In JoVE (1)

Other Publications (39)

Articles by Mary C. Mullins in JoVE

Other articles by Mary C. Mullins on PubMed

Modulation of BMP Activity in Dorsal-ventral Pattern Formation by the Chordin and Ogon Antagonists

Developmental Biology. May, 2002  |  Pubmed ID: 11969259

We analyzed the interactions between mutations in antagonistic BMP pathway signaling components to examine the roles that the antagonists play in regulating BMP signaling activity. The dorsalized mutants swirl/bmp2b, snailhouse/bmp7, lost-a-fin/alk8, and mini fin/tolloid were each analyzed in double mutant combinations with the ventralized mutants chordino/chordin and ogon, whose molecular nature is not known. Similar to the BMP antagonist chordino, we found that the BMP ligand mutants swirl/bmp2b and snailhouse/bmp7 are also epistatic to the putative BMP pathway antagonist, ogon, excluding a class of intracellular antagonists as candidates for ogon. In ogon;mini fin double mutants, we observed a mutual suppression of the ogon and mini fin mutant phenotypes, frequently to a wild type phenotype. Thus, the Tolloid/Mini fin metalloprotease that normally cleaves and inhibits Chordin activity is dispensable, when Ogon antagonism is reduced. These results suggest that Ogon encodes a Tolloid and Chordin-independent antagonistic function. By analyzing genes whose expression is very sensitive to BMP signaling levels, we found that the absence of Ogon or Chordin antagonism did not increase the BMP activity remaining in swirl/bmp2b or hypomorphic snailhouse/bmp7 mutants. These results, together with other studies, suggest that additional molecules or mechanisms are essential in generating the presumptive gastrula BMP activity gradient that patterns the dorsal-ventral axis. Lastly we observed a striking increased penetrance of the swirl/bmp2b dominant dorsalized phenotype, when Chordin function is also absent. Loss of the BMP antagonist Chordin is expected to increase BMP signaling levels in a swirl heterozygote, but instead we observed an apparent decrease in BMP signaling levels and a loss of ventral tail tissue. As has been proposed for the fly orthologue of chordin, short gastrulation, our paradoxical results can be explained by a model whereby Chordin both antagonizes and promotes BMP activity.

Building-blocks of Embryogenesis

Nature Genetics. Jun, 2002  |  Pubmed ID: 12040373

Hop is an Unusual Homeobox Gene That Modulates Cardiac Development

Cell. Sep, 2002  |  Pubmed ID: 12297045

Hop is a small, divergent homeodomain protein that lacks certain conserved residues required for DNA binding. Hop gene expression initiates early in cardiogenesis and continues in cardiomyocytes throughout embryonic and postnatal development. Genetic and biochemical data indicate that Hop functions directly downstream of Nkx2-5. Inactivation of Hop in mice by homologous recombination results in a partially penetrant embryonic lethal phenotype with severe developmental cardiac defects involving the myocardium. Inhibition of Hop activity in zebrafish embryos likewise disrupts cardiac development and results in severely impaired cardiac function. Hop physically interacts with serum response factor (SRF) and inhibits activation of SRF-dependent transcription by inhibiting SRF binding to DNA. Hop encodes an unusual homeodomain protein that modulates SRF-dependent cardiac-specific gene expression and cardiac development.

Dorsoventral Patterning in the Zebrafish: Bone Morphogenetic Proteins and Beyond

Results and Problems in Cell Differentiation. 2002  |  Pubmed ID: 12353487

Maternally Supplied Smad5 is Required for Ventral Specification in Zebrafish Embryos Prior to Zygotic Bmp Signaling

Developmental Biology. Oct, 2002  |  Pubmed ID: 12376102

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.

The Pro-BMP Activity of Twisted Gastrulation is Independent of BMP Binding

Development (Cambridge, England). Sep, 2003  |  Pubmed ID: 12874126

The determination of the vertebrate dorsoventral body axis is regulated in the extracellular space by a system of interacting secreted molecules consisting of BMP, Chordin, Tolloid and Twisted Gastrulation (Tsg). Tsg is a BMP-binding protein that forms ternary complexes with BMP and Chordin. We investigated the function of Tsg in embryonic patterning by generating point mutations in its two conserved cysteine-rich domains. Surprisingly, Tsg proteins with mutations in the N-terminal domain were unable to bind BMP, yet ventralized the embryo very effectively, indicating strong pro-BMP activity. This hyperventralizing Tsg activity required an intact C-terminal domain and could block the anti-BMP activity of isolated BMP-binding modules of Chordin (CRs) in embryonic assays. This activity was specific for CR-containing proteins as it did not affect the dorsalizing effects of Noggin or dominant-negative BMP receptor. The ventralizing effects of the xTsg mutants were stronger than the effect of Chordin loss-of-function in Xenopus or zebrafish. The results suggest that xTsg interacts with additional CR-containing proteins that regulate dorsoventral development in embryos.

