Ventral Zone Research Articles

Relationship of cervical spinal rootlets and the inferior vertebral notch.

The anatomic features of cervical spinal rootlets from C5 to T1 and their relationships to the inferior vertebral notches were studied. Fifteen fresh cadavers were dissected and the cervical spinal cord and spinal rootlets were exposed by posterior total laminectomy. The dorsal rootlet entry zone of each spinal root was located proximal to the inferior vertebral notch, with an increasing distance from 15 mm at C5 to 28 mm at T1. The angle sustained by the rootlets to the cord decreased from 45 degrees to 89 degrees at C5 and to 23 degrees to 41 degrees at T1. Ventral rootlet exit zones shared similar arrangements and orientations, but they could not be exposed with posterior laminectomy only. Spinal rootlets of a particular cervical spinal segment may be found medial to the pedicles or through the intervertebral foramen one level above. They may be exposed by foraminotomy or partial excision of the pedicles. The inferior vertebral notch, which is the inferior border of the pedicle, is a reliable landmark for location of the rootlets. The information is useful for safe surgical manipulation and instrumentation around the pedicles, and when reimplantation of spinal nerve roots is considered for total brachial palsy.

Unique developmental patterns of GABAergic neurons in rat spinal cord.

Gamma-aminobutyric acid (GABA)ergic neurons have been postulated to compose an important component of local circuits in the adult spinal cord, yet their identity and axonal projections have not been well defined. We have found that, during early embryonic ages (E12-E16), both glutamic acid decarboxylase 65 (GAD65) and GABA were expressed in cell bodies and growing axons, whereas at older ages (E17-P28), they were localized primarily in terminal-like structures. To determine whether these developmental changes in GAD65 and GABA were due to an intracellular shift in the distribution pattern of GAD proteins, we used a spinal cord slice model. Initial experiments demonstrated that the pattern of GABAergic neurons within organotypic cultures mimicked the expression pattern seen in embryos. Sixteen-day-old embryonic slices grown 1 day in vitro contained many GAD65- and GAD67-labeled somata, whereas those grown 4 days in vitro contained primarily terminal-like varicosities. When isolated E14-E16 slices were grown for 4 days in vitro, the width of the GAD65-labeled ventral marginal zone decreased by 40-50%, a finding that suggests these GABAergic axons originated from sources both intrinsic and extrinsic to the slices. Finally, when axonal transport was blocked in vitro, the developmental subcellular localization of GAD65 and GAD67 was reversed, so that GABAergic cell bodies were detected at all ages examined. These data indicate that an intracellular redistribution of both forms of GAD underlie the developmental changes observed in GABAergic spinal cord neurons. Taken together, our findings suggest a rapid translocation of GAD proteins from cell bodies to synaptic terminals following axonal outgrowth and synaptogenesis.

Cholinergic mechanism in the lateral septal area is involved in the stress-induced blood pressure increase in rats

Previously, we demonstrated that the rostral part of the ventral zone of the lateral septal area (LSV) was involved in the restraint stress-induced pressor response. It is suggested that there exist acetylcholine receptors responsible for blood pressure increase in the caudal part of the lateral septal area. In this study, we examined whether acetylcholine receptors responsible for pressor responses also exist in the rostral part of the LSV and whether these acetylcholine receptors are involved in the stress-induced pressor response in rats. Microinjection of either carbachol (10–100 pmol) or physostigmine (0.46 and 1.5 nmol) into the LSV caused a dose-dependent increase in blood pressure. The pressor response to carbachol (30 pmol) was inhibited by the M1 antagonist pirenzepine and the M3 antagonist 4-DAMP mustard but not by the M2 antagonist methoctramine injected into the LSV. Bilateral microinjections of the M1/M3 antagonist 4-DAMP (1 nmol) inhibited the restraint stress-induced pressor response. These findings suggest that M1/M3 muscarinic receptors responsible for blood pressure increase exist in the rostral part of the LSV and they are partly involved in the stress-induced pressor response.

