Krox20 Expression Research Articles

Progesterone stimulates Krox-20 gene expression in Schwann cells

The gene of the zinc finger transcription factor Krox-20 (Egr-2) is expressed in Schwann cells and plays an important role in myelination of peripheral nerves. We have shown that progesterone promotes myelination in the regenerating sciatic nerve and in cocultures of Schwann cells and sensory neurones. To determine whether progesterone regulates Krox-20 expression, we measured its effects on Krox-20 mRNA levels in the MSC80 mouse Schwann cell line by semi-quantitative RT-PCR. Although low levels of Krox-20 mRNA are detectable in MSC80 cells cultured in defined medium, treatment with 10 −6 M progesterone induces a rapid (15 min) and transient increase in the levels of Krox-20 mRNA. Lower doses of progesterone (10 −9, 10 −8 and 10 −7 M) are also effective in increasing Krox-20 mRNA. Other steroids including testosterone, dexamethasone, and estradiol are ineffective when added to the culture medium at 10 −6 M for 1 h. The induction of Krox-20 mRNA was also observed with the selective progesterone agonist Organon 2058 and was abolished by treating the MSC80 Schwann cells with the progesterone antagonist RU486, indicating that progesterone induces Krox-20 mRNA expression by binding to its intracellular receptor. The induction of Krox-20 by progesterone was also demonstrated in primary cultures of Schwann cells isolated from neonatal rat sciatic nerves, at the mRNA level by RT-PCR and at the protein level by immunohistochemistry. As Krox-20 is a necessary step for the initiation of myelin formation in peripheral nerves, its stimulation by progesterone suggests an important signalling function for this steroid in myelination.

Meis3 synergizes with Pbx4 and Hoxb1b in promoting hindbrain fates in the zebrafish.

Many Hox proteins are thought to require Pbx and Meis co-factors to specify cell identity during embryogenesis. Here we demonstrate that Meis3 synergizes with Pbx4 and Hoxb1b in promoting hindbrain fates in the zebrafish. We find that Hoxb1b and Pbx4 act together to induce ectopic hoxb1a expression in rhombomere 2 of the hindbrain. In contrast, Hoxb1b and Pbx4 acting together with Meis3 induce hoxb1a, hoxb2, krox20 and valentino expression rostrally and cause extensive transformation of forebrain and midbrain fates to hindbrain fates, including differentiation of excess rhombomere 4-specific Mauthner neurons. This synergistic effect requires that Hoxb1b and Meis3 have intact Pbx-interaction domains, suggesting that their in vivo activity is dependent on binding to Pbx4. In the case of Meis3, binding to Pbx4 is also required for nuclear access. Our results are consistent with Hoxb1b and Meis3 interacting with Pbx4 to form complexes that regulate hindbrain development during zebrafish embryogenesis.

The Krox-20 Null Mutation Differentially Affects the Development of Masticatory Muscles

The Krox-20 null mutation results in a loss of rhombomeres 3 and 5, which give rise to neurons that are essential to oral motor behaviors. Thus, the Krox-20 null mutant is an excellent model to investigate the development of oral motor circuitry. Our morphological examination of embryonic and neonatal Krox-20 null mutants revealed that a significant reduction of anterior digastric and mylohyoid muscles, the primary jaw openers, occurs between embryonic days 15 and 19. There are no gross morphological alterations in other masticatory muscles. These findings demonstrate that Krox-20 expression is critical for the normal development of the primary jaw opener musculature and they help explain previous studies documenting a reduction in jaw opening in Krox-20 null mutants. Since jaw opening is the power stroke of suckling behavior, our data help explain the reduction of colostrum/milk ingestion in Krox-20 null mutant neonates.

Jun, Fos and Krox in the hippocampus after noxious stimulation: simultaneous-input-dependent expression and nuclear speckling