Regulation of Msx Genes by a Bmp Gradient is Essential for Neural Crest Specification

Development (Cambridge, England). Dec, 2003  |  Pubmed ID: 14627721

There is evidence in Xenopus and zebrafish embryos that the neural crest/neural folds are specified at the border of the neural plate by a precise threshold concentration of a Bmp gradient. In order to understand the molecular mechanism by which a gradient of Bmp is able to specify the neural crest, we analyzed how the expression of Bmp targets, the Msx genes, is regulated and the role that Msx genes has in neural crest specification. As Msx genes are directly downstream of Bmp, we analyzed Msx gene expression after experimental modification in the level of Bmp activity by grafting a bead soaked with noggin into Xenopus embryos, by expressing in the ectoderm a dominant-negative Bmp4 or Bmp receptor in Xenopus and zebrafish embryos, and also through Bmp pathway component mutants in the zebrafish. All the results show that a reduction in the level of Bmp activity leads to an increase in the expression of Msx genes in the neural plate border. Interestingly, by reaching different levels of Bmp activity in animal cap ectoderm, we show that a specific concentration of Bmp induces msx1 expression to a level similar to that required to induce neural crest. Our results indicate that an intermediate level of Bmp activity specifies the expression of Msx genes in the neural fold region. In addition, we have analyzed the role that msx1 plays on neural crest specification. As msx1 has a role in dorsoventral pattering, we have carried out conditional gain- and loss-of-function experiments using different msx1 constructs fused to a glucocorticoid receptor element to avoid an early effect of this factor. We show that msx1 expression is able to induce all other early neural crest markers tested (snail, slug, foxd3) at the time of neural crest specification. Furthermore, the expression of a dominant negative of Msx genes leads to the inhibition of all the neural crest markers analyzed. It has been previously shown that snail is one of the earliest genes acting in the neural crest genetic cascade. In order to study the hierarchical relationship between msx1 and snail/slug we performed several rescue experiments using dominant negatives for these genes. The rescuing activity by snail and slug on neural crest development of the msx1 dominant negative, together with the inability of msx1 to rescue the dominant negatives of slug and snail strongly argue that msx1 is upstream of snail and slug in the genetic cascade that specifies the neural crest in the ectoderm. We propose a model where a gradient of Bmp activity specifies the expression of Msx genes in the neural folds, and that this expression is essential for the early specification of the neural crest.

Maternal Control of Vertebrate Development Before the Midblastula Transition: Mutants from the Zebrafish I

Developmental Cell. Jun, 2004  |  Pubmed ID: 15177026

Maternal factors control development prior to the activation of the embryonic genome. In vertebrates, little is known about the molecular mechanisms by which maternal factors regulate embryonic development. To understand the processes controlled by maternal factors and identify key genes involved, we embarked on a maternal-effect mutant screen in the zebrafish. We identified 68 maternal-effect mutants. Here we describe 15 mutations in genes controlling processes prior to the midblastula transition, including egg development, blastodisc formation, embryonic polarity, initiation of cell cleavage, and cell division. These mutants exhibit phenotypes not previously observed in zygotic mutant screens. This collection of maternal-effect mutants provides the basis for a molecular genetic analysis of the maternal control of embryogenesis in vertebrates.

Maternal Control of Development at the Midblastula Transition and Beyond: Mutants from the Zebrafish II

Developmental Cell. Jun, 2004  |  Pubmed ID: 15177027

Many maternal factors in the oocyte persist in the embryo. They are required to initiate zygotic transcription but also function beyond this stage, where they interact with zygotic gene products during embryonic development. In a four-generation screen in the zebrafish, we identified 47 maternal-effect and five paternal-effect mutants that manifest their phenotypes at the time of, or after, zygotic genome activation. We propagated a subset of 13 mutations that cause developmental arrest at the midblastula transition, defects in cell viability, embryonic morphogenesis, and establishment of the embryonic body plan. This diverse group of mutants, many not previously observed in vertebrates, demonstrates a substantial maternal contribution to the "zygotic" period of embryogenesis and a surprising degree of paternal control. These mutants provide powerful tools to dissect the maternal and paternal control of vertebrate embryogenesis.

Twisted Gastrulation Promotes BMP Signaling in Zebrafish Dorsal-ventral Axial Patterning

Development (Cambridge, England). Dec, 2004  |  Pubmed ID: 15525664

In vertebrates and invertebrates, the bone morphogenetic protein (BMP) signaling pathway patterns cell fates along the dorsoventral (DV) axis. In vertebrates, BMP signaling specifies ventral cell fates, whereas restriction of BMP signaling by extracellular antagonists allows specification of dorsal fates. In misexpression assays, the conserved extracellular factor Twisted gastrulation (Tsg) is reported to both promote and antagonize BMP signaling in DV patterning. To investigate the role of endogenous Tsg in early DV patterning, we performed morpholino (MO)-based knockdown studies of Tsg1 in zebrafish. We found that loss of tsg1 results in a moderately strong dorsalization of the embryonic axis, suggesting that Tsg1 promotes ventral fates. Knockdown of tsg1 combined with loss of function of the BMP agonist tolloid (mini fin) or heterozygosity for the ligand bmp2b (swirl) enhanced dorsalization, supporting a role for Tsg1 in specifying ventral cell fates as a BMP signaling agonist. Moreover, loss of tsg1 partially suppressed the ventralized phenotypes of mutants of the BMP antagonists Chordin or Sizzled (Ogon). Our results support a model in which zebrafish Tsg1 promotes BMP signaling, and thus ventral cell fates, during DV axial patterning.

Genetic Screens for Maternal-effect Mutations

Methods in Cell Biology. 2004  |  Pubmed ID: 15602904

Genetic Screens for Genes Controlling Motor Nerve-muscle Development and Interactions

Developmental Biology. Apr, 2005  |  Pubmed ID: 15766756

Motor growth cones navigate long and complex trajectories to connect with their muscle targets. Experimental studies have shown that this guidance process critically depends on extrinsic cues. In the zebrafish embryo, a subset of mesodermal cells, the adaxial cells, delineates the prospective path of pioneering motor growth cones. Genetic ablation of adaxial cells causes profound pathfinding defects, suggesting the existence of adaxial cell derived guidance factors. Intriguingly, adaxial cells are themselves migratory, and as growth cones approach they migrate away from the prospective axonal path to the lateral surface of the myotome, where they develop into slow-twitching muscle fibers. Genetic screens in embryos stained with an antibody cocktail identified mutants with specific defects in differentiation and migration of adaxial cells/slow muscle fibers, as well as mutants with specific defects in axonal pathfinding, including exit from the spinal cord and pathway selection. Together, the genes underlying these mutant phenotypes define pathways essential for nerve and muscle development and interactions between these two cell types.

