Biomedical subjects
G Eichele
Publications and source records attributed to G Eichele.
Developmental regulation of the orphan receptor COUP-TF II gene in spinal motor neurons.
Members of the steroid/thyroid hormone receptor superfamily are involved in the control of cell identity and of pattern formation during embryonic development. Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) can act as regulators of various steroid/thyroid hormone receptor pathways. To begin to study the role of COUP-TFs during embryogenesis, we cloned a chicken COUP-TF (cCOUP-TF II) which is highly homologous to human COUP-TF II. Northern analysis revealed high levels of cCOUP-TF II transcripts during organogenesis. Nuclear extracts from whole embryos and from embryonic spinal cords were used in electrophoretic mobility shift assays. These assays showed that COUP-TF protein is present in these tissues and is capable of binding to a COUP element (a direct repeat of AGGTCA with one base pair spacing). Analysis of cCOUP-TF expression by in situ hybridization revealed high levels of cCOUP-TF II mRNA in the developing spinal motor neurons. Since the ventral properties of the spinal cord, including the development of motor neurons, is in part established by inductive signals from the notochord, we transplanted an additional notochord next to the dorsal region of the neural tube in order to induce ectopic motor neurons. We observed that an ectopic notochord induced cCOUP-TF II gene expression in the dorsal spinal cord in a region coextensive with ectopic domains of SC1 and Islet-1, two previously identified motor neuron markers. Collectively, our studies raise the possibility that cCOUP-TF II is involved in motor neuron development.
Relationship between retinoic acid and sonic hedgehog, two polarizing signals in the chick wing bud.
Local application of all-trans-retinoic acid (RA) to the anterior margin of chick limb buds results in pattern duplications reminescent of those that develop after grafting cells from the zone of polarizing activity (ZPA). RA may act directly by conferring positional information to limb bud cells, or it may act indirectly by creating a polarizing region in the tissue distal to the RA source. Here we demonstrate that tissue distal to an RA-releasing bead acquires polarizing activity in a dose-dependent manner. Treatments with pharmacological (beads soaked in 330 micrograms/ml) and physiological (beads soaked in 10 micrograms/ml) doses of RA are equally capable of inducing digit pattern duplication. Additionally, both treatments induce sonic hedgehog (shh; also known as vertebrate hedgehog-1, vhh-1), a putative ZPA morphogen and Hoxd-11, a gene induced by the polarizing signal. However, tissue transplantation assays reveal that pharmacological, but not physiological, doses create a polarizing region. This differential response could be explained if physiological doses induced less shh than pharmacological doses. However, our in situ hybridization analyses demonstrate that both treatments result in similar amounts of mRNA encoding this candidate ZPA morphogen. We outline a model describing the apparently disparate effects of pharmacologic and physiological doses RA on limb bud tissue.
X-linked Kallmann syndrome. A neuronal targeting defect in the olfactory system?
Kallmann syndrome is a human genetic disorder characterized by the association of hypogonadism with the inability to smell, and is due to defects in the olfactory system development (i.e. incomplete migration of olfactory axons and of gonadotropin-releasing hormone producing neurons from the olfactory epithelium to the forebrain; aplasia or hypoplasia of olfactory bulbs and tracts). The human X-linked Kallmann syndrome gene and its chicken homologue have been cloned. Their protein products contain fibronectin type III repeats and a 'four-disulfide-core' domain also found in molecules that are involved in neural development. Consistent with the human phenotype, the chicken Kallmann gene is expressed in the developing olfactory bulb. At present the molecular and cellular mechanism of action of the Kallmann syndrome gene product is unknown. Based on expression studies and the characteristics domains of the predicted protein, it is hypothesized that the protein may be involved in targeting olfactory axons to the bulb. Alternatively, the Kallmann protein could be an extracellular matrix component required for the proper formation of the multilayered structure of the olfactory bulb.
Retinoids in embryonic development.
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Expression pattern of the Kallmann syndrome gene in the olfactory system suggests a role in neuronal targeting.
Kallmann syndrome is a genetic disorder characterized by a defect in olfactory system development, which appears to be due to an abnormality in the migration of olfactory axons and gonadotropin releasing hormone (Gn-RH) producing neurons. The X-linked Kallmann syndrome gene shares significant similarities with molecules involved in neural development. We have now isolated the evolutionarily conserved chicken homologue of the Kallmann gene. In the developing and adult chicken, high levels of expression were found in the mitral cells of the olfactory bulb (the target of olfactory axons) and in the Purkinje cells of the cerebellar cortex, both areas affected in patients with Kallmann syndrome. We propose a model in which the Kallmann syndrome gene product is a signal molecule required for neuronal targeting throughout life.
