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The activity of fumagillin, a mycotoxin produced by Aspergillus fumigatus, has not been studied in depth. In this study, we used a commercial fumagillin on cultures of two cell types (A549 pneumocytes and RAW 264.7 macrophages). This toxin joins its target, MetAP2 protein, inside cells and, as a result, significantly reduces the electron chain activity, the migration, and the proliferation ability on the A549 cells, or affects the viability and proliferation ability of the RAW 264.7 macrophages. However, the toxin stimulates the germination and double branch hypha production of fungal cultures, pointing out an intrinsic resistant mechanism to fumagillin of fungal strains. In this study, we also used a fumagillin non-producer A. fumigatus strain (∆fmaA) as well as its complemented strain (∆fmaA::fmaA) and we tested the fumagillin secretion of the fungal strains using an Ultra High-Performance Liquid Chromatography (UHPLC) method. Furthermore, fumagillin seems to protect the fungus against phagocytosis in vitro, and during in vivo studies using infection of immunosuppressed mice, a lower fungal burden in the lungs of mice infected with the ∆fmaA mutant was demonstrated. This research was funded by the Basque Government: grant number IT1362-19. X.G. and S.C.-S. received a Ph.D. fellowship from the Basque Government; and U.P.-C. from the University of the Basque Country.

In many animal species, germ cell specification requires the inheritance of germ plasm, a biomolecular condensate containing maternally derived RNAs and proteins. Most studies of germ plasm composition and function have been performed in widely evolutionarily divergent model organisms, such as Caenorhabditis elegans, Drosophila, Xenopus laevis, and Danio rerio (zebrafish). In zebrafish, 12 RNAs localize to germ plasm at the furrows of the early embryo. Here, we tested for the presence of these RNAs in three additional species within the Danionin clade: Danio kyathit, Danio albolineatus, and Devario aequipinnatus. By visualizing nanos RNA, we find that germ plasm segregation patterns during early embryogenesis are conserved across these species. Ten additional germ plasm RNAs exhibit localization at the furrows of early embryos in all three non-zebrafish Danionin species, consistent with germ plasm localization. One component of zebrafish germ plasm, ca15b, lacked specific localization in embryos of the more distantly related D. aequipinnatus. Our findings show that within a subset of closely related Danionin species, the vast majority of germ plasm RNA components are conserved. At the same time, the lack of ca15b localization in D. aequipinnatus germ plasm highlights the potential for the divergence of germ plasm composition across a restricted phylogenetic space.

The differentiation of primordial germ cells (PGCs) occurs during early embryonic development and is critical for the survival and fitness of sexually reproducing species. Here, we review the two main mechanisms of PGC specification, induction, and preformation, in the context of four model vertebrate species: mouse, axolotl,Xenopusfrogs, and zebrafish. We additionally discuss some notable molecular characteristics shared across PGC specification pathways, including the shared expression of products from three conserved germline gene families,DAZ(Deleted in Azoospermia) genes,nanos-related genes, andDEAD-box RNA helicases. Then, we summarize the current state of knowledge of the distribution of germ cell determination systems across kingdom Animalia, with particular attention to vertebrate species, but include several categories of invertebrates – ranging from the “proto-vertebrate” cephalochordates to arthropods, cnidarians, and ctenophores. We also briefly highlight ongoing investigations and potential lines of inquiry that aim to understand the evolutionary relationships between these modes of specification.

Background In zebrafish and many other organisms, specification of primordial germ cells (PGCs) requires the transmission of maternally-derived germ plasm. Zebrafish germ plasm ribonucleoparticles (RNPs) aggregate along the cleavage furrows during the first several cell cycles, segregate asymmetrically during the cleavage stages, and undergo cytoplasmic dispersal in the late blastula. Results For all tested germ plasm RNAs [carbonic anhydrase 15b (ca15b), deleted in azoospermia-like (dazl), dead end (dnd), nanos 3 (nos3), regulator of G-protein signaling14a (rgs14a), and vasa/DEAD box polypeptide 4 (vasa/ddx4)], RNPs are homotypic (containing a single RNA type), with RNPs packing tightly yet remaining distinct within germ plasm aggregates. Homotypic clustering of RNAs within RNPs is observed before aggregation in the cortex and is maintained through germ plasm recruitment, asymmetric segregation and RNP dispersal. We also identify a step of germ plasm fragmentation during the cleavage stages that precedes RNP dispersal. Conclusions Our findings suggest that germ plasm aggregates act as subcellular compartments that temporarily collect and carry single RNA-type RNPs from fertilization until their cytoplasmic dispersal in PGCs at the end of the blastula period, and describe a previously unknown fragmentation step that allows for an increase in the pool of germ plasm-carrying cells, presumably PGCs. Developmental Dynamics 248:306-318, 2019. © 2019 Wiley Periodicals, Inc.

We show that the zebrafish maternal-effect mutation too much information (tmi) corresponds to zebrafish prc1-like (prc1l), which encodes a member of the MAP65/Ase1/PRC1family of microtubule-associated proteins. Embryos from tmi/prc1l homozygous mutant mothers display cytokinesis defects in meiotic and mitotic divisions in the early embryo, indicating that tmi/prc1l has a role in midbody formation during cell division at the egg-to-embryo transition. Unexpectedly, maternal tmi/prc1l function is also essential for the reorganization of vegetal pole microtubules required for embryonic axis induction. While Prc1 is widely regarded to crosslink microtubules in an antiparallel conformation, our studies provide evidence for an additional function of Prc1 in the bundling of parallel microtubules in the vegetal cortex of the early embryo during cortical rotation and prior to mitotic cycling. These findings highlight common yet distinct aspects of microtubule reorganization that occur during the egg-to-embryo transition, driven by maternal product for the midbody component Prc1l and required for embryonic cell division and pattern formation.

