Notch signaling can regulate endoderm formation in zebrafish.

Early in vertebrate development, the processes of gastrulation lead to the formation of the three germ layers: ectoderm, mesoderm, and endoderm. The mechanisms leading to the segregation of the endoderm and mesoderm are not well understood. In mid-blastula stage zebrafish embryos, single marginal cells can give rise to both endoderm and mesoderm (reviewed by Warga and Stainier [2002] The guts of endoderm formation. In: Solnica-Krezel L, editor. Pattern formation in zebrafish. Berlin: Springer-Verlag. p 28-47). By the late blastula stage, however, single marginal cells generally give rise to either endoderm or mesoderm. To investigate this segregation of the blastoderm into cells with either endodermal or mesodermal fates, we analyzed the role of Notch signaling in this process. We show that deltaC, deltaD, and notch1 are expressed in the marginal domain of blastula stage embryos and that this expression is dependent on Nodal signaling. Activation of Notch signaling from an early stage leads to a reduction of endodermal cells, as assessed by sox17 and foxA2 expression. We further find that this reduction in endoderm formation by the activation of Notch signaling is preceded by a reduction in the expression of bonnie and clyde (bon) and faust/gata5, two genes necessary for endoderm formation (Reiter et al. [1999] Genes Dev 13:2983-2995; Reiter et al. [2001] Development 128:125-135; Kikuchi et al. [2001] Genes Dev 14:1279-1289). However, activation of Notch signaling in bon mutant embryos leads to a further reduction in endodermal cells, also arguing for a bon-independent role for Notch signaling in endoderm formation. Altogether, these results suggest that Notch signaling plays a role in the formation of the endoderm, possibly in its segregation from the mesoderm.     

Expression pattern for unc5b, an axon guidance gene in embryonic zebrafish development

Branching processes such as nerves and vessels share molecular mechanisms of path determination. Our study focuses on unc5b, a member of the unc5 axon guidance gene family. Here, we have cloned the full-length zebrafish ortholog of unc5b, mapped its chromosome location in the zebrafish genome, and compared its expression patterns to robo4, another axon guidance family member. In situ show that unc5b is expressed predominantly in sensory structures such as the eye, ear, and brain. Both unc5b and robo4 show robust expression in all three compartments of the embryonic brain, namely forebrain, midbrain, and hindbrain. In particular, the hindbrain rhombomere expression displays interesting patterns in that robo4 is expressed in medial rhombomere cell clusters when compared to unc5b expressed in lateral rhombomere clusters. A similar medial-lateral theme is observed in other neural structures such as the neural tube. Our expression analysis provides a starting point for studying the role of axon guidance genes in embryonic hindbrain patterning.     

lunatic fringe regulates Delta-Notch induction of hypochord in zebrafish.

Cells that occupy the midline of vertebrate embryos arise from a common population of precursors. Several lines of evidence indicate that Delta-Notch signaling regulates specification of midline precursors for different fates. We show that zebrafish midline precursors transiently express lunatic fringe, which encodes a glycosyltransferase that modifies Notch activity in response to its ligands, and that lunatic fringe function is required for Delta-Notch-mediated induction of hypochord cells at the lateral borders of the midline precursor domain.     

Rhombomere boundaries are Wnt signaling centers that regulate metameric patterning in the zebrafish hindbrain.

