Matthias Hammerschmidt

Large-scale mutagenesis in the zebrafish: in search of genes controlling development in a vertebrate

BACKGROUND In Drosophila melanogaster and Caenorhabditis elegans, the elucidation of developmental mechanisms has relied primarily on the systematic induction and isolation of mutations in genes with specific functions in development. Such an approach has not yet been possible in a vertebrate species, owing to the difficulty of analyzing and keeping a sufficiently high number of mutagenized lines of animals. RESULTS We have developed the methods necessary to perform large-scale saturation screens for mutations affecting embryogenesis in the zebrafish, Danio (Brachydanio) rerio. Firstly, a new aquarium system was developed to raise and keep large numbers of strains of genetically different fish safely and with little maintenance care. Secondly, by placing adult male fish in water containing the chemical mutagen, ethylnitrosourea, we induced point mutations in premeiotic germ cells with a rate of one to three mutations per locus per 1,000 mutagenized haploid genomes. This rate, which is similar to the mutagenesis rates produced by ethylmethanesulfonate in Drosophila, was determined for alleles at four different pigmentation genes. Finally, in a pilot screen in which mutagenized fish were inbred for two generations and scored for embryonic mutants, we isolated 100 recessive mutations with phenotypes visible in the homozygous embryos. CONCLUSION The high rate of induction and recovery of point mutations, in addition to an efficient aquarium system to house large numbers of mutagenized lines, means that it is now possible to perform saturation mutagenesis screens in a vertebrate, similar to those done in invertebrates.

The Zebrafish Glypican Knypek Controls Cell Polarity during Gastrulation Movements of Convergent Extension

Mutations in the zebrafish knypek locus impair gastrulation movements of convergent extension that narrow embryonic body and elongate it from head to tail. We demonstrate that knypek regulates cellular movements but not cell fate specification. Convergent extension movement defects in knypek are associated with abnormal cell polarity, as mutant cells fail to elongate and align medio-laterally. Positional cloning reveals that knypek encodes a member of the glypican family of heparan sulfate proteoglycans. Double mutant and overexpression analyses show that Knypek potentiates Wnt11 signaling, mediating convergent extension. These studies provide experimental and genetic evidence that glypican Knypek acts during vertebrate gastrulation as a positive modulator of noncanonical Wnt signaling to establish polarized cell behaviors underlying convergent extension movements.

N-cadherin mediates retinal lamination, maintenance of forebrain compartments and patterning of retinal neurites

The complex, yet highly ordered and predictable, structure of the neural retina is one of the most conserved features of the vertebrate central nervous system. In all vertebrate classes, retinal neurons are organized into laminae with each neuronal class adopting specific morphologies and patterns of connectivity. Using genetic analyses in zebrafish, we demonstrate that N-cadherin (Ncad) has several distinct and crucial functions during the establishment of retinal organization. Although the location of cell division is disorganized in embryos with reduced or no Ncad function, different classes of retinal neurons are generated. However, these neurons fail to organize into correct laminae, most probably owing to compromised adhesion between retinal cells. In addition, amacrine cells exhibit exuberant and misdirected outgrowth of neurites that contributes to severe disorganization of the inner plexiform layer. Retinal ganglion cells also exhibit defects in process outgrowth, with axons exhibiting fasciculation defects and adopting incorrect ipsilateral trajectories. At least some of these defects are likely to be due to a failure to maintain compartment boundaries between eye, optic nerve and brain. Although in vitro studies have implicated Fgf receptors in modulating the axon outgrowth promoting properties of Ncad, most aspects of the Ncad mutant phenotype are not phenocopied by treatments that block Fgf receptor function.

