One of the most significant problems facing developmental biologists who do not work on an organism with well-developed genetics - and even for some who do - is how to inhibit the action of a gene of interest during development so as to learn about its normal biological function. A widely adopted approach is to use antisense technologies, and especially morpholino antisense oligonucleotides. In this article, we review the use of such reagents and present examples of how they have provided insights into developmental mechanisms. We also discuss how the use of morpholinos can lead to misleading results, including off-target effects, and we suggest controls that will allow researchers to interpret morpholino experiments correctly.
]]>N-linked glycosylation is a prevalent protein modification in eukaryotic cells. Although glycosylation plays an important role in cell signaling during development, a role for N-linked glycosylation in embryonic patterning has not previously been described. In a screen for maternal factors involved in embryo patterning, we isolated mutations in Drosophila ALG5, a UDP-glucose:dolichyl-phosphate glucosyltransferase. Based on the embryonic cuticle phenotype, we designated the ALG5 locus wollknäuel (wol). Mutations in wol result in posterior segmentation phenotypes, reduced Dpp signaling, as well as impaired mesoderm invagination and germband elongation at gastrulation. The segmentation phenotype can be attributed to a post-transcriptional effect on expression of the transcription factor Caudal, whereas wol acts upstream of Dpp signalin by regulating dpp expression. The wol/ALG5 cDNA was able to partially complement the hypoglycosylation phenotype of alg5 mutant S. cerevisiae, whereas the two wol mutant alleles failed to complement. We show that reduced glycosylation in wol mutant embryos triggers endoplasmic reticulum stress and the unfolded protein response (UPR). As a result, phosphorylation of the translation...]]>
Maintenance of the stem cell population located at the apical meristems is essential for repetitive organ initiation during the development of higher plants. Here, we have characterized the roles of OBERON1 (OBE1) and its paralog OBERON2 (OBE2), which encode plant homeodomain finger proteins, in the maintenance and/or establishment of the meristems in Arabidopsis. Although the obe1 and obe2 single mutants were indistinguishable from wild-type plants, the obe1 obe2 double mutant displayed premature termination of the shoot meristem, suggesting that OBE1 and OBE2 function redundantly. Further analyses revealed that OBE1 and OBE2 allow the plant cells to acquire meristematic activity via the WUSCHEL-CLAVATA pathway, which is required for the maintenance of the stem cell population, and they function parallel to the SHOOT MERISTEMLESS gene, which is required for preventing cell differentiation in the shoot meristem. In addition, obe1 obe2 mutants failed to establish the root apical meristem, lacking both the initial cells and the quiescent center. In situ hybridization revealed that expression of PLETHORA and SCARECROW, which are...]]>
Fibroblast growth factor (FGF) signalling regulates essential developmental processes in vertebrates and invertebrates, but its role during early metazoan evolution remains obscure. Here, we analyse the function of FGF signalling in a non-bilaterian animal, the sea anemone Nematostella vectensis. We identified the complete set of FGF ligands and FGF receptors, of which two paralogous FGFs (NvFGFa1 and NvFGFa2) and one FGF receptor (NvFGFRa) are specifically coexpressed in the developing apical organ, a sensory structure located at the aboral pole of ciliated larvae from various phyla. Morpholino-mediated knockdown experiments reveal that NvFGFa1 and NvFGFRa are required for the formation of the apical organ, whereas NvFGFa2 counteracts NvFGFRa signalling to prevent precocious and ectopic apical organ development. Marker gene expression analysis shows that FGF signalling regulates local patterning in the aboral region. Furthermore, NvFGFa1 activates its own expression and that of the antagonistic NvFGFa2, thereby establishing positive- and negative-feedback loops. Finally, we show that loss of the apical organ upon NvFGFa1 knockdown blocks metamorphosis into polyps. We propose...]]>
Directed cell migration is crucial for development, but most of our current knowledge is derived from in vitro studies. We analyzed how neural crest (NC) cells migrate in the direction of their target during embryonic development. We show that the proteoglycan Syndecan-4 (Syn4) is expressed in the migrating neural crest of Xenopus and zebrafish embryos. Loss-of-function studies using an antisense morpholino against syn4 show that this molecule is required for NC migration, but not for NC induction. Inhibition of Syn4 does not affect the velocity of cell migration, but significantly reduces the directional migration of NC cells. Furthermore, we show that Syn4 and PCP signaling control the directional migration of NC cells by regulating the direction in which the cell protrusions are generated during migration. Finally, we perform FRET analysis of Cdc42, Rac and RhoA in vitro and in vivo after interfering with Syn4 and PCP signaling. This is the first time that FRET analysis of small GTPases has been performed in vivo....]]>
Apicobasal polarity plays an important role in regulating asymmetric cell divisions by neural progenitor cells (NPCs) in invertebrates, but the role of polarity in mammalian NPCs is poorly understood. Here, we characterize the function of the PDZ domain protein MALS-3 in the developing cerebral cortex. We find that MALS-3 is localized to the apical domain of NPCs. Mice lacking all three MALS genes fail to localize the polarity proteins PATJ and PALS1 apically in NPCs, whereas the formation and maintenance of adherens junctions appears normal. In the absence of MALS proteins, early NPCs progressed more slowly through the cell cycle, and their daughter cells were more likely to exit the cell cycle and differentiate into neurons. Interestingly, these effects were transient; NPCs recovered normal cell cycle properties during late neurogenesis. Experiments in which MALS-3 was targeted to the entire membrane resulted in a breakdown of apicobasal polarity, loss of adherens junctions, and a slowing of the cell cycle. Our results suggest that MALS-3 plays...]]>
Loss of Dkk1 results in ectopic WNT/β-catenin signalling activity in the anterior germ layer tissues and impairs cell movement in the endoderm of the mouse gastrula. The juxtaposition of the expression domains of Dkk1 and Wnt3 is suggestive of an antagonist-agonist interaction. The downregulation of Dkk1 when Wnt3 activity is reduced reveals a feedback mechanism for regulating WNT signalling. Compound Dkk1;Wnt3 heterozygous mutant embryos display head truncation and trunk malformation, which are not found in either Dkk1+/- or Wnt3+/- embryos. Reducing the dose of Wnt3 gene in Dkk1-/- embryos partially rescues the truncated head phenotype. These findings highlight that head development is sensitive to the level of WNT3 signalling and that DKK1 is the key antagonist that modulates WNT3 activity during anterior morphogenesis.
