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M C Mullins

Publications and source records attributed to M C Mullins.

At least 19 recordsLinked to original sources
PubMed2001

Genetic analysis of digestive physiology using fluorescent phospholipid reporters.

Zebrafish are a valuable model for mammalian lipid metabolism; larvae process lipids similarly through the intestine and hepatobiliary system and respond to drugs that block cholesterol synthesis in humans. After ingestion of fluorescently quenched phospholipids, endogenous lipase activity and rapid transport of cleavage products results in intense gall bladder fluorescence. Genetic screening identifies zebrafish mutants, such as fat free, that show normal digestive organ morphology but severely reduced phospholipid and cholesterol processing. Thus, fluorescent lipids provide a sensitive readout of lipid metabolism and are a powerful tool for identifying genes that mediate vertebrate digestive physiology.

Animals↗
PubMed2001

Lost-a-fin encodes a type I BMP receptor, Alk8, acting maternally and zygotically in dorsoventral pattern formation.

TGFbeta signaling pathways of the bone morphogenetic protein (BMP) subclass are essential for dorsoventral pattern formation of both vertebrate and invertebrate embryos. Here we determine by chromosomal mapping, linkage analysis, cDNA sequencing and mRNA rescue that the dorsalized zebrafish mutant lost-a-fin (laf) is defective in the gene activin receptor-like kinase 8 (alk8), which encodes a novel type I TGFbeta receptor. The alk8 mRNA is expressed both maternally and zygotically. Embyros that lack zygotic, but retain maternal Laf/Alk8 activity, display a weak dorsalization restricted to the tail and die by 3 days postfertilization. We rescued the laf dorsalized mutant phenotype by alk8 mRNA injection and generated homozygous laf/alk8 mothers to investigate the maternal role of Laf/Alk8 activity. Adult fish lacking Laf/Alk8 activity are fertile, exhibit a growth defect and are significantly smaller than their siblings. Embryos derived from homozygous females, which lack both maternal and zygotic Laf/Alk8 activity, display a strongly dorsalized mutant phenotype, no longer limited to the tail. These mutant embryos lack almost all gastrula ventral cell fates, with a concomitant expansion of dorsal cell types. During later stages, most of the somitic mesoderm and neural tissue circumscribe the dorsoventral axis of the embryo. Zygotic laf/alk8 mutants can be rescued by overexpression of the BMP signal transducer Smad5, but not the Bmp2b or Bmp7 ligands, consistent with the Laf/Alk8 receptor acting within a BMP signaling pathway, downstream of a Bmp2b/Bmp7 signal. Antibodies specific for the phosphorylated, activated form of Smad1/5, show that BMP signaling is nearly absent in gastrula lacking both maternal and zygotic Laf/Alk8 activity, providing further evidence that Laf/Alk8 transduces a BMP signal. In total, our work strongly supports the role of Laf/Alk8 as a type I BMP receptor required for the specification of ventral cell fates.

Activin Receptors↗
PubMed2000

Equivalent genetic roles for bmp7/snailhouse and bmp2b/swirl in dorsoventral pattern formation.

A bone morphogenetic protein (BMP) signaling pathway acts in the establishment of the dorsoventral axis of the vertebrate embryo. Here we demonstrate the genetic requirement for two different Bmp ligand subclass genes for dorsoventral pattern formation of the zebrafish embryo. From the relative efficiencies observed in Bmp ligand rescue experiments, conserved chromosomal synteny, and isolation of the zebrafish bmp7 gene, we determined that the strongly dorsalized snailhouse mutant phenotype is caused by a mutation in the bmp7 gene. We show that the original snailhouse allele is a hypomorphic mutation and we identify a snailhouse/bmp7 null mutant. We demonstrate that the snailhouse/bmp7 null mutant phenotype is identical to the presumptive null mutant phenotype of the strongest dorsalized zebrafish mutant swirl/bmp2b, revealing equivalent genetic roles for these two Bmp ligands. Double mutant snailhouse/bmp7; swirl/bmp2b embryos do not exhibit additional or stronger dorsalized phenotypes, indicating that these Bmp ligands do not function redundantly in early embryonic development. Furthermore, overexpression experiments reveal that Bmp2b and Bmp7 synergize in the ventralization of wild-type embryos through a cell-autonomous mechanism, suggesting that Bmp2b/Bmp7 heterodimers may act in vivo to specify ventral cell fates in the zebrafish embryo.

