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(A) Control stage 10 embryo showing Cek-8 expression in r3 and r5 and in neural crest derived from r6. (B) Control stage 10 embryo showing Krox-20 expression in r3 and r5 and in neural crest derived from r6. (C) Cek-8 is expressed with sharp boundaries in r3 and r5 in flat-mounted control stage 18 hindbrain. (D) r5 domain of Cek-8 expression is lost (arrowheads) 6 hours after addition of bead (b) at r4 level, r3 domain remains strong. Bead was damaged slightly by dissection. (E) After 48 hours, Cek-8 expression borders of r3 remain sharp and coincident with morphological boundaries. (F) High power of Cek-8 expression in retinoid-treated hindbrain that lacked caudal r3 boundary. Lack of morphological boundary coincides with fuzzy border to Cek-8 (short arrow), rostral border of expression (long arrow) is sharp and matches morphological boundary (b). (G) Krox-20 is downregulated in both r3 and r5 (arrows) when retinoic acid is applied at r4 and all expression is lost (arrows) by stage 14 (H). Bar is 500 µm in A, B, G and H; 200 µm in C-E; 50 µm in F.
Source publication
Rhombomeres are segmental units of the hindbrain that are separated from each other by a specialised zone of boundary cells. Retinoic acid application to a recently segmented hindbrain leads to disappearance of posterior rhombomere boundaries. Boundary loss is preceded by changes in segmental expression of Krox-20 and Cek-8 and followed by alterati...
Contexts in source publication
Context 1
... r3 and r5 from stage 10 onwards ( Fig In specimens where only two rhombomeres were clearly distinguishable, Cek-8 had a sharp expression boundary at the anterior part of r3, where a morphological boundary was still visible, but not at the posterior part, where Cek-8 expression was downregu- lated and the morphological boundary abolished (n=2/2 Fig. 2F). In two specimens, a r4/5 boundary was also present and, in both cases, this correlated with residual Cek-8 expression in r5, although this expression was not immediately adjacent to the ...
Context 2
... effect on Krox-20 expression varied with the position of the bead. At 6 hours, a bead placed at r4 level abolished Krox-20 expression in both r3 and r5 (n=1, Fig. 2G), and a bead placed at r5 or r6 abolished Krox-20 transcripts at r5 level only (n=2). Surprisingly beads placed at r2 or r3 level, had no affect on r3 expression but complete downregu- lation in r5 (n=2). Beads anterior to r2 or posterior to r6 did not affect Krox-20 expression. With beads placed at r4, Krox- 20 in both r3 and r5 ...
Context 3
... beads placed at r2 or r3 level, had no affect on r3 expression but complete downregu- lation in r5 (n=2). Beads anterior to r2 or posterior to r6 did not affect Krox-20 expression. With beads placed at r4, Krox- 20 in both r3 and r5 remains completely downregulated 24 hours after retinoic acid treatment when the embryos have reached stage 15/16 (Fig. 2H, n=4/5, the fifth embryo had weak expression in r3). Despite the loss of Krox-20, the morpho- logical boundaries of r3 were ...
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Transient segmentation in the hindbrain is a fundamental morphogenetic phenomenon in the vertebrate embryo, and the restricted expression of subsets of Hox genes in the developing rhombomeric units and their derivatives is linked with regional specification. Here we show that patterning of the vertebrate hindbrain involves the direct upregulation o...
Segmentation is a key feature of the development of the vertebrate hindbrain where it involves the generation of repetitive morphological units termed rhombomeres (r). Hox genes are likely to play an essential role in the specification of segmental identity and we have been investigating their regulation. We show here that the mouse and chicken Hox...
Citations
... [18][19][20][21][22] The rhombomeres contain repeating populations of neurons, which undergo differentiation in a coordinated rhombomere-specific manner to generate specialized properties, transcriptional programs, and functional connectivity for each segment. [23][24][25][26][27][28][29][30] Hence, hindbrain segmentation lays down the basic ground plan for organization and wiring of the vertebrate hindbrain, which underlies its conserved functional role as a complex coordination center in the central nervous system. ...
