An autoregulatory cell cycle timer integrates growth and specification in chick wing digit development.
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ABSTRACT:
SUBMITTER: Pickering J
PROVIDER: S-EPMC6777937 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Hoxd13, Tbx2, Tbx3, Sall1 and Sall3 genes are candidates for encoding antero-posterior positional values in the developing chick wing and specifying digit identity. In order to build up a detailed profile of gene expression patterns in cell lineages that give rise to each of the digits over time, we compared 3 dimensional (3D) expression patterns of these genes during wing development and related them to digit fate maps. 3D gene expression data at stages 21, 24 and 27 spanning early bud to digital plate formation, captured from in situ hybridisation whole mounts using Optical Projection Tomography (OPT) were mapped to reference wing bud models. Grafts of wing bud tissue from GFP chicken embryos were used to fate map regions of the wing bud giving rise to each digit; 3D images of the grafts were captured using OPT and mapped on to the same models. Computational analysis of the combined computerised data revealed that Tbx2 and Tbx3 are expressed in digit 3 and 4 progenitors at all stages, consistent with encoding stable antero-posterior positional values established in the early bud; Hoxd13 and Sall1 expression is more dynamic, being associated with posterior digit 3 and 4 progenitors in the early bud but later becoming associated with anterior digit 2 progenitors in the digital plate. Sox9 expression in digit condensations lies within domains of digit progenitors defined by fate mapping; digit 3 condensations express Hoxd13 and Sall1, digit 4 condensations Hoxd13, Tbx3 and to a lesser extent Tbx2. Sall3 is only transiently expressed in digit 3 progenitors at stage 24 together with Sall1 and Hoxd13; then becomes excluded from the digital plate. These dynamic patterns of expression suggest that these genes may play different roles in digit identity either together or in combination at different stages including the digit condensation stage.
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Project description:An often overlooked aspect of digit development is the special nature of the terminal phalanx, a specialized structure with characteristics distinct from other phalanges, for example the presence of ectodermal derivatives such as nails and claws. Here, we describe the unique ossification pattern of distal phalanges and characteristic gene expression in the digit tips of chick and duck embryos. Our results show that the distal phalanx of chick wing digit 1 is a genuine tip with a characteristic ossification pattern and expression of Bambi and Sp8; however, the terminal phalanx of digits 2* and 3 is not a genuine tip, and these are therefore truncated digits. Bambi and Sp8 expression in the chick wing provides a direct molecular assessment of digit identity changes after experimental manipulations of digit primordia. In contrast, digits 1 and 2 of the duck wing both possess true tips. Although chick wing-tip development was not rescued by application of Fgf8, this treatment induced the development of extra phalanges. Grafting experiments show that competence for tip formation, including nails, is latent in the interdigital tissue. Our results deepen understanding of the mechanisms of digit tip formation, highlighting its developmental autonomy and modular nature, with implications for digit reduction or loss during evolution. * Numbering of wing digits is 1, 2, 3 from anterior to posterior.
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Project description:Birth dating neurons with bromodeoxyuridine (BrdU) labeling is an established method widely employed by neurobiologists to study cell proliferation in embryonic, postnatal, and adult brain. Birth dating studies in the chick dorsal telencephalon and the mammalian striatum have suggested that these structures develop in a strikingly similar manner, in which neurons with the same birth date aggregate to form "isochronic clusters." Here we show that isochronic cluster formation in the chick dorsal telencephalon is an artifact. In embryos given standardly employed doses of BrdU, we observed isochronic clusters but found that clusters were absent with BrdU doses close to the limits of detection. In addition, in situ hybridization experiments established that neurons in the clusters display errors in cell type specification: BrdU cell clusters in nidopallium adopted a mesopallial neuronal fate, mesopallial clusters were misspecified as nidopallial cells, and in some instances, the BrdU clusters failed to express neuronal differentiation markers characteristic of the dorsal telencephalon. These results demonstrate that the chick dorsal telencephalon does not develop by isochronic cluster formation and highlight the need to test the integrity of BrdU-treated tissue with gene expression markers of regional and cell type identity.
