LRP2/megalin is required for patterning of the ventral telencephalon

Megalin is a low-density lipoprotein receptor-related protein (LRP2) expressed in the neuroepithelium and the yolk sac of the early embryo. Absence of megalin expression in knockout mice results in holoprosencephaly, indicating an essential yet unidentified function in forebrain development. We used mice with complete or conditional megalin gene inactivation in the embryo to demonstrate that expression of megalin in the neuroepithelium but not in the yolk sac is crucial for brain development. During early forebrain development, megalin deficiency leads to an increase in bone morphogenic protein (Bmp) 4 expression and signaling in the rostral dorsal neuroepithelium, and a subsequent loss of sonic hedgehog (Shh) expression in the ventral forebrain. As a consequence of absent SHH activity, ventrally derived oligodendroglial and interneuronal cell populations are lost in the forebrain of megalin-/- embryos. Similar defects are seen in models with enhanced signaling through BMPs, central regulators of neural tube patterning. Because megalin mediates endocytic uptake and degradation of BMP4, these findings indicate a role for megalin in neural tube specification, possibly by acting as BMP4 clearance receptor in the neuroepithelium.     

Hedgehog signalling is required for correct anteroposterior patterning of the zebrafish otic vesicle

Currently, few factors have been identified that provide the inductive signals necessary to transform the simple otic placode into the complex asymmetric structure of the adult vertebrate inner ear. We provide evidence that Hedgehog signalling from ventral midline structures acts directly on the zebrafish otic vesicle to induce posterior otic identity. We demonstrate that two strong Hedgehog pathway mutants, chameleon (con(tf18b)) and slow muscle omitted (smu(b641)) exhibit a striking partial mirror image duplication of anterior otic structures, concomitant with a loss of posterior otic domains. These effects can be phenocopied by overexpression of patched1 mRNA to reduce Hedgehog signalling. Ectopic activation of the Hedgehog pathway, by injection of sonic hedgehog or dominant-negative protein kinase A RNA, has the reverse effect: ears lose anterior otic structures and show a mirror image duplication of posterior regions. By using double mutants and antisense morpholino analysis, we also show that both Sonic hedgehog and Tiggy-winkle hedgehog are involved in anteroposterior patterning of the zebrafish otic vesicle.     

Anteroposterior patterning is required within segments for somite boundary formation in developing zebrafish

Somite formation involves the establishment of a segmental prepattern in the presomitic mesoderm, anteroposterior patterning of each segmental primordium and formation of boundaries between adjacent segments. How these events are co-ordinated remains uncertain. In this study, analysis of expression of zebrafish mesp-a reveals that each segment acquires anteroposterior regionalisation when located in the anterior presomitic mesoderm. Thus anteroposterior patterning is occurring after the establishment of a segmental prepattern in the paraxial mesoderm and prior to somite boundary formation. Zebrafish fss(-), bea(-), des(-) and aei(-) embryos all fail to form somites, yet we demonstrate that a segmental prepattern is established in the presomitic mesoderm of all these mutants and hox gene expression shows that overall anteroposterior patterning of the mesoderm is also normal. However, analysis of various molecular markers reveals that anteroposterior regionalisation within each segment is disturbed in the mutants. In fss(-), there is a loss of anterior segment markers, such that all segments appear posteriorized, whereas in bea(-), des(-) and aei(-), anterior and posterior markers are expressed throughout each segment. Since somite formation is disrupted in these mutants, correct anteroposterior patterning within segments may be a prerequisite for somite boundary formation. In support of this hypothesis, we show that it is possible to rescue boundary formation in fss(-) through the ectopic expression of EphA4, an anterior segment marker, in the paraxial mesoderm. These observations indicate that a key consequence of the anteroposterior regionalisation of segments may be the induction of Eph and ephrin expression at segment interfaces and that Eph/ephrin signalling subsequently contributes to the formation of somite boundaries.     

spalt-induced specification of distinct dorsal and ventral domains is required for Drosophila tracheal patterning

Morphogenesis of the Drosophila tracheal system relies on different signalling pathways that have distinct roles in specifying both the migration of the tracheal cells and the particular morphological features of the primary branches. The current view is that the tracheal cells are initially specified as an equivalent group of cells whose diversification depends on signals from the surrounding cells. In this work, we show that the tracheal primordia are already specified as distinct dorsal and ventral cell populations. This subdivision depends on the activity of the spalt (sal) gene and occurs prior to the activity of the signalling pathways that dictate the development of the primary branches. Finally, we show that the specification of these two distinct cell populations, which are not defined by cell lineage, are critical for proper tracheal patterning. These results indicate that tracheal patterning depends not only on signalling from surrounding cells but also in the different response of the tracheal cells depending on their allocation to the dorsal or ventral domains.     

