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Anand Chandrasekhar - One of the best experts on this subject based on the ideXlab platform.
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structural and temporal requirements of wnt pcp protein vangl2 function for convergence and extension movements and facial branchiomotor neuron migration in zebrafish
Mechanisms of Development, 2014Co-Authors: Vinoth Sittaramane, Suman Gurung, Anand ChandrasekharAbstract:Abstract Van gogh-like 2 (Vangl2), a core component of the Wnt/planar cell polarity (PCP) signaling pathway, is a four-pass transmembrane protein with N-terminal and C-terminal domains located in the cytosol, and is structurally conserved from flies to mammals. In vertebrates, Vangl2 plays an essential role in convergence and extension (CE) movements during gastrulation and in facial branchiomotor (FBM) neuron migration in the hindbrain. However, the roles of specific Vangl2 domains, of membrane association, and of specific extracellular and intracellular motifs have not been examined, especially in the context of FBM neuron migration. Through heat shock-inducible expression of various Vangl2 transgenes, we found that membrane associated functions of the N-terminal and C-terminal domains of Vangl2 are involved in regulating FBM neuron migration. Importantly, through temperature shift experiments, we found that the critical period for Vangl2 function coincides with the initial stages of FBM neuron migration out of Rhombomere 4. Intriguingly, we have also uncovered a putative nuclear localization motif in the C-terminal domain that may play a role in regulating CE movements.
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the pcp protein vangl2 regulates migration of hindbrain motor neurons by acting in floor plate cells and independently of cilia function
Developmental Biology, 2013Co-Authors: Vinoth Sittaramane, Hui Wang, Xiufang Pan, Derrick M Glasco, Peng Huang, Suman Gurung, Anagha S Bock, Koichi Kawakami, Michael P Matise, Anand ChandrasekharAbstract:Vangl2, a core component of the Planar Cell Polarity pathway, is necessary for the caudal migration of Facial Branchiomotor (FBM) neurons in the vertebrate hindbrain. Studies in zebrafish suggest that vangl2 functions largely non-cell autonomously to regulate FBM neuron migration out of Rhombomere 4 (r4), but the cell-type within which it acts is not known. Here, we demonstrate that vangl2 functions largely in floor plate cells to regulate caudal neuronal migration. Furthermore, FBM neurons fail to migrate caudally in the mouse Gli2 mutant that lacks the floor plate, suggesting an evolutionarily conserved role for this cell type in neuronal migration. Although hindbrain floor plate cilia are disorganized in vangl2 mutant embryos, cilia appear to be dispensable for neuronal migration. Notably, Vangl2 is enriched in the basolateral, but not apical, membranes of floor plate cells. Taken together, our data suggest strongly that Vangl2 regulates FBM neuron migration by acting in floor plate cells, independently of cilia function.
Robb Krumlauf - One of the best experts on this subject based on the ideXlab platform.
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expression of hoxa2 in Rhombomere 4 is regulated by a conserved cross regulatory mechanism dependent upon hoxb1
Developmental Biology, 2007Co-Authors: Stefan Tumpel, Robb Krumlauf, Elisabetta Ferretti, Francisco Cambronero, Leanne M Wiedemann, Francesco BlasiAbstract:The Hoxa2 gene is an important component of regulatory events during hindbrain segmentation and head development in vertebrates. In this study we have used sequenced comparisons of the Hoxa2 locus from 12 vertebrate species in combination with detailed regulatory analyses in mouse and chicken embryos to characterize the mechanistic basis for the regulation of Hoxa2 in Rhombomere (r) 4. A highly conserved region in the Hoxa2 intron functions as an r4 enhancer. In vitro binding studies demonstrate that within the conserved region three bipartite Hox/Pbx binding sites (PH1-PH3) in combination with a single binding site for Pbx-Prep/Meis (PM) heterodimers co-operate to regulate enhancer activity in r4. Mutational analysis reveals that these sites are required for activity of the enhancer, suggesting that the r4 enhancer from Hoxa2 functions in vivo as a Hox-response module in combination with the Hox cofactors, Pbx and Prep/Meis. Furthermore, this r4 enhancer is capable of mediating a response to ectopic HOXB1 expression in the hindbrain. These findings reveal that Hoxa2 is a target gene of Hoxb1 and permit us to develop a gene regulatory network for r4, whereby Hoxa2, along with Hoxb1, Hoxb2 and Hoxa1, is integrated into a series of auto- and cross-regulatory loops between Hox genes. These data highlight the important role played by direct cross-talk between Hox genes in regulating hindbrain patterning.