In animals, early development is dependent on a pool of maternal factors, both RNA and proteins, which are required for basic cellular process and cell differentiation until zygotic genome activation. The role of a majority of these maternally expressed factors in adult fertility and early development is not fully understood. By exploiting the biallelic editing ability of CRISPR-Cas9 and the benefits of the zebrafish model, we identify and characterize maternal-effect genes in a single generation, using a maternal crispant technique. We validated the ability to generate biallelic mutations in the germline by creating maternal crispants that phenocopied previously characterized maternal-effect genes:motley/birc5b,tmi/prc1l, andaura/mid1ip1. Additionally, by targeting maternally expressed genes of unknown function in zebrafish, we identified two new maternal-effect zebrafish genes,kpna7andfhcd3. The genetic identity of these maternal crispants was confirmed by sequencing haploid progeny from F0 females, which allowed the sequence analysis of newly induced lesions in the maternal germ line. Analysis of the induced lesions shows minimal genetic variation within a clutch, with an average of two edited alleles per clutch. These findings are consistent with biallelic editing events occurring in germ cells or their precursors of early CRISPR-Cas9-injected embryos, leading to maternal-effect phenotypes in the offspring. Our studies show that maternal crispants allow for the effective identification and primary characterization of maternal-effect genes in a single generation, facilitating the reverse genetics analysis of maternal factors that drive embryonic development.

Zebrafish embryos, with their large size (>0.5 mm) and accessibility, are valuable tools for investigating core cellular processes. Many of those processes, such as cell division, asymmetric inheritance of cellular components, and structural dynamics involved in cell motility and morphology, rely on cytoskeletal rearrangements and associated macromolecules. In addition to the protein-rich cytoskeleton, the early embryo is packed with maternally deposited RNA, which serves essential roles in establishing cell polarity, cell fate, and cell organization. Here, we present methods for visualizing endogenous RNA along with cytoskeletal structures, including microtubules and filamentous actin (F-actin) in the context of an intact vertebrate embryo. Each of the four protocols described herein (embryo fixation, RNA probe design/synthesis, double fluorescent in situ hybridization with tubulin immunofluorescence, and fluorescent in situ hybridization with phalloidin labeling of F-actin) are intended for optimal preservation and visualization of both the cytoskeleton and RNAs of interest. These methods can also be modified and applied to a broad range of other uses.

Heart regeneration in regeneration-competent organisms can be accomplished through the remodeling of gene expression in response to cardiac injury. This dynamic transcriptional response relies on the activities of tissue regeneration enhancer elements (TREEs); however, the mechanisms underlying TREEs are poorly understood. We dissected a cardiac regeneration enhancer in zebrafish to elucidate the mechanisms governing spatiotemporal gene expression during heart regeneration. Cardiac lepb regeneration enhancer (cLEN) exhibits dynamic, regeneration-dependent activity in the heart. We found that multiple injury-activated regulatory elements are distributed throughout the enhancer region. This analysis also revealed that cardiac regeneration enhancers are not only activated by injury, but surprisingly, they are also actively repressed in the absence of injury. Our data identified a short 22-bp DNA element containing a key repressive element. Comparative analysis across Danio species indicated that the repressive element is conserved in closely related species. The repression mechanism is not operational during embryogenesis and emerges when the heart begins to mature. Incorporating both activation and repression components into the mechanism of tissue regeneration constitutes a new paradigm that may be extrapolated to other regeneration scenarios.

The transmission of genetic information from one generation to another is crucial for survival of animal species. This is accomplished by the induction of primordial germ cells (PGCs) that will eventually establish the germline. In some animals the germline is induced by signals in gastrula, whereas in others it is specified by inheritance of maternal determinants, known as germ plasm. In zebrafish, aggregation and compaction of maternally derived germ plasm during the first several embryonic cell cycles is essential for generation of PGCs. These processes are controlled by cellular functions associated with the cellular division apparatus. Ribonucleoparticles containing germ plasm components are bound to both the ends of astral microtubules and a dynamic F-actin network through a mechanism integrated with that which drives the cell division program. In this chapter we discuss the role that modifications of the cell division apparatus, including the cytoskeleton and cytoskeleton-associated proteins, play in the regulation of zebrafish germ plasm assembly.

Recombination is a main source of genetic variability. However, the potential role of the variation generated by recombination in phenotypic traits, including diseases, remains unexplored as there is currently no method to infer chromosomal subpopulations based on recombination patterns differences. We developedrecombClust, a method that uses SNP-phased data to detect differences in historic recombination in a chromosome population. We validated our method by performing simulations and by using real data to accurately predict the alleles of well known recombination modifiers, including common inversions inDrosophila melanogasterand human, and the chromosomes under selective pressure at the lactase locus in humans. We then appliedrecombClustto the complex human 1q21.1 region, where nonallelic homologous recombination produces deleterious phenotypes. We discovered and validated the presence of two different recombination histories in these regions that significantly associated with the differential expression ofANKRD35in whole blood and that were in high linkage with variants previously associated with hypertension. By detecting differences in historic recombination, our method opens a way to assess the influence of recombination variation in phenotypic traits.

Introduction Bacterial sexually transmitted infections (STIs) have an important impact on reproductive health, highlighting the increase in Chlamydia trachomatis infection rates among young people. To reduce the costs of STI detection, the pooling strategy is beneficial for high-throughput tests in low-prevalence populations using non-invasive samples. Objectives (1) To describe the performance of a 7-STI PCR assay using the pooling of three urine samples to detect C. trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium; (2) to estimate the cost saving of the pooling strategy; (3) to describe the prevalence, risk factors and coinfections of C. trachomatis, N. gonorrhoeae and M. genitalium in young people ≤25 years in Catalonia. Methods cross-sectional prevalence study conducted in 2016 among young people ≤25 years of age seen in sexual and reproductive health centres throughout Catalonia from pools of three urine samples. A standardized questionnaire was used to collect clinical-epidemiological and behavioural variables. Results 1032 young people were tested. The prevalence of C. trachomatis, N. gonorrhoeae and M. genitalium was 8.5%, 0.6% and 3.5%, respectively. The pooling strategy provided a 33% savings in reagent costs. Conclusions The pooling strategy implemented for epidemiological studies in our context provides a savings that has an impact on the viability of STI detection programmes. In the same way, this study shows that C. trachomatis prevalence continues to increase in this population and, for the first time in Catalonia, the prevalence of M. genitalium in young people is shown.