CCR2 recruits an inflammatory macrophage subpopulation critical for angiogenesis in tissue repair

Monocytes/macrophages are critical in orchestrating the tissue-repair response. However, the mechanisms that govern macrophage regenerative activities during the sequential phases of repair are largely unknown. In the present study, we examined the dynamics and functions of diverse monocyte/macrophage phenotypes during the sequential stages of skin repair. By combining the analysis of a new CCR2-eGFP reporter mouse model with conditional mouse mutants defective in myeloid cell-restricted CCR2 signaling or VEGF-A synthesis, we show herein that among the large number of inflammatory CCR2(+)Ly6C(+) macrophages that dominate the early stage of repair, only a small fraction strongly expresses VEGF-A that has nonredundant functions for the induction of vascular sprouts. The switch of macrophage-derived VEGF-A during the early stage of tissue growth toward epidermal-derived VEGF-A during the late stage of tissue maturation was critical to achieving physiologic tissue vascularization and healing progression. The results of the present study provide new mechanistic insights into CCR2-mediated recruitment of blood monocyte subsets into damaged tissue, the dynamics and functional consequences of macrophage plasticity during the sequential repair phases, and the complementary role of macrophage-derived VEGF-A in coordinating effective tissue growth and vascularization in the context of tissue-resident wound cells. Our findings may be relevant for novel monocyte-based therapies to promote tissue vascularization.

BMP and Wnt Specify Hematopoietic Fate by Activation of the Cdx-Hox Pathway

The formation of blood in the embryo is dependent on bone morphogenetic protein (BMP), but how BMP signaling intersects with other regulators of hematopoietic development is unclear. Using embryonic stem (ES) cells, we show that BMP4 first induces ventral-posterior (V-P) mesoderm and subsequently directs mesodermal cells toward blood fate by activating Wnt3a and upregulating Cdx and Hox genes. When BMP signaling is blocked during this latter phase, enforced expression of either Cdx1 or Cdx4 rescues hematopoietic development, thereby placing BMP4 signaling upstream of the Cdx-Hox pathway. Wnt signaling cooperates in BMP-induced hemogenesis, and the Wnt effector LEF1 mediates BMP4 activation of Cdx genes. Our data suggest that BMP signaling plays two distinct and sequential roles during blood formation, initially as an inducer of mesoderm, and later to specify blood via activation of Wnt signaling and the Cdx-Hox pathway.

Interrelation of immunity and tissue repair or regeneration.

Although tremendous progress has been achieved in understanding the molecular basis of tissue repair and regeneration in diverse model organisms, the tendency of mammals for imperfect healing and scarring rather than regeneration remains unexplained. Moreover, conditions of impaired wound healing, e.g. non-healing skin ulcers associated with diabetes mellitus or vascular disease, as well as excessive scarring, represent major clinical and socio-economical problems. The development of innovative strategies to improve tissue repair and regeneration is therefore an important task that requires a more thorough understanding of the underlying molecular and cellular mechanisms. There is substantial evidence in different model organisms that the immune system is of primary importance in determining the quality of the repair response, including the extent of scarring, and the restoration of organ structure and function. Findings in diverse species support a correlation between the loss of regeneration capacity and maturation of immune competence. However, in recent years, there is increasing evidence on conditions where the immune response promotes repair and ensures local tissue protection. Hence, the relationship between repair and the immune response is complex and there is evidence for both negative and positive roles. We present an overview on recent evidence that highlights the immune system to be key to efficient repair or its failure. First, we summarize studies in different model systems that reveal both promoting and impeding roles of the immune system on the regeneration and repair capacity. This part is followed by a delineation of diverse inflammatory cell types, selected peptide growth factors and their receptors as well as signaling pathways controlling inflammation during tissue repair. Finally, we report on new mechanistic insights on how these inflammatory pathways impair healing under pathological conditions and discuss therapeutic implications.

