In the hematopoietic hierarchy, only stem cells are thought to be capable of long-term self-renewal. Erythroid progenitors derived from fetal or adult mammalian hematopoietic tissues are capable of short-term, or restricted (10(2)- to 10(5)-fold), ex vivo expansion in the presence of erythropoietin, stem cell factor, and dexamethasone. Here, we report that primary erythroid precursors derived from early mouse embryos are capable of extensive (10(6)- to 10(60)-fold) ex vivo proliferation. These cells morphologically, immunophenotypically, and functionally resemble proerythroblasts, maintaining both cytokine dependence and the potential, despite prolonged culture, to generate enucleated erythrocytes after 3-4 maturational cell divisions. This capacity for extensive erythroblast self-renewal is temporally associated with the emergence of definitive erythropoiesis in the yolk sac and its transition to the fetal liver. In contrast, hematopoietic stem cell-derived definitive erythropoiesis in the adult is associated almost exclusively with restricted ex vivo self-renewal. Primary primitive erythroid precursors, which lack significant expression of Kit and glucocorticoid receptors, lack ex vivo self-renewal capacity. Extensively self-renewing erythroblasts, despite their near complete maturity within the hematopoietic hierarchy, may ultimately serve as a renewable source of red cells for transfusion therapy.

Mechanisms for shaping and folding sheets of cells during development are poorly understood. An example is the complex reorganisation of the forebrain neural plate during neurulation, which must fold a sheet into a tube while evaginating two eyes from a single contiguous domain within the neural plate. We, for the first time, track these cell rearrangements to show that forebrain morphogenesis differs significantly from prior hypotheses. We postulate a new model for forebrain neurulation and demonstrate how mutations affecting two signalling pathways can generate cyclopic phenotypes by disrupting normal cell movements or introducing new erroneous behaviours.

Vertebrate locomotor circuitry contains distinct classes of ventral spinal cord neurons which each have particular functional properties. While we know some of the genes expressed by each of these cell types, we do not yet know how several of these neurons are specified. Here, we investigate the functions of Tal1, Gata2a, and Gata3 transcription factors in the development of two of these populations of neurons with important roles in locomotor circuitry: V2b neurons and cerebrospinal fluid-contacting Kolmer-Agduhr (KA) neurons (also called CSF-cNs). Our data provide the first demonstration, in any vertebrate, that Tal1 and Gata3 are required for correct development of KA and V2b neurons, respectively. We also uncover differences in the genetic regulation of V2b cell development in zebrafish compared to mouse. In addition, we demonstrate that Sox1a and Sox1b are expressed by KA and V2b neurons in zebrafish, which differs from mouse, where Sox1 is expressed by V2c neurons. KA neurons can be divided into ventral KA″ neurons and more dorsal KA′ neurons. Consistent with previous morpholino experiments, our mutant data suggest that Tal1 and Gata3 are required in KA′ but not KA″ cells, whereas Gata2a is required in KA″ but not KA′ cells, even though both of these cell types co-express all three of these transcription factors. In gata2a mutants, cells in the KA″ region of the spinal cord lose expression of most KA″ genes and there is an increase in the number of cells expressing V3 genes, suggesting that Gata2a is required to specify KA″ and repress V3 fates in cells that normally develop into KA″ neurons. On the other hand, our data suggest that Gata3 and Tal1 are both required for KA′ neurons to differentiate from progenitor cells. In the KA′ region of these mutants, cells no longer express KA′ markers and there is an increase in the number of mitotically-active cells. Finally, our data demonstrate that all three of these transcription factors are required for later stages of V2b neuron differentiation and that Gata2a and Tal1 have different functions in V2b development in zebrafish than in mouse.

