened pericycle cells found in dgt1-1 roots were unrelated to primordium formation. Auxin application stimulated this unusual proliferation, leading to formation of a multi-layered xylem-adjacent pericycle, but did not rescue the primordium formation. In contrast to wild type, auxin could not induce any cell divisions in the pericycle of the most distal dgt1-1 root-tip portion. In wild-type roots, the Dgt gene promoter was expressed strongly in lateral root primordia starting from their initiation, and on auxin treatment was induced in the primary root meristem. Auxin level and distribution were altered in dgt1-1 root tissues, as judged by direct auxin measurements, and the tissue-specific expression of an auxin-response reporter was altered in transgenic plants. Together, our data demonstrate that the Dgt gene product, a type-A cyclophilin, is essential for morphogenesis of lateral root primordia, and that the dgt mutations uncouple patterned cell division in lateral root initiation from proliferative cell division in the pericycle.

Properties of plate meristem of growing epigeal cotyledons in an experimental system

Plate meristem activity in epigeal cotyledons of plants, which are largely used in research, was comparatively studied. Formation of growing clusters of daughter cells is a common feature of cotyledon meristems. Species-specific differences connected with the number of cells entering division, the homogeneity of the palisade cell population, tissue specification of proliferation, the end number of daughter cells in clusters and the possibility to use clusters for simultaneous determination of the cell growth and division activity are shown. Exogenous cytokinin enlarges the number of cells entering proliferative divisions in the division competent tissues of the respective cotyledon and stimulates the growth of isolated cotyledons cultured in darkness. The analysis of morphological peculiarities substantiates the properties of the meristems of studied species in an experimental system and the possible light microscopy approach for using them in obtaining data on the number of divisions and accompanying cell growth. The first palisade layer of zucchini cotyledon was chosen as the most suitable experimental system reflecting the cellular behaviour during the growth of a plant organ and permitting easy and exact quantification of the cell divisions and growth simultaneously.

Phosphorylation of Neurogenin2 Specifies the Migration Properties and the Dendritic Morphology of Pyramidal Neurons in the Neocortex

The molecular mechanisms specifying the dendritic morphology of different neuronal subtypes are poorly understood. Here we demonstrate that the bHLH transcription factor Neurogenin2 (Ngn2) is both necessary and sufficient for specifying the dendritic morphology of pyramidal neurons in vivo by specifying the polarity of its leading process during the initiation of radial migration. The ability of Ngn2 to promote a polarized leading process outgrowth requires the phosphorylation of a single tyrosine residue at position 241, an event that is neither involved in Ngn2 direct transactivation properties nor its proneural function. Interestingly, the migration defect observed in the Ngn2 knockout mouse and in progenitors expressing the Ngn2(Y241F) mutation can be rescued by inhibiting the activity of the small-GTPase RhoA in cortical progenitors. Our results demonstrate that Ngn2 coordinates the acquisition of the radial migration properties and the unipolar dendritic morphology characterizing pyramidal neurons through molecular mechanisms distinct from those mediating its proneural activity.

Selective Lengthening of the Cell Cycle in the Neurogenic Subpopulation of Neural Progenitor Cells during Mouse Brain Development

During embryonic development of the mammalian brain, the average cell-cycle length of progenitor cells in the ventricular zone is known to increase. However, for any given region of the developing cortex and stage of neurogenesis, the length of the cell cycle is thought to be similar in the two coexisting subpopulations of progenitors [i.e., those undergoing (symmetric) proliferative divisions and those undergoing (either asymmetric or symmetric) neuron-generating divisions]. Using cumulative bromodeoxyuridine labeling of Tis21-green fluorescent protein knock-in mouse embryos, in which these two subpopulations of progenitors can be distinguished in vivo, we now show that at the onset as well as advanced stages of telencephalic neurogenesis, progenitors undergoing neuron-generating divisions are characterized by a significantly longer cell cycle than progenitors undergoing proliferative divisions. In addition, we find that the recently characterized neuronal progenitors dividing at the basal side of the ventricular zone and in the subventricular zone have a longer G(2) phase than those dividing at the ventricular surface. These findings are consistent with the hypothesis (Calegari and Huttner, 2003) that cell-cycle lengthening can causally contribute to neural progenitors switching from proliferative to neuron-generating divisions and may have important implications for the expansion of somatic stem cells in general.

Asymmetric distribution of the apical plasma membrane during neurogenic divisions of mammalian neuroepithelial cells.

At the onset of neurogenesis in the mammalian central nervous system, neuroepithelial cells switch from symmetric, proliferative to asymmetric, neurogenic divisions. In analogy to the asymmetric division of Drosophila neuroblasts, this switch of mammalian neuroepithelial cells is thought to involve a change in cleavage plane orientation from perpendicular (vertical cleavage) to parallel (horizontal cleavage) relative to the apical surface of the neuroepithelium. Here, we report, using TIS21-GFP knock-in mouse embryos to identify neurogenic neuroepithelial cells, that at the onset as well as advanced stages of neurogenesis the vast majority of neurogenic divisions, like proliferative divisions, show vertical cleavage planes. Remarkably, however, neurogenic divisions of neuroepithelial cells, but not proliferative ones, involve an asymmetric distribution to the daughter cells of the apical plasma membrane, which constitutes only a minute fraction (1-2%) of the entire neuroepithelial cell plasma membrane. Our results support a novel concept for the cell biological basis of asymmetric, neurogenic divisions of neuroepithelial cells in the mammalian central nervous system.