Amino Acid Sequence↗
PubMed2000

Dorsal and intermediate neuronal cell types of the spinal cord are established by a BMP signaling pathway.

We have studied the role of Bmp signaling in patterning neural tissue through the use of mutants in the zebrafish that disrupt three different components of a Bmp signaling pathway: swirl/bmp2b, snailhouse/bmp7 and somitabun/smad5. We demonstrate that Bmp signaling is essential for the establishment of the prospective neural crest and dorsal sensory Rohon-Beard neurons of the spinal cord. Moreover, Bmp signaling is necessary to limit the number of intermediate-positioned lim1+ interneurons of the spinal cord, as observed by the dramatic expansion of these prospective interneurons in many mutant embryos. Our analysis also suggests a positive role for Bmp signaling in the specification of these interneurons, which is independent of Bmp2b/Swirl activity. We found that a presumptive ventral signal, Hh signaling, acts to restrict the amount of dorsal sensory neurons and trunk neural crest. This restriction appears to occur very early in neural tissue development, likely prior to notochord or floor plate formation. A similar early role for Bmp signaling is suggested in the specification of dorsal neural cell types, since the bmp2b/swirl and bmp7/snailhouse genes are only coexpressed during gastrulation and within the tail bud, and are not found in the dorsal neural tube or overlying epidermal ectoderm. Thus, a gastrula Bmp2b/Swirl and Bmp7/Snailhouse-dependent activity gradient may not only act in the specification of the embryonic dorsoventral axis, but may also function in establishing dorsal and intermediate neuronal cell types of the spinal cord.

Animals↗
PubMed1999

Development of noradrenergic neurons in the zebrafish hindbrain requires BMP, FGF8, and the homeodomain protein soulless/Phox2a.

We report that the zebrafish mutation soulless, in which the development of locus coeruleus (LC) noradrenergic (NA) neurons failed to occur, disrupts the homeodomain protein Phox2a. Phox2a is not only necessary but also sufficient to induce Phox2b+ dopamine-beta-hydroxylase+ and tyrosine hydroxylase+ NA neurons in ectopic locations. Phox2a is first detected in LC progenitors in the dorsal anterior hindbrain, and its expression there is dependent on FGF8 from the mid/hindbrain boundary and on optimal concentrations of BMP signal from the epidermal ectoderm/future dorsal neural plate junction. These findings suggest that Phox2a coordinates the specification of LC in part through the induction of Phox2b and in response to cooperating signals that operate along the mediolateral and anteroposterior axes of the neural plate.

Amino Acid Sequence↗
PubMed1999

The role of tolloid/mini fin in dorsoventral pattern formation of the zebrafish embryo.

A highly conserved TGF-&bgr; signaling pathway is involved in the establishment of the dorsoventral axis of the vertebrate embryo. Specifically, Bone Morphogenetic Proteins (Bmps) pattern ventral tissues of the embryo while inhibitors of Bmps, such as Chordin, Noggin and Follistatin, are implicated in dorsal mesodermal and neural development. We investigated the role of Tolloid, a metalloprotease that can cleave Chordin and increase Bmp activity, in patterning the dorsoventral axis of the zebrafish embryo. Injection of tolloid mRNA into six dorsalized mutants rescued only one of these mutants, mini fin. Through chromosomal mapping, linkage and cDNA sequence analysis of several mini fin alleles, we demonstrate that mini fin encodes the tolloid gene. Characterization of the mini fin mutant phenotype reveals that Mini fin/Tolloid activity is required for patterning ventral tissues of the tail: the ventral fin, and the ventroposterior somites and vasculature. Gene expression studies show that mfn mutants exhibit reduced expression of ventrally restricted markers at the end of gastrulation, suggesting that the loss of ventral tail tissues is caused by a dorsalization occurring at the end of gastrulation. Based on the mini fin mutant phenotype and the expression of tolloid, we propose that Mini fin/Tolloid modifes the Bmp activity gradient at the end of gastrulation, when the ventralmost marginal cells of the embryo are in close proximity to the dorsal Chordin-expressing cells. At this time, unimpeded Chordin may diffuse to the most ventral marginal regions and inhibit high Bmp activity levels. In the presence of Mini fin/Tolloid, however, Chordin activity would be negatively modulated through proteolytic cleavage, thereby increasing Bmp signaling activity. This extracellular mechanism is amplified by an autoregulatory loop for bmp gene expression.

