Ameliorative effects of Magnolia sieboldii buds hexane extract on experimental reflux esophagitis.

Gastroesophageal reflux disease (GERD) is a disease that stomach contents continually refluxing into esophagus causes symptoms and/or complications. The study was working to find natural plant extracts with good effects and small side effects to treat reflux esophagitis (RE). The anti-inflammatory effects of hexane extract of Magnolia sieboldii (MsHE) were conducted on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. The ameliorative effects of MsHE on esophageal damage in rats induced by gastric acid reflux was explored in vivo. The results showed that MsHE decreased the production of nitric oxide (NO) and expression levels of iNOS, COX-2 and TNF-α on LPS-stimulated RAW 264.7 cells and MsHE treatment ameliorated the rats' esophageal tissue damage induced by gastric acid and inhibited the increase of inflammatory mediators and pro-inflammatory cytokines by regulating NF-κB signaling pathway. In addition, MsHE protected the function of barrier of epithelial cells against inflammatory conditions through increasing the expression of tight junctions. Furthermore, liquid chromatography-mass spectrometry analysis was used for determine the active ingredients contained in MsHE. The results show that MsHE can alleviate experimental rat RE by regulating NF-κB signaling pathway. In summary, MsHE may be used as a source material of drug candidate for the treatment of RE.

SOX13 exhibits a distinct spatial and temporal expression pattern during chondrogenesis, neurogenesis, and limb development.

SOX13 is a member of the SOX family of transcription factors. SOX proteins play essential roles in development, and some are associated with human genetic diseases. SOX13 maps to a multi-disease locus on chromosome 1q31-32, yet its function is unknown. Here we describe the temporal and spatial expression of SOX13 protein during mouse organogenesis. SOX13 is expressed in the three embryonic cell lineages, suggesting that it may direct various developmental processes. SOX13 is expressed in the developing central nervous system including the neural tube and the developing brain. Expression is also detected in the condensing mesenchyme and cartilage progenitor cells during endochondral bone formation in the limb as well as the somite sclerotome and its derivatives. SOX13 is also detected in the developing kidney, pancreas, and liver as well as in the visceral mesoderm of the extra-embryonic yolk sac and spongiotrophoblast layer of the placenta.

Nuclear import of cellular retinoic acid-binding protein type I in mouse embryonic cells.

Using confocal microscopy we show that cellular retinoic acid-binding protein type I (CRABP I), expressed in several embryonic cell types, displays a compartmentalized subcellular distribution. The protein was excluded from the nucleus in some cells, while in others it accumulated in the nucleus. In the rat cerebellar cell line ST15A, which expresses CRABP I, the protein was found in the cytoplasm with a prominent nuclear exclusion. Addition of retinoic acid to embryos in vivo and to ST15 A cells in vitro did not affect the localization of the protein. Localization of CRABP I and CRABP I fused to a nuclear localization signal expressed in transfected cells, suggested that cell-specific factors may regulate nuclear import of CRABP I. The potential role of a CRABP I-controlled nuclear import of retinoic acid is discussed.

Developmental expression patterns of mouse sFRP genes encoding members of the secreted frizzled related protein family.

Development of the metanephric kidney is an experimental model system to analyze interactions between mesenchymal and epithelial cells and mesenchymal-epithelial transition. To study the underlying genetic mechanisms we employed organ culture and differential display PCR to identify genes regulated upon induction of mesenchymal cells. One of the genes found encodes the secreted frizzled related protein 2 (sFRP2) that is upregulated within 2 days of in vitro development. In vivo sFRP2 expression was likewise found in mesenchymal condensates and subsequent epithelial structures. Detailed in situ hybridization analysis revealed sFRP2 expression during development of the eye, brain, neural tube, craniofacial mesenchyme, joints, testis, pancreas and below the epithelia of oesophagus, aorta and ureter where smooth muscles develop. In a comparative analysis transcripts of the related sFRP1 and sFRP4 genes were frequently found in the same tissues as sFRP2 with their expression domains overlapping in some instances, but mutually exclusive in others. While sFRP1 is specifically expressed in the embryonic metanephros, eye, brain, teeth, salivary gland and small intestine, there is only weak expression of sFRP4 except for the developing teeth, eye and salivary gland. The interpretation of the highly specific spatial and temporal expression patterns of sFRP genes will partly depend on a better functional understanding of the interaction between wnt, fz and sFRP family members. Nevertheless, sFRP genes must play quite distinct roles in the morphogenesis of several organ systems.