The vertebrate hindbrain develops from a series of segments (rhombomeres) distributed along the anteroposterior axis. We are studying the roles of Wnt and Delta-Notch signaling in maintaining rhombomere boundaries as organizing centers in the zebrafish hindbrain. Several wnt genes (wnt1, wnt3a, wnt8b, and wnt10b) show elevated expression at rhombomere boundaries, whereas several delta genes (dlA, dlB, and dlD) are expressed in transverse stripes flanking rhombomere boundaries. Partial disruption of Wnt signaling by knockdown of multiple wnt genes, or the Wnt mediator tcf3b, ablates boundaries and associated cell types. Expression of dlA is chaotic, and cell types associated with rhombomere centers are disorganized. Similar patterning defects are observed in segmentation mutants spiel-ohne-grenzen (spg) and valentino (val), which fail to form rhombomere boundaries due to faulty interactions between adjacent rhombomeres. Stripes of wnt expression are variably disrupted, with corresponding disturbances in metameric patterning. Mutations in dlA or mind bomb (mib) disrupt Delta-Notch signaling and cause a wide range of patterning defects in the hindbrain. Stripes of wnt1 are initially normal but subsequently dissipate, and metameric patterning becomes increasingly disorganized. Driving wnt1 expression using a heat-shock construct partially rescues metameric patterning in mib mutants. Thus, rhombomere boundaries act as Wnt signaling centers required for precise metameric patterning, and Delta signals from flanking cells provide feedback to maintain wnt expression at boundaries. Similar feedback mechanisms operate in the Drosophila wing disc and vertebrate limb bud, suggesting coaptation of a conserved signaling module that spatially organizes cells in complex organ systems.     

Manic fringe is not required for embryonic development,and fringe family members do not exhibit redundant functions in the axial skeleton,limb, or hindbrain

Tight regulation of Notch pathway signaling is important in many aspects of embryonic development. Notch signaling can be modulated by expression of fringe genes, encoding glycosyltransferases that modify EGF repeats in the Notch receptor. Although Lunatic fringe (Lfng) has been shown to play important roles in vertebrate segmentation, comparatively little is known regarding the developmental functions of the other vertebrate fringe genes, Radical fringe (Rfng) and Manic fringe (Mfng). Here we report that Mfng expression is not required for embryonic development. Further, we find that despite significant overlap in expression patterns, we detect no obvious synergistic defects in mice in the absence of two, or all three, fringe genes during development of the axial skeleton, limbs, hindbrain, and cranial nerves. Developmental Dynamics 238:1803–1812, 2009. © 2009 Wiley‐Liss, Inc.     

Localisation of members of the notch system and the differentiation of vibrissa hair follicles: receptors, ligands, and fringe modulators.

Hair vibrissa follicle morphogenesis involves several cell segregation phases, in the dermis as well as in the epidermis. The expression of Notch-related genes, which are well established mediators of multiple cell segregation events in Drosophila development, was studied by in situ hybridisation during embryonic mouse vibrissa follicle morphogenesis and the first adult hair cycle. The results show that two receptors, Notch1 and -2, three ligands, Delta1, Serrate1, and -2, and the three Fringe regulators, Lunatic, Manic, and Radical, are expressed in different locations and morphogenetic stages. First, the appearance of hair vibrissa primordia involves the expression of complementary patterns of Notch2, Delta1, and Lunatic Fringe in the dermis and of Notch1, Serrate2, and Lunatic Fringe in the epidermis. Second, this expression pattern is no longer found after stage 3 in the dermis. Meanwhile, in the epidermis, the expression of Notch1, Serrate2, and Lunatic Fringe before the formation of the placode may be involved in determining two populations of epidermal cells in the developing follicle. Third, complementary expression patterns for Notch1, Manic, and Lunatic Fringe, as well as Serrate1 and -2 as previously shown (Powell et al., 1998), are progressively established from stage 4 of embryonic development both in the outer root sheath and in the hair matrix. These patterns are consistent with the one found in the adult anagen phase. During the hair vibrissa cycle, Notch1 and Manic Fringe display temporal and spatial changes of expression, suggesting that they may intervene as modulators of trichocyte activities.     