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hoxb1 enhancer and control of Rhombomere 4 expression complex interplay between prep1 pbx1 hoxb1 binding sites
Molecular and Cellular Biology, 2005Co-Authors: Elisabetta Ferretti, Robb Krumlauf, Francisco Cambronero, Stefan Tumpel, Elena Longobardi, Leanne M Wiedemann, Francesco BlasiAbstract:The Hoxb1 autoregulatory enhancer directs segmental expression in vertebrate hindbrain. Three conserved repeats (R1, R2, and R3) in the enhancer have been described as Pbx-Hoxb1 (PH) binding sites, and one Pbx-Meinox (PM) binding site has also been characterized. We have investigated the importance and relative roles of PH and PM binding sites with respect to protein interactions and in vivo regulatory activity. We have identified a new PM site (PM2) and found that it cooperates with the R3 PH site to form ternary Prep1-Pbx1-Hoxb1 complexes. In vivo, the combination of the R3 and PM2 sites is sufficient to mediate transgenic reporter activity in the developing chick hindbrain. In both chicken and mouse transgenic embryos, mutations of the PM1 and PM2 sites reveal that they cooperate to modulate in vivo regulatory activity of the Hoxb1 enhancer. Furthermore, we have shown that the R2 motif functions as a strong PM site, with a high binding affinity for Prep1-Pbx1 dimers, and renamed this site R2/PM3. In vitro R2/PM3, when combined with the PM1 and R3 motifs, inhibits ternary complex formation mediated by these elements and in vivo reduces and restricts reporter expression in transgenic embryos. These inhibitory effects appear to be a consequence of the high PM binding activity of the R2/PM3 site. Taken together, our results demonstrate that the activity of the Hoxb1 autoregulatory enhancer depends upon multiple Prep1-Pbx1 (PM1, PM2, and PM3) and Pbx1-Hoxb1 (R1 and R3) binding sites that cooperate to modulate and spatially restrict the expression of Hoxb1 in r4 Rhombomere.
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genetic interactions between hoxa1 and hoxb1 reveal new roles in regulation of early hindbrain patterning
Development, 1998Co-Authors: Michele Studer, Filippo M Rijli, Pierre Chambon, Heather Marshall, Anthony Gavalas, Linda Arizamcnaughton, Robb KrumlaufAbstract:In the developing vertebrate hindbrain Hoxa1 and Hoxb1 play important roles in patterning segmental units (Rhombomeres). In this study, genetic analysis of double mutants demonstrates that both Hoxa1 and Hoxb1 participate in the establishment and maintenance of Hoxb1 expression in Rhombomere 4 through auto- and para-regulatory interactions. The generation of a targeted mutation in a Hoxb1 3′ retinoic acid response element (RARE) shows that it is required for establishing early high levels of Hoxb1 expression in neural ectoderm. Double mutant analysis with this Hoxb1(3′RARE) allele and other targeted loss-of-function alleles from both Hoxa1 and Hoxb1 reveals synergy between these genes. In the absence of both genes, a territory appears in the region of r4, but the earliest r4 marker, the Eph tyrosine kinase receptor EphA2, fails to be activated. This suggests a failure to initiate rather than maintain the specification of r4 identity and defines new roles for both Hoxb1 and Hoxa1 in early patterning events in r4. Our genetic analysis shows that individual members of the vertebrate labial-related genes have multiple roles in different steps governing segmental processes in the developing hindbrain.