Neuromuscular Synaptogenesis in Wild-type and Mutant Zebrafish

Developmental Biology. Sep, 2005  |  Pubmed ID: 16102744

Genetic screens for synaptogenesis mutants have been performed in many organisms, but few if any have simultaneously screened for defects in pre- and postsynaptic specializations. Here, we report the results of a small-scale genetic screen, the first in vertebrates, for defects in synaptogenesis. Using zebrafish as a model system, we identified seven mutants that affect different aspects of neuromuscular synapse formation. Many of these mutant phenotypes have not been previously reported in zebrafish and are distinct from those described in other organisms. Characterization of mutant and wild-type zebrafish, from the time that motor axons first arrive at target muscles through adulthood, has provided the new information about the cellular events that occur during neuromuscular synaptogenesis. These include insights into the formation and dispersal of prepatterned AChR clusters, the relationship between motor axon elongation and synapse size, and the development of precise appositions between presynaptic clusters of synaptic vesicles in nerve terminals and postsynaptic receptor clusters. In addition, we show that the mechanisms underlying synapse formation within the myotomal muscle itself are largely independent of those that underlie synapse formation at myotendinous junctions and that the outgrowth of secondary motor axons requires at least one cue not necessary for the outgrowth of primary motor axons, while other cues are required for both. One-third of the mutants identified in this screen did not have impaired motility, suggesting that many genes involved in neuromuscular synaptogenesis were missed in large scale motility-based screens. Identification of the underlying genetic defects in these mutants will extend our understanding of the cellular and molecular mechanisms that underlie the formation and function of neuromuscular and other synapses.

Tolloid Gets Sizzled Competing with Chordin

Developmental Cell. Feb, 2006  |  Pubmed ID: 16459293

The enigma of Sizzled, a secreted Frizzled-related protein, has been resolved in a recent study from the De Robertis lab ( [in the January 13 issue of Cell]). Sizzled, although homologous to other Wnt antagonists, does not function as such, nor does it function within a Wnt signaling pathway. Remarkably it functions as an antagonist of BMP signaling, competing with Chordin for binding to its inhibitor a Tolloid-related metalloprotease. This competition protects Chordin from cleavage, thus allowing it to bind and limit BMP signaling.

Temporal and Spatial Action of Tolloid (mini Fin) and Chordin to Pattern Tail Tissues

Developmental Biology. May, 2006  |  Pubmed ID: 16530746

In vertebrates, a bone morphogenetic protein (BMP) signaling pathway patterns all ventral cell fates along the embryonic axis. BMP activity is positively regulated by Tolloid, a metalloprotease, that can eliminate the activity of the BMP antagonist Chordin. A tolloid mutant in zebrafish, mini fin (mfn), exhibits a specific loss of ventral tail tissues. Here, we investigate the spatial and temporal requirements for Tolloid (Mfn) in dorsoventral patterning of the tail. Through chimeric analyses, we found that Tolloid (Mfn) functions cell non-autonomously in the ventral-most vegetal cells of the gastrula or their derivatives. We generated a tolloid transgene under the control of the inducible hsp70 promoter and demonstrate that tolloid (mfn) is first required at the completion of gastrulation. Although tolloid is expressed during gastrulation and dorsally and ventrally within the tail bud, our results indicate that Tolloid (Mfn) acts specifically in the ventral tail bud during a approximately 4 h period extending from the completion of gastrulation to early somitogenesis stages to regulate BMP signaling. Examination of the temporal requirements of Chordin activity by overexpression of the hsp70-tolloid transgene indicates that Chordin is required both during and after gastrulation for proper patterning of the tail, contrasting Tld's requirement only during post-gastrula stages. We hypothesize that the gastrula role of Chordin in tail patterning is to generate the proper size domains of cells to enter the ventral and dorsal tail bud, whereas post-gastrula Chordin activity patterns the derivatives of the tail bud. Thus, fine modulation of BMP signaling levels through the negative and positive actions of Chordin and Tolloid, respectively, patterns tail tissues.

The Zebrafish Homologue of Mammalian Chimerin Rac-GAPs is Implicated in Epiboly Progression During Development

Proceedings of the National Academy of Sciences of the United States of America. Apr, 2006  |  Pubmed ID: 16569702

In this paper, we report an in vivo model for the chimerins, a family of Rac GTPase-activating proteins (Rac-GAPs) that are uniquely regulated by the lipid second messenger diacylglycerol and have been implicated in the control of actin dynamics, migration, and proliferation. We cloned the zebrafish homologue of mammalian alpha2-chimerin (chn1) and determined that it possesses Rac-GAP activity and a C1 domain with phorbol ester/diacylglycerol-binding capability. chn1 morpholino knockdown embryos exhibit severe abnormalities, including the development of round somites, lack of yolk extension, and a kinked posterior notochord. These zebrafish morphants show Rac hyperactivation and progress faster through epiboly, leading to tailbud-stage embryos that have a narrow axis and an enlarged tailbud with expanded bmp4 and shh expression. Phenotypic rescue was achieved by mRNA microinjection of chn1 or an active chimerin Rac-GAP domain into the yolk syncytial layer but not by a chn1 mutant deficient in Rac-GAP activity, suggesting that the lack of chn1 Rac-GAP activity in the yolk syncytial layer was causative of the misbalance in morphogenetic movements. Our results reveal a crucial role for chn1 in early development and implicate Rac as a key regulator of morphogenetic movements during zebrafish epiboly.