The answer is blowing in the Wnt

Specification of cardiac tissue requires signals from the dorsal lip and the underlying endoderm during gastrulation in Xenopus laevis. Wnt-11 expression appears to match spatiotemporally to this specification. Kuhl and colleagues [1xWnt-11 activation of a non-canonical Wnt signalling pathway is required for cardiogenesis. Pandur, P. et al. Nature. 2002; 418: 636–641Crossref | PubMed | Scopus (382)See all References][1] now demonstrate that Wnt-11 is indeed necessary for cardiogenesis.The authors introduced a dominant-negative (dn) XWnt-11 construct into the presumptive cardiac region and found decreased expression of cardiac markers XNkx2.5 and cardiac troponin I. The organization of the endocardium and myocardium were disrupted. Similar effects were observed with XWnt-11 morpholino antisense oligonucleotides. RT-PCR showed that XWnt-11 expression alone was sufficient to induce the expression of cardiac genes (GATA-4, GATA-6, XNkx2.5, MHCα and TnIc) in animal caps. XWnt-11 enhanced the effect of activin in cardiac gene activation, and dnXWnt-11 RNA inhibited activin-induced cardiac gene expression. Overexpression of β-catenin induced the expression of XNkx2.5 in animal caps. Activation of both protein kinase C (PKC) and Jun N-terminal kinase (JNK) induced cardiac gene expression. A PKC, but not CamKII, inhibitor had the same effect as dnXWnt-11 on cardiac gene expression, and JNK could rescue the suppression by dnXWnt-11. They then showed that JNK-1 was activated by XWnt-11 by increased phosphorylation. However, XWnt-11 alone did not induce formation of beating tissue in animal cap explants but could do so in ventral marginal zone explants. Furthermore, dkk-1, an inhibitor of Wnt/β-catenin signaling, and XWnt-11 could induce a primary heart-tube-like structure. Finally, the authors demonstrated that mouse Wnt-11 could induce cardiomyogenesis in mouse P19 cell lines.It had been thought that signals from different tissues are required for heart development during embryogenesis. However, if tissue interaction also is required for cardiac muscle formation, it does not explain why P19 cells readily differentiate into cardiac muscle – presumably without intricate tissue interaction. It appears that stem cell differentiation uses a different mechanism even when using the same inducer.

Common and distinct signals specify the distribution of blood and vascular cell lineages in Xenopus laevis embryos.

In an effort to elucidate the regulatory mechanisms that determine the fate of blood cells and vascular cells in the ventral blood island mesoderm, the embryonic expression of Xtie-2, a Xenopus homolog of the tie-2 receptor tyrosine kinase, was examined. Whole-mount in situ hybridization analysis revealed that Xtie-2 mRNA is expressed at the late tailbud stage within the regions where endothelial precursor cells exist. On the ventral side of embryos, Xtie-2-positive cells are predominantly present just outside the boundary of alpha-globin-positive cells, thus the expression pattern of these two markers seems mutually exclusive. Further experiments revealed that there is a consistent and strong correlation between the induction of Xtie-2 and alpha-globin expression in embryos and explant tissues. First, these two markers displayed overlapping expression in embryos ventralized by the removal of a "dorsal determinant" from the vegetal cytoplasm at the 1-cell stage. Second, expression of both Xtie-2 and alpha-globin were markedly induced in ectodermal explants (animal caps) from embryos co-injected with activin and bone morphogenetic protein (BMP)-4 RNA. Furthermore, both Xtie-2 and alpha-globin messages were strongly positive in dorsal marginal zone explants that had been injected with BMP-4 RNA. In contrast, however, there was a clear distinction in the localization of these two transcripts in embryos dorsalized by LiCl treatment. Distinct localization was also found in the ventral marginal zone (VMZ) explants. Using the VMZ explant system, we demonstrate a role of fibroblast growth factor (FGF) signaling in enhancing the vascular cell marker and reducing the blood cell marker. The present study suggests that the early steps of blood and vascular cell differentiation are regulated by a common BMP-4-dependent signaling; however, distinct factor(s) such as FGF are involved in different distribution of these two cell lineages.