]]>Although germ cell formation has been relatively well understood in worms and insects, how germ cell-specific developmental programs are initiated is not clear. In Caenorhabditis elegans, translational activation of maternal nos-2 mRNA is the earliest known molecular event specific to the germline founder cell P4. Cis-elements in nos-2 3'UTR have been shown to mediate translational control; however, the trans-acting proteins are not known. Here, we provide evidence that four maternal RNA-binding proteins, OMA-1, OMA-2, MEX-3 and SPN-4, bind nos-2 3'UTR to suppress its translation, and POS-1, another maternal RNA-binding protein, relieves this suppression in P4. The POS-1: SPN-4 ratio in P4 increases significantly over its precursor, P3; and POS-1 competes with SPN-4 for binding to nos-2 RNA in vitro. We propose temporal changes in the relative concentrations of POS-1 and SPN-4, through their effect on the translational status of maternal mRNAs such as nos-2, initiate germ cell-specific developmental programs in C. elegans.
]]>The TGF-β superfamily of secreted signalling molecules plays a pivotal role in the regulation of early embryogenesis, organogenesis and adult tissue homeostasis. Here we report the identification of Xenopus N-acetylgalactosaminyltransferase-like 1 (xGalntl-1) as a novel important regulator of TGF-β signalling. N-acetylgalactosaminyltransferases mediate the first step of mucin-type glycosylation, adding N-acetylgalactose to serine or threonine side chains. xGalntl-1 is expressed in the anterior mesoderm and neural crest territory at neurula stage, and in the anterior neural crest, notochord and the mediolateral spinal cord at tailbud stage. Inhibition of endogenous xGalntl-1 protein synthesis, using specific morpholino oligomers, interfered with the formation of anterior neural crest, anterior notochord and the spinal cord. Xenopus and mammalian Galntl-1 inhibited Activin as well as BMP signalling in the early Xenopus embryo and in human HEK 293T cells. Gain- and loss-of-function experiments showed that xGalntl-1 interferes with the activity of the common TGF-β type II receptor ActR-IIB in vivo. In addition, our biochemical data demonstrated that xGalntl-1 specifically interferes with the binding of...]]>
Aortic smooth muscle cells (SMCs) have been proposed to derive from lateral plate mesoderm. It has further been suggested that induction of SMC differentiation is confined to the ventral side of the aorta, and that SMCs later migrate to the dorsal side. In this study, we investigate the origin of SMCs in the descending aorta using recombination-based lineage tracing in mice. Hoxb6-cre transgenic mice were crossed with Rosa 26 reporter mice to track cells of lateral plate mesoderm origin. The contribution of lateral plate mesoderm to SMCs in the descending aorta was determined at different stages of development. SMC differentiation was induced in lateral plate mesoderm-derived cells on the ventral side of the aorta at embryonic day (E) 9.0-9.5, as indicated by expression of the SMC-specific reporter gene SM22-lacZ. There was, however, no migration of SMCs from the ventral to the dorsal side of the vessel. Moreover, the lateral plate mesoderm-derived cells in the ventral wall of the aorta were replaced by somite-derived cells...]]>
Axons of retinal ganglion cells (RGCs) make a divergent choice at the optic chiasm to cross or avoid the midline in order to project to ipsilateral and contralateral targets, thereby establishing the binocular visual pathway. The zinc-finger transcription factor Zic2 and a member of the Eph family of receptor tyrosine kinases, EphB1, are both essential for proper development of the ipsilateral projection at the mammalian optic chiasm midline. Here, we demonstrate in mouse by functional experiments in vivo that Zic2 is not only required but is also sufficient to change the trajectory of RGC axons from crossed to uncrossed. In addition, our results reveal that this transcription factor regulates the expression of EphB1 in RGCs and also suggest the existence of an additional EphB1-independent pathway controlled by Zic2 that contributes to retinal axon divergence at the midline.