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cross regulation in the mouse hoxb complex the expression of hoxb2 in Rhombomere 4 is regulated by hoxb1
Genes & Development, 1997Co-Authors: Mark Maconochie, Michele Studer, Mh Sham, Stefan Nonchev, Siukwong Chan, Heike Popperl, Richard S Mann, Robb KrumlaufAbstract:Correct regulation of the segment-restricted patterns of Hox gene expression is essential for proper patterning of the vertebrate hindbrain. We have examined the molecular basis of restricted expression of Hoxb2 in Rhombomere 4 (r4), by using deletion analysis in transgenic mice to identify an r4 enhancer from the mouse gene. A bipartite Hox/Pbx binding motif is located within this enhancer, and in vitro DNA binding experiments showed that the vertebrate labial-related protein Hoxb1 will cooperatively bind to this site in a Pbx/Exd-dependent manner. The Hoxb2 r4 enhancer can be transactivated in vivo by the ectopic expression of Hoxb1, Hoxa1, and Drosophila labial in transgenic mice. In contrast, ectopic Hoxb2 and Hoxb4 are unable to induce expression, indicating that in vivo this enhancer preferentially responds to labial family members. Mutational analysis demonstrated that the bipartite Hox/Pbx motif is required for r4 enhancer activity and the responses to retinoids and ectopic Hox expression. Furthermore, three copies of the Hoxb2 motif are sufficient to mediate r4 expression in transgenic mouse embryos and a labial pattern in Drosophila embryos. This reporter expression in Drosophila embryos is dependent upon endogenous labial and exd, suggesting that the ability of this Hox/Pbx site to interact with labial-related proteins has been evolutionarily conserved. The endogenous Hoxb2 gene is no longer upregulated in r4 in Hoxb1 homozygous mutant embryos. On the basis of these experiments we conclude that the r4-restricted domain of Hoxb2 in the hindbrain is the result of a direct cross-regulatory interaction by Hoxb1 involving vertebrate Pbx proteins as cofactors. This suggests that part of the functional role of Hoxb1 in maintaining r4 identity may be mediated by the Hoxb2 gene.
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Ectopic Hoxa-1 induces Rhombomere transformation in mouse hindbrain
Development (Cambridge England), 1994Co-Authors: M. B. Zhang, Robb Krumlauf, Hee-joon Kim, Heather Marshall, M. Gendron-maguire, D. A. Lucas, A. Baron, Lorraine J. Gudas, T. Gridley, J F GrippoAbstract:Homeobox genes are expressed with a specific spatial and temporal order, which is essential for pattern formation during the early development of both invertebrates and vertebrates. Here we show that widespread ectopic expression of the Hoxa-1 (Hox 1.6) gene directed by a human beta-actin promoter in transgenic mice is embryolethal and produces abnormal phenotypes in a subset of domains primarily located in anterior regions. Interestingly, this abnormal development in the Hoxa-1 transgenic mice is associated with ectopic expression of the Hoxb-1 (Hox 2.9) gene in select hindbrain regions. At gestation day 9.5, two domains of strong Hoxb-1 expression are found in the anterior region of the hindbrains of Hoxa-1 transgenic embryos. One region represents the normal pattern of Hoxb-1 expression in Rhombomere 4 and its associated migrating neural crest cells, while another major domain of Hoxb-1 expression consistently appears in Rhombomere 2. Similar ectopic domains of beta-galactosidase activity are detected in dual transgenic embryos containing both beta-actin/Hoxa-1 transgene and a Hoxb-1/lacZ reporter construct. Expression of another lacZ reporter gene that directs beta-galactosidase activity predominantly in Rhombomere 2 is suppressed in the Hoxa-1 transgenic embryos. We have also detected weaker and variable ectopic Hoxb-1 expression in Rhombomeres 1, 3 and 6. No ectopic Hoxb-1 expression is detected in Rhombomere 5 and the expression of Hoxa-3 and Krox-20 in this region is unchanged in the Hoxa-1 transgenic embryos. While no obvious change in the morphology of the trigeminal or facial-acoustic ganglia is evident, phenotypic changes do occur in neurons that emanate from Rhombomeres 2 and 3 in the Hoxa-1 transgenic embryos. Additionally, alterations in the pattern of Hoxa-2 and Hoxb-1 expression in a subpopulation of neural crest cells migrating from the Rhombomere 2 region are detected in these transgenics. Taken together, these data suggest that ectopic Hoxa-1 expression can reorganize select regions of the developing hindbrain by inducing partial transformations of several Rhombomeres into a Rhombomere-4-like identity.