Synaptophysin in the Cochlear Nucleus Following Acoustic Trauma

Chinchillas are notable for a low-frequency hearing range similar to that of humans and a marked sensitivity to loud noise. A single noise exposure that produces cochlear damage may lead to progressive loss of synaptic endings in the cochlear nucleus, followed by new axonal growth. As an index of synaptic regulation during such changes, we have examined the expression of a synaptic vesicle protein, synaptophysin, in the cochlear nucleus following a damaging acoustic stimulus in adult chinchillas. With one ear protected by a plug, following a 3-h exposure to an octave-band noise of 108 dB sound pressure level, centered at 4 kHz, the unprotected cochlea and the cochlear nuclei exhibited degeneration of hair cells and axons over periods of 7, 14, 30, 90, and 150 days. Axonal degeneration, as revealed by a silver degeneration method, was heavy ipsilateral to the cochlear damage, but sparse degeneration also appeared on the contralateral, unexposed side. Synaptophysin immunostaining underwent a major, bilateral decline in the anteroventral and posteroventral cochlear nuclei, interrupted at intervening periods by transient increases in the numbers of stained structures. A distinction in staining between large perisomatic structures and smaller puncta in the neuropil and between the dorsal and the ventral zones of the ventral cochlear nuclei revealed some variations in the response and degree of recovery of synaptophysin staining. These findings could best be explained by degeneration of synaptic endings followed by new growth of terminals and by regulatory changes in the levels of synaptophysin expression and synaptic vesicle accumulation over time.

The Competence of Marginal Zone Cells to Become Spemann's Organizer Is Controlled by Xcad2

The organizer in vertebrate embryos is responsible for the formation of the primary body axis. In amphibian embryos, the organizer forms in the dorsal marginal zone (prospective dorsal mesoderm) at a location determined by the point of sperm entry. Using inducible versions of axis-inducing proteins, it has been shown that, irrespective of the mode of secondary axis induction, organizer formation in the ventral marginal zone is temporally restricted from the midblastula transition to the onset of gastrulation. Here, we show that the competence of marginal zone cells to respond to organizer-inducing signals is under temporal control, one of the regulators being the homeobox transcription factor Xcad2. Overexpression of Xcad2 restricts the temporal competence for axis induction, whereas partial loss of function expands this competence, supporting our suggestion. We propose that Xcad2 competes with putative axis-inducing signals within the marginal zone to prevent expression of organizer-specific genes. Elimination of endogenous Xcad2 allows for the activation of organizer genes beyond the normal competence window during early/mid-gastrulation. We conclude that Xcad2, through its early expression in the ventrolateral marginal zone, terminates the competence of this embryonic region to respond to organizer-inducing signals by preventing the activation of organizer-specific genes.

Symposium 8: Regulation of Oligodendrocyte Development. Guidance of oligodendrocyte precursor migration in the developing spinal cord

Oligodendrocyte precursors arise in restricted regions of the developing neuroepithelium due to local signals that include sonic hedgehog. In the spinal cord the founder cells of the oligodendrocyte lineage develop in a specific domain of the ventral ventricular zone. These cells or their progeny subsequently migrate long distances to populate the entire spinal cord and myelinate axons in the peripheral presumptive white matter. The majority of migration in the oligodendrocyte lineage is accomplished by immature precursors, which then stop, proliferate and differentiate in the appropriate location. Several distinct mechanisms appear to regulate this migration. The initial dispersal of cells from the ventral ventricular zone is guided by chemorepellent cues including netrin‐1 present in the ventral ventricular domain. Migratory precursors are arrested in particular locations within the developing spinal cord as the result of the localized expression of the chemokine, CXCL1 by astrocytes. This chemokine, signalling through the CXCR2 receptor combines with PDGF to inhibit cell migration and enhance cell proliferation thereby facilitating the local expansion of the oligodendrocyte lineage and myelination of all relevant axons.