Aspergillus fumigatus is a ubiquitous soil decomposer and an opportunistic pathogen that is characterized by its large metabolic machinery for acquiring nutrients from media. Lately, an ever-increasing number of genes involved in fungal nutrition has been associated with its virulence. Of these, nitrogen, iron, and zinc metabolism-related genes are particularly noteworthy, since 78% of them have a direct implication in virulence. In this review, we describe the sensing, uptake and regulation process of the acquisition of these nutrients, the connections between pathways and the virulence-implicated genes. Nevertheless, only 40% of the genes mentioned in this review have been assayed for roles in virulence, leaving a wide field of knowledge that remains uncertain and might offer new therapeutic and diagnostic targets. This research was funded by the Basque Government: grant number IT1362-19. U.P.-C. and S.C.-S. received a predoctoral fellowship from the University of the Basque Country and Basque Government, respectively.

Stress granules are cytoplasmic foci that directly respond to the protein synthesis status of the cell. Various environmental insults, such as oxidative stress or extreme heat, block protein synthesis; consequently, mRNA will stall in translation, and stress granules will immediately form and become enriched with mRNAs. P54 DEAD box RNA helicases are components of RNA granules such as P-bodies and stress granules. We studied the expression, in cytoplasmic foci, of both zebrafish P54 RNA helicases (P54a and P54b) during development and found that they are expressed in cytoplasmic granules under both normal conditions and stress conditions. In zebrafish embryos exposed to heat shock, some proportion of P54a and P54b helicases move to larger granules that exhibit the properties of genuine stress granules. Knockdown of P54a and/or P54b in zebrafish embryos produces developmental abnormalities restricted to the posterior trunk; further, these embryos do not form stress granules, and their survival upon exposure to heat-shock conditions is compromised. Our observations fit the model that cells lacking stress granules have no resilience or ability to recover once the stress has ended, indicating that stress granules play an essential role in the way organisms adapt to a changing environment., Summary: Stress granules are formed by mRNAs stalled in translation during stress conditions. P54 RNA helicases from zebrafish reside in cytoplasmic granules and are essential for heat-shock resilience.

Neural stem cells have become the focus of many studies as they have the potential to differentiate into all three neural lineages. This may be utilised to develop new and novel ways to treat neurological conditions such as spinal cord and brain injuries, especially if the stem cells can be modulated in vivo without additional invasive surgical procedures. This research is aimed at investigating the effects of the growth factors vascular endothelial growth factor, platelet-derived growth factor, brain-derived neurotrophic factor, and vascular endothelial growth factor/platelet-derived growth factor on hippocampal-derived neural stem cells. Cell growth and differentiation were assessed using immunohistochemistry and glutaminase enzyme assay. Cells were cultured for 14 days and treated with different growth factors at two different concentrations 20 ng/mL and 100 ng/mL. At 2 weeks, cells were fixed, and immunohistochemistry was conducted to determine cellular differentiation using antibodies against GFAP, nestin, OSP, and NF200. The cell medium supernatant was also collected during treatment to determine glutaminase levels secreted by the cells as an indicator of neural differentiation. VEGF/PDGF at 100 ng/mL had the greatest influence on cellular proliferation of HNSC, which also stained positively for nestin, OSP, and NF200. In comparison, HNSC in other treatments had poorer cell health and adhesion. HNSC in all treatment groups displayed some differentiation markers and morphology, but this is most significant in the 100 ng/ml VEGF/PDGF treatment. VEGF/PDGF combination produced the optimal effect on the HNSCs inducing the differentiation pathway exhibiting oligodendrocytic and neuronal markers. This is a promising finding that should be further investigated in the brain and spinal cord injury.

The ability to create targeted mutations in specific genes, and therefore a loss-of-function condition, provides essential information about their endogenous functions during development and homeostasis. The discovery that CRISPR-Cas9 can target specific sequences according to base-pair complementarity and readily create knockouts in a desired gene has elevated the implementation of genetic analysis in numerous organisms. As CRISPR-Cas9 has become a powerful tool in a number of species, multiple methods for designing, creating, and screening editing efficiencies have been published, each of which has unique benefits. This chapter presents a cost-efficient, accessible protocol for creating knockout mutants in zebrafish using insertions/deletions (INDELS), from target site selection to mutant propagation, using basic laboratory supplies. The presented approach can be adapted to other systems, including any vertebrate species.

Background: Whole genome duplication is a useful genetic tool because it allows immediate and complete genetic homozygosity in gynogenetic offspring. A whole genome duplication method in zebrafish, Heat Shock, involves a heat pulse in the period 13–15 min postfertilization (mpf) to inhibit cytokinesis of the first mitotic cycle. However, Heat Shock produces a relatively low yield of gynogenotes. Results: A heat pulse at a later time point during the first cell cycle (22 mpf, HS2) results in a high (>80%) frequency of embryos exhibiting a precise one-cell division stall during the second cell cycle, inducing whole genome duplication. Coupled with haploid production, HS2 generates viable gynogenetic diploids with yields up to 4 times higher than those achieved through standard Heat Shock. The cell cycle delay also causes blastomere cleavage pattern variations, supporting a role for cytokinesis in spindle orientation during the following cell cycle. Conclusions: Our studies provide a new tool for whole genome duplication, induced gynogenesis, and cleavage pattern alteration in zebrafish, based on a time period before the initiation of cell division that is sensitive to temperature-mediated interference with centrosome duplication. Targeting of this period may also facilitate genetic and developmental manipulations in other organisms. Developmental Dynamics 244:1300–1312, 2015. © 2015 Wiley Periodicals, Inc.