Charles G. Sagerström - One of the best experts on this subject based on the ideXlab platform.
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analysis of novel caudal hindbrain genes reveals different regulatory logic for gene expression in Rhombomere 4 versus 5 6 in embryonic zebrafish
Neural Development, 2018Co-Authors: Priyanjali Ghosh, Jennifer M Maurer, Charles G. SagerströmAbstract:Previous work aimed at understanding the gene regulatory networks (GRNs) governing caudal hindbrain formation identified morphogens such as Retinoic Acid (RA) and Fibroblast growth factors (FGFs), as well as transcription factors like hoxb1b, hoxb1a, hnf1ba, and valentino as being required for Rhombomere (r) r4-r6 formation in zebrafish. Considering that the caudal hindbrain is relatively complex – for instance, unique sets of neurons are formed in each Rhombomere segment – it is likely that additional essential genes remain to be identified and integrated into the caudal hindbrain GRN. By taking advantage of gene expression data available in the Zebrafish Information Network (ZFIN), we identified 84 uncharacterized genes that are expressed in r4-r6. We selected a representative set of 22 genes and assayed their expression patterns in hoxb1b, hoxb1a, hnf1b, and valentino mutants with the goal of positioning them in the caudal hindbrain GRN. We also investigated the effects of RA and FGF on the expression of this gene set. To examine whether these genes are necessary for r4-r6 development, we analyzed germline mutants for six of the genes (gas6, gbx1, sall4, eglf6, celf2, and greb1l) for defects in hindbrain development. Our results reveal that r4 gene expression is unaffected by the individual loss of hoxb1b, hoxb1a or RA, but is under the combinatorial regulation of RA together with hoxb1b. In contrast, r5/r6 gene expression is dependent on RA, FGF, hnf1ba and valentino – as individual loss of these factors abolishes r5/r6 gene expression. Our analysis of six mutant lines did not reveal Rhombomere or neuronal defects, but transcriptome analysis of one line (gas6 mutant) identified expression changes for genes involved in several developmental processes – suggesting that these genes may have subtle roles in hindbrain development. We conclude that r4-r6 formation is relatively robust, such that very few genes are absolutely required for this process. However, there are mechanistic differences in r4 versus r5/r6, such that no single factor is required for r4 development while several genes are individually required for r5/r6 formation.
