Katrin Henke

Microglia in the developing brain: from immunity to behaviour.

For decades, microglia, the resident macrophages of the brain, have been recognized mostly for their role in several, if not all, pathologies affecting the brain. However, several studies under physiological conditions demonstrate that microglial function is indispensable also in the healthy brain. Indeed, microglia implement key functions already during development, such as the clearance of the huge amount of neurons that are produced in large excess in the embryo and later die of apoptosis. Beside these classical functions, however, novel roles are emerging that strikingly link microglia with higher order brain functions and show that these cells can ultimately influence behaviour. Therefore a detailed understanding of microglia under physiological conditions may open unprecedented perspectives in the prevention and treatment of neuropsychiatric diseases.

Efficient Mapping and Cloning of Mutations in Zebrafish by Low-Coverage Whole-Genome Sequencing

The generation and analysis of mutants in zebrafish has been instrumental in defining the genetic regulation of vertebrate development, physiology, and disease. However, identifying the genetic changes that underlie mutant phenotypes remains a significant bottleneck in the analysis of mutants. Whole-genome sequencing has recently emerged as a fast and efficient approach for identifying mutations in nonvertebrate model organisms. However, this approach has not been applied to zebrafish due to the complicating factors of having a large genome and lack of fully inbred lines. Here we provide a method for efficiently mapping and detecting mutations in zebrafish using these new parallel sequencing technologies. This method utilizes an extensive reference SNP database to define regions of homozygosity-by-descent by low coverage, whole-genome sequencing of pooled DNA from only a limited number of mutant F2 fish. With this approach we mapped each of the five different zebrafish mutants we sequenced and identified likely causative nonsense mutations in two and candidate mutations in the remainder. Furthermore, we provide evidence that one of the identified mutations, a nonsense mutation in bmp1a, underlies the welded mutant phenotype.

Novel microcephalic primordial dwarfism disorder associated with variants in the centrosomal protein ninein.

CONTEXT Microcephalic primordial dwarfism (MPD) is a rare, severe form of human growth failure in which growth restriction is evident in utero and continues into postnatal life. Single causative gene defects have been identified in a number of patients with MPD, and all involve genes fundamental to cellular processes including centrosome functions. OBJECTIVE The objective of the study was to find the genetic etiology of a novel presentation of MPD. DESIGN The design of the study was whole-exome sequencing performed on two affected sisters in a single family. Molecular and functional studies of a candidate gene were performed using patient-derived primary fibroblasts and a zebrafish morpholino oligonucleotides knockdown model. PATIENTS Two sisters presented with a novel subtype of MPD, including severe intellectual disabilities. MAIN OUTCOME MEASURES NIN, encoding Ninein, a centrosomal protein critically involved in asymmetric cell division, was identified as a candidate gene, and functional impacts in fibroblasts and zebrafish were studied. RESULTS From 34,606 genomic variants, two very rare missense variants in NIN were identified. Both probands were compound heterozygotes. In the zebrafish, ninein knockdown led to specific and novel defects in the specification and morphogenesis of the anterior neuroectoderm, resulting in a deformity of the developing cranium with a small, squared skull highly reminiscent of the human phenotype. CONCLUSION We identified a novel clinical subtype of MPD in two sisters who have rare variants in NIN. We show, for the first time, that reduction of ninein function in the developing zebrafish leads to specific deficiencies of brain and skull development, offering a developmental basis for the myriad phenotypes in our patients.

Perspectives for identification of mutations in the zebrafish: Making use of next-generation sequencing technologies for forward genetic approaches

The ability to identify a phenotype causing mutation is essential for successful use of mutagenesis screens in many model organisms. Mapping mutations was for a long time a bottleneck in zebrafish research, as the standard method for mapping and identification of mutations was time consuming and expensive. The development of new sequencing technologies in the last couple of years has enabled the rapid and cost-effective sequencing of whole genomes. This has led to the establishment of new strategies for mapping and identification of mutations in several model organisms. The application of these techniques to the zebrafish model, with its large genome and the high level of variation in and between strains, was not trivial. Several techniques have been developed recently, taking the specific characteristics of the zebrafish genome into account. Here we give an overview on how to plan a mapping experiment, detail the critical parameters and discuss available tools for mapping and identification of mutations in zebrafish using next-generation sequencing. Using these methods, zebrafish mutants can now be mapped in a couple of weeks for a fraction of the costs. The increased efficiency of identification of mutations in the zebrafish broadens the utility of the model and allows for systematic analysis of gene function in a vertebrate model.