During the early embryonic cell cycles, zebrafish germ plasm ribonucleoparticles (RNPs) gradually multimerize and become recruited to the forming furrows. RNPs multimerization occurs prior to and during furrow initiation, as forming aggregates move outward through their association with the tips of growing interphase astral microtubules. Germ plasm RNPs are also associated with short cortical F-actin. We show that, in embryos mutant for the cytoskeletal regulator mid1ip1L, germ plasm RNPs fail to become recruited to the furrow, accumulating instead at the periphery of the blastodisc. RNP aggregates are associated with zones of mid1ip1L-dependent cyclical local cortical F-actin network enrichments as well as contractions at both the cortex and the contractile ring. F-actin inhibition in wild-type embryos mimics the RNP peripheral accumulation defect of mid1ip1L mutants. Our studies suggest that a common mechanism underlies distinct steps of germ plasm RNP segregation. At the cortex, this process attenuates microtubule-dependent outward RNP movement to retain RNPs in the blastodisc cortex and allow their recruitment to the furrows. F-actin network contraction likely also facilitates higher order germ plasm RNP multimerization.

Zebrafish germ plasm ribonucleoparticles (RNPs) become recruited to furrows of early zebrafish embryos through their association with astral microtubules ends. During the initiation of cytokinesis, microtubules are remodeled into a furrow microtubule array (FMA), thought to be analogous to the mammalian midbody involved in membrane abscission. During furrow maturation, RNPs and FMA tubules transition from their original distribution along the furrow to enrichments at the furrow distal ends, which facilitates germ plasm mass compaction. We show that nebel mutants exhibit reduced furrow-associated slow calcium waves (SCWs), caused at least in part by defective enrichment of calcium stores. RNP and FMA distal enrichment mirrors the medial-to-distal polarity of SCWs, and inhibition of calcium release or downstream mediators such as Calmodulin affects RNP and FMA distal enrichment. Blastomeres with reduced or lacking SWCs, such as early blastomeres in nebel mutants and wild-type blastomeres at later stages, exhibit medially-bundling microtubules similar to midbodies in other cell types. Our data indicates that SCWs provide medial-to-distal directionality along the furrow to facilitate germ plasm RNP enrichment at the furrow ends.

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Determination of primordial germ cells (PGCs) is one of the earliest decisions in animal embryogenesis. In many species, PGCs are determined through maternally-inherited germ plasm ribonucleoparticles (RNPs). In zebrafish, these are transmitted during oogenesis as dispersed RNPs, which after fertilization multimerize and become recruited as large aggregates at furrows for the first and second cell cycles. Here, we show that the number of recruited germ plasm RNPs is halved every cell cycle. We also show that germ plasm RNPs are recruited during the third cell cycle, but only transiently. Our data support a mechanism in which systematic local gathering of germ plasm RNPs during cytokinesis and threshold-dependent clearing contribute to forming germ plasm aggregates with the highest RNP number and germ cell-inducing potential.

The pattern of the earliest cell divisions in a vertebrate embryo lays the groundwork for later developmental events such as gastrulation, organogenesis, and overall body plan establishment. Understanding these early cleavage patterns and the mechanisms that create them is thus crucial for the study of vertebrate development. This chapter describes the early cleavage stages for species representing ray-finned fish, amphibians, birds, reptiles, mammals, and proto-vertebrate ascidians and summarizes current understanding of the mechanisms that govern these patterns. The nearly universal influence of cell shape on orientation and positioning of spindles and cleavage furrows and the mechanisms that mediate this influence are discussed. We discuss in particular models of aster and spindle centering and orientation in large embryonic blastomeres that rely on asymmetric internal pulling forces generated by the cleavage furrow for the previous cell cycle. Also explored are mechanisms that integrate cell division given the limited supply of cellular building blocks in the egg and several-fold changes of cell size during early development, as well as cytoskeletal specializations specific to early blastomeres including processes leading to blastomere cohesion. Finally, we discuss evolutionary conclusions beginning to emerge from the contemporary analysis of the phylogenetic distributions of cleavage patterns. In sum, this chapter seeks to summarize our current understanding of vertebrate early embryonic cleavage patterns and their control and evolution.

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

Axonal injury occurs during trauma when tissue-scale loads are transferred to individual axons. Computational models are used to understand this transfer and predict the circumstances that cause injury. However, these findings are limited by a lack of validating experimental work examining the mechanics of axons in their in situ state. As a first step towards validation for dynamic stretch, we use contactin-associated protein (Caspr), expressed at the nodes of Ranvier, as a fiduciary marker of quasistatic axonal stretch. We measured changes in the distance between immunolabled Caspr pairs along axons as a function of tissue-level stretch in chick embryo spinal cords harvested from different developmental periods. We then identified and characterized broken axons and adapted a kinematic model published previously by our group (Singh et al., 2015) to estimate average strain thresholds for axon mechanical failure. The distance between Caspr pairs increased with stretch, though not as much as predicted by simple continuum mechanics. For equivalent tissue stretch, greater numbers of broken axons were found at later stages of development. In adapting our kinematic model to predict a breaking threshold strain, we found that breaking thresholds decrease with development stage. When thresholds were split and classified based on kinematic behavior, non-affine, uncoupled axons had higher strain thresholds than affine, coupled axons, corroborating thresholds predicted from in vitro and in vivo preparations. These results provide a valuable launching point for generating more accurate multi-scale models in primary central nervous system injury.