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a gal4 uas system for conditional transgene expression in Rhombomere 4 of the zebrafish hindbrain
Developmental Dynamics, 2012Co-Authors: Seong-kyu Choe, Mako Nakamura, Franck Ladam, Letitiah Etheridge, Charles G. SagerströmAbstract:Background: The zebrafish is well established as a model organism for the study of vertebrate embryogenesis, but transgenic lines enabling restricted gene expression are still lacking for many tissues. Results: We first generated the hoxb1a(β−globin):eGFPum8 line that expresses eGFP in hindbrain Rhombomere 4 (r4), as well as in facial motor neurons migrating caudally from r4. Second, we generated the hoxb1a(β-globin) Gal4VP16um60 line to express the exogenous Gal4VP16 transcription factor in r4. Lastly, we prepared the UAS(β-actin):hoxa3aum61 line where the hoxa3a gene, which is normally expressed in r5 and r6, is under control of Gal4-regulated UAS elements. Crossing the hoxb1a(β−globin):Gal4VP16um60 line to the UAS(β-actin):hoxa3aum61 line drives robust hoxa3a expression in r4. We find that transgenic expression of hoxa3a in r4 does not affect hoxb1a expression, but has variable effects on migration of facial motorneurons and formation of Mauthner neurons. While cases of somatic transgene silencing have been reported in zebrafish, we have not observed such silencing to date, possibly because of our efforts to minimize repetitive sequences in the transgenic constructs. Conclusion: We have generated three transgenic lines that will be useful for future studies by permitting the labeling of r4-derived cells, as well as by enabling r4-specific expression of various transgenes. Developmental Dynamics 241:1125–1132, 2012. © 2012 Wiley Periodicals, Inc.
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a screen for hoxb1 regulated genes identifies ppp1r14al as a regulator of the Rhombomere 4 fgf signaling center
Developmental Biology, 2011Co-Authors: Seong-kyu Choe, Xiaolan Zhang, Nicolas Hirsch, Juerg R Straubhaar, Charles G. SagerströmAbstract:Segmentation of the vertebrate hindbrain into multiple Rhombomeres is essential for proper formation of the cerebellum, cranial nerves and cranial neural crest. Paralog group 1 (PG1) hox genes are expressed early in the caudal hindbrain and are required for Rhombomere formation. Accordingly, loss of PG1 hox function disrupts development of caudal Rhombomeres in model organisms and causes brainstem defects, associated with cognitive impairment, in humans. In spite of this important role for PG1 hox genes, transcriptional targets of PG1 proteins are not well characterized. Here we use ectopic expression together with embryonic dissection to identify novel targets of the zebrafish PG1 gene hoxb1b. Of 100 genes up-regulated by hoxb1b, 54 were examined and 25 were found to represent novel hoxb1b regulated hindbrain genes. The ppp1r14al gene was analyzed in greater detail and our results indicate that Hoxb1b is likely to directly regulate ppp1r14al expression in Rhombomere 4. Furthermore, ppp1r14al is essential for establishment of the earliest hindbrain signaling-center in Rhombomere 4 by regulating expression of fgf3.
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Meis3 synergizes with Pbx4 and Hoxb1b in promoting hindbrain fates in the zebrafish.
Development (Cambridge England), 2001Co-Authors: Nikolaos Vlachakis, Seong-kyu Choe, Charles G. SagerströmAbstract: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.
Vinoth Sittaramane - One of the best experts on this subject based on the ideXlab platform.
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structural and temporal requirements of wnt pcp protein vangl2 function for convergence and extension movements and facial branchiomotor neuron migration in zebrafish
Mechanisms of Development, 2014Co-Authors: Vinoth Sittaramane, Suman Gurung, Anand ChandrasekharAbstract:Abstract Van gogh-like 2 (Vangl2), a core component of the Wnt/planar cell polarity (PCP) signaling pathway, is a four-pass transmembrane protein with N-terminal and C-terminal domains located in the cytosol, and is structurally conserved from flies to mammals. In vertebrates, Vangl2 plays an essential role in convergence and extension (CE) movements during gastrulation and in facial branchiomotor (FBM) neuron migration in the hindbrain. However, the roles of specific Vangl2 domains, of membrane association, and of specific extracellular and intracellular motifs have not been examined, especially in the context of FBM neuron migration. Through heat shock-inducible expression of various Vangl2 transgenes, we found that membrane associated functions of the N-terminal and C-terminal domains of Vangl2 are involved in regulating FBM neuron migration. Importantly, through temperature shift experiments, we found that the critical period for Vangl2 function coincides with the initial stages of FBM neuron migration out of Rhombomere 4. Intriguingly, we have also uncovered a putative nuclear localization motif in the C-terminal domain that may play a role in regulating CE movements.