Background
The basic ground plan of vertebrate hindbrain is established through a process of segmentation, which generates eight transient lineage‐restricted cellular compartments called rhombomeres (r). The segments adopt distinct individual identities in response to axial patterning signals. It is unclear whether signaling between rhombomeres plays a conserved role in regulating segmental patterning during hindbrain development.
Results
Using tissue manipulations of rhombomeres in chicken embryos, we have uncovered roles for r2 and r4 in regulating the expression of EphA4 in r3 and r5. Perturbations of signaling pathways reveal that these regulatory inputs from r2 and r4 into EphA4 expression are mediated independent of inputs from Krox20 through cues involving fibroblast growth factor (FGF) signaling. These interactions are stage dependent and are set up in embryos with <10 somites.
Conclusions
We show that r2 and r4 function as temporally dynamic signaling centers in the early patterning of adjacent hindbrain segments and this activity is dependent upon the FGF pathway. These results reveal that inter‐rhombomeric signaling is a conserved feature of the regulatory networks that control the specification of individual rhombomere identities in vertebrate hindbrain segmentation. However, the timing of when restricted domains of FGF signaling are coupled to formation of r4 may vary between the species.
... Anterior-posterior gradients of retinoic acid, Fgfs, and Wnts roughly set boundaries in the hindbrain (Tuazon and Mullins, 2015), which are then sharpened and maintained by cell sorting mediated by Eph-ephrin and by cell identity switching (Calzolari et al., 2014;Cayuso et al., 2015;Xu et al., 1999;Addison et al., 2018). Finally, cells at the boundaries express specific marker genes and become elongated with increased extracellular space, which are supposed to stabilize the sharp boundaries for a long period of time (Kiecker and Lumsden, 2005;Nittenberg et al., 1997;Sela-Donenfeld et al., 2009;Weisinger et al., 2012). ...
Establishment of robust gene expression boundary is crucial for creating elaborate morphology during development. However, mechanisms underlying boundary formation have been extensively studied only in a few model systems. We examined the establishment of zic1/zic4-expression boundary demarcating dorsoventral boundary of the entire trunk of medaka fish (Oryzias latipes) and identified a subgroup of dermomyotomal cells called horizontal boundary cells (HBCs) as crucial players for the boundary formation. Embryological and genetic analyses demonstrated that HBCs play crucial roles in the two major events of the process, i.e., refinement and maintenance. In the refinement, HBCs could serve as a chemical barrier against Wnts from the neural tube by expressing Hhip. At later stages, HBCs participate in the maintenance of the boundary by differentiating into the horizontal myoseptum physically inhibiting cell mixing across the boundary. These findings reveal the mechanisms underlying the dorsoventral boundary in the teleost trunk by specialized boundary cells. : Abe et al. find horizontal boundary cells (HBCs) crucial players for the formation of the dorsoventral compartments of the teleost myotome. HBCs express Hhip, which is necessary for the refinement of the boundary, and they contribute to the horizontal myoseptum, an anatomical structure dividing the dorsoventral compartments. Keywords: boundary formation, boundary maintenance, boundary cell, dorsoventral patterning, somite, dermomyotome, canonical Wnt signaling, hedgehog signaling, SDF1/CXCR4 signaling, Hhip
... These results were later confirmed by pharmacologic or genetic disruption of RA in other species, where the posterior hindbrain became anteriorized [179,242,257,258]. Conversely, either inhibition of FGF8 or enhancement of RA activity led to the expansion of anterior Hox genes [256,259]. Knockout of FGF8 also resulted in midbrain Wnt1 expression expanding posteriorly into the isthmus and cerebellum [260]. These and other studies indicate that posterior RA and anterior FGF8 act in opposing manners to specify Hox-positive/negative domains to define the AP borders of the hindbrain. ...