Embryos from females homozygous for a recessive maternal-effect mutation in the gene aura exhibit defects including reduced cortical integrity, defective cortical granule (CG) release upon egg activation, failure to complete cytokinesis, and abnormal cell wound healing. Subcellular analysis shows that the cytokinesis defects observed in aura mutants are associated with aberrant cytoskeletal reorganization during furrow maturation, including abnormal F-actin enrichment and microtubule reorganization. Cortical F-actin prior to furrow formation fails to exhibit a normal transition into F-actin-rich arcs, and drug inhibition is consistent with aura function promoting F-actin polymerization and/or stabilization. In mutants, components of exocytic and endocytic vesicles, such as Vamp2, Clathrin and Dynamin, are sequestered in unreleased CGs, indicating a need for CG recycling in the normal redistribution of these factors. However, the exocytic targeting factor Rab11 is recruited to the furrow plane normally at the tip of bundling microtubules, suggesting an alternate anchoring mechanism independent of membrane recycling. A positional cloning approach indicates that the mutation in aura is associated with a truncation of Mid1 Interacting Protein 1L (Mid1ip1L), previously identified as an interactor of the X-linked Opitz G/BBB syndrome gene Mid1. A Cas9/CRISPR-induced mutant allele in mid1ip1L fails to complement the originally isolated aura maternal-effect mutation, confirming gene assignment. Mid1ip1L protein localizes to cortical F-actin aggregates, consistent with a direct role in cytoskeletal regulation. Our studies indicate that maternally provided aura/mid1ip1L acts during the reorganization of the cytoskeleton at the egg-to-embryo transition and highlight the importance of cytoskeletal dynamics and membrane recycling during this developmental period.

Animals specify primordial germ cells (PGCs) in two alternate modes: preformation and epigenesis. Epigenesis relies on signal transduction from the surrounding tissues to in‐ struct a group of cells to acquire PGC identity. Preformation, thought to be the more de‐ rived PGC specification mode, is instead based on the maternal inheritance of germ celldetermining factors. We use the zebrafish as a model system, in which PGCs are specified through maternal inheritance of germ plasm, to study this process in vertebrates. In ze‐ brafish, maternally inherited germ plasm ribonucleoparticles (RNPs) have co-opted the cytoskeletal machinery to reach progressive levels of multimerization, resulting in the formation of four large masses of aggregated germ plasm RNPs. At later stages, germ plasm masses continue to use components of the cell division machinery, such as the spindles, centrosomes, and/or subcellular organelles to segregate asymmetrically during cell division and subsequently induce germ cell fate. This chapter discusses the current knowledge of germ cell specification focusing on the zebrafish as a model system. We al‐ so provide a comparative analysis of the mechanism for germ plasm RNP segregation in zebrafish versus other known vertebrate systems of germ cell preformation, such as in amphibian and avian models.

Summary Background The centrosome has a well-established role as a microtubule organizer during mitosis and cytokinesis. In addition, it facilitates the union of parental haploid genomes following fertilization by nucleating a microtubule aster along which the female pronucleus migrates toward the male pronucleus. Stable associations between the sperm aster and the pronuclei are essential during this directed movement. Results Our studies reveal that the zebrafish gene futile cycle ( fue ) is required in the zygote for male pronucleus-centrosome attachment and female pronuclear migration. We show that fue encodes a novel, maternally-provided long form of lymphoid-restricted membrane protein (lrmp ), a vertebrate-specific gene of unknown function. Both maternal lrmp messenger RNA (mRNA) and protein are highly localized in the zygote, in a largely overlapping pattern at nuclear membranes, centrosomes, and spindles. Truncated Lrmp-EGFP fusion proteins identified subcellular targeting signals in the C terminus of Lrmp; however, endogenous mRNA localization is likely important to ensure strict spatial expression of the protein. Localization of both Lrmp protein and lrmp RNA is defective in fue mutant embryos, indicating that correct targeting of lrmp gene products is dependent on Lrmp function. Conclusions Lrmp is a conserved vertebrate gene whose maternally inherited products are essential for nucleus-centrosome attachment and pronuclear congression during fertilization. Precise subcellular localization of lrmp products also suggests a requirement for strict spatiotemporal regulation of their function in the early embryo.

Chordin is the prototype of a group of cysteine-rich domain-containing proteins that bind and modulate signaling of various TGFβ-like ligands. Chordin-like 1 and 2 (CHL1 and 2) are two members of this group that have been described in human, mouse, and chick. However, in vivo roles for CHL1 and 2 in early development are unknown due to lack of loss-of-function analysis. Here we identify and characterize zebrafish, Danio rerio, CHL (Chl). The chl gene is on a region of chromosome 21 syntenic with the area of murine chromosome 7 bearing the CHL2 gene. Inability to identify a separate zebrafish gene corresponding to the mammalian CHL1 gene suggests that Chl may serve roles in zebrafish distributed between CHL1 and CHL2 in other species. Chl is a maternal factor that is also zygotically expressed later in development and has spatiotemporal expression patterns that differ from but overlap those of zebrafish chordin (Chd), suggesting differences but also possible overlap in developmental roles of the two proteins. Chl, like Chd, dorsalizes embryos upon overexpression and is cleaved by BMP1, which antagonizes this activity. Loss-of-function experiments demonstrate that Chl serves as a BMP antagonist with functions that overlap and are redundant with those of Chd in forming the dorsoventral axis.

Genes for tetrapod fibrillar procollagen chains can be divided into two clades, A and B, based on sequence homologies and differences in protein domain and gene structures. Although the major fibrillar collagen types I-III comprise only clade A chains, the minor fibrillar collagen types V and XI comprise both clade A chains and the clade B chains pro-alpha1(V), pro-alpha3(V), pro-alpha1(XI) and pro-alpha2(XI), in which defects can underlie various genetic connective tissue disorders. Here we characterize the clade B procollagen chains of zebrafish. We demonstrate that in contrast to the four tetrapod clade B chains, zebrafish have six clade B chains, designated here as pro-alpha1(V), pro-alpha3(V)a and b, pro-alpha1(XI)a and b, and pro-alpha2(XI), based on synteny, sequence homologies, and features of protein domain and gene structures. Spatiotemporal expression patterns are described, as are conserved and non-conserved features that provide insights into the function and evolution of the clade B chain types. Such features include differential alternative splicing of NH(2)-terminal globular sequences and the first case of a non-triple helical imperfection in the COL1 domain of a clade B, or clade A, fibrillar procollagen chain. Evidence is also provided for previously unknown and evolutionarily conserved alternative splicing within the pro-alpha1(V) C-propeptide, which may affect selectivity of collagen type V/XI chain associations in species ranging from zebrafish to human. Data presented herein provide insights into the nature of clade B procollagen chains and should facilitate their study in the zebrafish model system.