The SLC7A7 Transporter Identifies Microglial Precursors prior to Entry into the Brain

During development, macrophages invade organs to establish phenotypically and transcriptionally distinct tissue-resident populations. How they invade and colonize these organs is unclear. In particular, it remains to be established whether they arise from naive equivalents that colonize organs randomly or whether there are committed macrophages that follow pre-determined migration paths. Here, by using a combination of genetics and imaging approaches in the zebrafish embryo, we have addressed how macrophages colonize the brain to become microglia. Identification and cloning of a mutant that lacks microglia has shown that Slc7a7, a Leucine/Arginine transporter, defines a restricted macrophage sub-lineage and is necessary for brain colonization. By taking a photoconversion approach, we show that these macrophages give rise to microglia. This study provides direct experimental evidence for the existence of sub-lineages among embryonic macrophages.

Genetic Screen for Postembryonic Development in the Zebrafish (Danio rerio): Dominant Mutations Affecting Adult Form

Large-scale forward genetic screens have been instrumental for identifying genes that regulate development, homeostasis, and regeneration, as well as the mechanisms of disease. The zebrafish, Danio rerio, is an established genetic and developmental model used in genetic screens to uncover genes necessary for early development. However, the regulation of postembryonic development has received less attention as these screens are more labor intensive and require extensive resources. The lack of systematic interrogation of late development leaves large aspects of the genetic regulation of adult form and physiology unresolved. To understand the genetic control of postembryonic development, we performed a dominant screen for phenotypes affecting the adult zebrafish. In our screen, we identified 72 adult viable mutants showing changes in the shape of the skeleton as well as defects in pigmentation. For efficient mapping of these mutants and mutation identification, we devised a new mapping strategy based on identification of mutant-specific haplotypes. Using this method in combination with a candidate gene approach, we were able to identify linked mutations for 22 out of 25 mutants analyzed. Broadly, our mutational analysis suggests that there are key genes and pathways associated with late development. Many of these pathways are shared with humans and are affected in various disease conditions, suggesting constraint in the genetic pathways that can lead to change in adult form. Taken together, these results show that dominant screens are a feasible and productive means to identify mutations that can further our understanding of gene function during postembryonic development and in disease.

Identification of Mutations in Zebrafish Using Next-Generation Sequencing

Whole‐genome sequencing (WGS) has been used in many invertebrate model organisms as an efficient tool for mapping and identification of mutations affecting particular morphological or physiological processes. However, the application of WGS in highly polymorphic, larger genomes of vertebrates has required new experimental and analytical approaches. As a consequence, a wealth of different analytical tools has been developed. As the generation and analysis of data stemming from WGS can be unwieldy and daunting to researchers not accustomed to many common bioinformatic analyses and Unix‐based computational tools, we focus on how to manage and analyze next‐generation sequencing datasets without an extensive computational infrastructure and in‐depth bioinformatic knowledge. Here we describe methods for the analysis of WGS for use in mapping and identification of mutations in the zebrafish. We stress key elements of the experimental design and the analytical approach that allow the use of this method across different sequencing platforms and in different model organisms with annotated genomes. Curr. Protoc. Mol. Biol. 104:7.13.1‐7.13.33.

Utility of quantitative micro-computed tomographic analysis in zebrafish to define gene function during skeletogenesis