The hindbrain is a key relay hub of the central nervous system (CNS), linking the bilaterally symmetric half-sides of lower and upper CNS centers via an extensive network of neural pathways. Dedicated neural assemblies within the hindbrain control many physiological processes, including respiration, blood pressure, motor coordination and different sensations. During early development, the hindbrain forms metameric segmented units known as rhombomeres along the antero-posterior (AP) axis of the nervous system. These compartmentalized units are highly conserved during vertebrate evolution and act as the template for adult brainstem structure and function. TALE and HOX homeodomain family transcription factors play a key role in the initial induction of the hindbrain and its specification into rhombomeric cell fate identities along the AP axis. Signaling pathways, such as canonical-Wnt, FGF and retinoic acid, play multiple roles to initially induce the hindbrain and regulate Hox gene-family expression to control rhombomeric identity. Additional transcription factors including Krox20, Kreisler and others act both upstream and downstream to Hox genes, modulating their expression and protein activity. In this review, we will examine the earliest embryonic signaling pathways that induce the hindbrain and subsequent rhombomeric segmentation via Hox and other gene expression. We will examine how these signaling pathways and transcription factors interact to activate downstream targets that organize the segmented AP pattern of the embryonic vertebrate hindbrain.
... Unlike in chick (Fraser et al., 1990), this study did not detect intermingling between prospective segments at early stages (Calzolari et al., 2014), perhaps reflecting that the transgenic reporters are not expressed until after lineage restriction has been established. Since blocking of boundary cell formation does not affect border sharpness, it seems unlikely that these specialized cells serve as a barrier to cell movement (Nittenberg et al., 1997). Rather, there is an immiscibility of adjacent rhombomeres, which transplantation experiments in chick suggest involves distinct properties of odd versus even numbered segments (Guthrie et al., 1993). ...
The formation of sharp borders, across which cell intermingling is restricted, has a crucial role in the establishment and maintenance of organized tissues. Signaling of Eph receptors and ephrins underlies formation of a number of boundaries between and within tissues during vertebrate development. Eph-ephrin signaling can regulate several types of cell response-adhesion, repulsion and tension-that can in principle underlie the segregation of cells and formation of sharp borders. Recent studies have implicated each of these cell responses as having important roles at different boundaries: repulsion at the mesoderm-ectoderm border, decreased adhesion at the notochord-presomitic mesoderm border, and tension at boundaries within the hindbrain and forebrain. These distinct responses to Eph receptor and ephrin activation may in part be due to the adhesive properties of the tissue.
Copyright © 2014. Published by Elsevier Inc.
... Misexpression of Hox4 proteins suppresses r6/r7 and more anterior boundaries We next used exogenous retinoic acid (RA) as an indirect way of misexpressing Hoxb4 and Hoxd4 across the r6/r7 interface. RA suppresses varying numbers of hindbrain boundaries, depending upon the treatment stage (Wood et al., 1994;Nittenberg et al., 1997;Dupé and Lumsden, 2001). At E9.25, it expands murine Hox4 expression ectopically into the anterior hindbrain (Conlon and Rossant, 1992;Morrison et al., 1997). ...
Hox genes encode a conserved family of homeodomain transcription factors regulating development along the major body axis. During embryogenesis, Hox proteins are expressed in segment-specific patterns and control numerous different segment-specific cell fates. It has been unclear, however, whether Hox proteins drive the epithelial cell segregation mechanism that is thought to initiate the segmentation process. Here, we investigate the role of vertebrate Hox proteins during the partitioning of the developing hindbrain into lineage-restricted units called rhombomeres. Loss-of-function mutants and ectopic expression assays reveal that Hoxb4 and its paralogue Hoxd4 are necessary and sufficient for cell segregation, and for the most caudal rhombomere boundary (r6/r7). Hox4 proteins regulate Eph/ephrins and other cell-surface proteins, and can function in a non-cell-autonomous manner to induce apical cell enlargement on both sides of their expression border. Similarly, other Hox proteins expressed at more rostral rhombomere interfaces can also regulate Eph/ephrins, induce apical remodelling and drive cell segregation in ectopic expression assays. However, Krox20, a key segmentation factor expressed in odd rhombomeres (r3 and r5), can largely override Hox proteins at the level of regulation of a cell surface target, Epha4. This study suggests that most, if not all, Hox proteins share a common potential to induce cell segregation but in some contexts this is masked or modulated by other transcription factors.
... The conservative mode of medio-lateral intercalation we report here (by which few cells move into or out of labeled cohorts) is also supported by high-magnification timelapse imaging of cell behaviors during neural plate convergent extension Elul and Keller (2000). Such behavior could be maintained by multiple specialized boundary populations similar to those populations found between rhombomeres in the chick hindbrain Nittenberg et al. (1997). One boundary has already been proposed to exist at the Xenopus sulcus limitans, where cell movement appears restricted across a small population of F-Cadherin-expressing cells Espeseth et al. (1998). ...