In the earliest stages of animal development prior to the commencement of zygotic transcription, all critical cellular processes are carried out by maternally-provided molecular products accumulated in the egg during oogenesis. Disruption of these maternal products can lead to defective embryogenesis. In this review, we focus on maternal genes with roles in the fundamental processes of fertilization, cell division, centrosome regulation, and germ cell development with emphasis on findings from the zebrafish, as this is a unique and valuable model system for vertebrate reproduction.

Forward genetics remains an important approach for the unbiased identification of factors involved in biological pathways. Forward genetic analysis in the zebrafish has until now largely been restricted to the developmental period from zygotic genome activation through the end of embryogenesis. However, the use of the zebrafish as a model system for the analysis of late larval, juvenile and adult traits, including fertility and maternal and paternal effects, continues to gain momentum. Here, we describe two approaches, based on an F3-extended family and gynogenetic methods, 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, map, and molecularly clone the identified mutations.

In zebrafish embryos, factors involved in both axis induction and primordial germ cell (PGC) development are localized to the vegetal pole of the egg. However, upon egg activation axis induction factors experience an asymmetric off-center shift whereas PGC factors undergo symmetric animally-directed movement. We examined the spatial relationship between the proposed dorsal genes wnt8a and grip2a and the PGC factor dazl at the vegetal cortex. We find that RNAs for these genes localize to different cortical depths, with the RNA for the PGC factor dazl at a deeper cortical level than those for axis-inducing factors. In addition, and in contrast to the role of microtubules in the long-range transport of dorsal determinants, we find that germ line determinant transport depends on the actin cytoskeleton. Our results support a model in which vegetal cortex differential RNA transport behavior is facilitated by RNA localization along cortical depth and differential coupling to cortical transport.

Drosophila metalloproteinase Tolloid (TLD) is responsible for cleaving the antagonist Short gastrulation (SOG), thereby regulating signaling by the bone morphogenetic protein (BMP) Decapentaplegic (DPP). In mice there are four TLD-related proteinases, two of which, BMP1 and mammalian Tolloid-like 1 (mTLL1), are responsible for cleaving the SOG orthologue Chordin, thereby regulating signaling by DPP orthologues BMP2 and 4. However, although TLD mutations markedly dorsalize Drosophila embryos, mice doubly homozygous null for BMP1 and mTLL1 genes are not dorsalized in early development. Only a single TLD-related proteinase has previously been reported for zebrafish, and mutation of the zebrafish TLD gene (mini fin) results only in mild dorsalization, manifested by loss of the most ventral cell types of the tail. Here we identify and map the zebrafish BMP1 gene bmp1. Knockdown of BMP1 expression results in a mild tail phenotype. However, simultaneous knockdown of mini fin and bmp1 results in severe dorsalization resembling the Swirl (swr) and Snailhouse (snh) phenotypes; caused by defects in major zebrafish ventralizing genes bmp2b and bmp7, respectively. We conclude that bmp1 and mfn gene products functionally overlap and are together responsible for a key portion of the Chordin processing activity necessary to formation of the zebrafish dorsoventral axis.

In animal species, early developmental processes are driven by maternally derived factors. Here, we describe a forward genetics approach to identify recessive mutations in genes encoding such maternal factors in the zebrafish. We used a gynogenesis-based approach to identify 14 recessive maternal-effect mutations. Homozygosity for these mutations in adult females leads to the inviability of their offspring. Confocal microscopy of embryos labeled with a DNA dye and a membrane marker allowed us to further analyze mutant embryos for defects in nuclear and cellular divisions. The mutations result in a range of defects in early developmental processes, including egg activation, early nuclear events, mitosis, cytokinesis, axial patterning, and gastrulation. Our effort constitutes a systematic attempt to identify maternal-effect genes in a vertebrate species. The sample of mutations that we have identified reflects the diversity of maternally driven functions in early development and underscores the importance of maternal factors in this process.

Maternal homozygosity for three independent mutant hecate alleles results in embryos with reduced expression of dorsal organizer genes and defects in the formation of dorsoanterior structures. A positional cloning approach identified all hecate mutations as stop codons affecting the same gene, revealing that hecate encodes the Glutamate receptor interacting protein 2a (Grip2a), a protein containing multiple PDZ domains known to interact with membrane-associated factors including components of the Wnt signaling pathway. We find that grip2a mRNA is localized to the vegetal pole of the oocyte and early embryo, and that during egg activation this mRNA shifts to an off-center vegetal position corresponding to the previously proposed teleost cortical rotation. hecate mutants show defects in the alignment and bundling of microtubules at the vegetal cortex, which result in defects in the asymmetric movement of wnt8a mRNA as well as anchoring of the kinesin-associated cargo adaptor Syntabulin. We also find that, although short-range shifts in vegetal signals are affected in hecate mutant embryos, these mutants exhibit normal long-range, animally directed translocation of cortically injected dorsal beads that occurs in lateral regions of the yolk cortex. Furthermore, we show that such animally-directed movement along the lateral cortex is not restricted to a single arc corresponding to the prospective dorsal region, but occur in multiple meridional arcs even in opposite regions of the embryo. Together, our results reveal a role for Grip2a function in the reorganization and bundling of microtubules at the vegetal cortex to mediate a symmetry-breaking short-range shift corresponding to the teleost cortical rotation. The slight asymmetry achieved by this directed process is subsequently amplified by a general cortical animally-directed transport mechanism that is neither dependent on hecate function nor restricted to the prospective dorsal axis., Author Summary One of the earliest and most crucial events in animal development is the establishment of the embryonic dorsal axis. In amphibians and fish, this event depends on the transport of so-called “dorsal determinants” from one region of the egg, at the pole opposite from the site where the oocyte nucleus lies, towards the site of axis induction. There, the dorsal determinant activates the Wnt signaling pathway, which in turn triggers dorsal gene expression. Dorsal determinant transport is mediated by the reorganization of a cellular network composed of microtubules. We determine that hecate, a zebrafish gene active during egg formation that is essential for embryonic axis induction, is required for an early step in this microtubule reorganization. We find that hecate corresponds to glutamate receptor interacting protein 2a, which participates in other animal systems in Wnt-based pathways. We also show that the microtubule reorganization dependent on hecate results in a subtle symmetry-breaking event that subsequently becomes amplified by a more general transport process independent of hecate function. Our data reveal new links between glutamate receptor interacting protein 2a, Wnt signaling and axis induction, and highlights basic mechanisms by which small changes early in development translate into global changes in the embryo.