Zebrafish As a "biosensor"? Effects of Ionizing Radiation and Amifostine on Embryonic Viability and Development

Cancer Research. Aug, 2006  |  Pubmed ID: 16912196

The zebrafish (Danio rerio) has emerged as a popular vertebrate model system for cancer and treatment-related research. Benefits include ease of care, rapid development, optical clarity of embryos, which allows visualization of major organ systems, and opportunities for genetic manipulation. However, specific parameters of radiation sensitivity have not been systematically documented. We investigated the effects of radiation and a radiomodifier on zebrafish viability and embryonic development. Embryos were exposed to gamma-radiation (5, 10, or 20 Gy) at sequential times postfertilization and serially assessed for viability and morphologic abnormalities. As expected, lethality and morphologic perturbations were more pronounced earlier in embryogenesis and with higher radiation doses and were partially reversed by amifostine. The effects of radiation and concurrent treatment with amifostine on the developmental organization of the eye and brain were striking. Radiation resulted in hypocellularity and disorganization of the cellular layers of the retina, effects partially reversed by amifostine, as well as lens opacification. Radiation strikingly reduced the volume of brain, but the volume loss was substantially blocked by amifostine. Increased terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling signal was noted in both the irradiated eye and brain, but reduced by amifostine. Finally, irradiating embryos resulted in caspase activation detectable in 96-well microplates, which was proportional to the number of embryos and radiation dose; the degree of activation was markedly reduced by amifostine. These results together suggest the power and versatility of the zebrafish in assessing the effects of radiation and radiomodifiers on organ and tissue development.

Extracellular Modulation of BMP Activity in Patterning the Dorsoventral Axis

Birth Defects Research. Part C, Embryo Today : Reviews. Sep, 2006  |  Pubmed ID: 17061292

Signaling via bone morphogenetic proteins (BMPs) regulates a vast array of diverse biological processes in the developing embryo and in postembryonic life. Many insights into BMP signaling derive from studies of the BMP signaling gradients that pattern cell fates along the embryonic dorsal-ventral (DV) axis of both vertebrates and invertebrates. This review examines recent developments in the field of DV patterning by BMP signaling, focusing on extracellular modulation as a key mechanism in the formation of BMP signaling gradients in Drosophila, Xenopus, and zebrafish.

Bmp and Fgf Signaling Are Essential for Liver Specification in Zebrafish

Development (Cambridge, England). Jun, 2007  |  Pubmed ID: 17507405

Based on data from in vitro tissue explant and ex vivo cell/bead implantation experiments, Bmp and Fgf signaling have been proposed to regulate hepatic specification. However, genetic evidence for this hypothesis has been lacking. Here, we provide in vivo genetic evidence that Bmp and Fgf signaling are essential for hepatic specification. We utilized transgenic zebrafish that overexpress dominant-negative forms of Bmp or Fgf receptors following heat-shock induction. These transgenes allow one to bypass the early embryonic requirements for Bmp and Fgf signaling, and also to completely block Bmp or Fgf signaling. We found that the expression of hhex and prox1, the earliest liver markers in zebrafish, was severely reduced in the liver region when Bmp or Fgf signaling was blocked just before hepatic specification. However, hhex and prox1 expression in adjacent endodermal and mesodermal tissues appeared unaffected by these manipulations. Additional genetic studies indicate that the endoderm maintains competence for Bmp-mediated hepatogenesis over an extended window of embryonic development. Altogether, these data provide the first genetic evidence that Bmp and Fgf signaling are essential for hepatic specification, and suggest that endodermal cells remain competent to differentiate into hepatocytes for longer than anticipated.

Smad1 and Smad5 Differentially Regulate Embryonic Hematopoiesis

Blood. Dec, 2007  |  Pubmed ID: 17761518

The bone morphogenetic protein (BMP) signaling pathway regulates multiple steps of hematopoiesis, mediated through receptor-regulated Smads, including Smad1 and Smad5. Here, we use loss-of-function approaches in zebrafish to compare the roles of Smad1 and Smad5 during embryonic hematopoiesis. We show that knockdown of Smad1 or Smad5 generates distinct and even opposite hematopoietic phenotypes. Embryos depleted for Smad1 have an increased number of primitive erythrocytes, but fail to produce mature embryonic macrophages. In contrast, Smad5-depleted embryos are defective in primitive erythropoiesis, yet have normal numbers of macrophages. Loss of either Smad1 or Smad5 causes a failure in the generation of definitive hematopoietic progenitors. To investigate the mechanism behind these phenotypes, we used rescue experiments and found that Smad5 is unable to rescue the Smad1 loss-of-function phenotype, indicating that the 2 highly related proteins have inherently distinct activities. Microarray experiments revealed that the 2 proteins redundantly regulate the key initiators of the hemato-vascular program, including scl, lmo2, and gfi1. However, each also regulates a remarkably distinct genetic program, with Smad5 uniquely regulating the BMP signaling pathway itself. Our results suggest that specificity of BMP signaling output, with respect to hematopoiesis, can be explained by differential functions of Smad1 and Smad5.