Apoptosis and proliferation in the trigeminal placode

The neurogenic trigeminal placode develops from the crescent-shaped panplacodal primordium which delineates the neural plate anteriorly. We show that, in Tupaia belangeri, the trigeminal placode is represented by a field of focal ectodermal thickenings which over time changes positions from as far rostral as the level of the forebrain to as far caudal as opposite rhombomere 3. Delamination proceeds rostrocaudally from the ectoderm adjacent to the rostral midbrain, and contributes neurons to the trigeminal ganglion as well as to the ciliary ganglion/oculomotor complex. Proliferative events are centered on the field prior to the peak of delamination. They are preceded, paralleled and, finally, outnumbered by apoptotic events which proceed rostrocaudally from non-delaminating to delaminating parts of the field. Apoptosis persists upon regression of the placode, thereby exhibiting a massive “wedge” of apoptotic cells which includes the postulated position of the “ventrolateral postoptic placode” (Lee et al. in Dev Biol 263:176–190, 2003), merges with groups of lens-associated apoptotic cells, and disappears upon lens detachment. In conjunction with earlier work (Washausen et al. in Dev Biol 278:86–102, 2005) our findings suggest that apoptosis contributes repeatedly to the disintegration of the panplacodal primordium, to the elimination of subsets of premigratory placodal neuroblasts, and to the regression of placodes.     

The epicardium and epicardially derived cells (EPDCs) as cardiac stem cells

After its initial formation the epicardium forms the outermost cell layer of the heart. As a result of an epithelial-to-mesenchymal transformation (EMT) individual cells delaminate from this primitive epicardial epithelium and migrate into the subepicardial space (Pérez-Pomares et al., Dev Dyn 1997; 210:96-105; Histochem J 1998a;30:627-634). Several studies have demonstrated that these epicardially derived cells (EPDCs) subsequently invade myocardial and valvuloseptal tissues (Mikawa and Fischman, Proc Natl Acad Sci USA 1992;89:9504-9508; Mikawa and Gourdie, Dev Biol 1996;174:221-232; Dettman et al., Dev Biol 1998;193:169-181; Gittenberger de Groot et al., Circ Res 1998;82:1043-1052; Manner, Anat Rec 1999;255:212-226; Pérez-Pomares et al., Dev. Biol. 2002b;247:307-326). A subset of EPDCs continue to differentiate in a variety of different cell types (including coronary endothelium, coronary smooth muscle cells (CoSMCs), interstitial fibroblasts, and atrioventricular cushion mesenchymal cells), whereas other EPDCs remain in a more or less undifferentiated state. Based on its specific characteristics, we consider the EPDC as the ultimate 'cardiac stem cell'. In this review we briefly summarize what is known about events that relate to EPDC development and differentiation while at the same time identifying some of the directions where EPDC-related research might lead us in the near future.     

Chordin affects pronephros development in Xenopus embryos by anteriorizing presomitic mesoderm.

Spemann's organizer emits signals that pattern the mesodermal germ layer during Xenopus embryogenesis. In a previous study, we demonstrated that FGFR1 activity within the organizer is required for the production of both the somitic muscle- and pronephros-patterning signals by the organizer and the expression of chordin, an organizer-specific secreted protein (Mitchell and Sheets [2001] Dev. Biol. 237:295-305). Studies from others in both chicken and Xenopus embryos provide compelling evidence that pronephros forms by means of secondary induction signals emitted from anterior somites (Seufert et al. [1999] Dev. Biol. 215:233-242; Mauch et al. [2000] Dev. Biol. 220:62-75). Here we provide several lines of evidence in support of the hypothesis that chordin influences pronephros development by directing the formation of anterior somites. Chordin mRNA was absent in ultraviolet (UV) -irradiated embryos lacking pronepheros (average DAI<2) but was always found in UV-irradiated embryos that retain pronepheros (average DAI>2). Furthermore, ectopic expression of chordin in embryos and in tissue explants leads to the formation of anterior somites and pronephros. In these experiments, pronephros was only observed in association with muscle. Chordin diverted somatic muscle cells to more anterior positions within the somite file in chordin-induced secondary trunks and induced the expression of the anterior myogenic gene myf5. Finally, depletion of chordin mRNA with DEED antisense oligonucleotides substantially reduced somitic muscle and pronephric tubule and duct formation in whole embryos. These data and previous studies on ectoderm and endoderm (Sasai et al. [1995] Nature 377:757) support the idea that chordin functions as an anteriorizing signal in patterning the germ layers during vertebrate embryogenesis. Our data support the hypothesis that chordin directs the formation of anterior somites that in turn are necessary for pronephros development.     