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Project description:The displacement of the mitotic spindle to one side of a cell is important for many cells to divide unequally. While recent progress has begun to unveil some of the molecular mechanisms of mitotic spindle displacement, far less is known about how spindle displacement is precisely timed. A conserved mitotic progression mechanism is known to time events in dividing cells, although this has never been linked to spindle displacement. This mechanism involves the anaphase-promoting complex (APC), its activator Cdc20/Fizzy, its degradation target cyclin, and cyclin-dependent kinase (CDK). Here we show that these components comprise a previously unrecognized timer for spindle displacement. In the Caenorhabditis elegans zygote, mitotic spindle displacement begins at a precise time, soon after chromosomes congress to the metaphase plate. We found that reducing the function of the proteasome, the APC, or Cdc20/Fizzy delayed spindle displacement. Conversely, inactivating CDK in prometaphase caused the spindle to displace early. The consequence of experimentally unlinking spindle displacement from this timing mechanism was the premature displacement of incompletely assembled components of the mitotic spindle. We conclude that in this system, asymmetric positioning of the mitotic spindle is normally delayed for a short time until the APC inactivates CDK, and that this delay ensures that the spindle does not begin to move until it is fully assembled. To our knowledge, this is the first demonstration that mitotic progression times spindle displacement in the asymmetric division of an animal cell. We speculate that this link between the cell cycle and asymmetric cell division might be evolutionarily conserved, because the mitotic spindle is displaced at a similar stage of mitosis during asymmetric cell divisions in diverse systems.
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Project description:The insect wing is a key evolutionary innovation that was essential for insect diversification. Yet despite its importance, there is still debate about its evolutionary origins. Two main hypotheses have been proposed: the paranotal hypothesis, which suggests that wings evolved as an extension of the dorsal thorax, and the gill-exite hypothesis, which proposes that wings were derived from a modification of a pre-existing branch at the dorsal base (subcoxa) of the leg. Here, we address this question by studying how wing fates are initially specified during Drosophila embryogenesis, by characterizing a cis-regulatory module (CRM) from the snail (sna) gene, sna-DP (for dorsal primordia). sna-DP specifically marks the early primordia for both the wing and haltere, collectively referred to as the DP. We found that the inputs that activate sna-DP are distinct from those that activate Distalless, a marker for leg fates. Further, in genetic backgrounds in which the leg primordia are absent, the DP are still partially specified. However, lineage-tracing experiments demonstrate that cells from the early leg primordia contribute to both ventral and dorsal appendage fates. Together, these results suggest that the wings of Drosophila have a dual developmental origin: two groups of cells, one ventral and one more dorsal, give rise to the mature wing. We suggest that the dual developmental origins of the wing may be a molecular remnant of the evolutionary history of this appendage, in which cells of the subcoxa of the leg coalesced with dorsal outgrowths to evolve a dorsal appendage with motor control.
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Project description:BackgroundHighly diverse butterfly wing patterns have emerged as a powerful system for understanding the genetic basis of phenotypic variation. While the genetic basis of this pattern variation is being clarified, the precise developmental pathways linking genotype to phenotype are not well understood. The gene aristaless, which plays a role in appendage patterning and extension, has been duplicated in Lepidoptera. One copy, aristaless1, has been shown to control a white/yellow color switch in the butterfly Heliconius cydno, suggesting a novel function associated with color patterning and pigmentation. Here we investigate the developmental basis of al1 in embryos, larvae, and pupae using new antibodies, CRISPR/Cas9, RNAi, qPCR assays of downstream targets, and pharmacological manipulation of an upstream activator.ResultsWe find that Al1 is expressed at the distal tips of developing embryonic appendages consistent with its ancestral role. In developing wings, we observe Al1 accumulation within developing scale cells of white H. cydno during early pupation while yellow scale cells exhibit little Al1 at this time point. Reduced Al1 expression is also associated with yellow scale development in al1 knockouts and knockdowns. We propose that Al1 expression in future white scales might be related to an observed downregulation of the enzyme Cinnabar and other genes that synthesize and transport the yellow pigment, 3-hydroxykynurenine (3-OHK). Finally, we provide evidence that Al1 activation is under the control of Wnt signaling.ConclusionsWe propose a model in which high levels of Al1 during early pupation, which are mediated by Wnt, are important for melanic pigmentation and specifying white portions of the wing while reduced levels of Al1 during early pupation promote upregulation of proteins needed to move and synthesize 3-OHK, promoting yellow pigmentation. In addition, we discuss how the ancestral role of aristaless in appendage extension may be relevant in understanding the cellular mechanism behind color patterning in the context of the heterochrony hypothesis.