The BMP Signaling Gradient Patterns Dorsoventral Tissues in a Temporally Progressive Manner Along the Anteroposterior Axis

Developmental Cell. Jan, 2008  |  Pubmed ID: 18194657

Patterning of the vertebrate anteroposterior (AP) axis proceeds temporally from anterior to posterior. How dorsoventral (DV) axial patterning relates to AP temporal patterning is unknown. We examined the temporal activity of BMP signaling in patterning ventrolateral cell fates along the AP axis, using transgenes that rapidly turn "off" or "on" BMP signaling. We show that BMP signaling patterns rostral DV cell fates at the onset of gastrulation, whereas progressively more caudal DV cell fates are patterned at progressively later intervals during gastrulation. Increased BMP signal duration is not required to pattern more caudal DV cell fates; rather, distinct temporal intervals of signaling are required. This progressive action is regulated downstream of, or in parallel to, BMP signal transduction at the level of Smad1/5 phosphorylation. We propose that a temporal cue regulates a cell's competence to respond to BMP signaling, allowing the acquisition of a cell's DV and AP identity simultaneously.

Bucky Ball Functions in Balbiani Body Assembly and Animal-vegetal Polarity in the Oocyte and Follicle Cell Layer in Zebrafish

Developmental Biology. Sep, 2008  |  Pubmed ID: 18582455

The Balbiani body is an evolutionarily conserved asymmetric aggregate of organelles that is present in early oocytes of all animals examined, including humans. Although first identified more than 150 years ago, genes acting in the assembly of the Balbiani body have not been identified in a vertebrate. Here we show that the bucky ball gene in the zebrafish is required to assemble this universal aggregate of organelles. In the absence of bucky ball the Balbiani body fails to form, and vegetal mRNAs are not localized in oocytes. In contrast, animal pole localized oocyte markers are expanded into vegetal regions in bucky ball mutants, but patterning within the expanded animal pole remains intact. Interestingly, in bucky ball mutants an excessive number of cells within the somatic follicle cell layer surrounding the oocyte develop as micropylar cells, an animal pole specific cell fate. The single micropyle permits sperm to fertilize the egg in zebrafish. In bucky ball mutants, excess micropyles cause polyspermy. Thus bucky ball provides the first genetic access to Balbiani body formation in a vertebrate. We demonstrate that bucky ball functions during early oogenesis to regulate polarity of the oocyte, future egg and embryo. Finally, the expansion of animal identity in oocytes and somatic follicle cells suggests that somatic cell fate and oocyte polarity are interdependent.

Regulation of Hematopoiesis by the BMP Signaling Pathway in Adult Zebrafish

Experimental Hematology. Dec, 2008  |  Pubmed ID: 18973974

The zebrafish is an established model system for studying the embryonic emergence of tissues and organs, including the hematopoietic system. We hypothesized that key signaling pathways controlling embryonic hematopoiesis continue to be important in the adult, and we sought to develop approaches to test this in zebrafish, focused on the bone morphogenetic protein (BMP) signaling pathway. Functions for this pathway in adult hematopoiesis have been challenging to probe in other models.

Tbx2b is Required for Ultraviolet Photoreceptor Cell Specification During Zebrafish Retinal Development

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2009  |  Pubmed ID: 19179291

The vertebrate rod and cone photoreceptors are highly specialized sensory neurons that transduce light into the chemical and electrical signals of the nervous system. Although the physiological properties of cones and rods are well known, only a handful of genes have been identified that regulate the specification of photoreceptor subtypes. Taking advantage of the mosaic organization of photoreceptors in zebrafish, we report the isolation of a mutation resulting in a unique change in photoreceptor cell fate. Mutation of the lots-of-rods (lor) locus results in a near one-for-one transformation of UV-cone precursors into rods. The transformed cells exhibit morphological characteristics and a gene-expression pattern typical of rods, but differentiate in a temporal and spatial pattern consistent with UV-cone development. In mutant larvae and adults, the highly ordered photoreceptor mosaic is maintained and degeneration is not observed, suggesting that lor functions after the specification of the other photoreceptor subtypes. In genetic chimeras, lor functions cell-autonomously in the specification of photoreceptor cell fate. Linkage analysis and genetic-complementation testing indicate that lor is an allele of tbx2b/fby (from beyond). fby was identified by a pineal complex phenotype, and carries a nonsense mutation in the T-box domain of the tbx2b transcription factor. Homozygous fby mutant larvae and lor/fby transheterozygotes also display the lots-of-rods phenotype. Based upon these data, we propose a previously undescribed function for tbx2b in photoreceptor cell precursors, to promote the UV cone fate by repressing the rod differentiation pathway.

A Late Role for Bmp2b in the Morphogenesis of Semicircular Canal Ducts in the Zebrafish Inner Ear

PloS One. 2009  |  Pubmed ID: 19190757

The Bone Morphogenetic Protein (BMP) genes bmp2 and bmp4 are expressed in highly conserved patterns in the developing vertebrate inner ear. It has, however, proved difficult to elucidate the function of BMPs during ear development as mutations in these genes cause early embryonic lethality. Previous studies using conditional approaches in mouse and chicken have shown that Bmp4 has a role in semicircular canal and crista development, but there is currently no direct evidence for the role of Bmp2 in the developing inner ear.