Spatio-temporal expression of FGFR 1, 2 and 3 genes during human embryo-fetal ossification.

Mutations in FGFR 1-3 genes account for various human craniosynostosis syndromes, while dwarfism syndromes have been ascribed exclusively to FGFR 3 mutations. However, the exact role of FGFR 1-3 genes in human skeletal development is not understood. Here we describe the expression pattern of FGFR 1-3 genes during human embryonic and fetal endochondral and membranous ossification. In the limb bud, FGFR 1 and FGFR 2 are initially expressed in the mesenchyme and in epidermal cells, respectively, but FGFR 3 is undetectable. At later stages, FGFR 2 appears as the first marker of prechondrogenic condensations. In the growing long bones, FGFR 1 and FGFR 2 transcripts are restricted to the perichondrium and periosteum, while FGFR 3 is mainly expressed in mature chondrocytes of the cartilage growth plate. Marked FGFR 2 expression is also observed in the periarticular cartilage. Finally, membranous ossification of the skull vault is characterized by co-expression of the FGFR 1-3 genes in preosteoblasts and osteoblasts. In summary, the simultaneous expression of FGFR 1-3 genes in cranial sutures might explain their involvement in craniosynostosis syndromes, whereas the specific expression of FGFR 3 in chondrocytes does correlate with the involvement of FGFR 3 mutations in inherited defective growth of human long bones.

Hedgehog can drive terminal differentiation of amniote slow skeletal muscle.

BACKGROUND: Secreted Hedgehog (Hh) signalling molecules have profound influences on many developing and regenerating tissues. Yet in most vertebrate tissues it is unclear which Hh-responses are the direct result of Hh action on a particular cell type because Hhs frequently elicit secondary signals. In developing skeletal muscle, Hhs promote slow myogenesis in zebrafish and are involved in specification of medial muscle cells in amniote somites. However, the extent to which non-myogenic cells, myoblasts or differentiating myocytes are direct or indirect targets of Hh signalling is not known. RESULTS: We show that Sonic hedgehog (Shh) can act directly on cultured C2 myoblasts, driving Gli1 expression, myogenin up-regulation and terminal differentiation, even in the presence of growth factors that normally prevent differentiation. Distinct myoblasts respond differently to Shh: in some slow myosin expression is increased, whereas in others Shh simply enhances terminal differentiation. Exposure of chick wing bud cells to Shh in culture increases numbers of both muscle and non-muscle cells, yet simultaneously enhances differentiation of myoblasts. The small proportion of differentiated muscle cells expressing definitive slow myosin can be doubled by Shh. Shh over-expression in chick limb bud reduces muscle mass at early developmental stages while inducing ectopic slow muscle fibre formation. Abundant later-differentiating fibres, however, do not express extra slow myosin. Conversely, Hh loss of function in the limb bud, caused by implanting hybridoma cells expressing a functionally blocking anti-Hh antibody, reduces early slow muscle formation and differentiation, but does not prevent later slow myogenesis. Analysis of Hh knockout mice indicates that Shh promotes early somitic slow myogenesis. CONCLUSIONS: Taken together, the data show that Hh can have direct pro-differentiative effects on myoblasts and that early-developing muscle requires Hh for normal differentiation and slow myosin expression. We propose a simple model of how direct and indirect effects of Hh regulate early limb myogenesis.

Reduced-folate carrier (RFC) is expressed in placenta and yolk sac, as well as in cells of the developing forebrain, hindbrain, neural tube, craniofacial region, eye, limb buds and heart.