Animals↗
PubMed1998

Ventral and lateral regions of the zebrafish gastrula, including the neural crest progenitors, are established by a bmp2b/swirl pathway of genes.

A bone morphogenetic protein (BMP) signaling pathway is implicated in dorsoventral patterning in Xenopus. Here we show that three genes in the zebrafish, swirl, snailhouse, and somitabun, function as critical components within a BMP pathway to pattern ventral regions of the embryo. The dorsalized mutant phenotypes of these genes can be rescued by overexpression of bmp4, bmp2b, an activated BMP type I receptor, and the downstream functioning Smad1 gene. Consistent with a function as a BMP ligand, swirl functions cell nonautonomously to specify ventral cell fates. Chromosomal mapping of swirl and cDNA sequence analysis demonstrate that swirl is a mutation in the zebrafish bmp2b gene. Interestingly, our analysis suggests that the previously described nonneural/neural ectodermal interaction specifying the neural crest occurs through a patterning function of swirl/bmp2b during gastrulation. We observe a loss in neural crest progenitors in swirl/bmp2b mutant embryos, while somitabun mutants display an opposite, dramatic expansion of the prospective neural crest. Examination of dorsally and ventrally restricted markers during gastrulation reveals a successive reduction and reciprocal expansion in nonneural and neural ectoderm, respectively, in snailhouse, somitabun, and swirl mutant embryos, with swirl/bmp2b mutants exhibiting almost no nonneural ectoderm. Based on the alterations in tissue-specific gene expression, we propose a model whereby swirl/bmp2b acts as a morphogen to specify different cell types along the dorsoventral axis.

Animals↗
PubMed1997

Differential regulation of chordin expression domains in mutant zebrafish.

Patterning along the dorsal-ventral (D-V) axis of Xenopus and Drosophila embryos is believed to occur through a conserved molecular mechanism, with homologous proteins Chordin and Short gastrulation (Sog) antagonizing signaling by bone morphogenetic protein 4 (BMP-4) and Decapentaplegic (Dpp), respectively. We have isolated a zebrafish gene that is highly homologous to chordin and sog within cysteine-rich domains and exhibits conserved aspects of expression and function. As in Xenopus embryos, zebrafish chordin is expressed in the organizer region and transiently in axial mesoderm. Injection of zebrafish chordin mRNA to the ventral side of Xenopus embryos induced secondary axes. Ectopic overexpression in zebrafish resulted in an expansion of paraxial mesoderm and neurectoderm at the expense of more lateral and ventral derivatives, producing a range of defects similar to those of dorsalized zebrafish mutants (Mullins et al., 1996). In accordance with the proposed function of chordin in D-V patterning, dorsalized zebrafish mutants showed expanded domains of chordin expression by midgastrulation, while some ventralized mutants had reduced expression; however, in all mutants examined, early organizer expression was unaltered. In contrast to Xenopus, zebrafish chordin is also expressed in paraxial mesoderm and ectoderm and in localized regions of the developing brain, suggesting that there are additional roles for chordin in zebrafish embryonic development. Surprisingly, paraxial mesodermal expression of chordin appeared unaltered in spadetail mutants that later lack trunk muscle (Kimmel et al., 1989), while axial mesodermal expression was affected. This finding reveals an unexpected function for spadetail in midline mesoderm and in differential regulation of chordin expression during gastrulation.

Amino Acid Sequence↗
PubMed1996

The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio.