Bucky Ball Organizes Germ Plasm Assembly in Zebrafish

Current Biology : CB. Mar, 2009  |  Pubmed ID: 19249209

In many animals, gamete formation during embryogenesis is specified by maternal cytoplasmic determinants termed germ plasm. During oogenesis, germ plasm forms a distinct cellular structure such as pole plasm in Drosophila or the Balbiani body, an aggregate of organelles also found in mammals. However, in vertebrates, the key regulators of germ plasm assembly are largely unknown. Here, we show that, at the beginning of zebrafish oogenesis, the germ plasm defect in bucky ball (buc) mutants precedes the loss of polarity, indicating that Buc primarily controls Balbiani body formation. Moreover, we molecularly identify the buc gene, which is exclusively expressed in the ovary with a novel, dynamic mRNA localization pattern first detectable within the Balbiani body. We find that a Buc-GFP fusion localizes to the Balbiani body during oogenesis and with the germ plasm during early embryogenesis, consistent with a role in germ plasm formation. Interestingly, overexpression of buc seems to generate ectopic germ cells in the zebrafish embryo. Because we discovered buc homologs in many vertebrate genomes, including mammals, these results identify buc as the first gene necessary and sufficient for germ plasm organization in vertebrates.

A Novel Role for MAPKAPK2 in Morphogenesis During Zebrafish Development

PLoS Genetics. Mar, 2009  |  Pubmed ID: 19282986

One of the earliest morphogenetic processes in the development of many animals is epiboly. In the zebrafish, epiboly ensues when the animally localized blastoderm cells spread, thin over, and enclose the vegetally localized yolk. Only a few factors are known to function in this fundamental process. We identified a maternal-effect mutant, betty boop (bbp), which displays a novel defect in epiboly, wherein the blastoderm margin constricts dramatically, precisely when half of the yolk cell is covered by the blastoderm, causing the yolk cell to burst. Whole-blastoderm transplants and mRNA microinjection rescue demonstrate that Bbp functions in the yolk cell to regulate epiboly. We positionally cloned the maternal-effect bbp mutant gene and identified it as the zebrafish homolog of the serine-threonine kinase Mitogen Activated Protein Kinase Activated Protein Kinase 2, or MAPKAPK2, which was not previously known to function in embryonic development. We show that the regulation of MAPKAPK2 is conserved and p38 MAP kinase functions upstream of MAPKAPK2 in regulating epiboly in the zebrafish embryo. Dramatic alterations in calcium dynamics, together with the massive marginal constrictive force observed in bbp mutants, indicate precocious constriction of an F-actin network within the yolk cell, which first forms at 50% epiboly and regulates epiboly progression. We show that MAPKAPK2 activity and its regulator p38 MAPK function in the yolk cell to regulate the process of epiboly, identifying a new pathway regulating this cell movement process. We postulate that a p38 MAPKAPK2 kinase cascade modulates the activity of F-actin at the yolk cell margin circumference allowing the gradual closure of the blastopore as epiboly progresses.

Bone Morphogenetic Protein Heterodimers Assemble Heteromeric Type I Receptor Complexes to Pattern the Dorsoventral Axis

Nature Cell Biology. May, 2009  |  Pubmed ID: 19377468

Patterning the embryonic dorsoventral axis of both vertebrates and invertebrates requires signalling through bone morphogenetic proteins (BMPs). Although a well-studied process, the identity of the physiologically relevant BMP signalling complex in the Drosophila melanogaster embryo is controversial, is generally inferred from cell culture studies and has not been investigated in vertebrates. Here, we demonstrate that dorsoventral patterning in zebrafish, Danio rerio, requires two classes of non-redundant type I BMP receptors, Alk3/6 and Alk8 (activin-like kinases 3/6 and 8). We show, under physiological conditions in the embryo, that these two type I receptor classes form a complex in a manner that depends on Bmp2 and Bmp7. We found that both Bmp2-7 heterodimers, as well as Bmp2 and Bmp7 homodimers, form in the embryo. However, only recombinant ligand heterodimers can activate BMP signalling in the early embryo, whereas a combination of Bmp2 and Bmp7 homodimers cannot. We propose that only heterodimers, signalling through two distinct classes of type I receptor, possess sufficient receptor affinity in an environment of extracellular antagonists to elicit the signalling response required for dorsoventral patterning.

The Maternal-effect Gene Cellular Island Encodes Aurora B Kinase and is Essential for Furrow Formation in the Early Zebrafish Embryo

PLoS Genetics. Jun, 2009  |  Pubmed ID: 19543364

Females homozygous for a mutation in cellular island (cei) produce embryos with defects in cytokinesis during early development. Analysis of the cytoskeletal events associated with furrow formation reveal that these defects include a general delay in furrow initiation as well as a complete failure to form furrow-associated structures in distal regions of the blastodisc. A linkage mapping-based candidate gene approach, including transgenic rescue, shows that cei encodes the zebrafish Aurora B kinase homologue. Genetic complementation analysis between the cei mutation and aurB zygotic lethal mutations corroborate gene assignment and reveal a complex nature of the maternal-effect cei allele, which appears to preferentially affect a function important for cytokinesis in the early blastomeres. Surprisingly, in cei mutant embryos a short yet otherwise normal furrow forms in the center of the blastodisc. Furrow formation is absent throughout the width of the blastodisc in cei mutant embryos additionally mutant for futile cycle, which lack a spindle apparatus, showing that the residual furrow signal present in cei mutants is derived from the mitotic spindle. Our analysis suggests that partially redundant signals derived from the spindle and astral apparatus mediate furrow formation in medial and distal regions of the early embryonic blastomeres, respectively, possibly as a spatial specialization to achieve furrow formation in these large cells. In addition, our data also suggest a role for Cei/AurB function in the reorganization of the furrow-associated microtubules in both early cleavage- and somite-stage embryos. In accordance with the requirement for cei/aurB in furrow induction in the early cleavage embryo, germ plasm recruitment to the forming furrow is also affected in embryos lacking normal cei/aurB function.