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the pcp protein vangl2 regulates migration of hindbrain motor neurons by acting in floor plate cells and independently of cilia function
Developmental Biology, 2013Co-Authors: Vinoth Sittaramane, Hui Wang, Xiufang Pan, Derrick M Glasco, Peng Huang, Suman Gurung, Anagha S Bock, Koichi Kawakami, Michael P Matise, Anand ChandrasekharAbstract:Vangl2, a core component of the Planar Cell Polarity pathway, is necessary for the caudal migration of Facial Branchiomotor (FBM) neurons in the vertebrate hindbrain. Studies in zebrafish suggest that vangl2 functions largely non-cell autonomously to regulate FBM neuron migration out of Rhombomere 4 (r4), but the cell-type within which it acts is not known. Here, we demonstrate that vangl2 functions largely in floor plate cells to regulate caudal neuronal migration. Furthermore, FBM neurons fail to migrate caudally in the mouse Gli2 mutant that lacks the floor plate, suggesting an evolutionarily conserved role for this cell type in neuronal migration. Although hindbrain floor plate cilia are disorganized in vangl2 mutant embryos, cilia appear to be dispensable for neuronal migration. Notably, Vangl2 is enriched in the basolateral, but not apical, membranes of floor plate cells. Taken together, our data suggest strongly that Vangl2 regulates FBM neuron migration by acting in floor plate cells, independently of cilia function.
Sarah Guthrie - One of the best experts on this subject based on the ideXlab platform.
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facial visceral motor neurons display specific Rhombomere origin and axon pathfinding behavior in the chick
The Journal of Neuroscience, 2000Co-Authors: John Jacob, Sarah GuthrieAbstract:In the chick embryo, facial motor neurons comprise branchiomotor and visceral motor subpopulations, which innervate branchial muscles and parasympathetic ganglia, respectively. Although facial motor neurons are known to develop within hindbrain Rhombomere 4 (r4) and r5, the precise origins of branchiomotor and visceral motor neuron subpopulations are unclear. We investigated the organization and axon pathfinding of these motor neurons using axonal tracing and Rhombomere transplantation in quail-chick chimeras. Our results show that a large majority of branchiomotor neurons originate in r4 but that a cohort of these neurons undergoes a caudal migration from r4 into r5. By contrast, visceral motor neurons develop exclusively in r5. We found that a striking property of facial visceral motor neurons is the ability of their axons to navigate back to appropriate ganglionic targets in the periphery after heterotopic transplantation. These results complement previous studies in which heterotopic facial branchiomotor neurons sent axons to their correct, branchial arch, target. By contrast, when trigeminal branchiomotor neurons were transplanted heterotopically, we found that they were unable to pathfind correctly, and instead projected to an inappropriate target region. Thus, facial and trigeminal motor neuron populations have different axon pathfinding characteristics.
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Rhombomere origin plays a role in the specificity of cranial motor axon projections in the chick.
The European journal of neuroscience, 1999Co-Authors: Joanne Warrilow, Sarah GuthrieAbstract:Guidance of cranial motor axons to their targets conforms to a segmental plan in the chick embryo. Trigeminal motor neurons lie within Rhombomeres 2 and 3 and project via an exit point in Rhombomere 2 to innervate the first branchial arch. Facial motor neurons lie within Rhombomeres 4 and 5 and grow out via an exit point in Rhombomere 4 to innervate the second branchial arch. We have investigated the axial level-specific matching of motor neurons and branchial arches using donor to host transplantation in avian embryos. Previous work has shown that rostrocaudal reversal of a single hindbrain segment (Rhombomere 3) leads to misprojection of a contingent of trigeminal axons via the facial nerve exit point. Using the same experimental manipulation in chick embryos and quail–chick chimaeras, we have analysed the pathways of these aberrant projections. We have found that in the majority of embryos analysed from stage 19 to 31, trigeminal axons from the transplanted Rhombomere projected towards second branchial arch muscles, in addition to their normal first arch muscle targets. However, from stage 32 to 36, aberrant projections to second arch-derived muscles were detected only in a small minority of embryos. These experiments show that trigeminal motor neurons show a lack of specificity in their early projection into the periphery but that inappropriate projections may be later eliminated. This suggests that segmental mechanisms intrinsic to the hindbrain specify motor neurons with respect to their eventual innervation pattern.