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.

In animals, females deposit gene products into developing oocytes, which drive early cellular events in embryos immediately after fertilization. As maternal gene products are present before fertilization, the functional manipulation of maternal genes is often challenging to implement, requiring gene expression or targeting during oogenesis. Maternal expression can be achieved through transgenesis, but transgenic approaches are time consuming and subject to undesired epigenetic effects. Here, we have implemented in vitro culturing of experimentally manipulated immature oocytes to study maternal gene contribution to early embryonic development in the zebrafish. We demonstrate phenotypic rescue of a maternal-effect mutation by expressing wild-type product in cultured oocytes. We also generate loss-of-function phenotypes in embryos through either the expression of a dominant-negative transcript or injection of translation-blocking morpholino oligonucleotides. Finally, we demonstrate subcellular localization during the early cell divisions immediately after fertilization of an exogenously provided maternal product fused to a fluorescent protein. These manipulations extend the potential to carry out genetic and imaging studies of zebrafish maternal genes during the egg-to-embryo transition.

The tropical fresh water minnow, Danio rerio, more commonly known as zebrafish, has emerged rapidly over the last decade as a powerful tool for developmental geneticists. External fertilization, high fecundity, a short generation time, and optical transparency of embryos during early development combined with the amenability to a variety of genetic manipulations constitute in the zebrafish the convergence of several unique advantages for a vertebrate model system. Traditional forward genetic screens, which employ the use of a chemical mutagen such as N-ethyl-N-nitrosourea to induce mutations in the male genome, have also proven to be highly successful in the zebrafish. This chapter provides experimental approaches to successfully induce pre-meiotic mutations in the male zebrafish germline and genetic strategies to recover and maintain such mutations in subsequent generations (Section 3.1). Though discussed specifically in the context of zebrafish research in this chapter, many of these genetic approaches may also be broadly applicable in other model systems. We also discuss experimental techniques to manipulate the ploidy of zebrafish embryos, which when used in combination with the standard mutagenesis protocol significantly expedite the identification of the induced mutations (Section 3.2). Additional stand-alone procedures are provided in Section 3.3, which are also required for the execution of the experiments discussed in its preceding sections.

The effect of bezafibrate, at doses of 200 mg three times weekly throughout a period of 10 weeks, on lipoprotein (a) and triglyceride-rich lipoproteins including intermediate-density lipoproteins (IDL) has been studied in 12 patients with chronic renal failure receiving haemodialysis. No side-effects were observed, and serum creatine phosphate kinase values remained within normal limits throughout the duration of the study. Serum cholesterol, triglycerides and apoprotein (apo) B significantly decreased after bezafibrate, whereas apo A-I increased significantly. Serum lipoprotein (a) decreased after bezafibrate, although differences reached no statistical significance. The very-low-density lipoproteins (VLDL) cholesterol and the VLDL triglycerides decreased from 0.93 +/- 0.45 mmol/l (Mean +/- SD) to 0.54 +/- 0.27 mmol/l (P less than 0.05) and from 1.50 +/- 0.58 mmol/l to 0.91 +/- 0.35 mmol/l (P less than 0.01) respectively. The IDL cholesterol and IDL triglycerides decreased from 0.44 +/- 0.35 mmol/l to 0.20 +/- 0.18 mmol/l (P = NS) and from 0.46 +/- 0.35 mmol/l to 0.17 +/- 0.12 mmol/l (P less than 0.05) respectively. These data, with a substantial improvement of the lipoprotein profile in patients with chronic renal failure on haemodialysis after treatment with bezafibrate, suggest a decrease in the coronary heart disease risk.

BackgroundBrisken et al (2000) showed that Wnt4 null mammary glands were deficient in early lobuloalveolar mammary outgrowth during pregnancy, and implicated Wnt4 as an effector for the progesterone-induced mammary growth program. Though ectopic Wnt1 signaling is known to be mitogenic and oncogenic, no endogenously expressed Wnt ligands have ever been directly implicated in mammary growth and morphogenesis. Therefore, we generated conditional transgenic mice to test whether Wnt4 can stimulate mammary epithelial cell growth.ResultsWe found that despite pregnancy-associated expression levels of Wnt4, mammary glands did not display the side-branching typical of early pregnancy. Control experiments designed to test the Wnt4 construct in zebrafish reproduced other studies that demonstrated Wnt4-specific phenotypes distinct from Wnt1-induced phenotypes. Indeed, using qPCR-based array analyses, we found that a specific transcriptional target of Wnt4, namely Wnt16, was induced in Wnt4-expressing transgenic glands, to levels equivalent to that of early pregnant glands.ConclusionTaken together, we propose that Wnt4 is necessary, but not sufficient, to induce side-branch development.

