Effect of lectins and sugars on primary sperm attachment in the horseshoe crab, Limulus polyphemus L

The in vitro differentiation of quail neural crest cells into serotoninergic neurons is reported. Serotoninergic neurons were identified by two independent methods, formaldehyde-induced histofluorescence and indirect staining with antiserotonin antibodies. Serotonin-positive cells first appeared on the third day in culture, simultaneously, or slightly prior to the first pigmented cells and adrenergic neurons. Comparable numbers of serotoninergic cells were found in crest cell cultures derived from vagal, thoracic/upper lumbar, and lumbosacral levels of the neuraxis. The neural crest origin of the serotonin neurons was further corroborated by the demonstration that cultures of somites, notochords, and neural tubes (three tissues adjacent to the neural crest and thus the most likely contaminants of crest cell cultures) did not contain serotonin-producing cells, and that mast cells were absent in crest cell cultures. The identification of serotoninergic neurons in quail neural crest cell cultures makes an important addition to the number of neural crest derivatives that are capable of differentiating in culture. Furthermore, it suggests that the in vitro culture system will prove a valid approach to the elucidation of the cellular and molecular mechanisms that govern neural crest cell differentiation.     

Characterization of stage-specific embryonic antigen-1 (SSEA-1) expression during early development of the turkey embryo.

SSEA-1 is a carbohydrate epitope associated with cell adhesion, migration and differentiation. In the present study, SSEA-1 expression was characterized during turkey embryogenesis with an emphasis on its role in primordial germ cell development. During hypoblast formation, SSEA-1 positive cells were identified in the blastocoel and hypoblast and later in the germinal crescent. Based on location and morphology, these cells were identified, as PGCs. Germ cells circulating through embryonic blood vessels were also SSEA-1 positive. During the active phase of migration, PGCs in the dorsal mesentery and gonad could no longer be identified using the SSEA-1 antibody. The presence of PGCs at corresponding stages was verified using periodic acid Schiff stain. Pretreatment of PGCs with trypsin, alpha-galactosidase and neuraminidase did not restore immunoreactivity to SSEA-1. In general, expression was not limited to the germ cell lineage. SSEA-1 was also detected on the ectoderm, yolk sac endoderm, gut and mesonephric tubules. During neural tube closure, SSEA-1 was expressed by the neural epithelium of the fusing neural folds. Later SSEA-1 was detected in regions of the developing spinal cord. Enzyme pretreatment unmasked the epitope on some neural crest cells and cells in the sympathetic ganglion. The temporal and spatial distribution of SSEA-1 in the turkey embryo suggests a role in early germ cell and neural cell development. The absence of SSEA-1 on turkey gonadal germ cells was different from that observed for the chick. Therefore, while features of avian germ cell development appear to be conserved, expression of SSEA-1 can vary with the species.     

Experimental analysis of the migration and differentiation of neuroblasts of the autonomic nervous system and of neurectodermal mesenchymal derivatives, using a biological cell marking technique

By isotopic and isochronic transplantations of fragments of quail neural tube into chick, it has been previously shown that enteric ganglion cells arise from the “vagal” (somites 1–7) and the “lumbo-sacral” (behind somite 28) levels of the neural crest, while the trunk region (somites 8–28) gives rise to orthosympathetic ganglion chain and adrenomedullary cells. The latter originate precisely from the neural crest corresponding to somites 18–24 (i.e., “adrenomedullary” level of the crest). Heterotopic transplantations of fragments of quail neural tube into chick have been carried out in the present work. When the “adrenomedullary” level of the quail neural tube is grafted into the “vagal” region of a chick, the crest cells colonize the gut and differentiate into enteric ganglia of Auerbach's and Meissner's plexi. If quail cephalic neural crest is transplanted in the “adrenomedullary” level of a chick, quail cells migrate into the suprarenal glands and differentiate into adrenomedullary cells. Mesectodermal cells migrate laterally, and differentiate into cartilage, dermis and connective tissues. Thus it appears that preferential pathways located at precise levels of the embryo lead crest cells to their definitive sites. On the other hand the differentiation of the autonomic neuroblasts is controlled by the environment in which crest cells are localized at the end of their migration. On the contrary, mesenchymal derivatives of the cephalic neural crest appear to be early determined since they differentiate according to their presumptive fate when transplanted into the trunk.     