BACKGROUND: Folate is essential for cellular proliferation and tissue regeneration. As mammalian cells cannot synthesize folates de novo, tightly regulated cellular uptake processes have evolved to sustain sufficient levels of intracellular tetrahydrofolate cofactors to support biosynthesis of purines, pyrimidines, and some amino acids (serine, methionine). Though reduced-folate carrier (RFC) is one of the major proteins mediating folate transport, knowledge of the developmental expression of RFC is lacking. We utilized in situ hybridization and immunolocalization to determine the developmental distribution of RFC message and protein, respectively. RESULTS: In the mouse, RFC transcripts and protein are expressed in the E10.0 placenta and yolk sac. In the E9.0 to E11.5 mouse embryo RFC is widely detectable, with intense signal localized to cell populations in the neural tube, craniofacial region, limb buds and heart. During early development, RFC is expressed throughout the eye, but by E12.5, RFC protein becomes localized to the retinal pigment epithelium (RPE). CONCLUSIONS: Clinical studies show a statistical decrease in the number of neural tube defects, craniofacial abnormalities, cardiovascular defects and limb abnormalities detected in offspring of female patients given supplementary folate during pregnancy. The mechanism, however, by which folate supplementation ameliorates the occurrence of developmental defects is unclear. The present work demonstrates that RFC is present in placenta and yolk sac and provides the first evidence that it is expressed in the neural tube, craniofacial region, limb buds and heart during organogenesis. These findings suggest that rapidly dividing cells in the developing neural tube, craniofacial region, limb buds and heart may be particularly susceptible to folate deficiency.

Extra-pituitary growth hormone in peripheral tissues of early chick embryos.

Early embryonic growth is independent of pituitary growth hormone (GH), since it occurs prior to the differentiation of pituitary somatotrophs. Embryogenesis is therefore thought to be regulated by local growth factors. As GH is now known to be produced in many extrapituitary sites, in which it acts in an autocrine or paracrine manner, the possibility that extra-pituitary GH may participate in embryogenesis and organogenesis was assessed by determining the immunocytochemical presence and location of GH- and GH-receptor (GHR)-like proteins in the peripheral tissues of chick embryos during their 21-day incubation period. Immunoreactive (IR)-GH, detectable by a monoclonal and two polyclonal antibodies for chicken GH, was specifically and ubiquitously present in tissues of 3-day-old embryos. At embryonic day (ED) 5, IR-GH was widespread in ectodermal, mesodermal and endodermal tissues, but it was not present in every cell of each tissue. IR-GH was particularly abundant i! n the neural tube, notochord, limb bud, somites, heart, stomach, liver, kidney, Wolffian duct and the amnion. By ED8, IR-GH was still widespread and was now present in limb bud cartilage, although the heart and liver were no longer GH immunoreactive. GH receptor immunoreactivity was also present in most tissues and cells of ED3-ED8 embryos. These results demonstrate that extrapituitary GH is abundantly present during early embryogenesis, prior to the differentiation of pituitary somatotrophs (at ED12). Since GH- and GHR-like proteins are present in most tissues of the chick embryo, it is proposed that extrapituitary GH may act as a local growth factor during embryonic development.

The development of vesicular acetylcholine transporter immunoreactivity in the hindlimbs of the opossum Monodelphis domestica.

Vesicular acetylcholine transporter (VAChT) was revealed immunohistochemically in light microscopy on hindlimb sections of developing opossums, Monodelphis domestica. In the immobile hindlimbs of the newborn, which comprise cartilaginous bones and loose, unstriated myofibers, scant immunolabeled nerve segments and small spherical terminals, presumably growth cones or immature neuromuscular junctions, are found in the muscle tissue of the thigh, leg and proximal foot, decreasing in number and size proximodistally. When the hindlimbs start moving at 1 week, terminals are more numerous and larger, still decreasing proximodistally, and occur in the newly formed interosseous foot muscles. At 4 weeks, when the hindlimbs start supporting weight and quadrupedal locomotion appears, terminals are more numerous, flattened and in comparable size and density in thigh, leg and foot muscles. By 7 weeks, large and completely flat terminals are observed in groups of 3 to 4 at regular intervals along muscle fibers. VAChT expression develops largely postnatally in the opossum hindlimbs, along a proximodistal gradient that parallels somatic and reflex development.

Characterization of chondrogenesis in cells isolated from limb buds in mouse.