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selective dispersal of avian Rhombomere cells in orthotopic and heterotopic grafts
Development, 1993Co-Authors: Sarah Guthrie, Victoria E Prince, Andrew LumsdenAbstract:During hindbrain development, cells become segregated into segmental groups, Rhombomeres, by mechanisms that are presently unknown. One contributory factor early in development may be an alternating periodicity in cell surface properties down the neuraxis. This possibility was previously suggested by experiments in which tissue from different segmental levels was apposed in the absence of a boundary. New boundaries were regenerated only when Rhombomeres from adjacent positions or positions three Rhombomeres distant from one another were apposed. Combinations of two odd-numbered or two even-numbered Rhombomeres usually failed to generate a boundary. In order to pursue this phenomenon to the cellular level, we have used two approaches, both involving donor-to-host transplantation. First, quail Rhombomeres were grafted at various hindbrain levels of a chick host. Apposition of Rhombomere 4 (r4) with r3 was concomitant with negligible cell mixing across the interface. By contrast, combinations of r3 with r5 or with r3 tissue led to cell mixing that was more extensive in combinations of identical Rhombomeres (r3 with r3) than between two alternate ones (r3 with r5). Secondly, we grafted small pieces of fluorescently prelabelled chick Rhombomere tissue at various hindbrain levels of chick hosts. In most cases, cells dispersed widely when transplanted orthopically or two segments distant from that of their origin. Cells transplanted into an adjacent segment, however, showed a tendency to remain undispersed. Among the different graft combinations, furthermore, there was a variation in the extent of dispersal that showed an additional level of complexity not revealed in boundary regeneration experiments. The possibility is raised that the early partitioning of Rhombomeres involves a hierarchy in the adhesive preferences of cell-cell interactions along the neuraxis.
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neuroectodermal autonomy of hox 2 9 expression revealed by Rhombomere transpositions
Nature, 1992Co-Authors: Sarah Guthrie, Robb Krumlauf, Heather Marshall, Atsushi Kuroiwa, Ian Muchamore, Andrew LumsdenAbstract:INVOLVEMENT of the Hox genes in regional specifications of the vertebrate body axis is suggested by sequence similarity with the homeotic selector genes of Drosophila, the conservation of a collinear relationship between genomic organization and site of expression, and mutational analysis1–5. Subdivision of vertebrate embryo hindbrain neuroepithelium into lineage compartments6 (Rhombomeres7,8) underlies segmental patterning of neuronal differentiation9. The Rhombomere boundaries delimit domains of expression of Hox genes10–12, presumed to be determinants of Rhombomere phenotype, suggesting that Hox genes confer positional value13; the formation of Rhombomere 4 (r4) is followed by strong expression of Hox-2.9within its confines14. If the Hox genes are determinants, their expression should be autonomous from the developmental stage at which regional commitment becomes fixed and irreversible. We have transplanted the future r4 region (from state-9 – chick embryos) into the more anterior position of r2 and probed for Hox-2.9 transcripts. We report here that Hox-2.9 was expressed in the ectopic r4 as strongly as in the normal r4, whereas reciprocal grafts of future r2 to r4 position did not express Hox-2.9. The phenotype of ectopic Rhombomeres developed according to their original position, as demonstrated by retrograde tracing of efferent cranial nerve nuclei. As early as stage-9 – (six somites), both Hox-2.9 expression and segment identity are autonomous in the chick embryo hindbrain, independent both of position in the neuroepithelium and of signals from the underlying mesoderm15.