Fgf4 is required for left-right patterning of visceral organs in zebrafish

Fgf signaling plays essential roles in many developmental events. To investigate the roles of Fgf4 signaling in zebrafish development, we generated Fgf4 knockdown embryos by injection with Fgf4 antisense morpholino oligonucleotides. Randomized LR patterning of visceral organs including the liver, pancreas, and heart was observed in the knockdown embryos. Prominent expression of Fgf4 was observed in the posterior notochord and Kupffer's vesicle region in the early stages of segmentation. Lefty1, lefty2, southpaw, and pitx2 are known to play crucial roles in LR patterning of visceral organs. Fgf4 was essential for the expression of lefty1, which is necessary for the asymmetric expression of southpaw and pitx2 in the lateral plate mesoderm, in the posterior notochord, and the expression of lefty2 and lefty1 in the left cardiac field. Fgf8 is also known to be crucial for the formation of Kupffer's vesicle, which is needed for the LR patterning of visceral organs. In contrast, Fgf4 was required for the formation of cilia in Kupffer's vesicle, indicating that the role of Fgf4 in the LR patterning is quite distinct from that of Fgf8. The present findings indicate that Fgf4 plays a unique role in the LR patterning of visceral organs in zebrafish.     

The CNS midline cells and spitz class genes are required for proper patterning of Drosophila ventral neuroectoderm.

The Drosophila embryonic central nervous system (CNS) develops from sets of neuroblasts (NBs) which segregate from the ventral neuroectoderm during early embryogenesis. It is not well established how each individual NB in the neuroectoderm acquires its characteristic identity along the dorsal-ventral axis. Since it is known that CNS midline cells and spitz class genes (pointed, rhomboid, single-minded, spitz and Star) are required for the proper patterning of ventral CNS and epidermis originated from the ventral neuroectoderm, this study was carried out to determine the functional roles of the CNS midline cells and spitz class genes in the fate determination of ventral NBs and formation of mature neurons and their axon pathways. Several molecular markers for the identified NBs, neurons, and axon pathways were employed to examine marker gene expression profile, cell lineage and axon pathway formation in the spitz class mutants. This analysis showed that the CNS midline cells specified by single-minded gene as well as spitz class genes are required for identity determination of a subset of ventral NBs and for formation of mature neurons and their axon pathways. This study suggests that the CNS midline cells and spitz class genes are necessary for proper patterning of the ventral neuroectoderm along the dorsal-ventral axis.     

Geminin is required for left-right patterning through regulating Kupffer's vesicle formation and ciliogenesis in zebrafish

Geminin plays an important role in coordinating the cell cycle with anterior–posterior patterning during embryonic development. However, whether it is involved in the regulation of left–right (LR) patterning remains unknown. Here, we reported that geminin is required for setting up heart and visceral laterality during zebrafish development. Defective heart and visceral laterality was observed in geminin morphants. Further study demonstrated that the left-sided nodal/spaw in the lateral plate mesoderm (LPM) as well as the sideness of its downstream targets lefty2 and lefty1 was perturbed in geminin morphants. Upstream of the left-sided Nodal signal along the regulatory cascade of LR asymmetry, knock down of geminin resulted in defective Kupffer’s vesicle (KV) formation and ciliogenesis rather than middle line defects. Predominant distribution of an antisense morpholino against geminin in dorsal forerunner cells (DFCs) led to defective KV morphogenesis and perturbed LR asymmetry, similar to those of geminin morphants, indicating a cell-autonomous role of geminin in regulating KV formation and ciliogenesis. Our results demonstrate that geminin is required for proper KV formation and ciliogenesis, thus playing an important part in setting up LR asymmetry.     

Role of the floor plate in axonal patterning in the zebrafish CNS.

To determine the role of the floor plate (FP) in CNS development, I have used labeling techniques, including immunolabeling, to analyze cyclops mutant embryos, which lack the FP. Except for the anterior brain, the mutant phenotype is almost exclusively confined to the vicinity of the ventral CNS midline. In the midbrain, the number of ventral neurons is reduced and cell patterning is disturbed. In contrast, the neuronal arrangement in the spinal cord is almost normal, including in particular both primary and secondary motoneurons. Longitudinal axonal bundles are disorganized in both the brain and spinal cord. Laser ablating the FP in wild-type embryos locally phenocopies cyclops axonal disturbances, and transplanting wild-type FP precursor cells into mutants locally rescues the disturbances. These results demonstrate a significant role for the FP in pathfinding and fasciculation by axons in situ, especially during their longitudinal courses.     