In a large-scale screen, we isolated mutants displaying a specific visible phenotype in embryos or early larvae of the zebrafish, Danio rerio. Males were mutagenized with ethylnitrosourea (ENU) and F2 families of single pair matings between sibling F1 fish, heterozygous for a mutagenized genome, were raised. Egg lays were obtained from several crosses between F2 siblings, resulting in scoring of 3857 mutagenized genomes. F3 progeny were scored at the second, third and sixth day of development, using a stereomicroscope. In a subsequent screen, fixed embryos were analyzed for correct retinotectal projection. A total of 4264 mutants were identified. Two thirds of the mutants displaying rather general abnormalities were eventually discarded. We kept and characterized 1163 mutants. In complementation crosses performed between mutants with similar phenotypes, 894 mutants have been assigned to 372 genes. The average allele frequency is 2.4. We identified genes involved in early development, notochord, brain, spinal cord, somites, muscles, heart, circulation, blood, skin, fin, eye, otic vesicle, jaw and branchial arches, pigment pattern, pigment formation, gut, liver, motility and touch response. Our collection contains alleles of almost all previously described zebrafish mutants. From the allele frequencies and other considerations we estimate that the 372 genes defined by the mutants probably represent more than half of all genes that could have been discovered using the criteria of our screen. Here we give an overview of the spectrum of mutant phenotypes obtained, and discuss the limits and the potentials of a genetic saturation screen in the zebrafish.

Animals↗
PubMed1996

Mutations affecting the formation of the notochord in the zebrafish, Danio rerio.

In a large scale screen for mutants with defects in the embryonic development of the zebrafish we identified mutations in four genes,floating head (flh), momo (mom), no tail (ntl), and doc, that are required for early notochord formation. Mutations in flh and ntl have been described previously, while mom and doc are newly identified genes. Mutant mom embryos lack a notochord in the trunk, and trunk somites from the right and left side of the embryo fuse underneath the neural tube. In this respect mom appears similar to flh. In contrast, notochord precursor cells are present in both ntl and doc embryos. In order to gain a greater understanding of the phenotypes, we have analysed the expression of several axial mesoderm markers in mutant embryos of all four genes. In flh and mom, Ntl expression is normal in the germ ring and tailbud, while the expression of Ntl and other notochord markers in the axial mesodermal region is disrupted. Ntl expression is normal in doc embryos until early somitic stages, when there is a reduction in expression which is first seen in anterior regions of the embryo. This suggests a function for doc in the maintenance of ntl expression. Other notochord markers such as twist, sonic hedgehog and axial are not expressed in the axial mesoderm of ntl embryos, their expression parallels the expression of ntl in the axial mesoderm of mutant doc, flh and mom embryos, indicating that ntl is required for the expression of these markers. The role of doc in the expression of the notochord markers appears indirect via ntl. Floor plate formation is disrupted in most regions in flh and mom mutant embryos but is present in mutant ntl and doc embryos. In mutant embryos with strong ntl alleles the band of cells expressing floor plate markers is broadened. A similar broadening is also observed in the axial mesoderm underlying the floor plate of ntl embryos, suggesting a direct involvement of the notochord precursor cells in floor plate induction. Mutations in all of these four genes result in embryos lacking a horizontal myoseptum and muscle pioneer cells, both of which are thought to be induced by the notochord. These somite defects can be traced back to an impairment of the specification of the adaxial cells during early stages of development. Transplantation of wild-type cells into mutant doc embryos reveals that wild-type notochord cells are sufficient to induce horizontal myoseptum formation in the flanking mutant tissue. Thus doc, like flh and ntl, acts cell autonomously in the notochord. In addition to the four mutants with defects in early notochord formation, we have isolated 84 mutants, defining at least 15 genes, with defects in later stages of notochord development. These are listed in an appendix to this study.

Animals↗
PubMed1996

Mutations affecting development of the midline and general body shape during zebrafish embryogenesis.