Rhombomere transplantation repatterns the segmental organization of cranial nerves and reveals cell-autonomous expression of a homeodomain protein.
The developing vertebrate hindbrain consists of segmental units known as rhombomeres. Hindbrain neuroectoderm expresses 3' Hox 1 and 2 cluster genes in characteristic patterns whose anterior limit of expression coincides with rhombomere boundaries. One particular Hox gene, referred to as Ghox 2.9, is initially expressed throughout the hindbrain up to the anterior border of rhombomere 4 (r4). Later, Ghox 2.9 is strongly upregulated in r4 and Ghox 2.9 protein is found in all neuroectodermal cells of r4 and in the hyoid crest cell population derived from this rhombomere. Using a polyclonal antibody, Ghox 2.9 was immunolocalized after transplanting r4 within the hindbrain. Wherever r4 was transplanted, Ghox 2.9 expression was cell-autonomous, both in the neuroectoderm of the graft and in the hyoid crest cell population originating from the graft. In all vertebrates, rhombomeres and cranial nerves (nerves V, VII+VIII, IX, X) exhibit a stereotypic relationship: nerve V arises at the level of r2, nerve VII+VIII at r4 and nerves IX-X extend caudal to r6. To examine how rhombomere transplantation affects this pattern, operated embryos were stained with monoclonal antibodies E/C8 (for visualization of the PNS and of even-numbered rhombomeres) and HNK-1 (to detect crest cells and odd-numbered rhombomeres). Upon transplantation, rhombomeres did not change E/C8 or HNK-1 expression or their ability to produce crest cells. For example, transplanted r4 generated a lateral stream of crest cells irrespective of the site into which it was grafted. Moreover, later in development, ectopic r4 formed an additional cranial nerve root. In contrast, transplantation of r3 (lacks crest cells) into the region of r7 led to inhibition of nerve root formation in the host. These findings emphasize that in contrast to spinal nerve segmentation, which entirely depends on the pattern of somites, cranial nerve patterning is brought about by factors intrinsic to rhombomeres and to the attached neural crest cell populations. The patterns of the neuroectoderm and of the PNS are specified early in hindbrain development and cannot be influenced by tissue transplantation. The observed cell-autonomous expression of Ghox 2.9 (and possibly also of other Hox genes) provides further evidence for the view that Hox gene expression underlies, at least in part, the segmental specification within the hindbrain neuroectoderm.
9-cis-retinoic acid, a potent inducer of digit pattern duplications in the chick wing bud.
The effects of retinoids are mediated by two types of receptors, the retinoic acid receptors (RARs) and the retinoid-X-receptors (RXRs). The physiological ligand of the RARs is all-trans-retinoic acid whereas RXRs have high affinity for 9-cis-retinoic acid, a naturally occurring retinoid isomer. RXRs are broadly expressed in embryonic and adult tissues, and they are capable of forming homodimers as well as heterodimers with RARs and other nuclear hormone receptors. The role of 9-cis-retinoic acid in regulating the activity of RXR homodimers and RXR-containing heterodimers is poorly understood in vivo. To begin to explore the function of 9-cis-retinoic acid in morphogenesis, we have examined the activity of this isomer in the chick wing. Using reverse transcriptase polymerase chain reaction analyses, we show that RXR gamma is expressed in stage 20 wing buds. Similar to all-trans-retinoic acid, the 9-cis-isomer induces pattern duplications when locally applied to chick wing buds, but the 9-cis isomer is about 25 times more potent than the all-trans form. Furthermore, applied all-trans-retinoic acid is converted to the 9-cis isomer in the wing bud. The ratio of 9-cis to all-trans-retinoic acid established in the tissue is approximately 1:25. This quantitative agreement between the degree of conversion and the 25-fold higher efficacy of the 9-cis isomer, raises the possibility that, at least in part, the effects of all-trans-retinoic acid on the wing pattern result from a conversion to the 9-cis isomer.(ABSTRACT TRUNCATED AT 250 WORDS)
Evidence that Hensen's node is a site of retinoic acid synthesis.
Hensen's node of amniotes, like the Spemann organizer of amphibians, can induce a second body axis when grafted into a host embryo. The avian node, as well as several midline structures originating from it (notochord, floor plate), can also induce digit pattern duplications when grafted into the chick wing bud. We report here that the equivalent of Hensen's node from mouse is an effective inducer of digits in the chick wing bud. Tissues anterior and posterior to the node also evoke pattern duplications, but with a significantly lower efficiency. The finding that the murine node operates in an avian wing bud suggests that the same inducing agent(s) function in both primary and secondary embryonic fields and have been conserved during vertebrate evolution. Digit pattern duplications are also evoked by local administration of all-trans-retinoic acid. This similarity raises the possibility that Hensen's node is a source of retinoic acid. The mouse node is capable of synthesizing retinoic acid from its biosynthetic precursor all-trans-retinol at a substantially higher rate than either anterior or posterior tissues.