Contrasting Effects of Basic Fibroblast Growth Factor and Neurotrophin 3 on Cell Cycle Kinetics of Mouse Cortical Stem Cells

Basic fibroblast growth factor (bFGF) exerts a mitogenic effect on cortical neuroblasts, whereas neurotrophin 3 (NT3) promotes differentiation in these cells. Here we provide evidence that both the mitogenic effect of bFGF and the differentiation-promoting effect of NT3 are linked with modifications of cell cycle kinetics in mouse cortical precursor cells. We adapted an in vitro assay, which makes it possible to evaluate (1) the speed of progression of the cortical precursors through the cell cycle, (2) the duration of individual phases of the cell cycle, (3) the proportion of proliferative versus differentiative divisions, and (4) the influence on neuroglial differentiation. Contrary to what has been claimed previously, bFGF promotes proliferation via a change in cell cycle kinetics by simultaneously decreasing G1 duration and increasing the proportion of proliferative divisions. In contrast, NT3 lengthens G1 and promotes differentiative divisions. We investigated the molecular foundations of these effects and show that bFGF downregulates p27(kip1) and upregulates cyclin D2 expression. This contrasts with NT3, which upregulates p27(kip1) and downregulates cyclin D2 expression. Neither bFGF nor NT3 influences the proportion of glia or neurons in short to medium term cultures. The data point to links between the length of the G1 phase and the type of division of cortical precursors: differentiative divisions are correlated with long G1 durations, whereas proliferative divisions correlate with short G1 durations. The present results suggest that concerted mechanisms control the progressive increase in the cell cycle duration and proportion of differentiative divisions that is observed as corticogenesis proceeds.

Contrasting effects of basic fibroblast growth factor and neurotrophin 3 on cell cycle kinetics of mouse cortical stem cells.

Basic fibroblast growth factor (bFGF) exerts a mitogenic effect on cortical neuroblasts, whereas neurotrophin 3 (NT3) promotes differentiation in these cells. Here we provide evidence that both the mitogenic effect of bFGF and the differentiation-promoting effect of NT3 are linked with modifications of cell cycle kinetics in mouse cortical precursor cells. We adapted an in vitro assay, which makes it possible to evaluate (1) the speed of progression of the cortical precursors through the cell cycle, (2) the duration of individual phases of the cell cycle, (3) the proportion of proliferative versus differentiative divisions, and (4) the influence on neuroglial differentiation. Contrary to what has been claimed previously, bFGF promotes proliferation via a change in cell cycle kinetics by simultaneously decreasing G1 duration and increasing the proportion of proliferative divisions. In contrast, NT3 lengthens G1 and promotes differentiative divisions. We investigated the molecular foundations of these effects and show that bFGF downregulates p27(kip1) and upregulates cyclin D2 expression. This contrasts with NT3, which upregulates p27(kip1) and downregulates cyclin D2 expression. Neither bFGF nor NT3 influences the proportion of glia or neurons in short to medium term cultures. The data point to links between the length of the G1 phase and the type of division of cortical precursors: differentiative divisions are correlated with long G1 durations, whereas proliferative divisions correlate with short G1 durations. The present results suggest that concerted mechanisms control the progressive increase in the cell cycle duration and proportion of differentiative divisions that is observed as corticogenesis proceeds.

Anisomorphic Cell Division by African Trypanosomes

In the bloodstream of a mammalian host, African trypanosomes are pleomorphic; the shorter, non-proliferative, stumpy forms arise from longer, proliferative, slender forms with differentiation occurring via a range of morphological intermediates. In order to investigate how the onset of morphological change is co-ordinated with exit from the cell cycle we first characterized slender form cell division. Outgrowth of the new flagellum was found to occur at a linear rate, so by using outgrowth of the new flagellum as a temporal marker of the cell cycle we were able determine the order in which single copy organelles (nucleus, kinetoplast and mitochondrion) were segregated. We also found that flagellar length was an effective marker of the slender to stumpy differentiation and were, therefore, able to study both cell division and differentiation. When these differentiating cells were compared to cells undergoing proliferative cell division, they were found to be anisomorphic--showing discernible differences not only in the length of their new flagella but also in the shape and size of the cells and their nuclei.

Cell division patterns of the protoderm and root cap in the "closed" root apical meristem of Arabidopsis thaliana.

The peripheral root cap and protoderm in Arabidopsis thaliana are organized into modular packets of cells derived from formative T-divisions of the root cap/protoderm (RCP) initials and subsequent proliferative divisions of their daughter cells. Each module consists of protoderm and peripheral root cap packets derived from the same periclinal T-division event of an RCP initial. Anatomical analyses are used to interpret the history of extensively coordinated cell divisions producing this modular construction. Within a given layer of root cap, the columella and RCP initials divided in a centrifugal sequence from the innermost columella initials toward the RCP initials. All RCP initials in the lineages around the circumference of the root divided nearly simultaneously in "waves" to form one module prior to the next wave of initial divisions forming a younger module. The peripheral root cap and protoderm packets within each module completed four rounds of proliferative divisions in the axial plane to produce, on average, 16 cells per packet in the basalmost modules in axial view. Peripheral root cap and protoderm cells predominantly in the T-type (trichoblast) lineages also underwent radial divisions as they were displaced basipetally. The regularity in the cellular pattern within the modules suggests a timing mechanism controlling highly coordinated cell division in the initials and their daughter cells.