Micromass cultures of mesenchymal cells isolated from limb buds of 11.5-day-old mouse fetuses were used to study chondrogenesis. After 3 days of culture, dense cell aggregates were observed. They then were converted into macroscopically visible cartilage foci during the following 2-4 days. Comparison of 2-, 4- and 7-day-old cultures has shown that the cells first expressed collagen type I, then switched to collagen type II expression as shown by immunohistochemistry and in situ hybridization. At day 7, proteoglycans were synthesized centrally in the foci. At the same time, most cells expressed collagen type II, with the highest expression in the periphery of the aggregates. The oncogene c-fos and homeodomain protein FS-1 were found in the cells expressing collagen type II, indicating that these transcription factors may be involved in the regulation of cell differentiation. The expression of alkaline phosphatase was detected first in mature cartilage foci (day 4) and increased during culture. Early in culture, DNA-replicating cells were uniformly distributed. With differentiation, the proliferating cells were present predominantly between the aggregates and their total number became significantly reduced. Our results indicate that the process of chondrogenesis in micromass cultures of mesenchymal cells mimics the differentiation process occurring during fetal development in vivo and can be directly studied by in situ hybridization, immunohistochemical and histochemical methods.

Development of collagen fibres and lysyl oxidase expression in the presumptive dermis of chick limb bud.

Lysyl oxidase (LOX) plays a critical role in the formation of cross-linkages in extracellular matrix molecules. Thus, it is essential for the biogenesis and homeostasis of the connective tissue matrix. During development, collagen fibres and elastic system fibres emerge and accumulate in a temporospatial manner in the presumptive dermis of chicks. In this study, we investigated LOX mRNA expression by laser capture microdissection and RT-qPCR and LOX protein localization by immunohistochemistry. The picrosirius polarization method was used to investigate a relation between collagen accumulation and LOX expression. PCR analysis showed that the expression of LOX mRNA in the presumptive dermis became apparent at embryonic day 13 and increased considerably by ED17. Immunohistochemical staining for LOX in the dermis was very low at all stages of development. Accumulation of collagen fibres was seen in the dermis on ED10, and higher wavelengths of birefringence became evident by ED13. Our findings suggest that the temporal pattern of LOX mRNA expression correlates with collagen fibre accumulation in the dermis of the developing chick limb bud, whereas LOX expression was relatively constant at the protein level.

Microarray analysis of Leflunomide-induced limb malformations in CD-1 mice.

The immuno-suppressant Leflunomide, a potent inhibitor of dihydroorotate dehydrogenase (DHODH) and tyrosine kinases, is teratogenic in laboratory animals. To better understand its teratogenic mechanism, pregnant mice (CD-1) received a single dose of 70mg/kg Leflunomide, or vehicle control, by gastric intubation on gestation day 10. Gene expression was evaluated in the pooled fore- and hindlimb buds of embryos 4 and 24h post-treatment. The down-regulation of cholesterol biosynthesis-related genes was observed but could not be correlated with teratogenicity, since Leflunomide did not alter cholesterol concentration in limb bud. Leflunomide inhibited the mitosis of limb mesenchymal cells, which may be linked to DHODH inhibition as well as a potential effect on tyrosine kinases that mediate cytokine and growth factor signaling and that may be responsible for the Leflunomide's teratogenicity.

Mohawk is a novel homeobox gene expressed in the developing mouse embryo.

Homeodomain-containing proteins comprise a superfamily of transcription factors that participate in the regulation of almost all aspects of embryonic development. Here, we describe the mouse embryonic expression pattern of Mohawk, a new member of the TALE superclass of atypical homeobox genes that is most-closely related to the Iroquois class. During mouse development, Mohawk was transcribed in cell lineages derived from the somites. As early as embryonic day 9.0, Mohawk was expressed in an anterior to posterior gradient in the dorsomedial and ventrolateral lips of the dermomyotome of the somites that normally give rise to skeletal muscle. Mohawk transcription in the dorsomedial region required the expression of the transcription factor paraxis. As somites matured, Mohawk transcription was observed in the tendon-specific syndetome and the sclerotome-derived condensing mesenchyme that prefigures the proximal ribs and vertebral bodies. In the limbs, Mohawk was expressed in a pattern consistent with the developing tendons that form along the dorsal and ventral aspect of the phalanges. Finally, Mohawk was detectable in the tips of the ureteric buds in the metanephric kidneys and the testis cords of the male gonad. Together, these observations suggest that Mohawk is an important regulator of vertebrate development.

The possible differentiation of osteogenic elements in vitro from chick limb mesodermal cells. I. Morphological evidence.