Stimulation of sensory C-fibres produces extensive expression of the Fos, Jun and Krox families of inducible transcription factors (ITFs) in many nociceptive CNS areas [28]. In the hippocampus, however, c-Fos is only weakly induced by such stimulation, and expression of the other ITFs has not been studied. Here we examine the effects of single, repeated and simultaneous C-fibre inputs on ITF expressions in the rat hippocampus. A brief, strong electrical stimulation of sciatic nerve C-fibres induced little or no expression of c-Fos or Krox-20. In contrast, FosB was induced and continued to rise in all areas, whereas the basal expressions of c-Jun and Krox-24 were initially reduced but then returned during the subsequent 36 h. A weak noxious cutaneous stimulus applied to one hindpaw induced only weak expressions of the ITFs. However, if the sciatic stimulation was applied contralaterally and 6 h beforehand, this weak stimulus strongly induced Krox-24, but not other ITFs, i.e. there was a potentiation of Krox-24 expression. When these two stimuli were applied simultaneously a few c-Fos labelled cells did appear, and there was and an increased Krox-24 expression. There was also a strong potentiation of FosB and a strong reduction in c-Jun expression. This simultaneous stimulation was the only type of stimulation to induce expression of Krox-20. Also after simultaneous stimulation the majority of the nuclear labelling for FosB, but not of the other ITFs, had a speckled appearance. MK-801 blocked these changes in ITF expressions, but it could also cause the C-fibre stimulations to induce c-Fos and c-Jun in specific areas of the hippocampus. Thus C-fibre stimulation does affect transcription factor activity in the hippocampus; and the strong responses of some ITFs to simultaneous inputs points to their having a role as ‘genetic coincidence detectors’ in the hippocampus.

Krox-20 patterns the hindbrain through both cell-autonomous and non cell-autonomous mechanisms.

The Krox-20 gene encodes a zinc finger transcription factor, which has been shown previously, by targeted inactivation in the mouse, to be required for the development of rhombomeres (r) 3 and 5 in the segmented embryonic hindbrain. In the present work, Krox-20 was expressed ectopically in the developing chick hindbrain by use of electroporation. We demonstrate that Krox-20 expression is sufficient to confer odd-numbered rhombomere characteristics to r2, r4, and r6 cells, presumably in a cell-autonomous manner. Therefore, Krox-20, appears as the major determinant of odd-numbered identity within the hindbrain. In addition, we provide evidence for the existence of a non cell-autonomous autoactivation mechanism allowing recruitment of Krox-20-positive cells from even-numbered territories by neighboring Krox-20-expressing cells. On the basis of these observations, we propose that Krox-20 regulates multiple, intertwined steps in segmental patterning: Initial activation of Krox-20 in a few cells leads to the segregation, homogenization, and possibly expansion of territories to which Krox-20 in addition confers an odd-numbered identity.

Transcription of krox-20/egr-2 is upregulated after exposure to fibrous particles and adhesion in rat alveolar macrophages.

Alveolar macrophages meet various types of particulate substances deposited deep in the lung. We report differences in biologic responses of alveolar macrophages between phagocytosis of fine spherical and fibrous particles. Although titanium dioxide (TiO(2)) is thought to be biologically inert, the cytotoxicity of fibrous TiO(2) (F-TiO(2)) was much higher than spherical TiO(2) (S-TiO(2)). Differential display and the subsequent Northern blot analysis indicated that transcription of krox-20/egr-2 gene was slightly and greatly upregulated in S- and F-TiO(2)-exposed alveolar macrophages, respectively. The messenger RNA (mRNA) level of krox-20/egr-2 increased up to 8 h in F-TiO(2)-exposed alveolar macrophages, whereas krox-20/egr-2 mRNA level was transiently increased in response to adhesion to the culture dish. Stimulation with lipopolysaccharide also increased krox-20/egr-2 mRNA level transiently, although the mRNA level rebounded after 8 h. The analysis with 5' rapid amplification of complementary DNA ends suggested that there is a heterogeneity in the upstream region of this gene (krox-20/egr-2 and krox-20H1; accession numbers AB032420 and AB032419, respectively). The polymerase chain reaction analysis with specific primers for krox-20/egr-2 and krox-20H1 indicated that both genes were almost equally upregulated after either adhesion to the plastic dish or phagocytosis of F-TiO(2). These results suggest that both krox-20/egr-2 and krox-20H1 are implicated in adhesion and phagocytosis, and that the expression of krox-20 may reflect interaction with foreign substances and adhesion in alveolar macrophages.

Pattern of expression of the transcription factor Krox-20 in mouse hair follicle

We examined the pattern of expression in hair follicles of the transcription factor gene Krox-20. During embryogenesis, Krox-20 is first expressed in the upper portion of the hair bud, then in the hair canal, in the sebaceous glands and in the outer root sheath. In the mature follicles, Krox-20, like Shh and TGFβRII, is also expressed in a sub-population of matrix keratinocytes located in a ring-like fashion in vibrissal follicles and clustered on one side of the papilla in pelage follicles. This polarized pattern rotates around the papilla as a result of sequential gene expression by individual groups of matrix cells. This peculiar pattern is not linked to follicle angling.