Dorsally derived BMP4 inhibits the induction of spinal cord oligodendrocyte precursors

During development oligodendrocyte precursors arise in a distinct domain of the ventral ventricular zone in the spinal cord that they share with motor neurons. The localized appearance of oligodendrocyte and motor neuron precursors is the result of local inductive signals including sonic hedgehog (Shh). Previous studies suggested that inhibitory signals from dorsal spinal cord act to sharpen the boundaries of the Shh induced region. Here we show that the dorsal spinal cord contains BMP4 during the developmental period when oligodendrocyte precursors first appear. In dissociated cultures of embryonic spinal cord cells, BMP4 competitively blocks the induction of oligodendrocyte precursors by Shh. Similarly, in embryonic slice preparations addition of BMP4 inhibited the appearance of oligodendrocyte precursors in the ventral spinal cord while addition of Shh enhanced their appearance. In vivo, transplantation of a BMP4 coated bead adjacent to the dorsal spinal cord inhibited ventral oligodendrogenesis while transplantation of a Shh coated bead enhanced ventral oligodendrogenesis. These data suggest that the initial localization of oligodendrocytes in the ventral spinal cord reflects the neutralization of dorsally‐derived BMP4 inhibition by locally supplied Shh.

Antimorphic PV.1 Causes Secondary Axis by Inducing Ectopic Organizer

Xenopus homeobox gene, PV.1 ventralizes activin-induced dorsal mesoderm and inhibits neuralization of ectoderm in animal cap when overexpressed. Here we generated PV.1/engrailed fusion construct (N-PV1-EnR) to perform loss-of-function study for this transcription factor. N-PV1-EnR showed an extremely antimorphic effect, causing a partial secondary embryonic axis when expressed at ventral marginal zone of blastula. In ventral marginal zone cells, this chimeric protein induced organizer genes and suppressed ventral markers mimicking those effects reported for dominant negative BMP-4 receptor (DNBR). Moreover, N-PV1-EnR rescued the ventralized embryos caused by the ectopic dorsal expression of PV.1 but not by that of Xvent-2. These results suggested that PV.1 functions at downstream of BMP-4 as a ventralizing effector which acts separately from Xvent-2 and the dominant negative effect gained by this specific mutant is applicable for the further studies of BMP-4 downstream pathway.

Descending supraspinal pathways in amphibians: III. Development of descending projections to the spinal cord in Xenopus laevis with emphasis on the catecholaminergic inputs.

In developmental stages of the clawed toad, Xenopus laevis, we describe the ontogeny of descending supraspinal connections, catecholaminergic projections in particular, by means of retrograde tracing techniques with dextran amines. Already at embryonic stages (stage 40), spinal projections from the reticular formation, raphe nuclei, Mauthner neurons, vestibular nuclei, the locus coeruleus, the interstitial nucleus of the medial longitudinal fasciculus, the posterior tubercle, and the periventricular nucleus of the zona incerta are well developed. At the beginning of the premetamorphic period (stage 46), spinal projections arise from the suprachiasmatic nucleus, the torus semicircularis, the pretectal region, and the ventral telencephalon. After stage 48, tectospinal and cerebellospinal projections develop, with spinal projections from the preoptic area following at stage 51. Rubrospinal projections are present at stage 50. During the prometamorphic period, spinal projections arise in the nucleus of the solitary tract, the lateral line nucleus, and the mesencephalic trigeminal nucleus. With in vitro double-labeling methods, based on retrograde tracing of dextran amines in combination with tyrosine hydroxylase (TH) immunohistochemistry, we show that at stage 40/41, catecholaminergic (CA) neurons in the posterior tubercle are the first to project to the spinal cord. Subsequently, at stage 43, new projections arise in the periventricular nucleus of the zona incerta and the locus coeruleus. The last CA projection to the spinal cord originates from neurons in the nucleus of the solitary tract at the beginning of prometamorphosis (stage 53). Our data show a temporal, rostrocaudal sequence in the development of the CA cell groups projecting to the spinal cord. Moreover, the early appearance of CA fibers, preterminals and terminal-like structures in dorsal, intermediate, and ventral zones of the embryonic spinal cord, suggests an important role for catecholamines during development in nociception, autonomic functions, and motor control at the spinal level.

