The Independent Probabilistic Firing of Transcription Factors: A Paradigm for Clonal Variability in the Zebrafish Retina.

Early retinal progenitor cells (RPCs) in vertebrates produce lineages that vary greatly both in terms of cell number and fate composition, yet how this variability is achieved remains unknown. One possibility is that these RPCs are individually distinct and that each gives rise to a unique lineage. Another is that stochastic mechanisms play upon the determinative machinery of equipotent early RPCs to drive clonal variability. Here we show that a simple model, based on the independent firing of key fate-influencing transcription factors, can quantitatively account for the intrinsic clonal variance in the zebrafish retina and predict the distributions of neuronal cell types in clones where one or more of these fates are made unavailable.BDS and SR acknowledge the support of the Wellcome Trust (098357/Z/12/Z). WAH and HB also acknowledge the support of the Wellcome Trust (100329/Z/12/Z). HB was also supported by the Swedish Research Council (2011-7054).This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.devcel.2015.08.01

Hop Mice Display Synchronous Hindlimb Locomotion and a Ventrally Fused Lumbar Spinal Cord Caused by a Point Mutation in Ttc26

Identifying the spinal circuits controlling locomotion is critical for unravelling the mechanisms controlling the production of gaits. Development of the circuits governing left-right coordination relies on axon guidance molecules such as ephrins and netrins. To date, no other class of proteins have been shown to play a role during this process. Here, we have analyzed hop mice, which walk with a characteristic hopping gait using their hindlimbs in synchrony. Fictive locomotion experiments suggest that a local defect in the ventral spinal cord contributes to the aberrant locomotor phenotype. Hop mutant spinal cords had severe morphologic defects, including the absence of the ventral midline and a poorly defined border between white and gray matter. The hop mice represent the first model where, exclusively found in the lumbar domain, the left and right components of the central pattern generators (CPGs) are fused with a synchronous hindlimb gait as a functional consequence. These defects were associated with abnormal developmental processes, including a misplaced notochord and reduced induction of ventral progenitor domains. Whereas the underlying mutation in hop mice has been suggested to lie within the Ttc26 gene, other genes in close vicinity have been associated with gait defects. Mouse embryos carrying a CRISPR replicated point mutation within Ttc26 displayed an identical morphologic phenotype. Thus, our data suggest that the assembly of the lumbar CPG network is dependent on fully functional TTC26 protein

The Rose-comb Mutation in Chickens Constitutes a Structural Rearrangement Causing Both Altered Comb Morphology and Defective Sperm Motility

Rose-comb, a classical monogenic trait of chickens, is characterized by a drastically altered comb morphology compared to the single-combed wild-type. Here we show that Rose-comb is caused by a 7.4 Mb inversion on chromosome 7 and that a second Rose-comb allele arose by unequal crossing over between a Rose-comb and wild-type chromosome. The comb phenotype is caused by the relocalization of the MNR2 homeodomain protein gene leading to transient ectopic expression of MNR2 during comb development. We also provide a molecular explanation for the first example of epistatic interaction reported by Bateson and Punnett 104 years ago, namely that walnut-comb is caused by the combined effects of the Rose-comb and Pea-comb alleles. Transient ectopic expression of MNR2 and SOX5 (causing the Pea-comb phenotype) occurs in the same population of mesenchymal cells and with at least partially overlapping expression in individual cells in the comb primordium. Rose-comb has pleiotropic effects, as homozygosity in males has been associated with poor sperm motility. We postulate that this is caused by the disruption of the CCDC108 gene located at one of the inversion breakpoints. CCDC108 is a poorly characterized protein, but it contains a MSP (major sperm protein) domain and is expressed in testis. The study illustrates several characteristic features of the genetic diversity present in domestic animals, including the evolution of alleles by two or more consecutive mutations and the fact that structural changes have contributed to fast phenotypic evolution

Reconciling competence and transcriptional hierarchies with stochasticity in retinal lineages

Recent advances suggest that there is a stochastic contribution to the proliferation and fate choice of retinal progenitors. How does this stochasticity fit with the progression of temporal competence and the transcriptional hierarchies that also influence cell division and cell fate in the developing retina? Where may stochasticity arise in the system and how do we make progress in this field when we may never fully explain the behavior of individual progenitor cells

Copy Number Variation in Intron 1 of SOX5 Causes the Pea-comb Phenotype in Chickens

Pea-comb is a dominant mutation in chickens that drastically reduces the size of the comb and wattles. It is an adaptive trait in cold climates as it reduces heat loss and makes the chicken less susceptible to frost lesions. Here we report that Pea-comb is caused by a massive amplification of a duplicated sequence located near evolutionary conserved non-coding sequences in intron 1 of the gene encoding the SOX5 transcription factor. This must be the causative mutation since all other polymorphisms associated with the Pea-comb allele were excluded by genetic analysis. SOX5 controls cell fate and differentiation and is essential for skeletal development, chondrocyte differentiation, and extracellular matrix production. Immunostaining in early embryos demonstrated that Pea-comb is associated with ectopic expression of SOX5 in mesenchymal cells located just beneath the surface ectoderm where the comb and wattles will subsequently develop. The results imply that the duplication expansion interferes with the regulation of SOX5 expression during the differentiation of cells crucial for the development of comb and wattles. The study provides novel insight into the nature of mutations that contribute to phenotypic evolution and is the first description of a spontaneous and fully viable mutation in this developmentally important gene