Early Zebrafish Development: It's in the Maternal Genes

Current Opinion in Genetics & Development. Aug, 2009  |  Pubmed ID: 19608405

The earliest stages of embryonic development in all animals examined rely on maternal gene products that are generated during oogenesis and supplied to the egg. The period of maternal control of embryonic development varies among animals according to the onset of zygotic transcription and the persistence of maternal gene products. This maternal regulation has been little studied in vertebrates, owing to the difficulty in manipulating maternal gene function and lack of basic molecular information. However, recent maternal-effect screens in the zebrafish have generated more than 40 unique mutants that are providing new molecular entry points to the maternal control of early vertebrate development. Here we discuss recent studies of 12 zebrafish mutant genes that illuminate the maternal molecular controls on embryonic development, including advances in the regulation of animal-vegetal polarity, egg activation, cleavage development, body plan formation, tissue morphogenesis, microRNA function and germ cell development.

HnRNP I is Required to Generate the Ca2+ Signal That Causes Egg Activation in Zebrafish

Development (Cambridge, England). Sep, 2009  |  Pubmed ID: 19666827

Egg activation is an important cellular event required to prevent polyspermy and initiate development of the zygote. Egg activation in all animals examined is elicited by a rise in free Ca(2+) in the egg cytosol at fertilization. This Ca(2+) rise is crucial for all subsequent egg activation steps, such as cortical granule exocytosis, which modifies the vitelline membrane to prevent polyspermy. The cytosolic Ca(2+) rise is primarily initiated by inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from the endoplasmic reticulum. The genes involved in regulating the IP(3)-mediated Ca(2+) release during egg activation remain largely unknown. Here we report on a zebrafish maternal-effect mutant, brom bones, which is defective in the cytosolic Ca(2+) rise and subsequent egg activation events, including cortical granule exocytosis and cytoplasmic segregation. We show that the egg activation defects in brom bones can be rescued by providing Ca(2+) or the Ca(2+)-release messenger IP(3), suggesting that brom bones is a regulator of IP(3)-mediated Ca(2+) release at fertilization. Interestingly, brom bones mutant embryos also display defects in dorsoventral axis formation accompanied by a disorganized cortical microtubule network, which is known to be crucial for dorsal axis formation. We provide evidence that the impaired microtubule organization is associated with non-exocytosed cortical granules from the earlier egg activation defect. Positional cloning of the brom bones gene reveals that a premature stop codon in the gene encoding hnRNP I (referred to here as hnrnp I) underlies the abnormalities. Our studies therefore reveal an important new role of hnrnp I in regulating the fundamental process of IP(3)-mediated Ca(2+) release at egg activation.

The Fibrodysplasia Ossificans Progressiva R206H ACVR1 Mutation Activates BMP-independent Chondrogenesis and Zebrafish Embryo Ventralization

The Journal of Clinical Investigation. Nov, 2009  |  Pubmed ID: 19855136

Patients with classic fibrodysplasia ossificans progressiva, a disorder characterized by extensive extraskeletal endochondral bone formation, share a recurrent mutation (R206H) within the glycine/serine-rich domain of ACVR1/ALK2, a bone morphogenetic protein type I receptor. Through a series of in vitro assays using several mammalian cell lines and chick limb bud micromass cultures, we determined that mutant R206H ACVR1 activated BMP signaling in the absence of BMP ligand and mediated BMP-independent chondrogenesis that was enhanced by BMP. We further investigated the interaction of mutant R206H ACVR1 with FKBP1A, a glycine/serine domain-binding protein that prevents leaky BMP type I receptor activation in the absence of ligand. The mutant protein exhibited reduced binding to FKBP1A in COS-7 simian kidney cell line assays, suggesting that increased BMP pathway activity in COS-7 cells with R206H ACVR1 is due, at least in part, to decreased binding of this inhibitory factor. Consistent with these findings, in vivo analyses of zebrafish embryos showed BMP-independent hyperactivation of BMP signaling in response to the R206H mutant, resulting in increased embryonic ventralization. These data support the conclusion that the mutant R206H ACVR1 receptor in FOP patients is an activating mutation that induces BMP signaling in a BMP-independent and BMP-responsive manner to promote chondrogenesis, consistent with the ectopic endochondral bone formation in these patients.

Microtubule Actin Crosslinking Factor 1 Regulates the Balbiani Body and Animal-vegetal Polarity of the Zebrafish Oocyte

PLoS Genetics. Aug, 2010  |  Pubmed ID: 20808893

Although of fundamental importance in developmental biology, the genetic basis for the symmetry breaking events that polarize the vertebrate oocyte and egg are largely unknown. In vertebrates, the first morphological asymmetry in the oocyte is the Balbiani body, a highly conserved, transient structure found in vertebrates and invertebrates including Drosophila, Xenopus, human, and mouse. We report the identification of the zebrafish magellan (mgn) mutant, which exhibits a novel enlarged Balbiani body phenotype and a disruption of oocyte polarity. To determine the molecular identity of the mgn gene, we positionally cloned the gene, employing a novel DNA capture method to target region-specific genomic DNA of 600 kb for massively parallel sequencing. Using this technique, we were able to enrich for the genomic region linked to our mutation within one week and then identify the mutation in mgn using massively parallel sequencing. This is one of the first successful uses of genomic DNA enrichment combined with massively parallel sequencing to determine the molecular identity of a gene associated with a mutant phenotype. We anticipate that the combination of these technologies will have wide applicability for the efficient identification of mutant genes in all organisms. We identified the mutation in mgn as a deletion in the coding sequence of the zebrafish microtubule actin crosslinking factor 1 (macf1) gene. macf1 is a member of the highly conserved spectraplakin family of cytoskeletal linker proteins, which play diverse roles in polarized cells such as neurons, muscle cells, and epithelial cells. In mgn mutants, the oocyte nucleus is mislocalized; and the Balbiani body, localized mRNAs, and organelles are absent from the periphery of the oocyte, consistent with a function for macf1 in nuclear anchoring and cortical localization. These data provide the first evidence for a role for spectraplakins in polarization of the vertebrate oocyte and egg.