Coexpression of sensory and autonomic neurotransmitter traits by avian neural crest cells in vitro

This study shows that explants of quail neural crest cultured in a medium containing serum and chick embryo extract give rise to large numbers of cells expressing immunoreactivity for substance P (SP), a neuropeptide found in sensory neurons. These cells arise from cycling precursors, but do not appear to divide after expressing SP. The SP-positive cells in cranial neural crest cultures express both neurofilament and the Q211 antigen, but those in trunk cultures express only the Q211 antigen. In both cranial and trunk cultures, large subpopulations of the SP-positive cells express tyrosine hydroxylase and/or choline acetyltransferase, neurotransmitter markers characteristic of autonomic neurons. This finding argues against the idea that SP expression necessarily indicates commitment to the sensory neuron lineage. I further show that embryonic dorsal root ganglion (DRG) cells retain the ability to coexpress SP and tyrosine hydroxylase in vitro although to a lesser extent than do neural crest cells.     

Coexpression of sensory and autonomic neurotransmitter traits by avian neural crest cells in vitro.

This study shows that explants of quail neural crest cultured in a medium containing serum and chick embryo extract give rise to large numbers of cells expressing immunoreactivity for substance P (SP), a neuropeptide found in sensory neurons. These cells arise from cycling precursors, but do not appear to divide after expressing SP. The SP-positive cells in cranial neural crest cultures express both neurofilament and the Q211 antigen, but those in trunk cultures express only the Q211 antigen. In both cranial and trunk cultures, large subpopulations of the SP-positive cells express tyrosine hydroxylase and/or choline acetyltransferase, neurotransmitter markers characteristic of autonomic neurons. This finding argues against the idea that SP expression necessarily indicates commitment to the sensory neuron lineage. I further show that embryonic dorsal root ganglion (DRG) cells retain the ability to coexpress SP and tyrosine hydroxylase in vitro, although to a lesser extent than do neural crest cells.     

Delta signaling mediates segregation of neural crest and spinal sensory neurons from zebrafish lateral neural plate

We examined the role of Delta signaling in specification of two derivatives in zebrafish neural plate: Rohon-Beard spinal sensory neurons and neural crest. deltaA-expressing Rohon-Beard neurons are intermingled with premigratory neural crest cells in the trunk lateral neural plate. Embryos homozygous for a point mutation in deltaA, or with experimentally reduced delta signalling, have supernumerary Rohon-Beard neurons, reduced trunk-level expression of neural crest markers and lack trunk neural crest derivatives. Fin mesenchyme, a putative trunk neural crest derivative, is present in deltaA mutants, suggesting it segregates from other neural crest derivatives as early as the neural plate stage. Cranial neural crest derivatives are also present in deltaA mutants, revealing a genetic difference in regulation of trunk and cranial neural crest development.     

Cell division in the ciliary ganglion of quail embryos in situ and after back-transplantation into the neural crest migration pathways of chick embryos

Embryonic 4- to 15-day-old quail ciliary ganglia (CG) were grafted into the neural crest migration pathway of 2-day-old chick embryos at the adrenomedullary level of the neural axis. This back-transplantation results in dispersion of cells of the implanted ganglion, their migration in the host embryo, and subsequent promotion of their differentiation into a variety of neural-crest-derived cell types including adrenergic cells of the sympathetic ganglia and adrenal medulla. These cells can be recognized in the host through the nuclear marker that they carry. Here, we have analyzed quantitatively the expansion of CG-derived cell population after the graft, and compared cell division in CG after back-transplantation and during normal in situ development over the same period of time. Tritiated-thymidine [( 3H]TdR) incorporation showed that grafted CG cells proliferated during their migration and, to a greater extent, after they had homed to the host structures. Furthermore, proliferative activity of quail cells in the graft was found to be significantly higher than the growth rate of the CG cells in situ during the same period of development. In the quail donor embryo, the birthdate of the CG neurons occurred early in development; from 6 days onward, only nonneuronal cells were still dividing. When back-transplanted, the 4- to 5-day-old CG provided numerous quail cells located in autonomic structures of the host embryo. However, this increase of the total quail cell population and of cell division was reduced when CG were taken from quail donors at progressively later developmental stages. Postmitotic neurons from mature CG were found not to survive under the graft conditions. It is proposed that back-transplantation of the CG stimulates cell division and modifies the developmental programme of still undifferentiated precursor cells which then can give rise to a variety of cell types belonging either to the glial or the autonomic nerve and paraganglionic cell phenotypes, to the exclusion of sensory neurons which never derive from CG grafts.     