Tissues of the dorsal midline of vertebrate embryos, such as notochord and floor plate, have been implicated in inductive interactions that pattern the neural tube and somites. In our screen for embryonic visible mutations in the zebrafish we found 113 mutations in more than 27 genes with altered body shape, often with additional defects in CNS development. We concentrated on a subgroup of mutations in ten genes (the midline-group) that cause defective development of the floor plate. By using floor plate markers, such as the signaling molecule sonic hedgehog, we show that the schmalspur (sur) gene is needed for early floor plate development, similar to one-eyed-pinhead (oep) and the previously described cyclops (cyc) gene. In contrast to oep and cyc, sur embryos show deletions of ventral CNS tissue restricted to the mid- and hindbrain, whereas the forebrain appears largely unaffected. In the underlying mesendodermal tissue of the head, sur is needed only for development of the posterior prechordal plate, whereas oep and cyc are required for both anterior and posterior prechordal plate development. Our analysis of sur mutants suggests that defects within the posterior prechordal plate may cause aberrant development of ventral CNS structures in the mid- and hindbrain. Later development of the floor plate is affected in mutant chameleon, you-too, sonic-you, iguana, detour, schmalhans and monorail embryos; these mutants often show additional defects in tissues that are known to depend on signals from notochord and floor plate. For example, sur, con and yot mutants show reduction of motor neurons; median deletions of brain tissue are seen in sur, con and yot embryos; and cyc, con, yot, igu and dtr mutants often show no or abnormal formation of the optic chiasm. We also find fusions of the ventral neurocranium for all midline mutants tested, which may reveal a hitherto unrecognized function of the midline in influencing differentiation of neural crest cells at their destination. As a working hypothesis, we propose that midline-group genes may act to maintain proper structure and inductive function of zebrafish midline tissues.

Animals↗
PubMed1996

Mutations affecting morphogenesis during gastrulation and tail formation in the zebrafish, Danio rerio.

We have identified several genes that are required for various morphogenetic processes during gastrulation and tail formation. Two genes are required in the anterior region of the body axis: one eyed pinhead (oep) and dirty nose (dns).oep mutant embryos are defective in prechordal plate formation and the specification of anterior and ventral structures of the central nervous system. In dns mutants, cells of the prechordal plate, such as the prospective hatching gland cells, fail to specify. Two genes are required for convergence and extension movements. In mutant trilobite embryos, extension movements on the dorsal side of the embryo are affected, whereas in the formerly described spadetail mutants, for which two new alleles have been isolated, convergent movements of ventrolateral cells to the dorsal side are blocked. Two genes are required for the development of the posterior end of the body axis. In pipetail mutants, the tailbud fails to move ventrally on the yolk sac after germ ring closure, and the tip of the tail fails to detach from the yolk tube. Mutants in kugelig (kgg) do not form the yolk tube at the posterior side of the yolk sac.

Animals↗
PubMed1996

Mutations affecting somite formation and patterning in the zebrafish, Danio rerio.

Somitogenesis is the basis of segmentation of the mesoderm in the trunk and tail of vertebrate embryos. Two groups of mutants with defects in this patterning process have been isolated in our screen for zygotic mutations affecting the embryonic development of the zebrafish (Danio rerio). In mutants of the first group, boundaries between individual somites are invisible early on, although the paraxial mesoderm is present. Later, irregular boundaries between somites are present. Mutations in fused somites (fss) and beamter (bea) affect all somites, whereas mutations in deadly seven (des), after eight (aei) and white tail (wit) only affect the more posterior somites. Mutants of all genes but wit are homozygous viable and fertile. Skeletal stainings and the expression pattern of myoD and snail1 suggest that anteroposterior patterning within individual somites is abnormal. In the second group of mutants, formation of the horizontal myoseptum, which separates the dorsal and ventral part of the myotome, is reduced. Six genes have been defined in this group (you-type genes). you-too mutants show the most severe phenotype; in these the adaxial cells, muscle pioneers and the primary motoneurons are affected, in addition to the horizontal myoseptum. The horizontal myoseptum is also missing in mutants that lack a notochord. The similarity of the somite phenotype in mutants lacking the notochord and in the you-type mutants suggests that the genes mutated in these two groups are involved in a signaling pathway from the notochord, important for patterning of the somites.