Mutations in Zebrafish Lrp2 Result in Adult-onset Ocular Pathogenesis That Models Myopia and Other Risk Factors for Glaucoma

PLoS Genetics. Feb, 2011  |  Pubmed ID: 21379331

The glaucomas comprise a genetically complex group of retinal neuropathies that typically occur late in life and are characterized by progressive pathology of the optic nerve head and degeneration of retinal ganglion cells. In addition to age and family history, other significant risk factors for glaucoma include elevated intraocular pressure (IOP) and myopia. The complexity of glaucoma has made it difficult to model in animals, but also challenging to identify responsible genes. We have used zebrafish to identify a genetically complex, recessive mutant that shows risk factors for glaucoma including adult onset severe myopia, elevated IOP, and progressive retinal ganglion cell pathology. Positional cloning and analysis of a non-complementing allele indicated that non-sense mutations in low density lipoprotein receptor-related protein 2 (lrp2) underlie the mutant phenotype. Lrp2, previously named Megalin, functions as an endocytic receptor for a wide-variety of bioactive molecules including Sonic hedgehog, bone morphogenic protein 4, retinol-binding protein, vitamin D-binding protein, and apolipoprotein E, among others. Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals--but not all--develop high IOP and severe myopia with obviously enlarged eye globes. This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis. Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease.

SnapShot: BMP Signaling in Development

Cell. May, 2011  |  Pubmed ID: 21565618

All-in-one Live: Genes Trapped, Tagged and Conditionally Broken

Nature Methods. Jun, 2011  |  Pubmed ID: 21623352

Genetic Screens for Mutations Affecting Adult Traits and Parental-effect Genes

Methods in Cell Biology. 2011  |  Pubmed ID: 21924158

Forward genetic analysis in the zebrafish has largely until now been restricted to the developmental period from the time of zygotic genome activation through the end of embryogenesis. However, the use of the zebrafish as a model system for the analysis of larval, juvenile and adult traits, including fertility and maternal and paternal effects, is gaining momentum. Here, we describe two different approaches, an F3-extended family and a gynogenesis-based approach, that allow genetic screening for and recovery of mutations affecting post-embryonic stages, including adult traits, fertility, and parental effects. For each approach, we also describe strategies to maintain and map the identified mutations.

Maternal and Zygotic Control of Zebrafish Dorsoventral Axial Patterning

Annual Review of Genetics. 2011  |  Pubmed ID: 21942367

Vertebrate development begins with precise molecular, cellular, and morphogenetic controls to establish the basic body plan of the embryo. In zebrafish, these tightly regulated processes begin during oogenesis and proceed through gastrulation to establish and pattern the axes of the embryo. During oogenesis a maternal factor is localized to the vegetal pole of the oocyte that is a determinant of dorsal tissues. Following fertilization this vegetally localized dorsal determinant is asymmetrically translocated in the egg and initiates formation of the dorsoventral axis. Dorsoventral axis formation and patterning is then mediated by maternal and zygotic factors acting through Wnt, BMP (bone morphogenetic protein), Nodal, and FGF (fibroblast growth factor) signaling pathways, each of which is required to establish and/or pattern the dorsoventral axis. This review addresses recent advances in our understanding of the molecular factors and mechanisms that establish and pattern the dorsoventral axis of the zebrafish embryo, including establishment of the animal-vegetal axis as it relates to formation of the dorsoventral axis.

An Intermediate Level of BMP Signaling Directly Specifies Cranial Neural Crest Progenitor Cells in Zebrafish

PloS One. 2011  |  Pubmed ID: 22102893

The specification of the neural crest progenitor cell (NCPC) population in the early vertebrate embryo requires an elaborate network of signaling pathways, one of which is the Bone Morphogenetic Protein (BMP) pathway. Based on alterations in neural crest gene expression in zebrafish BMP pathway component mutants, we previously proposed a model in which the gastrula BMP morphogen gradient establishes an intermediate level of BMP activity establishing the future NCPC domain. Here, we tested this model and show that an intermediate level of BMP signaling acts directly to specify the NCPC. We quantified the effects of reducing BMP signaling on the number of neural crest cells and show that neural crest cells are significantly increased when BMP signaling is reduced and that this increase is not due to an increase in cell proliferation. In contrast, when BMP signaling is eliminated, NCPC fail to be specified. We modulated BMP signaling levels in BMP pathway mutants with expanded or no NCPCs to demonstrate that an intermediate level of BMP signaling specifies the NCPC. We further investigated the ability of Smad5 to act in a graded fashion by injecting smad5 antisense morpholinos and show that increasing doses first expand the NCPCs and then cause a loss of NCPCs, consistent with Smad5 acting directly in neural crest progenitor specification. Using Western blot analysis, we show that P-Smad5 levels are dose-dependently reduced in smad5 morphants, consistent with an intermediate level of BMP signaling acting through Smad5 to specify the neural crest progenitors. Finally, we performed chimeric analysis to demonstrate for the first time that BMP signal reception is required directly by NCPCs for their specification. Together these results add substantial evidence to a model in which graded BMP signaling acts as a morphogen to pattern the ectoderm, with an intermediate level acting in neural crest specification.

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