Expression of basic fibroblast growth factor in the nervous system of early avian embryos

Basic fibroblast growth factor (bFGF) promotes the survival of a subpopulation of non-neuronal cells developing from trunk neural crest. It was therefore important to determine whether this factor is present in the nervous system at early developmental stages. Immunocytochemistry using specific polyclonal and monoclonal antibodies was combined with three highly sensitive assays: bFGF-induced proliferation of bovine adrenal cortex-derived capillary endothelial cells (ACE), a radioimmunoassay for bFGF (RIA) and Western blot analysis. bFGF immunoreactivity was localized to the cytoplasm of neuroepithelial cells derived from embryonic day 2 (E2) quail neural tubes and cultured for one day in a chemically defined medium. Specific staining was observed in young sensory neurons in cultures of neural crest clusters as well as in a subpopulation of non-neuronal cells. In cultured E7 dorsal root ganglia, immunostaining was confined to neuronal cell bodies and fibers. In situ, staining of spinal cord and ganglionic neurons appeared on E6 and increased in intensity towards E10. Various mesoderm-derived structures such as the limb buds, the mesenchyme dorsal to the neural tube, the vertebral muscles and cartilage showed specific staining patterns in addition to neural tissue. In agreement with the results of immunocytochemical studies, 1.4ng bFGF per mg protein was detected in spinal cord extracts by RIA as early as E3, its concentration increased to 8.0 ng mg-1 on E5 and then to a maximum of 18.0 ng mg-1 protein on E10, this was followed by a subsequent decrease in concentration in older embryos. On the other hand, high levels of bFGF were present in vertebral tissues from E10 onwards. Extracts of immunopositive tissues were subjected to heparin-Sepharose affinity chromatography and eluted in a stepwise salt gradient. Fractions that eluted from the columns at 2 M NaCl contained a bFGF-like protein as revealed by their ability to stimulate the proliferation of ACE cells and by Western blot analysis. These data demonstrate that bFGF is expressed during early nervous system development in both central and peripheral neurons.     

The developmental potentials of the caudalmost part of the neural crest are restricted to melanocytes and glia

The avian spinal cord is characterized by an absence of motor nerves and sensory nerves and ganglia at its caudalmost part. Since peripheral sensory neurons derive from neural crest cells, three basic mechanisms could account for this feature: (i) the caudalmost neural tube does not generate any neural crest cells; (ii) neural crest cells originating from the caudal part of the neural tube cannot give rise to dorsal root ganglia or (iii) the caudal environment is not permissive for the formation of dorsal root ganglia. To solve this problem, we have first studied the pattern of expression of ventral (HNF3beta) and dorsal (slug) marker genes in the caudal region of the neural tube; in a second approach, we have recorded the emergence of neural crest cells using the HNK1 monoclonal antibody; and finally, we have analyzed the developmental potentials of neural crest cells arising from the caudalmost part of the neural tube in avian embryo in in vitro culture and by means of heterotopic transplantations in vivo. We show here that neural crest cells arising from the neural tube located at the level of somites 47-53 can differentiate both in vitro and in vivo into melanocytes and Schwann cells but not into neurons. Furthermore, the neural tube located caudally to the last pair of somites (i.e. the 53rd pair) does not give rise to neural crest cells in any of the situations tested. The specific anatomical aspect of the avian spinal cord can thus be accounted for by limited developmental potentials of neural crest cells arising from the most caudal part of the neural tube.     