Signaling specificities of fibroblast growth factor receptors in early Xenopus embryo.

Formation of mesoderm and posterior structures in early Xenopus embryos is dependent on fibroblast growth factor (FGF) signaling. Although several FGF receptors (FGFRs) are expressed in the early embryo, their respective role in these processes remains poorly understood. We provide evidence that FGFR-1 and FGFR-4 signals elicit distinct responses both in naive and neuralized ectodermal cells. We show that naive ectodermal cells expressing a constitutively active chimeric torso-FGFR-1 (t-R1) are converted into mesoderm in a Ras-dependent manner, while those expressing torso-FGFR-4 (t-R4) differentiate into epidermis without significant activation of Erk-1. In neuralized ectoderm, expression of t-R4 causes the up-regulation of the midbrain markers En-2 and Wnt-1, but not of the hindbrain nor the spinal cord markers Krox20 and Hoxb9. Mutation of tyr(776) in the phospholipase C-(gamma) binding consensus sequence YLDL of t-R4 completely abolishes En-2 and Wnt-1 induction. In contrast to t-R4, platelet derived growth factor (PDGF)-dependent FGFR-1 activation in neuralized ectodermal cells expressing a chimeric PDGFR-FGFR-1 receptor results in the expression of Krox20 and Hoxb9. A similar effect is observed when an inducible form of oncogenic Raf is expressed, therefore implicating FGFR-1 and Raf in the transduction of FGF-caudalizing signals in neural tissue. Our results suggest that FGFR-1 and FGFR-4 transduce distinct signals in embryonic cells, and mainly differ in their ability to activate the Ras/MAPK pathway.

A distal Schwann cell-specific enhancer mediates axonal regulation of the Oct-6 transcription factor during peripheral nerve development and regeneration.

The POU domain transcription factor Oct-6 is a major regulator of Schwann cell differentiation and myelination. During nerve development and regeneration, expression of Oct-6 is under the control of axonal signals. Identification of the cis-acting elements necessary for Oct-6 gene regulation is an important step in deciphering the complex signalling between Schwann cells and axons governing myelination. Here we show that a fragment distal to the Oct-6 gene, containing two DNase I-hypersensitive sites, acts as the Oct-6 Schwann cell-specific enhancer (SCE). The SCE is sufficient to drive spatially and temporally correct expression, during both normal peripheral nerve development and regeneration. We further demonstrate that a tagged version of Oct-6, driven by the SCE, rescues the peripheral nerve phenotype of Oct-6-deficient mice. Thus, our isolation and characterization of the Oct-6 SCE provides the first description of a cis-acting genetic element that responds to converging signalling pathways to drive myelination in the peripheral nervous system.

Positional Regulation of Krox-20 and mafB/kr Expression in the Developing Hindbrain: Potentialities of Prospective Rhombomeres

Krox-20 and mafB/kr encode transcription factors involved in the control of hindbrain development and are expressed in rhombomeres (r) 3 and 5 and 5 and 6, respectively. To analyse the regulation of the expression of these genes by positional cues, focusing on the stages just preceding the formation of rhombomeres, we have performed ectopic grafts involving single prospective rhombomeres (pr) or couples of pr on 4–6 somite avian embryos. Transplantation of pr6 in the pr5 position leads to Krox-20 activation and grafting of pr7 in the pr5 position results in mafB/kr activation. Furthermore, pr6 grafted in the pr5 position develops an r5-like cytoarchitecture. These data establish that rostral transplantation can lead to anteriorization within the hindbrain. However, additional experiments indicate that the competence of the transplanted tissue for such anteriorization appears limited and that transformations corresponding to shifts of a single rhombomere are favoured. We also show that caudal transplantation of pr5 into the pr6 position can lead to a down-regulation of Krox-20 expression consistent with posteriorization, suggesting that caudalizing influences are present within the nonsomitic hindbrain after the 4- to 6-somite stage. Finally, combinations of extirpation and grafting experiments suggest that the regulation of mafB/kr expression in the r6–r7 region may involve anteriorizing influences in addition to previously identified posteriorizing signals from the somitic region.

Nab proteins mediate a negative feedback loop controlling Krox-20 activity in the developing hindbrain.