9-cis retinoic acid is a high affinity ligand for the retinoid X receptor.
All-trans retinoic acid (RA) has previously been shown to modulate the transcriptional properties of the retinoic acid receptor (RAR) and retinoid X receptor (RXR). The inability of all-trans RA to bind to RXR suggests that it may be metabolized to a more active high affinity ligand. We report here an experimental approach that has identified 9-cis RA as an RXR ligand. It is up to 40-fold more potent than all-trans RA in transfection assays and binds with high affinity. The production of 9-cis RA in cultured cells and the identification of this molecule in liver and kidney demonstrates the existence of this molecule in living organisms. The discovery of this novel hormone points to the key role retinoid metabolism may have in generating new signaling pathways.
An early marker of axial pattern in the chick embryo and its respecification by retinoic acid.
Chick Ghox 2.9 protein, a homeodomain-containing polypeptide, is first detected in the mid-gastrula stage embryo and its levels increase rapidly in the late gastrula. At this time, the initially narrow band of expression along the primitive streak expands laterally to form a shield-like domain that encompasses almost the entire posterior region of the embryo and extends anteriorly as far as Hensen's node. We have found that this expression domain co-localizes with a morphological feature that consists of a stratum of refractile, thickened mesoderm. Antibody-staining indicates that Ghox 2.9 protein is present in all cells of this mesodermal region. In contrast, expression within the ectoderm overlying the region of refractile mesoderm varies considerably. The highest levels of expression are found in ectoderm near the streak and surrounding Hensen's node, regions that recent fate mapping studies suggest that primarily destined to give rise to neurectoderm. At the definitive streak stage (Hamburger and Hamilton stage 4) the chick embryo is especially sensitive to the induction of axial malformations by retinoic acid. Four hours after the treatment of definitive streak embryos with a pulse of retinoic acid the expression of Ghox 2.9 protein is greatly elevated. This ectopic expression occurs in tissues anterior to Hensen's node, including floor plate, notochord, presumptive neural plate and lateral plate mesoderm, but does not occur in the anteriormost region of the embryo. The ectopic induction of Ghox 2.9 is strongest in ectoderm, and weaker in the underlying mesoderm. Endoderm throughout the embryo is unresponsive. At stage 11, Ghox 2.9 is normally expressed at high levels within rhombomere 4 of the developing hindbrain. In retinoic-acid-treated embryos which have developed to this stage, typical rhombomere boundaries are largely absent. Nevertheless, Ghox 2.9 is still expressed as a discrete band, but one that is widened and displaced to a more anterior position.
Temporal and regional differences in the expression pattern of distinct retinoic acid receptor-beta transcripts in the chick embryo.
Retinoic acid (RA) is a signaling molecule apparently involved in a variety of morphogenetic processes, such as patterning of developing and regenerating vertebrate limbs. RA binds to specific intracellular receptors that constitute a multigene family. RA receptors (RAR) bind to the regulatory region of specific target genes and thereby control the expression of these genes. Here we report the sequence and spatiotemporal expression pattern of RAR-beta from chick. Northern blots of RNA from whole embryos and from limb buds reveal the presence of transcripts of 3.2, 3.4, and 4.6 kb in size. Using two riboprobes, one that hybridizes to all three RAR-beta mRNAs and a second one, specific for the 4.6 kb transcript, we found by in situ hybridization a differential distribution of RAR-beta transcripts in limb bud mesenchyme, in craniofacial mesenchyme and in hindbrain neuroectoderm. In the hindbrain the 4.6 kb mRNA exhibits an anterior boundary of expression at the level of the constriction between rhombomeres 5 and 6. Examination of neural plate stage embryos by in situ hybridization indicates that this boundary of expression is already defined by this stage. In addition to having several RA receptors that are expressed with distinct spatial patterns in the embryo, our data indicate that the expression pattern of transcripts derived from a single receptor gene can also be differentially expressed, thus providing another level for regulating RA action.
Isolation of 3,4-didehydroretinoic acid, a novel morphogenetic signal in the chick wing bud.