Inhibition of the adrenergic phenotype in cultured neural crest cells by norepinephrine uptake inhibitors

Tricyclic antidepressants in combination with in vitro clonal analysis of quail neural crest cells were used to examine the role the norepinephrine uptake mechanism might play in the development of adrenergic neural crest derivatives. Norepinephrine (NE) uptake inhibitors blocked expression of the adrenergic phenotype by neural crest cells. The degree of inhibition of phenotypic expression correlated with the potency and specificity of the uptake inhibitors. The drugs acted during the early phase of in vitro development, i.e., several days before overt expression of the adrenergic phenotype in clonal culture. They were nontoxic, and a chronic exposure of the cells to NE uptake inhibitors was necessary to cause an effect. These observations suggest that norepinephrine and possibly related neurotransmitters play a direct or indirect role in the expression of the adrenergic phenotype by neural crest cells and that tricyclic antidepressants may affect neurogenesis during sensitive stages of embryonic development. The data may reflect in vivo mechanisms, since there are neurotransmitters present in the migratory pathway of presumptive sympathetic neurons and the norepinephrine uptake system is expressed in the embryo by these cells before they synthesize and accumulate catecholamines.     

The sympathoadrenal lineage in avian embryos. II. Effects of glucocorticoids on cultured neural crest cells

The neural crest-derived precursors of the sympathoadrenal lineage depend on environmental cues to differentiate as sympathetic neurons and pheochromocytes. We have used the monoclonal antibody A2B5 as a marker for neuronal differentiation and antisera against catecholamine synthesis enzymes to investigate the differentiation of catecholaminergic cells in cultures of quail neural crest cells. Cells corresponding phenotypically to sympathetic neurons and pheochromocytes can be identified in neural crest cell cultures after 5-6 days in vitro. Expression of the A2B5 antigen precedes expression of immunocytochemically detectable levels of tyrosine hydroxylase in cultured neural crest cells. Glucocorticoid treatment decreases the proportion of TH+ neural crest cells that express neuronal traits. We conclude that environmental cues normally encountered by sympathoadrenal precursors in vivo can influence the differentiation of a subpopulation of cultured neural crest cells in the sympathoadrenal lineage.     

In vivo and in vitro studies on the segregation of autonomic and sensory cell lineages

Analysis of interspecific quail/chick chimaeras (made by grafting neural primordium from one species to the other) has demonstrated that the neural crest cell population, which gives rises to a large number of derivatives, including the great majority of peripheral ganglion cells, is pluripotential. When peripheral ganglia themselves are transplanted, it can be shown that many of the developmental potentialities of the parent structure are retained, their ultimate expression depending on the microenvironment in which they become located. One of the conclusions obtained from these in vivo studies, that sensory ganglia contain dormant precursors with autonomic potentialities, has been confirmed and extended by the results of in vitro investigations with dissociated 9- to 15-day embryonic quail dorsal root ganglia. Undetectable during normal embryonic development, adrenergic properties (tyrosine hydroxylase immunoreactivity, radio- and cytochemically demonstrable catecholamine production) develop in a population of small, multipolar cells after four days in culture. This differentiation is strongly dependent on the presence of chick embryo extract in the medium. Unlike the postmitotic primary sensory neurons of the ganglia, many of the adrenergic cells were found to incorporate 3H-thymidine during the culture period. These results support the contention that the latent autonomic percursors belong to the non-neuronal compartment of sensory ganglia.     

Pax3-expressing trigeminal placode cells can localize to trunk neural crest sites but are committed to a cutaneous sensory neuron fate

The cutaneous sensory neurons of the ophthalmic lobe of the trigeminal ganglion are derived from two embryonic cell populations, the neural crest and the paired ophthalmic trigeminal (opV) placodes. Pax3 is the earliest known marker of opV placode ectoderm in the chick. Pax3 is also expressed transiently by neural crest cells as they emigrate from the neural tube, and it is reexpressed in neural crest cells as they condense to form dorsal root ganglia and certain cranial ganglia, including the trigeminal ganglion. Here, we examined whether Pax3+ opV placode-derived cells behave like Pax3+ neural crest cells when they are grafted into the trunk. Pax3+ quail opV ectoderm cells associate with host neural crest migratory streams and form Pax3+ neurons that populate the dorsal root and sympathetic ganglia and several ectopic sites, including the ventral root. Pax3 expression is subsequently downregulated, and at E8, all opV ectoderm-derived neurons in all locations are large in diameter, and virtually all express TrkB. At least some of these neurons project to the lateral region of the dorsal horn, and peripheral quail neurites are seen in the dermis, suggesting that they are cutaneous sensory neurons. Hence, although they are able to incorporate into neural crest-derived ganglia in the trunk, Pax3+ opV ectoderm cells are committed to forming cutaneous sensory neurons, their normal fate in the trigeminal ganglion. In contrast, Pax3 is not expressed in neural crest-derived neurons in the dorsal root and trigeminal ganglia at any stage, suggesting either that Pax3 is expressed in glial cells or that it is completely downregulated before neuronal differentiation. Since Pax3 is maintained in opV placode-derived neurons for some considerable time after neuronal differentiation, these data suggest that Pax3 may play different roles in opV placode cells and neural crest cells.     