The developing vertebrate hindbrain is transiently subdivided along the anterior-posterior axis into metameric units, called rhombomeres (r). These segments constitute units of lineage restriction and display specific gene expression patterns. The transcription factor gene Krox-20 is restricted to r3 and r5, and is required for the development of these rhombomeres. We present evidence that Krox-20 transcriptional activity is under the control of a negative feedback mechanism in the hindbrain. This regulatory loop involves two closely related proteins, Nabl and Nab2, previously identified as antagonists of Krox-20 transcriptional activity in cultured cells. Here we show that in the mouse hindbrain, Nab1 and Nab2 recapitulate the Krox-20 expression pattern and that their expression is dependent on Krox-20 function. Furthermore, misexpression of Nab1 or Nab2 in zebrafish embryos leads to alterations in the expression patterns of several hindbrain markers, consistent with an inhibition of Krox-20 activity. Taken together, these data indicate that Krox-20 positively regulates the expression of its own antagonists and raise the possibility that this negative feedback regulatory loop may play a role in the control of hindbrain development.

Hindbrain respecification in the retinoid-deficient quail

We report here the development and rescue of the truncated hindbrain of retinoid-deprived quail embryos. The embryo is completely rescued by an injection of retinol into the egg; this confirms retinol, or a related retinoid, as a required molecule in hindbrain development. Staging the retinoid replacement enabled us to determine that the 3–4 somite stage is the period when retinoids are required for normal development. Analysis of the development of the retinoid-deprived hindbrain phenotype through somitogenesis has revealed a pathway of retinoid action in early hindbrain regionalisation. The hindbrain of the retinoid-deprived embryo is normal in size, during early somitogenesis, but has a respecified pattern of Krox-20 expression. From the earliest expression of Krox-20, at the 5 somite stage, the rhombomere 3 stripe fills the caudal third of the developing hindbrain to the level of the first somite. Morphologically only 2, instead of the normal 5, rhombomere bulges form. These 2 bulges express genes and, later, develop morphology characteristic of rhombomeres 1 and 2 and rhombomere 3. Posterior hindbrain specific genes, Hoxb-1, Fgf3, MafB, and the rhombomere 5 stripe of Krox-20 are never expressed in the head neuroepithelium of these embryos. From the initial formation of the neural plate, there is no evidence of rhombomere 4–7 specific characteristics. These results indicate the specification of the posterior hindbrain is lost and its cells participate in the formation of an enlarged anterior hindbrain. In our previous study, we reported the absence of the posterior hindbrain in retinoid-deprived quails (Maden, M., Gale, E., Kostetskii, I., Zile, M., 1996. Vitamin A-deficient quail embryos have half a hindbrain and other neural defects. Curr. Biol. 6, 417–426). Here, we show this phenotype to be the result of respecification of the hindbrain cells. This provides evidence for a region specific response to a single stimulus, retinol, which suggests a pre-rhombomeric regionalisation of the hindbrain.

A Role for xGCNF in Midbrain–Hindbrain Patterning in Xenopus laevis

Cells in the presumptive neural ectoderm of Xenopus are committed to neural fate through a process called neural induction, which may involve proteins that antagonize BMP signaling pathways. To identify genes that are induced by the BMP antagonists and that may be involved in subsequent neural patterning, we used a suppression PCR-based subtraction screen. Here we investigate the prospective activities and functions of one of the genes, a nuclear orphan receptor previously described as xGCNF. In animal cap assays, xGCNF synergizes with ectopic chordin to induce the midbrain–hindbrain marker engrailed-2 (En-2). In Keller explants, which rely on endogenous factors for neural induction, similar increases in En-2 are observed. Expression in embryos of a dominant interfering form of xGCNF reduces the expression of endogenous En-2 and Krox-20. These gain-of-function and prospective loss-of-function experiments, taken with the observation that xGCNF is expressed in the early neural plate and is elevated in the prospective midbrain–hindbrain region, which subsequently expresses En-2, suggest that xGCNF may play a role in regulating En-2 and thus midbrain–hindbrain identity.

Segmental expression of the EphA4 (Sek-1) receptor tyrosine kinase in the hindbrain is under direct transcriptional control of Krox-20.

Segmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres (r) with distinct identities. Recent studies have uncovered regulatory links between transcription factors governing this process, but little is known of how these relate to molecules mediating cell-cell signalling. The Eph receptor tyrosine kinase gene EphA4 (Sek-1) is expressed in r3 and r5, and function-blocking experiments suggest that it is involved in restricting intermingling of cells between odd- and even-numbered rhombomeres. We have analysed the cis-acting regulatory sequences of the EphA4 gene in transgenic mice and identified a 470 bp enhancer element that drives specific expression in r3 and r5. Within this element, we have identified eight binding sites for the Krox-20 transcription factor that is also expressed in r3 and r5. Mutation of these binding sites abolishes r3/r5 enhancer activity and ectopic expression of Krox-20 leads to ectopic activation of the enhancer. These data indicate that Krox-20 is a direct transcriptional activator of EphA4. Together with evidence that Krox-20 regulates Hox gene expression, our findings reveal a mechanism by which the identity and movement of cells are coupled such that sharply restricted segmental domains are generated.

Inhibition of retinoic acid receptor-mediated signalling alters positional identity in the developing hindbrain.

Retinoids regulate gene expression via nuclear retinoic acid receptors, the RARs and RXRs. To investigate the functions of retinoid receptors during early neural development, we expressed a dominant negative RARbeta in early Xenopus embryos. We obtained evidence that dominant negative RARbeta specifically inhibits RAR/RXR heterodimer-mediated, but not RXR homodimer-mediated, transactivation. Both all-trans- and 9-cis-RA-induced teratogenesis were, however, efficiently opposed by ectopic expression of dominant negative RARbeta, indicating that only RAR/RXR transactivation is required for retinoid teratogenesis by each of these ligands. Experiments with two RXR-selective ligands confirmed that activation of RXR homodimers does not cause retinoid teratogenesis. Dominant negative RARbeta thus specifically interferes with the retinoid signalling pathway that is responsible for retinoid teratogenesis. Dominant negative RARbeta-expressing embryos had a specific developmental phenotype leading to disorganization of the hindbrain. Mauthner cell multiplications in the posterior hindbrain, and (both anteriorly and posteriorly) expanded Krox-20 expression domains indicated (partial) transformation of a large part of the hindbrain into (at least partial) rhombomere 3, 4 and/or 5 identity. In contrast, the fore- and midbrain and spinal cord appeared to be less affected. These data indicate that RARs play a role in patterning the hindbrain.

Regulation of dorsal fate in the neuraxis by Wnt-1 and Wnt-3a.

Members of the Wnt family of signaling molecules are expressed differentially along the dorsal-ventral axis of the developing neural tube. Thus we asked whether Wnt factors are involved in patterning of the nervous system along this axis. We show that Wnt-1 and Wnt-3a, both of which are expressed in the dorsal portion of the neural tube, could synergize with the neural inducers noggin and chordin in Xenopus animal explants to generate the most dorsal neural structure, the neural crest, as determined by the expression of Krox-20, AP-2, and slug. Overexpression of Wnt-1 or Wnt-3a in the neuroectoderm of whole embryos led to a dramatic increase of slug and Krox-20-expressing cells, but the hindbrain expression of Krox-20 remained unaffected. Enlargement in the neural crest population could occur even when cell proliferation was inhibited. Wnt-5A and Wnt-8, neither of which is expressed in the dorsal neuroectoderm, failed to induce neural crest markers. Overexpression of glycogen synthase kinase 3, known to antagonize Wnt signaling, blocked the neural-crest-inducing activity of Wnt-3a in animal explants and inhibited neural crest formation in whole embryos. We suggest that Wnt-1 and Wnt-3a have a role in patterning the neural tube along its dorsoventral axis and function in the differentiation of the neural crest.

XenopusHindbrain Patterning Requires Retinoid Signaling

We have asked how posterior neural tissue is patterned inXenopusby assaying the involvement of endogenous retinoic acid (RA) in this process and by using the labial Hox gene, HoxD1, as a posterior marker. Although RA is able to inhibit anterior gene expression and activate expression of more posterior genes, the normal role of retinoids in anteroposterior (A/P) patterning is unclear. HoxD1 is an early posterior neurectodermal marker, expressed from midgastrula with a later anterior expression limit in the future hindbrain. We previously showed that HoxD1 was induced as an immediate early response to retinoic acid in naive ectoderm (animal caps). Here, we use a truncated RARα2.2 receptor (RARΔ) to dominantly interfere with retinoid signaling. In embryos injected with RARΔ expression of HoxD1 is eliminated. Conjugates of ectoderm and dorsolateral mesoderm show that retinoid receptors are required in the ectoderm for HoxD1 induction. Further, expression of Krox-20 in r3 and r5 of the presumptive hindbrain is compressed into a single stripe that suggests elimination of r5. RARα2.2 expression almost precisely overlaps that of HoxD1, suggesting that this receptor may normally activate HoxD1. Expression of neither more anterior genes including cement gland, forebrain, and midbrain markers nor a more posterior spinal cord marker is affected by RARΔ. These data suggest that the posterior hindbrain is the region of the nervous system most sensitive to retinoid loss. Finally, we compare the ability of RA and fibroblast growth factor (FGF) to posteriorize isolated anterior neurectoderm and show that both factors can act directly on this substrate. RA acts in a more anterior domain than does FGF; however, neither factor is equivalent to the natural posteriorizing capacity of the posterior mesoderm. We propose that endogenous retinoid and FGF signals pattern largely nonoverlapping regions along the A/P axis and that posterior neural patterning requires multiple inducers.