Frizzled-10, up-regulated in primary colorectal cancer, is a positive regulator of the WNT - β-catenin - TCF signaling pathway

WNT signaling pathway is implicated in embryogenesis as well as in carcinogenesis. We have previously cloned and characterized Frizzled-1 (FZD1), FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, and FZD10, encoding seven-transmembrane-type WNT receptors. Here, expression of FZD10 mRNA in various types of human cancer and effects of FZD10 mRNA microinjection into Xenopus early embryos were investigated. Northern blot analyses revealed relatively high-level expression of 4.0-kb FZD10 mRNA in cervical cancer cell lines HeLa S3, SKG-I, SKG-IIIa, and in a glioblastoma cell line A-172. Matched tumor/normal expression array analysis revealed significant up-regulation of FZD10 mRNA in 2 cases of primary colon cancer. Function of FZD10 was next investigated by using Xenopus axis duplication assay, in which positive regulators of the WNT - beta-catenin - TCF signaling pathway induce axis duplication. Injection of wild-type FZD10 mRNA into the ventral marginal zone of 4-cell-stage Xenopus embryos induced partial axis duplication in 40% of embryos. Ventral injection of Thr579Ala FZD10 mRNA or Val581Leu FZD10 mRNA with mutations in the C-terminal Ser/Thr-X-Val motif also induced partial axis duplication in about 40% of embryos. Furthermore, ventral injection of FZD10 mRNA significantly augmented the potential of co-injected Xenopus wnt-8 (Xwnt-8) mRNA to induce complete axis duplication. These results suggest that up-regulation of FZD10 mRNA in several types of human cells might lead to carcinogenesis through activation of the beta-catenin - TCF signaling pathway synergistically with some class of WNTs.

Limb muscle development.

Skeletal muscle precursors for the limbs originate from the epithelial layer of the somites, the dermomyotomes. We summarize the steps of limb muscle development from the specification of precursor cells in the dermomyotome, the directed migration of these cells to and within the limb buds to muscle growth and differentiation. All steps are controlled by local signaling between embryonic structures. In dermomyotome development, signals from the neural tube, the ectoderm and the intermediate and lateral mesoderm result in a medio-lateral patterning. Only the lateral portions of the dermomyotomes give rise to muscle precursor cells destined to enter the limb buds. As a prerequisite for migration, precursor cells have to de-epithelialize as a result of interactions between SF/HGF and its receptor c-met. Precursor cells adopt a mesenchymal morphology without losing their myogenic specification. This is achieved by the expression of the transcription factors Pax3, Pax7 and myf5. During migration, premature differentiation has to be kept at bay to enable motility and proliferation. After having reached their target sites, the dorsal and ventral myogenic zones, myogenesis is initiated by the activation of the muscle determination factors MyoD, myogenin and MRF4. Finally, we briefly summarize the process of muscle hypertrophy and regeneration during which aspects of developmental processes are reinitiated.

Identity, distribution, and development of polydendrocytes: NG2-expressing glial cells.

Cells that express the NG2 proteoglycan (NG2+ cells) comprise a unique population of glial cells in the central nervous system. While there is no question that some NG2+ cells differentiate into oligodendrocytes during development, the persistence of numerous NG2+ cells in the mature CNS has raised questions about their identity, relation to other CNS cell types, and functions besides their progenitor role. NG2+ cells also express the alpha receptor for platelet-derived growth factor (PDGF alphaR), a receptor that mediates oligodendrocyte progenitor proliferation during development. Antigenically, NG2+ cells are distinct from fibrous and protoplasmic astrocytes, resting microglia, and mature oligodendrocytes. Therefore, we propose the term polydendrocytes to refer to all NG2-expressing glial cells in the CNS parenchyma. This distinguishes them from the classical glial cell types and identifies them as the fourth major glial population in the CNS. Recent observations suggest that polydendrocytes are complex cells that physically and functionally interact with other cell types in the CNS. Committed oligodendrocyte progenitor cells arise from restricted foci in the ventral ventricular zone in both spinal cord and brain. It remains to be clarified whether there are multiple sources of oligodendrocytes, and if so whether polydendrocytes (NG2+ cells) represent progenitor cells of all oligodendrocyte lineages. Proliferation of NG2+ cells during early development appears to be dependent on PDGF, but the regulatory mechanisms that govern NG2+ cell proliferation in the mature CNS remain unknown. Pulse-chase labeling with bromodeoxyuridine indicates that polydendrocytes that proliferate in the postnatal spinal cord differentiate into oligodendrocytes. Novel experimental approaches are being developed to further elucidate the functional properties and differentiation potential of polydendrocytes.