Monoclonal antibodies raised against pre-migratory neural crest reveal population heterogeneity during crest development

In order to address the problem of when heterogeneity arises within premigratory and early migratory neural crest cell populations, mouse monoclonal antibodies were raised against quail premigratory neural crest. Due to the limited availability of immunogen an intrasplenic route for immunization was used. Three monoclonal antibodies (referred to as LH2D4, LH5D3 and LH6C2) were subsequently isolated which recognized subpopulations in 24 h cultures of both quail and chick mesencephalic and trunk neural crest in immunocytochemical studies. Subsequent investigations using a range of six antibodies, including LH2D4, LH5D3 and LH6C2, showed that population heterogeneity (which was not cell cycle related) could be detected as early as 15 h following mesencephalic crest explantation, a stage at which all the neural crest cells were morphologically identical. However, premigratory neural crest from the same axial level of origin was homogeneous, as judged by immunoreactivity patterns with these antibodies. Significant differences were found in the proportion of immunoreactive cells between populations of mesencephalic and trunk neural crest cultures. Double immunofluorescence studies revealed the existence of at least four separate cell populations within individual crest cultures, each identified by their unique antibody reactivity pattern, thus providing some insight into the underlying complexity of subpopulation composition within the neural crest. Immunocytochemical studies on quail embryos from stages 7-22 showed that the epitopes detected by LH2D4, LH5D3 and LH6C2 were not necessarily confined to the neural crest or to cells of crest derivation. All three epitopes displayed a spatiotemporal regulation in their expression during early avian ontogeny. Since the differential epitope expression described in this investigation was detectable as early as 15 h after premigratory neural crest explantation, took place in vitro in the absence of any other cell type and changed progressively with time, we conclude that a certain degree of population heterogeneity can be generated very early in neural crest ontogeny and independently of the tissue interactions that normally ensue in vivo.     

Differential development of cholinergic neurons from cranial and trunk neural crest cells in vitro

Several studies have suggested that the development of cholinergic properties in cranial parasympathetic neurons is determined by these cells' axial level of origin in the neural crest. All cranial parasympathetic neurons normally derive from cranial neural crest. Trunk neural crest cells give rise to sympathetic neurons, most of which are noradrenergic. To determine if there is an intrinsic difference in the ability of cranial and trunk neural crest cells to form cholinergic neurons, we have compared the development of choline acetyltransferase (ChAT)-immunoreactive cells in explants of quail cranial and trunk neural crest in vitro. Both cranial and trunk neural crest explants gave rise to ChAT-immunoreactive cells in vitro. In both types of cultures, some of the ChAT-positive cells also expressed immunoreactivity for the catecholamine synthetic enzyme tyrosine hydroxylase. However, several differences were seen between cranial and trunk cultures. First, ChAT-immunoreactive cells appeared two days earlier in cranial than in trunk cultures. Second, cranial cultures contained a higher proportion of ChAT-immunoreactive cells. Finally, a subpopulation of the ChAT-immunoreactive cells in cranial cultures exhibited neuronal traits, including neurofilament immunoreactivity. In contrast, neurofilament-immunoreactive cells were not seen in trunk cultures. These results suggest that premigratory cranial and trunk neural crest cells differ in their ability to form cholinergic neurons.     

Mapping of the early neural primordium in quail-chick chimeras. II. The prosencephalic neural plate and neural folds: implications for the genesis of cephalic human congenital abnormalities

Mapping of the avian neural primordium was carried out at the early somitic stages by substituting definite regions of the chick embryo by their quail counterpart. The quail nuclear marker made it possible to identify precisely the derivatives of the grafted areas within the chimeric cephalic structures. A fate map of the prosencephalic neural plate and neural folds is presented. Moreover the origin of the forebrain meninges from the pro- and mesencephalic neural crest is demonstrated. In the light of the data resulting from these experiments, we present a rationale for the genesis of malformations of the face and brain and of congenital endocrine abnormalities occurring in man.     