Background:
During Xenopus laevis neurulation, neural ectodermal cells of the spinal cord are patterned at the same time that they intercalate mediolaterally and radially, moving within and between two cell layers. Curious if these rearrangements disrupt early cell identities, we lineage-traced cells in each layer from neural plate stages to the closed neural tube, and used in situ hybridization to assay gene expression in the moving cells.
Results:
Our biotin and fluorescent labeling of deep and superficial cells reveals that mediolateral intercalation does not disrupt cell cohorts; in other words, it is conservative. However, outside the midline notoplate, later radial intercalation does displace superficial cells dorsoventrally, radically disrupting cell cohorts. The tube roof is composed almost exclusively of superficial cells, including some displaced from ventral positions; gene expression in these displaced cells must now be surveyed further. Superficial cells also flank the tube's floor, which is, itself, almost exclusively composed of deep cells.
Conclusions:
Our data provide: (1) a fate map of superficial- and deep-cell positions within the Xenopus neural tube, (2) the paths taken to these positions, and (3) preliminary evidence of re-patterning in cells carried out of one environment and into another, during neural morphogenesis.
... Les frontières inter-rhombomériques ne sont pas directement impliquées puisqu'elles n'apparaissent que secondairement, après contact entre cellules des rhombomères pairs et cellules des rhombomères impairs (Guthrie and Lumsden, 1991). En outre, l'ablation des cellules frontières ne modifie pas la restriction de lignage (Nittenberg et al., 1997). Les frontières sont en fait constituées de cellules spécialisées impliquées dans la différenciation neuronale à des stades ultérieurs (Amoyel et al., 2005). ...
During embryonic development, the cell fates are specified by the expression of lineage factors whose expression must be finely regulated to ensure proper organ formation. To get insight into the control of lineage factors expression, we used the vertebrate hindbrain (rhombencephalon) as a system model. During development, the hindbrain is subdivided into seven segments, termed rhombomeres and noted from r1 to r7. Each segment corresponds to a coherent cell lineage. In particular, specification of r3 and r5 lineages is controlled by the zinc-finger transcription factor Krox20. When Krox20 function is abolished, r3 and r5 cells are not properly specified, hence modified neuronal fate. In the present PhD work, we aimed at deciphering the processes that control the number of Krox20-expressing cells, i.e. the size of r3 and r5. We focused in particular on the transcriptional control of Krox20 expression by studying the activity of Krox20 cis-regulatory elements. Two elements, termed B and C, are responsible for the initiation of Krox20 expression; a third one, noted A, amplifies and prolongs it through an autoregulatory activity. We showed (i) that cells commit to the r3/r5 fate only if they activate the element A, (ii) that the element A functions as a bistable switch, determined by the level of Krox20 initiation. These results were obtained by the analysis of a computational model, constrained by quantitative data derived from experiments on zebrafish embryos. Our model is built at the cell level and implement molecular events occuring at the level of element A. It thus establishes the relationship between enhancer activity and tissue-scale patterning
... Some examples in the CNS include the zona limitans intrathalamica (ZLI), which serves as an interface between the thalamic and prethalamic primordia, and expressed sonic hedgehog (SHH), Wnts and fibroblast growth factors (FGFs) (Guinazu et al., 2007;Lim and Golden, 2007;Scholpp et al., 2006;Shimamura et al., 1995), and the midbrainhindbrain boundary (MHB), which expresses FGF8 and regulates the fates of the mesencephalon and anterior hindbrain (Lekven et al., 2003;Mason et al., 2000;Wassef and Joyner, 1997;Zervas et al., 2005). In hindbrain boundaries of multiple species, various signaling factors have also been shown to be expressed, such as Wnts in zebrafish (Amoyel et al., 2005;Riley et al., 2004), and FGFs, as well as the TGFb inhibitor follistatin, in chick and mice (Connolly et al., 1995;Kurose et al., 2004;Mahmood et al., 1995;Nittenberg et al., 1997;Sela-Donenfeld et al., 2009;Weisinger et al., 2008). However, what role these factors may play in boundaries remains vague. ...