The expression of SCDGF/ PDGF-C/ fallotein and SCDGF-B/ PDGF-D in the rat central nervous system

We examined the expression patterns of the two homologous genes, spinal cord-derived growth factor ( SCDGF)/ platelet-derived growth factor ( PDGF)- C/ fallotein and SCDGF-B/ PDGF-D in the rat central nervous system. In the spinal cord, SCDGF/ PDGF-C/ fallotein was expressed in the floor plate at embryonic day (E) 11 and also in the ventricular zone at E16 but not in adult. However, SCDGF-B/ PDGF-D was prominently expressed in the adult motoneurons, although faint expression was observed in the ventral ventricular zone at E16. Also in the brain, the expression of SCDGF/ PDGF-C/ fallotein was more remarkable at E16 than at adult. It was highly expressed in the cortex, pontine area and choroid plexus at E16. Contrary to SCDGF/ PDGF-C/ fallotein, SCDGF-B/ PDGF-D expression was notable in several nuclei at adult.

WNT2B2 mRNA, Up-Regulated in Primary Gastric Cancer, Is a Positive Regulator of the WNT– β-Catenin–TCF Signaling Pathway

Genetic alterations of WNT signaling molecules lead to carcinogenesis through activation of the β-catenin–TCF signaling pathway. We have previously cloned and characterized WNT2B/WNT13 gene on human chromosome 1p13, which is homologous to proto-oncogene WNT2 on human chromosome 7q31. WNT2B1 and WNT2B2 mRNAs, generated from the WNT2B gene due to alternative splicing of the alternative promoter type, encode almost identical polypeptides with divergence in the N-terminal region. WNT2B2 mRNA rather than WNT2B1 mRNA is preferentially expressed in NT2 cells with the potential of neuronal differentiation. Here, we describe our investigations of expression of WNT2B mRNAs in various types of human primary cancer. Matched tumor/normal expression array analysis revealed that WNT2B mRNAs were significantly up-regulated in 2 of 8 cases of primary gastric cancer. WNT2B2 mRNA rather than WNT2B1 mRNA was found to be preferentially up-regulated in a case of primary gastric cancer (signet ring cell carcinoma). Function of WNT2B1 mRNA and that of WNT2B2 mRNA were investigated by using Xenopus axis duplication assay. Injection of synthetic WNT2B1 mRNA into the ventral marginal zone of fertilized Xenopus eggs at the 4-cell stage did not induce axis duplication. In contrast, ventral injection of synthetic WNT2B2 mRNA induced axis duplication in 90% of embryos (complete axis duplication, 24%). These results strongly suggest that WNT2B2 up-regulation in some cases of gastric cancer might lead to carcinogenesis through activation of the β-catenin–TCF signaling pathway.

The lateral septal area is involved in mediation of immobilization stress-induced blood pressure increase in rats

Immobilization stress increased the number of neurons with Fos immunoreactivity, mainly in the ventral zone of the rostral part of the lateral septal nucleus (LSV) in rats. Immobilization stress caused an increase in blood pressure, and the stress-induced pressor response was inhibited by the GABAA receptor agonist, muscimol (8 and 80 pmol), injected bilaterally into the rostral part of the LSV in a dose-dependent manner. Intracerebroventricular injection of muscimol (16 pmol) did not affect the immobilization stress-induced pressor response. These findings suggest that the rostral part of the LSV is involved in mediation of the stress-induced pressor response.