The transmembrane inner ear (tmie) gene contributes to vestibular and lateral line development and function in the zebrafish (Danio rerio)

The inner ear is a complex organ containing sensory tissue, including hair cells, the development of which is not well understood. Our long-term goal is to discover genes critical for the correct formation and function of the inner ear and its sensory tissue. A novel gene, transmembrane inner ear (Tmie), was found to cause hearing-related disorders when defective in mice and humans. A homologous tmie gene in zebrafish was cloned and its expression characterized between 24 and 51 hours post-fertilization. Embryos injected with morpholinos (MO) directed against tmie exhibited circling swimming behavior (approximately 37%), phenocopying mice with Tmie mutations; semicircular canal formation was disrupted, hair cell numbers were reduced, and maturation of electrically active lateral line neuromasts was delayed. As in the mouse, tmie appears to be required for inner ear development and function in the zebrafish and for hair cell maturation in the vestibular and lateral line systems as well.     

Effects of antisense misexpression of CFC on downstream flectin protein expression during heart looping.

Dextral looping of the heart is regulated on multiple levels. In humans, mutations of the genes CFC and Pitx2/RIEG result in laterality-associated cardiac anomalies. In animal models, a common read-out after the misexpression of laterality genes is heart looping direction. Missing in these studies is how laterality genes impact on downstream morphogenetic processes to coordinate heart looping. Previously, we showed that Pitx2 indirectly regulates flectin protein by regulating the timing of flectin expression in one heart field versus the other (Linask et al. [2002] Dev. Biol. 246:407-417). To address this question further we used a reported loss-of-function approach to interfere with chick CFC expression (Schlange et al. [2001] Dev. Biol. 234:376-389) and assaying for flectin expression during looping. Antisense CFC treatment results in abnormal heart looping or no looping. Our results show that regardless of the sidedness of downstream Pitx2 expression, it is the sidedness of predominant flectin protein expression in the extracellular matrix of the dorsal mesocardial folds and splanchnic mesoderm apposed to the foregut wall that is associated directly with looping direction. Thus, Pitx2 can be experimentally uncoupled from heart looping. The flectin asymmetry continues to be maintained in the secondary heart field during looping.     

Neurogenic phenotype of mind bomb mutants leads to severe patterning defects in the zebrafish hindbrain.

Failure of Notch signaling in zebrafish mind bomb (mib) mutants results in a neurogenic phenotype where an overproduction of early differentiating neurons is accompanied by the loss of later-differentiating cell types. We have characterized in detail the hindbrain phenotype of mib mutants. Hindbrain branchiomotor neurons (BMNs) are reduced in number but not missing in mib mutants. In addition, BMN clusters are frequently fused across the midline in mutants. Mosaic analysis indicates that the BMN patterning and fusion defects in the mib hindbrain arise non-cell autonomously. Ventral midline signaling is defective in the mutant hindbrain, in part due to the differentiation of some midline cells into neural cells. Interestingly, while early hindbrain patterning appears normal in mib mutants, subsequent rhombomere-specific gene expression is completely lost. The defects in ventral midline signaling and rhombomere patterning are accompanied by an apparent loss of neuroepithelial cells in the mutant hindbrain. These observations suggest that, by regulating the differentiation of neuroepithelial cells into neurons, Notch signaling preserves a population of non-neuronal cells that are essential for maintaining patterning mechanisms in the developing neural tube.     

Dosage-sensitive requirement for mouse Dll4 in artery development

Involvement of the Notch signaling pathway in vascular development has been demonstrated by both gain- and loss-of-function mutations in humans, mice, and zebrafish. In zebrafish, Notch signaling is required for arterial identity by suppressing the venous fate in developing artery cells. In mice, the Notch4 receptor and the Delta-like 4 (Dll4) ligand are specifically expressed in arterial endothelial cells, suggesting a similar role. Here we show that the Dll4 ligand alone is required in a dosage-sensitive manner for normal arterial patterning in development. This implicates Dll4 as the specific mammalian endothelial ligand for autocrine endothelial Notch signaling, and suggests that Dll4 may be a suitable target for intervention in arterial angiogenesis.