Axis specification in the spider embryo: dpp is required for radial-to-axial symmetry transformation and sog for ventral patterning

The mechanism by which Decapentaplegic (Dpp) and its antagonist Short gastrulation (Sog) specify the dorsoventral pattern in Drosophila embryos has been proposed to have a common origin with the mechanism that organizes the body axis in the vertebrate embryo. However, Drosophila Sog makes only minor contributions to the development of ventral structures that hypothetically correspond to the vertebrate dorsum where the axial notochord forms. In this study, we isolated a homologue of the Drosophila sog gene in the spider Achaearanea tepidariorum, and characterized its expression and function. Expression of sog mRNA initially appeared in a radially symmetrical pattern and later became confined to the ventral midline area, which runs axially through the germ band. RNA interference-mediated depletion of the spider sog gene led to a nearly complete loss of ventral structures, including the axial ventral midline and the central nervous system. This defect appeared to be the consequence of dorsalization of the ventral region of the germ band. By contrast, the extra-embryonic area formed normally. Furthermore, we showed that embryos depleted for a spider homologue of dpp failed to break the radial symmetry, displaying evenly high levels of sog expression except in the posterior terminal area. These results suggest that dpp is required for radial-to-axial symmetry transformation of the spider embryo and sog is required for ventral patterning. We propose that the mechanism of spider ventral specification largely differs from that of the fly. Interestingly, ventral specification in the spider is similar to the process in vertebrates in which the antagonism of Dpp/BMP signaling plays a central role in dorsal specification.     

Gdf6a is required for the initiation of dorsal–ventral retinal patterning and lens development

Dorsal–ventral patterning of the vertebrate retina is essential for accurate topographic mapping of retinal ganglion cell (RGC) axons to visual processing centers. Bone morphogenetic protein (Bmp) growth factors regulate dorsal retinal identity in vertebrate models, but the developmental timing of this signaling and the relative roles of individual Bmps remain unclear. In this study, we investigate the functions of two zebrafish Bmps, Gdf6a and Bmp4, during initiation of dorsal retinal identity, and subsequently during lens differentiation. Knockdown of zebrafish Gdf6a blocks initiation of retinal Smad phosphorylation and dorsal marker expression, while knockdown of Bmp4 produces no discernable retinal phenotype. These data, combined with analyses of embryos ectopically expressing Bmps, demonstrate that Gdf6a is necessary and sufficient for initiation of dorsal retinal identity. We note a profound expansion of ventral retinal identity in gdf6a morphants, demonstrating that dorsal BMP signaling antagonizes ventral marker expression. Finally, we demonstrate a role for Gdf6a in non-neural ocular tissues. Knockdown of Gdf6a leads to defects in lens-specific gene expression, and when combined with Bmp signaling inhibitors, disrupts lens fiber cell differentiation. Taken together, these data indicate that Gdf6a initiates dorsal retinal patterning independent of Bmp4, and regulates lens differentiation.     

Maternally controlled (beta)-catenin-mediated signaling is required for organizer formation in the zebrafish

We have identified and characterized a zebrafish recessive maternal effect mutant, ichabod, that results in severe anterior and dorsal defects during early development. The ichabod mutation is almost completely penetrant, but exhibits variable expressivity. All mutant embryos fail to form a normal embryonic shield; most fail to form a head and notochord and have excessive development of ventral tail fin tissue and blood. Abnormal dorsal patterning can first be observed at 3.5 hpf by the lack of nuclear accumulation of (beta)-catenin in the dorsal yolk syncytial layer, which also fails to express bozozok/dharma/nieuwkoid and znr2/ndr1/squint. At the onset of gastrulation, deficiencies in expression of dorsal markers and expansion of expression of markers of ventral tissues indicate a dramatic alteration of dorsoventral identity. Injection of (beta)-catenin RNA markedly dorsalized ichabod embryos and often completely rescued the phenotype, but no measurable dorsalization was obtained with RNAs encoding upstream Wnt pathway components. In contrast, dorsalization was obtained when RNAs encoding either Bozozok/Dharma/Nieuwkoid or Znr2/Ndr1/Squint were injected. Moreover, injection of (beta)-catenin RNA into ichabod embryos resulted in activation of expression of these two genes, which could also activate each other. RNA injection experiments strongly suggest that the component affected by the ichabod mutation acts on a step affecting (beta)-catenin nuclear localization that is independent of regulation of (beta)-catenin stability. This work demonstrates that a maternal gene controlling localization of (beta)-catenin in dorsal nuclei is necessary for dorsal yolk syncytial layer gene activity and formation of the organizer in the zebrafish.     