In the developing chick limb bud, myogenic, fibrogenic, chondrogenic, and osteogenic tissues are derived from embryonic mesenchyme. Previous reports show that when stage 24 limb mesenchymal cells are cultured in vitro, chondrocytes, fibrocytes, and myocytes can be identified on the basis of morphological and biochemical parameters. The studies reported here clearly demonstrate that, in similar cultures, crystalline calcium phosphate material is deposited in the chondrocytic and fibrocytic matrices. Such crystalline material is not observed before Day 10 of culture life. Subsequent to Day 10 of culture, first amorphous, then crystalline calcium phosphate is observed. On the basis of light and electron microscopic analysis, it appears that the in vitro calcification phenomenon closely resembles the morphological and temporal sequence of osteogenesis observed in vivo.

Immunolocalization of fibronectin and its receptors integrin alpha 3 and alpha 5 subunits in the rat limb development.

In the rat hind limb bud aged between prenatal days 14 and 16, immunoreactions of fibronectin in the apical ectodermal ridge were localized on the plasma membranes of epidermal cells and cytoplasmic projections of the underlying mesenchymal cells, which are in contact with the basal lamina. Those of integrin alpha 3 and alpha 5 subunits also appeared on such areas. Definite immunoreactions of fibronectin and both integrin subunits were seen in cell to cell contact areas of mesenchymal cells which are associated with the marginal vein, or with each other forming solid cell cords, and appeared on the basal plasma membrane of endothelial cells of the growing capillaries arising from the marginal vein. These findings suggest that fibronectin may work as a ligand for alpha 3 beta 1 and/or alpha 5 beta 1 integrins expressed by the mesenchymal and vasoformative cells in developing limb bud.

Genetic analysis of a Hoxd-12 regulatory element reveals global versus local modes of controls in the HoxD complex.

Vertebrate Hoxd genes are essential determinants of limb morphogenesis. In order to understand the genetic control of their complex expression patterns, we have used a combined approach involving interspecies sequence alignments in parallel with transgenic analyses, followed by in vivo mutagenesis. Here, we report on the identification of a regulatory element that is located in the vicinity of the Hoxd-12 gene. While this element is well conserved in tetrapods, little sequence similarity was scored when compared to the cognate fish DNA. The regulatory potential of this region XI (RXI) was first assayed in the context of a Hoxd-12/lacZ reporter transgene and shown to direct reporter gene expression in posterior limb buds. A deletion of this region was generated by targeted mutagenesis in ES cells and introduced into mice. Analyses of animals homozygous for the HoxDRXI mutant allele revealed the function of this region in controlling Hoxd-12 expression in the presumptive posterior zeugopod where it genetically interacts with Hoxa-11. Downregulation of Hoxd-12 expression was also detected in the trunk suggesting that RXI may mediate a rather general function in the activation of Hoxd-12. These results support a model whereby global as well as local regulatory influences are necessary to build up the complex expression patterns of Hoxd genes during limb development.

Conservation in hedgehog signaling: induction of a chicken patched homolog by Sonic hedgehog in the developing limb.

Hedgehog genes have been implicated in inductive signaling during development in a variety of organisms. A key element of the hedgehog signaling system is encoded by the gene patched. In Drosophila hedgehog regulates gene expression by antagonizing the action of patched. In addition, patched is itself a transcriptional target of hedgehog signaling. We have isolated a chicken patched homolog and find it to be strongly expressed adjacent to all tissues where members of the hedgehog family are expressed. As in Drosophila, ectopic expression of Sonic hedgehog leads to ectopic induction of chicken Patched. Based on this regulatory conservation, vertebrate Patched is likely to be directly downstream of Sonic hedgehog signaling. An important role of Sonic hedgehog is the regulation of anterior/posterior pattern in the developing limb bud. Since Patched is directly downstream of the hedgehog signal, the extent of high level Patched expression provides a measure of the distance that Sonic hedgehog diffuses and directly acts. On this basis, we find that Sonic hedgehog directly acts as a signal over only the posterior third of the limb bud. During limb patterning, secondary signals are secreted in both the mesoderm (e.g. Bone Morphogenetic Protein-2) and apical ectodermal ridge (e.g. Fibroblast Growth Factor-4) in response to Sonic hedgehog. Thus knowing which is the direct target tissue is essential for unraveling the molecular patterning of the limb. The expression of Patched provides a strong indication that the mesoderm and not the ectoderm is the direct target of Sonic hedgehog signaling in the limb bud. Finally we demonstrate that induction of Patched requires Sonic hedgehog but, unlike Bone Morphogenetic Protein-2 and Hox genes, does not require Fibroblast Growth Factor as a co-inducer. It is therefore a more direct target of Sonic hedgehog than previously reported patterning genes.