The zebrafish is a powerful experimental model to investigate the genetic and morphologic basis of vertebrate development. Analysis of skeletogenesis in this fish is challenging as a result of the small size of the developing and adult zebrafish. Many of the bones of small fishes such as the zebrafish and medaka are quite thin, precluding many standard assays of bone quality and morphometrics commonly used on bones of larger animals. Microcomputed tomography (microCT) is a common imaging technique used for detailed analysis of the skeleton of the zebrafish and determination of mutant phenotypes. However, the utility of this modality for analysis of the zebrafish skeleton, and the effect of inherent variation among individual zebrafish, including variables such as sex, age and strain, is not well understood. Given the increased use and accessibility of microCT, we set out to define the sensitivity of microCT methods in developing and adult zebrafish. We assessed skeletal shape and density measures in the developing vertebrae and parasphenoid of the skull base. We found most skeletal variables are tightly correlated to standard length, but that at later growth stages (>3months) there are age dependent effects on some skeletal measures. Further we find modest strain but not sex differences in skeletal measures. These data suggest that the appropriate control for assessing mutant phenotypes should be age and strain matched, ideally a wild-type sibling. By analyzing two mutants exhibiting skeletal dysplasia, we show that microCT imaging can be a sensitive method to quantify distinct skeletal parameters of adults. Finally, as developing zebrafish skeletons remain difficult to resolve by radiographic means, we define a contrast agent specific for bone that enhances resolution at early stages, permitting detailed morphometric analysis of the forming skeleton. This increased capability for detection extends the use of this imaging modality to leverage the zebrafish model to understand the development causes of skeletal dysplasias.

Latent developmental potential to form limb-like skeletal structures in zebrafish

The evolution of fins into limbs was a key transition in vertebrate history. A hallmark of this transition is the addition of multiple long bones to the proximal-distal axis of paired appendages. Whereas limb skeletons are often elaborate and diverse, teleost pectoral fins retain a simple endoskeleton. Fins and limbs share many core developmental processes, but how these programs were reshaped to produce limbs from fins during evolution remains enigmatic. Here we identify zebrafish mutants that form supernumerary long bones along the proximal-distal axis of pectoral fins with limb-like patterning. These new skeletal elements are integrated into the fin, as they are connected to the musculature, form joints, and articulate with neighboring bones. This phenotype is caused by activating mutations in previously unrecognized regulators of appendage development, vav2 and waslb, which we show function in a common pathway. We find that this pathway functions in appendage development across vertebrates, and loss of Wasl in developing limbs results in patterning defects identical to those seen in Hoxall knockout mice. Concordantly, formation of supernumerary fin long bones requires the function of hoxall paralogs, indicating developmental homology with the forearm and the existence of a latent functional Hox code patterning the fin endoskeleton. Our findings reveal an inherent limb-like patterning ability in fins that can be activated by simple genetic perturbation, resulting in the elaboration of the endoskeleton.

Zebrafish type I collagen mutants faithfully recapitulate human type I collagenopathies

Significance Type I collagenopathies are a heterogenous group of connective tissue disorders, caused by genetic defects in type I collagen. Inherent to these disorders is a large clinical variability, of which the underlying molecular basis remains undefined. By systematically analyzing skeletal phenotypes in a large set of type I collagen zebrafish mutants, we show that zebrafish models are able to both genocopy and phenocopy different forms of human type I collagenopathies, arguing for a similar pathogenetic basis. This study illustrates the future potential of zebrafish as a tool to further dissect the molecular basis of phenotypic variability in human type I collagenopathies, to improve diagnostic strategies as well as promote the discovery of new targetable pathways for pharmacological intervention of these disorders. The type I collagenopathies are a group of heterogeneous connective tissue disorders, that are caused by mutations in the genes encoding type I collagen and include specific forms of osteogenesis imperfecta (OI) and the Ehlers–Danlos syndrome (EDS). These disorders present with a broad disease spectrum and large clinical variability of which the underlying genetic basis is still poorly understood. In this study, we systematically analyzed skeletal phenotypes in a large set of zebrafish, with diverse mutations in the genes encoding type I collagen, representing different genetic forms of human OI, and a zebrafish model resembling human EDS, which harbors a number of soft connective tissues defects, typical of EDS. Furthermore, we provide insight into how zebrafish and human type I collagen are compositionally and functionally related, which is relevant in the interpretation of human type I collagen-related disease models. Our studies reveal a high degree of intergenotype variability in phenotypic expressivity that closely correlates with associated OI severity. Furthermore, we demonstrate the potential for select mutations to give rise to phenotypic variability, mirroring the clinical variability associated with human disease pathology. Therefore, our work suggests the future potential for zebrafish to aid in identifying unknown genetic modifiers and mechanisms underlying the phenotypic variability in OI and related disorders. This will improve diagnostic strategies and enable the discovery of new targetable pathways for pharmacological intervention.