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., Author Summary The molecular details driving the splitting of cells in two during cell division, known as cytokinesis, remain incompletely understood, as is the coordination of this process with events that pattern the early animal embryo. The dearth of our knowledge in this subject is particularly evident in the case of vertebrate embryonic development. This is in large part because of challenges associated with the identification and characterization in vertebrate systems of the responsible genes, which are expressed during oogenesis. We have carried out genetic screens to identify in an unbiased manner maternal-effect genes required for early zebrafish development. Here we report the molecular genetic analysis of one such gene, cellular island. We show that cellular island encodes the chromosomal passenger complex protein Aurora B kinase and that its function is essential for furrow induction and maturation in the zebrafish embryo. Genetic analysis involving mutations in this gene and a maternal-effect gene required for spindle formation shows the presence of spatial and temporal specializations in furrow induction signals and cellular island/aurora B kinase function in the early embryo.

A female-sterile zebrafish maternal-effect mutation in cellular atoll (cea) results in defects in the initiation of cell division starting at the second cell division cycle. This phenomenon is caused by defects in centrosome duplication, which in turn affect the formation of a bipolar spindle. We show that cea encodes the centriolar coiled-coil protein Sas-6, and that zebrafish Cea/Sas-6 protein localizes to centrosomes. cea also has a genetic paternal contribution, which when mutated results in an arrested first cell division followed by normal cleavage. Our data supports the idea that, in zebrafish, paternally inherited centrosomes are required for the first cell division while maternally derived factors are required for centrosomal duplication and cell divisions in subsequent cell cycles. DNA synthesis ensues in the absence of centrosome duplication, and the one-cycle delay in the first cell division caused by cea mutant sperm leads to whole genome duplication. We discuss the potential implications of these findings with regards to the origin of polyploidization in animal species. In addition, the uncoupling of developmental time and cell division count caused by the cea mutation suggests the presence of a time window, normally corresponding to the first two cell cycles, which is permissive for germ plasm recruitment.

Signaling pathways that rely on the controlled release and/or accumulation of calcium ions are important in a variety of developmental events in the vertebrate embryo, affecting cell fate specification and morphogenesis. One such major developmentally important pathway is the Wnt/calcium signaling pathway, which, through its antagonism of Wnt/β-catenin signaling, appears to regulate the formation of the early embryonic organizer. In addition, the Wnt/calcium pathway shares components with another non-canonical Wnt pathway involved in planar cell polarity, suggesting that these two pathways form a loose network involved in polarized cell migratory movements that fashion the vertebrate body plan. Furthermore, left–right axis determination, neural induction and somite formation also display dynamic calcium release, which may be critical in these patterning events. Finally, we summarize recent evidence that propose a role for calcium signaling in stem cell biology and human developmental disorders.

Cytokinesis in early zebrafish embryos involves coordinated changes in the f-actin- and microtubule-based cytoskeleton, and the recruitment of adhesion junction components to the furrow. We show that exposure to inhibitors of non-muscle myosin II function does not affect furrow ingression during the early cleavage cycles but interferes with the recruitment of pericleavage f-actin and cortical β-catenin aggregates to the furrow, as well as the remodeling of the furrow microtubule array. This remodeling is in turn required for the distal aggregation of the zebrafish germ plasm. Embryos with reduced myosin activity also exhibit at late stages of cytokinesis a stabilized contractile ring apparatus that appears as a ladder-like pattern of short f-actin cables, supporting a role for myosin function in the disassembly of the contractile ring after furrow formation. Our studies support a role for myosin function in furrow maturation that is independent of furrow ingression and which is essential for the recruitment of furrow components and the remodeling of the cytoskeleton during cytokinesis.

A zebrafish maternal effect mutation, in the gene hecate, results in embryos that have defects in the formation of dorsoanterior structures and altered calcium release. hecate mutant embryos lack nuclear accumulation of beta-catenin and have reduced expression of genes specific to the dorsal organizer. We found that hecate mutant embryos exhibit a nearly 10-fold increase in the frequency of intracellular Ca2+ transients normally present in the enveloping layer during the blastula stages. Inhibition of Ca2+ release leads to ectopic expression of dorsal genes in mutant embryos suggesting that Ca2+ transients are important in mediating dorsal gene expression. Inhibition of Ca2+ release also results in the expression of dorsal-specific genes in the enveloping layer in a beta-catenin-independent manner, which suggests an additional function for the Ca2+ transients in this cellular layer. The mutant phenotype can be reversed by the expression of factors that activate Wnt/beta-catenin signaling, suggesting that the Wnt/beta-catenin pathway, at least as activated by an exogenous Wnt ligand, is intact in hec mutant embryos. Our results are consistent with a role for the hecate gene in the regulation of Ca2+ release during the cleavage stages, which in turn influences dorsal gene expression in both marginal cells along the dorsoventral axis and in the enveloping layer.

Publisher Summary This chapter describes approaches and methodologies to carry out genetic screens for maternal-effect mutations. Two approaches have been used to identify de novo recessive maternal-effect mutations in zebrafish: (1) an F4 screen based solely on natural crosses and (2) an F3 screen based on gynogenesis, specifically the technique of Early Pressure. These alternatives differ in various important ways, which are summarized in and described throughout this chapter. Once females are identified as exhibiting a maternal-effect phenotype, the mutation needs to be recovered. The observed maternal-effect phenotypes are expected to be caused by maternal homozygosity for recessive mutations, because dominant mutations are unlikely to be propagated through the generations that occur prior to screening. Because homozygosity for recessive maternal-effect mutations in females leads to the inviability of their progeny, a genetic scheme has to allow the recovery of the mutations through genetically related individuals. Fine mapping a maternal-effect mutation can be performed much more efficiently than the initial mapping. Narrowing down the location of a mutation through fine mapping is necessary to identify the molecular nature of the mutated gene through either candidate gene or positional cloning approaches.