Expression of the cell surface proteoglycan glypican-5 is developmentally regulated in kidney, limb, and brain.

Heparan sulfate is ubiquitous at the cell surface, where it is expressed predominantly on proteoglycans of either the transmembrane syndecan family or the glycosylphosphatidylinositol (GPI)-anchored glypican family, and has been proposed to function as a "coreceptor" for a number of "heparin-binding" growth factors. Although little is known about functional differences between individual members of the glypican gene family, mutations in both the Drosophila gene dally and the human gene for glypican-3 strongly suggest that at least some glypicans do function in cellular growth control and morphogenesis. In particular, deletion of the human glypican-3 gene is responsible for Simpson-Golabi-Behmel syndrome, and its associated pre- and postnatal tissue overgrowth, increased risk of embryonal tumors during early childhood, and numerous visceral and skeletal anomalies. We have identified and characterized, by sequencing of EST clones and products of rapid amplification of cDNA ends (RACE), an mRNA that encodes a 572-amino-acid member of the glypican gene family (glypican-5) that is most related (50% amino acid similarity, 39% identity) to glypican-3. Glypican-5 mRNA is detected as a 3.9- and 4.4-kb transcript in adult and neonatal mouse brain total RNA, and in situ hybridization results localize transcript primarily to restricted regions of the developing central nervous system, limb, and kidney in patterns consistent with a role in the control of cell growth or differentiation. Interestingly, glypican-5 localizes to 13q31-32 of the human genome, deletions of which are associated with human 13q- syndrome, a developmental disorder with a pattern of defects that shows significant overlap with the pattern of glypican-5 expression.

Comparative analysis of AP-2 alpha and AP-2 beta gene expression during murine embryogenesis.

Transcription factor AP-2 has been identified as playing important roles during embryonic development of the neural tube, neural crest derivatives, skin, and urogenital tissues. Recently, we isolated a second AP-2 transcription factor, AP-2 beta, which is 76% homologous to the previously known AP-2 alpha gene, and showed that both genes are coexpressed in murine embryos at day 13.5 and 15.5 post coitum (pc). In the current study, we used specific cRNA probes to study comparatively AP-2 alpha and AP-2 beta expression by in situ hybridization of murine embryonic tissue sections. Our results reveal that expression of both genes starts at day 8 pc in the lateral head mesenchyme and extraembryonic trophoblast. The expression pattern was identical until day 10 pc but diverged significantly during later stages of development. From day 11 forward, specific expression patterns of AP-2 alpha and AP-2 beta mRNA were observed. Specific AP-2 beta signals were detected in the midbrain, sympathetic ganglia, adrenal medulla, and cornea. Specific AP-2 alpha signals were present in the limb buds, dorsal root ganglia, tooth germs, and Moll's and Meibom's glands. In contrast, expression of both genes occurred in skin, facial mesenchyme, spinal cord, cerebellum, and renal tubular epithelia. Our results indicate that both genes are expressed with different temporal and spatial patterns during embryonic development.

Intracellular collagen-like material in mouse embryonic mesenchymal cells incubated with TNF-alpha and IL-1 beta in organoid culture.

Blastemal cells of embryonic mouse limb buds (day 12) were cultivated in organoid cultures in the presence of the human recombinant cytokines interleukin-1 beta (IL-1 beta) and tumour necrosis factor alpha (TNF-alpha). The effects of both cytokines (applied alone or together) on mesenchymal cells were demonstrated by electron microscopy. Cultures treated with TNF-alpha (alone or in combination with IL-1 beta) showed several mesenchymal cells with numerous irregularly shaped membrane-bordered cavities containing thick bundled tannic-acid-positive fibrillar structures that resembled loosened collagen fibrils, whereas cells exposed to IL-1 beta alone did not exhibit such changes. These findings are discussed in the light of two hypotheses: the phagocytosis of extracellular collagen fibrils, and fibrillogenesis resulting from incongruity of synthesis and secretion rates of procollagen; our results favour the former.