Animals↗
PubMed1996

Mutations in zebrafish genes affecting the formation of the boundary between midbrain and hindbrain.

Mutations in two genes affect the formation of the boundary between midbrain and hindbrain (MHB): no isthmus (noi) and acerebellar (ace). noi mutant embryos lack the MHB constriction, the cerebellum and optic tectum, as well as the pronephric duct. Analysis of noi mutant embryos with neuron-specific antibodies shows that the MHB region and the dorsal and ventral midbrain are absent or abnormal, but that the rostral hindbrain is unaffected with the exception of the cerebellum. Using markers that are expressed during its formation (eng, wnt1 and pax-b), we find that the MHB region is already misspecified in noi mutant embryos during late gastrulation. The tectum is initially present and later degenerates. The defect in ace mutant embryos is more restricted: MHB and cerebellum are absent, but a tectum is formed. Molecular organisation of the tectum and tegmentum is disturbed, however, since eng, wnt1 and pax-b marker gene expression is not maintained. We propose that noi and ace are required for development of the MHB region and of the adjacent mid- and hindbrain, which are thought to be patterned by the MHB region. Presence of pax-b RNA, and absence of pax-b protein, together with the observation of genetic linkage and the occurrence of a point mutation, show that noi mutations are located in the pax-b gene. pax-b is a vertebrate orthologue of the Drosophila gene paired, which is involved in a pathway of cellular interactions at the posterior compartment boundary in Drosophila. Our results confirm and extend a previous report, and show that at least one member of this conserved signalling pathway is required for formation of the boundary between midbrain and hindbrain in the zebrafish.

Animals↗
PubMed1996

Genes involved in forebrain development in the zebrafish, Danio rerio.

We identified four zebrafish mutants with defects in forebrain induction and patterning during embryogenesis. The four mutants define three genes: masterblind (mbl), silberblick (slb), and knollnase (kas). In mbl embryos, the anterior forebrain acquires posterior forebrain characteristics: anterior structures such as the eyes, olfactory placodes and the telencephalon are missing, whereas the epiphysis located in the posterior forebrain is expanded. In slb embryos, the extension of the embryonic axis is initially delayed and eventually followed by a partial fusion of the eyes. Finally, in kas embryos, separation of the telencephalic primordia is incomplete and dorsal midline cells fail to form a differentiated roof plate. Analysis of the mutant phenotypes indicates that we have identified genes essential for the specification of the anterior forebrain (mbl), positioning of the eyes (slb) and differentiation of the roof plate (kas). In an appendix to this study we list mutants showing alterations in the size of the eyes and abnormal differentiation of the lenses.

Animals↗
PubMed1996

Mutations affecting neurogenesis and brain morphology in the zebrafish, Danio rerio.

In a screen for embryonic mutants in the zebrafish a large number of mutants were isolated with abnormal brain morphology. We describe here 26 mutants in 13 complementation groups that show abnormal development of large regions of the brain. Early neurogenesis is affected in white tail (wit). During segmentation stages, homozygous wit embryos display an irregularly formed neural keel, particularly in the hindbrain. Using a variety of molecular markers, a severe increase in the number of various early differentiating neurons can be demonstrated. In contrast, late differentiating neurons, radial glial cells and some nonneural cell types, such as the neural crest-derived melanoblasts, are much reduced. Somitogenesis appears delayed. In addition, very reduced numbers of melanophores are present posterior to the mid-trunk. The wit phenotype is reminiscent of neurogenic mutants in Drosophila, such as Notch or Delta. In mutant parachute (pac) embryos the general organization of the hindbrain is disturbed and many rounded cells accumulate loosely in the hindbrain and midbrain ventricles. Mutants in a group of 6 genes, snakehead(snk), natter (nat), otter (ott), fullbrain (ful), viper (vip) and white snake (wis) develop collapsed brain ventricles, before showing signs of general degeneration. atlantis (atl), big head (bid), wicked brain (win), scabland (sbd) and eisspalte (ele) mutants have different malformation of the brain folds. Some of them have transient phenotypes, and mutant individuals may grow up to adults.

Animals↗