Differential regulation of the zinc finger genes Krox-20 and Krox-24 (Egr-1) suggests antagonistic roles in Schwann cells.

Krox-20 and Krox-24 (Egr-1) encode closely related zinc finger transcription factors, which interact with the same DNA target sequences. Krox-20 is required for myelination in the peripheral nervous system. Using lacZ knock-in mutant mouse lines as well as immunohistochemical analyses, we have studied the expression of Krox-20 and Krox-24 in the Schwann cell lineage during normal development and following nerve lesion in the mouse and in human neuropathies. During embryogenesis, the two genes are expressed in a successive and mutually exclusive manner, Krox-24 being restricted to Schwann cell precursors and Krox-20 to mature Schwann cells. At birth, Krox-24 is reactivated and the two genes are coexpressed. In the adult, Krox-20 is expressed in myelinating cells, while Krox-24 is restricted to nonmyelinating cells. Following nerve lesion, Krox-24 is strongly induced in Schwann cells, reinforcing the link between its expression and the nonmyelinating and/or proliferative state, whereas Krox-20 is downregulated. These data are consistent with Krox-20 and Krox-24 playing antagonistic roles during the development of the Schwann cell lineage. In particular, their balance of expression might participate in the choice between myelinating and nonmyelinating pathways.

Differential regulation of the zinc finger genes Krox20 and Krox24 Egr1 suggests antagonistic roles in Schwann cells

Krox-20 and Krox-24 (Egr-1) encode closely related zinc finger transcription factors, which interact with the same DNA target sequences. Krox-20 is required for myelination in the peripheral nervous system. Using lacZ knock-in mutant mouse lines as well as immunohistochemical analyses, we have studied the expression of Krox-20 and Krox-24 in the Schwann cell lineage during normal development and following nerve lesion in the mouse and in human neuropathies. During embryogenesis, the two genes are expressed in a successive and mutually exclusive manner, Krox-24 being restricted to Schwann cell precursors and Krox-20 to mature Schwann cells. At birth, Krox-24 is reactivated and the two genes are coexpressed. In the adult, Krox-20 is expressed in myelinating cells, while Krox-24 is restricted to nonmyelinating cells. Following nerve lesion, Krox-24 is strongly induced in Schwann cells, reinforcing the link between its expression and the nonmyelinating and/or proliferative state, whereas Krox-20 is downregulated. These data are consistent with Krox-20 and Krox-24 playing antagonistic roles during the development of the Schwann cell lineage. In particular, their balance of expression might participate in the choice between myelinating and nonmyelinating pathways. J. Neurosci. Res. 50:702–712, 1997. © 1997 Wiley-Liss, Inc.

Heterogeneous Expression of Multiple Putative Patterning Genes by Single Cells from the Chick Hindbrain

The metameric organization of the vertebrate hindbrain into rhombomeres appears to result from the patterned expression of several transcription factors and putative signaling molecules. We have applied a refined single-cell reverse transcription-polymerase chain reaction strategy to examine the molecular logic proposed to pattern the hindbrain at the single-cell level. This technique allows analysis of the concurrent expression of several genes within an individual cell at higher sensitivity than byin situhybridization. Our results demonstrate that cells in rhombomere (r) 4 and r5 are heterogeneous in their expression of Hoxa-3, Hoxb-2, Sek-1, and Krox-20, suggesting that single cells are dynamically regulating their rhombomere-specific gene-expression profiles. Furthermore, the strong correlation between Sek-1 and Krox-20 expression at stage 12 was greatly diminished by stage 16, suggesting that the proposed interdependence of these two genes is present only at early stages of hindbrain development.