There is increasing evidence that retinoic acid is a morphogen involved in vertebrate development. This evidence comes in part from studies of the chick wing bud, in which local application of all-trans-retinoic acid results in a duplication of the digit pattern along the anteroposterior axis. Retinoic acid may be only one of several morphogenetic signalling compounds required for limb pattern formation. To identify novel morphogenetically active compounds, fractionated extracts of whole chick embryos were tested for their ability to induce digit pattern duplications. We describe here the isolation of a new activity present in the limb bud, which we have identified as all-trans-3,4-didehydroretinoic acid. The 3,4-didehydroretinoic acid is generated in situ from retinol through a 3,4-didehydroretinol intermediate. We show that 3,4-didehydroretinoic acid and retinoic acid are equipotent in evoking digit duplications. These findings suggest that there are at least two endogenous retinoids with morphogenetic properties in the chick limb.
Polarizing activity and retinoid synthesis in the floor plate of the neural tube.
In many developing organisms the establishment of axial polarity and the patterning of cells depend on local signals that derive from restricted regions of the embryo. In vertebrate embryos, the origins of tissue polarity have been examined extensively in the developing limb. The anteroposterior pattern of the chick limb seems to be controlled by a morphogen, possibly retinoic acid, that is enriched in a region of the limb known as the zone of polarizing activity (ZPA). Certain tissues other than the ZPA have also shown polarizing activity experimentally in the chick limb, raising the possibility that signalling molecules involved in pattern formation in different embryonic tissues are conserved. Here we provide evidence that a similar polarizing activity is also present in a restricted region of the developing central nervous system (CNS). We show that a specialized group of neural cells termed the floor plate, but not other regions of the CNS, mimics the ZPA in respecifying the digit pattern in the developing chick limb. In addition, using an in vitro biochemical assay, we show that the floor plate can synthesize retinoic acid and 3,4-didehydroretinol, the precursor of a second morphogenetically active retinoid, 3,4-didehydroretinoic acid. These results show that the floor plate is a local source of a ZPA-like polarizing signal, possibly a retinoid, which may regulate the pattern of cell differentiation in the developing CNS.
Targeted slow-release of retinoids into chick embryos.
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Pattern formation in vertebrate limbs.
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A homeo domain protein reveals the metameric nature of the developing chick hindbrain.
The segmented embryonic hindbrain of vertebrates develops by sequential constriction of the neural tube into eight metameric units known as rhombomeres. The cellular and molecular basis of this segmentation process is largely unknown. Using an antibody, we analyzed the expression pattern of the chick homeo domain-containing protein Ghox-lab in the developing chick hindbrain. At the neural plate stage, prior to the appearance of rhombomeres, Ghox-lab is expressed within a single domain that extends anteriorly up to the site where rhombomere 4 will later form. After rhombomere 4 has appeared and as hindbrain segmentation progresses, the level of Ghox-lab protein increases significantly within the fourth rhombomere. This intensification, accompanied by the elimination of Ghox-lab protein in rhombomeres 5 and 6, eventually results in the formation of a distinct island of expression in rhombomere 4. All cells in the newly formed rhombomere 4 express Ghox-lab, except for the cells of the floor plate. In addition, neural crest cells migrating from the fourth rhombomere are also Ghox-lab-positive. These data raise the possibility that Ghox-lab protein might be one of the factors involved in the specification of the metameric pattern of the vertebrate hindbrain.
Region-specific expression in early chick and mouse embryos of Ghox-lab and Hox 1.6, vertebrate homeobox-containing genes related to Drosophila labial.
A chick gene homologous to the Drosophila homeobox gene labial has been cloned and sequenced. Regions of additional sequence identity outside of the homeobox reveal a close relationship to the mouse gene Hox 1.6. Northern blot analysis demonstrates that Ghox-lab and Hox 1.6 transcripts are both present at high levels during early stages of chick and mouse development, with a subsequent decline in abundance to very low levels by the time limb mesenchyme begins to differentiate. In situ hybridization analysis of chick embryos shows intense expression of Ghox-lab mRNA by Hamburger and Hamilton stage 4 (avian 'mid gastrula') and by stage 6 (pre-somitic neural plate) with expression decreasing shortly thereafter. The pattern of Ghox-lab RNA expression in these early embryos divides the embryo into an anterior and a posterior compartment. At stage 6, considerable signal is observed in the posterior two thirds of the embryo, while none is detected in the anterior third which is fated to become the head. This pattern is purely regional in nature, and does not follow boundaries defined by known tissue types. In situ hybridization of Hox 1.6 probes to mouse embryos of day 7.5 or 8.0 indicate that the Hox 1.6 transcript has a temporal and spatial distribution very similar to that of Ghox-lab in the chick embryo.