Coagulation factor III (tissue factor) is required for vascularization in zebrafish embryos

Tissue factor (coagulation factor III) is a cell surface receptor for coagulation factor VII/VIIa; it was initially recognized as an initiator of the extrinsic coagulation pathway. Recently, the zebrafish tissue factor gene (TF) has been cloned. Paralogs encode coagulation factors IIIa and IIIb; both show remarkable sequence identity to the human and mouse coagulation factor III gene. It has been reported that TF could have additional properties that are essential for normal embryonic development, since knockout of the murine coagulation factor III gene resulted in 90% embryonic lethality. We examined the role of coagulation factor IIIb (f3b) during zebrafish embryonic development. Expression analysis revealed that endogenous f3b was chronologically expressed in the pectoral fins and in the vicinity of the pharynx. Knockout of f3b by injection of an f3b morpholino at the one-to-two cell stage caused distinctive morphological defects in embryos, including edema in the fourth brain ventricle at early embryonic stages and occasional bleeding at later stages. Furthermore, f3b morphants displayed abnormal vascular patterning. We conclude that f3b is required for brain vascular development and for development of part of the somatic vasculature during embryogenesis in the zebrafish.     

Maternally supplied Smad5 is required for ventral specification in zebrafish embryos prior to zygotic Bmp signaling

We have previously shown that the maternal effect dorsalization of zebrafish embryos from sbn(dtc24) heterozygous mothers is caused by a dominant negative mutation in Smad5, a transducer of ventralizing signaling by the bone morphogenetic proteins Bmp2b and Bmp7. Since sbn(dtc24) mutant Smad5 protein not only blocks wild-type Smad5, but also other family members like Smad1, it remained open to what extent Smad5 itself is required for dorsoventral patterning. Here, we report the identification of novelsmad5 alleles: three new isolates coming from a dominant enhancer screen, and four former isolates initially assigned to the cpt and pgy complementation groups. Overexpression analyses demonstrate that three of the new alleles, m169, fr5, and tc227, are true nulls (amorphs), whereas the initial dtc24 allele is both antimorphic and hypomorphic. We rescued m169 mutant embryos by smad5 mRNA injection. Although adult mutants are smaller than their siblings, the eggs laid by m169(-/-) females are larger than normal eggs. Embryos lacking maternal Smad5 function (Mm169(-/-) embryos) are even more strongly dorsalized thanbmp2b or bmp7 null mutants. They do not respond to injected bmp2b mRNA, indicating that Smad5 is absolutely essential for ventral development and Bmp2/7 signaling. Most importantly, Mm169(-/-) embryos display reducedbmp7 mRNA levels during blastula stages, when bmp2b and bmp7 mutants are still normal. This indicates that maternally supplied Smad5 is already required to mediate ventral specification prior to zygotic Bmp2/7 signaling to establish the initial dorsoventral asymmetry.     

Hedgehog-mediated patterning of the mammalian embryo requires transporter-like function of dispatched

The dispatched (disp) gene is required for long-range Hedgehog (Hh) signaling in Drosophila. Here, we demonstrate that one of two murine homologs, mDispA, can rescue disp function in Drosophila and is essential for all Hh patterning activities examined in the early mouse embryo. Embryonic fibroblasts lacking mDispA respond normally to exogenously provided Sonic hedgehog (Shh) signal, but are impaired in stimulation of other responding cells when expressing Shh. We have developed a biochemical assay that directly measures the activity of Disp proteins in release of soluble Hh proteins. This activity is disrupted by alteration of residues functionally conserved in Patched and in a related family of bacterial transmembrane transporters, thus suggesting similar mechanisms of action for all of these proteins.     

HrT is required for cardiovascular development in zebrafish

The recently identified zebrafish T-box gene hrT is expressed in the developing heart and in the endothelial cells forming the dorsal aorta. Orthologs of hrT are expressed in cardiovascular cells from Drosophila to mouse, suggesting that the function of hrT is evolutionarily conserved. The role of hrT in cardiovascular development, however, has not thus far been determined in any animal model. Using morpholino antisense oligonucleotides, we show that zebrafish embryos lacking hrT function have dysmorphic hearts and an absence of blood circulation. Although the early events in heart formation were normal in hrT morphant embryos, subsequently the hearts failed to undergo looping, and late onset defects in chamber morphology and gene expression were observed. In particular, we found that the loss of hrT function led to a dramatic upregulation of tbx5, a gene required for normal heart morphogenesis. Conversely, we show that overexpression of hrT causes a significant downregulation of tbx5, indicating that one key role of hrT is to regulate the levels of tbx5. Secondly, we found that HrT is required to inhibit the expression of the blood lineage markers gata1 and gata2 in the most posterior lateral plate mesoderm. Finally, we show that HrT is required for vasculogenesis in the trunk, leading to similar vascular defects to those observed in midline mutants such as floating head. hrT expression in the vascular progenitors depends upon midline mesoderm, indicating that this expression is one important component of the response to a midline-derived signal during vascular morphogenesis.