]]>The preservation of a pool of neural precursors is a prerequisite for proper establishment and maintenance of a functional central nervous system (CNS). Both Notch signaling and SoxB1 transcription factors have been ascribed key roles during this process, but whether these factors use common or distinct mechanisms to control progenitor maintenance is unsettled. Here, we report that the capacity of Notch to maintain neural cells in an undifferentiated state requires the activity of SoxB1 proteins, whereas the mechanism by which SoxB1 block neurogenesis is independent of Notch signaling. A common feature of Notch signaling and SoxB1 proteins is their ability to inhibit the activity of proneural bHLH proteins. Notch represses the transcription of proneural bHLH genes, while SoxB1 proteins block their neurogenic capacity. Moreover, E-proteins act as functional partners of proneural proteins and the suppression of E-protein expression is an important mechanism by which Notch counteracts neurogenesis. Interestingly, in contrast to the Hes-dependent repression of proneural genes, suppression of E-protein occurs in a Hes-independent fashion....]]>
Development of the vertebrate blood lineages is complex, with multiple waves of hematopoietic precursors arising in different embryonic locations. Monopotent, or primitive, precursors first give rise to embryonic macrophages or erythrocytes. Multipotent, or definitive, precursors are subsequently generated to produce the adult hematopoietic lineages. In both the zebrafish and the mouse, the first definitive precursors are committed erythromyeloid progenitors (EMPs) that lack lymphoid differentiation potential. We have previously shown that zebrafish EMPs arise in the posterior blood island independently from hematopoietic stem cells (HSCs). In this report, we demonstrate that a fourth wave of hematopoietic precursors arises slightly later in the zebrafish aorta/gonad/mesonephros (AGM) equivalent. We have identified and prospectively isolated these cells by CD41 (itga2b) and cmyb expression. Unlike EMPs, CD41+ AGM cells colonize the thymus to generate rag2+ T lymphocyte precursors. Timelapse imaging and lineage tracing analyses demonstrate that AGM-derived precursors use a previously undescribed migration pathway along the pronephric tubules to initiate adult hematopoiesis in the developing kidney, the teleostean equivalent of mammalian bone marrow....]]>
Much of the peripheral nervous system of the head is derived from ectodermal thickenings, called placodes, that delaminate or invaginate to form cranial ganglia and sense organs. The trigeminal ganglion, which arises lateral to the midbrain, forms via interactions between the neural tube and adjacent ectoderm. This induction triggers expression of Pax3, ingression of placode cells and their differentiation into neurons. However, the molecular nature of the underlying signals remains unknown. Here, we investigate the role of PDGF signaling in ophthalmic trigeminal placode induction. By in situ hybridization, PDGF receptor β is expressed in the cranial ectoderm at the time of trigeminal placode formation, with the ligand PDGFD expressed in the midbrain neural folds. Blocking PDGF signaling in vitro results in a dose-dependent abrogation of Pax3 expression in recombinants of quail ectoderm with chick neural tube that recapitulate placode induction. In ovo microinjection of PDGF inhibitor causes a similar loss of Pax3 as well as the later placodal marker, CD151, and failure of neuronal...]]>
β-Catenin, as an important effector of the canonical Wnt signaling pathway and as a regulator of cell adhesion, has been demonstrated to be involved in multiple developmental processes and tumorigenesis. β-Catenin expression was found mainly on the Sertoli cell membrane starting from embryonic day 15.5 in the developing testes. However, its potential role in Sertoli cells during testis formation has not been examined. To determine the function of β-catenin in Sertoli cells during testis formation, we either deleted β-catenin or expressed a constitutively active form of β-catenin in Sertoli cells. We found that deletion caused no detectable abnormalities. However, stabilization caused severe phenotypes, including testicular cord disruption, germ cell depletion and inhibition of Müllerian duct regression. β-Catenin stabilization caused changes in Sertoli cell identity and misregulation of inter-Sertoli cell contacts. As Wt1 conditional knockout in Sertoli cells causes similar phenotypes to our stabilized β-catenin mutants, we then investigated the relationship of Wt1 and β-catenin in Sertoli cells and found Wt1 inhibits β-catenin signaling in these cells...]]>
Septation of the mammalian heart into four chambers requires the orchestration of multiple tissue progenitors. Abnormalities in this process can result in potentially fatal atrioventricular septation defects (AVSD). The contribution of extracardiac cells to atrial septation has recently been recognized. Here, we use a genetic marker and novel magnetic resonance microscopy techniques to demonstrate the origins of the dorsal mesenchymal protrusion in the dorsal mesocardium, and its substantial contribution to atrioventricular septation. We explore the functional significance of this tissue to atrioventricular septation through study of the previously uncharacterized AVSD phenotype of Shh-/- mutant mouse embryos. We demonstrate that Shh signaling is required within the dorsal mesocardium for its contribution to the atria. Failure of this addition results in severe AVSD. These studies demonstrate that AVSD can result from a primary defect in dorsal mesocardium, providing a new paradigm for the understanding of human AVSD.
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