Generation of Retinal Neurons : Focus on the Proliferation and Differentiation of the Horizontal Cells and their Subtypes

We have used the chicken retina as a model for investigating cell cycle regulation and cell fate commitment during central nervous system development. This thesis focuses on the characterization of and commitment to the horizontal cell fate in the retina. Horizontal cells are interneurons that provide intraretinal signal processing prior to information relay to the brain. We have identified molecular markers that selectively distinguish the three subtypes of horizontal cells, previously described in the chicken retina based on morphology. Subtype specific birth-dating revealed that horizontal cell subtypes are generated consecutively by biased progenitors that are sensitive to the inhibitory effects of follistatin. Follistatin stimulates proliferation in progenitors by repressing the differentiation signal of activin. Initially, injection of follistatin led to a decrease in committed horizontal cells but as the inhibitory effect dissipated it resulted in an increased number of horizontal cells. During development committed horizontal cell progenitors migrate to the vitreal side of the retina where they become arrested in G2-phase for approximately two days. When the arrest is overcome the horizontal cell progenitors undergo ectopic mitosis followed by migration to their designated layer. The G2-phase arrest is not triggered or maintained by any of the classic G2-arrest pathways such as DNA damage or stress. Nevertheless, we show that the cyclin B1-Cdk1 complex has a central role in maintaining this G2-phase arrest. Two transcription factors, FoxN4 and Ptf1a, are required for the generation of horizontal cells. We show that these factors are also sufficient to promote horizontal cell fate. Overexpression of FoxN4 and Ptf1a resulted in an overproduction of horizontal- and amacrine cells at the expense of ganglion- and photoreceptor cells. We identified Atoh7, a transcription factor required for the generation of ganglion cells, as a Ptf1a transcriptional target for downregulation. Our data support a common horizontal/amacrine lineage separated from the ganglion/photoreceptor lineage by the action of Ptf1a. In conclusion, these data describe several novel characteristics of horizontal cells enhancing our understanding of neural development and cell fate commitment

A deep-dive into fictive locomotion - a strategy to probe cellular activity during speed transitions in fictively swimming zebrafish larvae

Fictive locomotion is frequently used to study locomotor output in paralyzed animals. We have evaluated the character of swim episodes elicited by different strategies in zebrafish. Motor output was measured on both sides of a body segment using electrodes and a pipeline for synchronizing stimulation and recording, denoising data and peak-finding was developed. The optomotor response generated swims most equivalent to spontaneous activity, while electrical stimulation and NMDA application caused various artefacts. Our optimal settings, optomotor stimulation using 5-day-old larvae, were combined with calcium imaging and optogenetics to validate the setup's utility. Expression of GCaMP5G by the mnx1 promoter allowed correlation of calcium traces of dozens of motor neurons to the fictive locomotor output. Activation of motor neurons through channelrhodopsin produced aberrant locomotor episodes. This strategy can be used to investigate novel neuronal populations in a high-throughput manner to reveal their role in shaping motor output. This article has an associated First Person interview with the first author of the paper

Horizontal Cells, the Odd Ones Out in the Retina, Give Insights into Development and Disease

Thorough investigation of a neuronal population can help reveal key aspects regarding the nervous system and its development. The retinal horizontal cells have several extraordinary features making them particularly interesting for addressing questions regarding fate assignment and subtype specification. In this review we discuss and summarize data concerning the formation and diversity of horizontal cells, how morphology is correlated to molecular markers, and how fate assignment separates the horizontal lineage from the lineages of other retinal cell types. We discuss the novel and unique features of the final cell cycle of horizontal cell progenitors and how they may relate to retinoblastoma carcinogenesis

Deterministic fate assignment of Müller glia cells in the zebrafish retina suggest a clonal backbone during development

The optic cup houses multipotent retinal progenitor cells that proliferate and differentiate to form the mature retina, containing five main types of neurons and a single glial cell type, the Müller cell. Progenitors of the zebrafish optic cup generate clones that vary regarding the number and types of neurons, a process we previously showed could be described by stochastic models. Here, we present data indicating that each retinal progenitor cell, in the 24 hrs post‐fertilization optic cup, is predestined to form a single Müller cell. This striking fate assignment of Müller cells reveals a dual nature of retinal lineages where stochastic mechanisms produce variable numbers of neurons while there is a strong deterministic component governing the formation of glia cells. A possible mechanism for this stereotypic fate assignment could be the maintenance of a clonal backbone during retina development, which would be similar to invertebrate and rodent cortical neurogenesis