Calcium mediates dorsoventral patterning of mesoderm in Xenopus

Calcium signals participate in the differentiation of electrically excitable and nonexcitable cells; one example of this differentiation is the acquisition of mature neuronal phenotypes [1]. For example, transient elevations of the intracellular calcium concentration have been recorded in the ectoderm of early embryos, and this elevation has been proposed to participate in neural induction [2–5]. Here, we present molecular evidence indicating that voltage-sensitive calcium channels (VSCC) are involved in early developmental processes leading to the establishment of the dorsoventral (D-V) patterning of a vertebrate embryo. We report that α1S VSCC are expressed selectively in the dorsal marginal zone at the early gastrula stage. The expression of the VSCC correlates with elevated intracellular calcium levels, as evaluated by the fluorescence of the intracellular calcium indicator Fluo-3. Misexpression of VSCC leads to a strong dorsalization of the ventral marginal zone and induction of the secondary axis but no direct neuralization of the ectoderm. Moreover, specific inhibition of VSCC by the use of calcicludine results in ventralization of the dorsal mesoderm. Together, these results indicate that calcium channels regulate mesodermal patterning by specificating the D-V identity of the mesodermal cells. The D-V patterning of the mesoderm has been shown to depend on a gradient of BMPs activity. We discuss the possibility that VSCC affect or act downstream of BMPs activity.

Neptune, a Krüppel-like transcription factor that participates in primitive erythropoiesis in Xenopus

The specification of the erythroid lineage from hematopoietic stem cells requires the expression and activity of lineage-specific transcription factors. One transcription factor family that has several members involved in hematopoiesis is the Krüppel-like factor (KLF) family [1]. For example, erythroid KLF (EKLF) regulates β-globin expression during erythroid differentiation [2–6]. KLFs share a highly conserved zinc finger-based DNA binding domain (DBD) that mediates binding to CACCC-box and GC-rich sites, both of which are frequently found in the promoters of hematopoietic genes. Here, we identified a novel Xenopus KLF gene, neptune, which is highly expressed in the ventral blood island (VBI), cranial ganglia, and hatching and cement glands. neptune expression is induced in response to components of the BMP-4 signaling pathway in injected animal cap explants. Similar to its family member, EKLF, Neptune can bind CACCC-box and GC-rich DNA elements. We show that Neptune cooperates with the hematopoietic transcription factor XGATA-1 to enhance globin induction in animal cap explants. A fusion protein comprised of Neptune's DBD and the Drosophila engrailed repressor domain suppresses the induction of globin in ventral marginal zones and in animal caps. These studies demonstrate that Neptune is a positive regulator of primitive erythropoiesis in Xenopus.

Structure and expression of Xenopus tropicalis BMP-2 and BMP-4 genes.

The Xenopus tropicalis BMP-2 and BMP-4 genes have been cloned and sequenced. A comparison with the corresponding genes from X. laevis reveals that the BMP-4 genes are conserved at a higher extent than the BMP-2 genes. This is especially evident for the intron sequences, but is also reflected by the exon sequences. While the amino acids of X. tropicalis and X. laevis BMP-4 proteins diverge at about 4%, those of BMP-2 proteins diverge at about 7%. By reverse transcriptase polymerase chain reaction analyses and whole mount in situ hybridizations, we demonstrate the temporal and spatial expression patterns of X. tropicalis BMP-2 and BMP-4 genes. BMP-2 is found to be expressed maternally, and later in development, in migrating heart progenitor cells as well as in the final heart, within the pineal gland and the olfactory placodes. BMP-4 is zygotically activated within the ventral marginal zone and later found in the eye, the otic vesicle, the heart and within blood islands. Although the overall patterns are very similar to those found in X. laevis, there is some distinct difference which might result from the accelerated development in X. tropicalis.