Adenohypophysis formation in the zebrafish and its dependence on sonic hedgehog

Formation of the adenohypophysis in mammalian embryos occurs via an invagination of the oral ectoderm to form Rathkes pouch, which becomes exposed to opposing dorsoventral gradients of signaling proteins governing specification of the different hormone-producing pituitary cell types. One signal promoting pituitary cell proliferation and differentiation to ventral cell types is Sonic hedgehog (Shh) from the oral ectoderm. To study pituitary formation and patterning in zebrafish, we cloned four cDNAs encoding different pituitary hormones, prolactin (prl), proopiomelancortin (pomc), thyroid stimulating hormone (tsh), and growth hormone (gh), and analyzed their expression patterns relative to that of the pituitary marker lim3. prl and pomc start to be expressed at the lateral edges of the lim3 expression domain, before pituitary cells move into the head. This indicates that patterning of the pituitary anlage and terminal differentiation of pituitary cells starts while cells are still organized in a placodal fashion at the anterior edge of the developing brain. Following the expression pattern of prl and pomc during development, we show that no pituitary-specific invagination equivalent to Rathkes pouch formation takes place. Rather, pituitary cells move inwards together with stomodeal cells during oral cavity formation, with medial cells of the placode ending up posterior and lateral cells ending up anterior, resulting in an anterior-posterior, rather than a dorsoventral, patterning of the adenohypophysis. Carrying out loss- and gain-of-function experiments, we show that Shh from the ventral diencephalon plays a crucial role during induction, patterning, and growth of the zebrafish adenohypophysis. The phenotypes are very similar to those obtained upon pituitary-specific inactivation or overexpression of Shh in mouse embryo, suggesting that the role of Shh during pituitary development has been largely conserved between fish and mice, despite the different modes of pituitary formation in the two vertebrate classes.

Bmp and Fgf signaling are essential for liver specification in zebrafish

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.

Zebrafish in Endocrine Systems: Recent Advances and Implications for Human Disease

Since its introduction as a genetic vertebrate model system approximately 30 years ago, the focus of zebrafish research has increasingly shifted to questions that are also relevant for human development and disease. Here, we review the potential of the zebrafish as a model for human endocrine systems. A recent review compared the functions of the different endocrine systems and glands in zebrafish with those in other vertebrates, including humans, coming to the conclusion that major aspects are conserved. Here, we present an updated overview of this rapidly growing field of zebrafish research, focusing on the hypothalamo-pituitary axis, which links the central nervous system with the endocrine systems, and on major processes that are under (neuro)endocrine control and are the subject of intensive current research in other endocrine model organisms, such as feeding circuits and energy homeostasis, sleep, stress, reproduction, osmoregulation, and calcium homeostasis. Finally, we summarize the strengths and weaknesses of zebrafish as a model for studying endocrine systems.

Has2 is required upstream of Rac1 to govern dorsal migration of lateral cells during zebrafish gastrulation.

The large extracellular polysaccharide Hyaluronan (HA) and its synthesizing enzymes (Has) have been implicated in regulating the migratory potential of metastatic cancer cells. Here, we analyze the roles of zebrafish Has2 in normal development. Antisense morpholino oligonucleotide (MO)-mediated knockdown of zebrafish Has2 leads to the loss of HA, and severe migratory defects during gastrulation, somite morphogenesis and primordial germ cell migration. During gastrulation, ventrolateral cells of has2 morphant embryos fail to develop lamellipodia and to migrate dorsally, resulting in a blockage of dorsal convergence, whereas extension of the dorsal axis is normal. The effect is cell autonomous, suggesting that HA acts as an autocrine signal to stimulate the migration of HA-generating cells. Upon ectopic expression in axial cells, has2 causes the formation of supernumerary lamellipodia and a blockage of axis extension. Epistasis analyses with constitutively active and dominant-negative versions of the small GTPase Rac1 suggest that HA acts by Rac1 activation, rather than as an essential structural component of the extracellular matrix. Together, our data provide evidence that convergence and extension are separate morphogenetic movements of gastrulation. In addition, they suggest that the same HA pathways are active to auto-stimulate cell migration during tumor invasion and vertebrate embryogenesis.