Compartment boundaries act as organizing centers that segregate adjacent areas into domains of gene expression and regulation, and control their distinct fates via the secretion of signalling factors. During hindbrain development, a specialized cell-population forms boundaries between rhombomeres. These boundary cells demonstrate unique morphological properties and express multiple genes that differs them from intra-rhombomeric cells. Yet, little is known regarding the mechanisms that controls the expression or function of these boundary markers.
Multiple components of the FGF signaling system, including ligands, receptors, downstream effectors as well as proteoglycans are shown to localize to boundary cells in the chick hindbrain. These patterns raise the possibility that FGF signaling plays a role in regulating boundary properties. We provide evidence to the role of FGF signaling, particularly the boundary-derived FGF3, in regulating the expression of multiple markers at hindbrain boundaries. These findings enable further characterization of the unique boundary-cell population, and expose a new function for FGFs as regulators of boundary-gene expression in the chick hindbrain.
... Morphologically visible boundaries form between rhombomeres during hindbrain development, and boundary formation may depend on the distinct properties of successive odd and evennumbered segments [37]. Based on our finding that marker gene expression is dramatically reduced in odd-numbered segments and variably retained in even-numbered segments we asked whether rhombomere boundaries are formed in kto mutant embryos. ...
Rhombomere boundaries form during hindbrain segmentation and are critical for maintaining segmental integrity and regulating migration in the hindbrain. Some genetic models affecting hindbrain boundary formation have been described, but involvement of components of the transcriptional mediator complex in boundary formation has not reported so far.
The kto/med12 mutant zebrafish, which affects the Mediator component Med12, causes specific loss of rhombomere boundary cells even though segmentation of the hindbrain takes place at least in part. In kto mutant embryos, cells forming rhombomere boundaries were largely absent as indicated by the use of several marker genes. While no obvious increase in cell death was observed, we found a notable reduction of cell proliferation in the hindbrain of kto mutant zebrafish.
The kto/med12 mutation results in specific defects of boundary cell formation in the zebrafish hindbrain.
... The established rhombomere boundary regions regulate broad aspects of the subsequent hindbrain development. The rhombomere boundary cells secrete a variety of signaling molecules, such as FGF, BMP inhibitor and Wnts (Mahmood et al., 1995(Mahmood et al., , 1996Nittenberg et al., 1997;Cheng et al., 2004;Riley et al., 2004;Amoyel et al., 2005;Sela-Donenfeld et al., 2009). These signaling molecules influence patterning and neuronal specification in the non-boundary regions (Amoyel et al., 2005;Weisinger et al., 2008). ...
The vertebrate neuroepithelium can be subdivided into non-boundary and boundary regions. The boundary regions act as signaling centers for regional specification and neurogenesis in the neighboring non-boundary regions. An important question is how boundary regions are specified and maintained during brain development. In this study, we report that Pax6, a homeodomain transcription factor, regulates boundary-cell specification between rhombomeres of the developing rat hindbrain. We compared expression patterns of four boundary-cell markers, including PLZF (Zbtb16), Ring1A (Ring1), Wnt5a, and cadherin7 (Cdh7), in wild-type and Pax6 loss-of-function mutant hindbrains and found that the expression of Zbtb16, Ring1, and Wnt5a was down-regulated in the rhombomere boundaries, while Cdh7 expression was up-regulated in the non-boundary regions of the Pax6 mutant hindbrain. Morphological observations revealed that the boundary regions were larger and that the interface between the boundary and non-boundary regions was obscured in the Pax6 mutant hindbrain. We also found ectopic neuronal differentiation in the boundary cells of the Pax6 mutant hindbrain. In addition, we observed that Hes5 was down-regulated and that Neurogenin2 (Neurog2) was up-regulated in the boundary regions of the Pax6 mutant hindbrain. Because Hes genes have been shown to inhibit neuronal differentiation by repressing proneural genes, Pax6 may act through this pathway to prevent neurogenesis in the boundary cells. Taken together, Pax6 seems to be required for coordinating boundary-cell specification and reducing neurogenesis within the hindbrain boundary region.