Differentiation of peptidergic neurones in quail-chick chimaeric embryos

The problem raised in this work was whether peptidergic neurones with vasoactive intestinal peptide (VIP)-and substance P-like immunoreactivity could develop in chimaeric embryos in which quail neural crest cells had been implanted into chick at an early developmental stage. Differentiation of peptide-containing nerve somas was looked for in different situations: i) when the quail neural primordium had been grafted orthotopically and isochronically into the chick host either at the adrenomedullary (level of somites 18-24) or at the vagal (level of somites 1-7) levels of the neural axis; ii) when the quail adrenomedullary neural primordium had been heterotopically implanted at the vagal level of the chick host. In all conditions, VIP- and substance P-like immunoreactivity were observed in a number of quail neurones located either in the peripheral ganglia of the trunk at the level of the graft (in orthotopic grafts of the adrenomedullary neural primordium) or in the enteric ganglia of the chick gut (in the other types of grafts). The developmental stage at which the first neurones become detectable in the host conforms to the genetic characteristics of the effector cells, i.e. they differentiate at the same stage in normal quail neuroblasts and in quail neuroblasts transplanted into the chick host. In contrast, the distribution of the peptidergic neurones in the host depends on the tissue into which the neural crest cells migrate and not on their origin in the neural axis and their fate in normal development.     

Developmental potentialities in the nonneuronal population of quail sensory ganglia

Sensory ganglia taken from quail embryos at E4 to E7 were back-transplanted into the vagal neural crest migration pathway (i.e., at the level of somites 1 to 6) of 8- to 10-somite stage chick embryos. Three types of sensory ganglia were used: (i) proximal ganglia of cranial sensory nerves IX and X forming the jugular-superior ganglionic complex, whose neurons and nonneuronal cells both arise from the neural crest; (ii) distal ganglia of the same nerves, i.e., the petrosal and nodose ganglia in which the neurons originate from epibranchial placodes and the nonneuronal cells from the neural crest; (iii) dorsal root ganglia taken in the truncal region between the fore- and hindlimb levels. The question raised was whether cells from the graft would be able to yield the neural crest derivatives normally arising from the hindbrain and vagal crest, such as carotid body type I and II cells, enteric ganglia, Schwann cells located along the local nerves, and the nonneuronal contingent of cells in the host nodose ganglion. All the grafted cephalic ganglia provided the host with the complete array of these cell types. In contrast, grafted dorsal root ganglion cells gave rise only to carotid body type I and II cells, to the nonneuronal cells of the nodose ganglion, and to Schwann cells; the ganglion-derived cells did not invade the gut and therefore failed to contribute to the host's enteric neuronal system. Coculture on the chorioallantoic membrane of aneural chick gut directly associated with quail sensory ganglia essentially reinforced these results. These data demonstrate that the capacity of peripheral ganglia to provide enteric plexuses varies according to the level of the neuraxis from which they originate.     

Partial restriction in the developmental potential of late emigrating avian neural crest cells.

Trunk neural crest cells migrate along two major pathways: a ventral pathway through the somites whose cells form neuronal derivatives and dorsolateral pathway underneath the ectoderm whose cells become pigmented. In avian embryos, the latest emigrating neural crest cells move only along the dorsolateral pathway. To test whether late emigrating neural crest cells are more restricted in developmental potential than early migrating cells, cultures were prepared from the neural tubes of embryos at various stages of neural crest cell migration. "Early" and "middle" aged neural crest cells differentiated into many derivatives including pigmented cells, neurofilament-immunoreactive cells, and adrenergic cells. In contrast, "late" neural crest cells differentiated into pigment cells and neurofilament-immunoreactive cells, but not into adrenergic cells even after 10-14 days. To further challenge the developmental potential of early and late emigrating neural crest cells, they were transplanted into embryos during the early phases of neural crest cell migration, known to be permissive for adrenergic neuronal differentiation. The cells were labeled with the vital dye, DiI, and injected onto the ventral pathway at stages 14-17. Two and three days after injection, some early neural crest cells were found to express catecholamines, suggesting they were adrenergic neuroblasts. In contrast, DiI-labeled late neural crest cells never became catecholamine-positive. These results suggest that the late emigrating neural crest cell population has a more restricted developmental potential than the early migrating neural crest cell population.