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Project description:Chick definitive endoderm is an important source of signals that pattern the early embryo forming a central structure around which the body plan is constructed. Although the origin of definitive endoderm has been mapped in the chick, arising principally from rostral streak at elongating streak stages, it is not known when this layer first becomes fully committed to its germ layer fate, an important issue to resolve in light of its critical role in subsequent patterning of the early embryo.Through gene expression screening of chick gastrula, we identified molecular markers of definitive endoderm restricted to rostral (Sox17) and caudal (Gata5/6) regions, suggesting that at least two subpopulations of definitive endodermal cells exist during ingression. We show (1) that presumptive mesoderm cells migrate to the middle layer and remain mesenchymal when transplanted to rostral primitive streak, and prospective endoderm cells enter the lower layer and become epithelial when transplanted to caudal primitive streak; and (2) that presumptive endoderm cells and mesoderm cells lose normal gene expression (Sox17 and Wnt8c, respectively) when transplanted outside of their normal position of origin. Moreover, when rostral or caudal primitive streak segments are transplanted into rostral blastoderm isolates (RBIs), both types of transplants express Sox17 4-6 hours later--consistent with their new position, regardless of their presumptive germ layer origin--and prospective mesoderm transplants, which normally express Wnt8c, turn off expression, suggesting that signals within the rostral blastoderm induce endoderm gene expression, and repress mesoderm gene expression, during gastrulation.Our results demonstrate that germ layer identity is fixed at the time populations of endoderm and mesoderm cells ingress through the primitive streak, whereas their gene expression patterns remain labile. In addition, our results show that inductive and repressive signals are present, and that these signals regulate gene expression of both ingressed endoderm and mesoderm cells. Thus, gastrula cells display elements of both pre-patterning and plasticity, with endoderm the first germ layer becoming committed to its fate during early gastrulation stages.
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Project description:The longstanding view of how proliferative outgrowth terminates following the patterning phase of limb development involves the breakdown of reciprocal extrinsic signalling between the distal mesenchyme and the overlying epithelium (e-m signalling). However, by grafting distal mesenchyme cells from late stage chick wing buds to the epithelial environment of younger wing buds, we show that this mechanism is not required. RNA sequencing reveals that distal mesenchyme cells complete proliferative outgrowth by an intrinsic cell cycle timer in the presence of e-m signalling. In this process, e-m signalling is required permissively to allow the intrinsic cell cycle timer to run its course. We provide evidence that a temporal switch from BMP antagonism to BMP signalling controls the intrinsic cell cycle timer during limb outgrowth. Our findings have general implications for other patterning systems in which extrinsic signals and intrinsic timers are integrated.
| S-EPMC6143340 | biostudies-literature
Project description:BackgroundRetinoic acid is implicated in the induction of the gene encoding Sonic hedgehog (Shh) that specifies anteroposterior positional values and promotes growth of the developing limb bud. However, because retinoic acid is involved in limb initiation, it has been difficult to determine if it could have additional roles in anteroposterior patterning. To investigate this, we implanted retinoic acid-soaked beads to the anterior margin of the chick wing bud and performed microarray analyses prior to onset of Shh expression.ResultsRetinoic acid up-regulates expression of Hoxd11-13 that encode transcription factors implicated in inducing Shh transcription and that are involved in digit development. In our assay, retinoic acid induces Shh transcription and, consequently, a new pattern of digits at a much later stage than anticipated. Retinoic acid represses many anteriorly expressed genes, including Bmp4, Lhx9, Msx2, and Alx4. We provide evidence that retinoic acid influences transcription via induction of dHAND and inhibition of Gli3 to establish a new anteroposterior pre-pattern. We show that transient exposure to retinoic acid can suppress distal development and expedite cells to transcriptionally respond to Shh.ConclusionsOur findings reveal how retinoic acid and Shh signaling could cooperate in anteroposterior patterning of the limb. Developmental Dynamics 246:682-690, 2017. © 2017 Wiley Periodicals, Inc.
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