BackgroundFor neurons to function correctly in neuronal circuitry they must utilize appropriate neurotransmitters. However, even though neurotransmitter specificity is one of the most important and defining properties of a neuron we still do not fully understand how neurotransmitter fates are specified during development. Most neuronal properties are determined by the transcription factors that neurons express as they start to differentiate. While we know a few transcription factors that specify the neurotransmitter fates of particular neurons, there are still many spinal neurons for which the transcription factors specifying this critical phenotype are unknown. Strikingly, all of the transcription factors that have been identified so far as specifying inhibitory fates in the spinal cord act through Pax2. Even Tlx1 and Tlx3, which specify the excitatory fates of dI3 and dI5 spinal neurons work at least in part by down-regulating Pax2.MethodsIn this paper we use single and double mutant zebrafish embryos to identify the spinal cord functions of Evx1 and Evx2.ResultsWe demonstrate that Evx1 and Evx2 are expressed by spinal cord V0v cells and we show that these cells develop into excitatory (glutamatergic) Commissural Ascending (CoSA) interneurons. In the absence of both Evx1 and Evx2, V0v cells still form and develop a CoSA morphology. However, they lose their excitatory fate and instead express markers of a glycinergic fate. Interestingly, they do not express Pax2, suggesting that they are acquiring their inhibitory fate through a novel Pax2-independent mechanism.ConclusionsEvx1 and Evx2 are required, partially redundantly, for spinal cord V0v cells to become excitatory (glutamatergic) interneurons. These results significantly increase our understanding of the mechanisms of neuronal specification and the genetic networks involved in these processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13064-016-0059-9) contains supplementary material, which is available to authorized users.

Wolbachia are maternally inherited intracellular bacteria known to manipulate the reproduction of their arthropod hosts. Wolbachia commonly affect the sperm of infected arthropods. Wolbachia-modified sperm cannot successfully fertilize unless the female is infected with the same Wolbachia type. A study of spermatogenesis in the parasitic wasp Nasonia vitripennis reveals that Wolbachia are not required in individual spermatocytes or spermatids to modify sperm. In N. vitripennis, Wolbachia modify nearly all sperm, but are found only in B28% of developing sperm, and are also found in surrounding cyst and sheath cells. In the beetle Chelymorpha alternans, Wolbachia can modify up to 90% of sperm, but were never observed within the developing sperm or within the surrounding cyst cells; they were abundant within the outer testis sheath. We conclude that the residence within a developing sperm is not a prerequisite for Wolbachia-induced sperm modification, suggesting that Wolbachia modification of sperm may occur across multiple tissue membranes or act upstream of spermiogenesis.

The transcription factor Evx1 is expressed in the joints between individual lepidotrichia (bony ray) segments and at the distal tips of the lepidotrichia in developing zebrafish fins. It is also expressed in the apical growth zone in regenerating fins. However, so far there is no functional evidence that addresses whether Evx1 is required for any aspect of fin development or regeneration. In this study, we use a novel mutation in evx1 to address this. We find that Evx1 is not required for either fin outgrowth or regeneration. All of the fins form normally in evx1 mutants, and there are no significant changes in fin length. In contrast, Evx1 is required for lepidotrichia joint formation during both fin development and regeneration. This is a very specific phenotype as both lepidotrichia hemisegment separations and lepidotrichia bifurcations still form normally in evx1 mutant fins, as do joints in the more proximal endoskeletal radials. Developmental Dynamics 240:1240-1248,

SUMMARYIn mouse, Hedgehog (Hh) signalling is required for most ventral spinal neurons to form. Here, we analyse the spinal cord phenotype of zebrafish maternal-zygotic smoothened (MZsmo) mutants that completely lack Hh signalling. We find that most V3 domain cells and motoneurons are lost, whereas medial floorplate still develops normally and V2, V1 and V0v cells form in normal numbers. This phenotype resembles that of mice that lack both Hh signalling and Gli repressor activity. Ventral spinal cord progenitor domain transcription factors are not expressed at 24 hpf in zebrafish MZsmo mutants. However, pMN, p2 and p1 domain markers are expressed at early somitogenesis stages in these mutants. This suggests that Gli repressor activity does not extend into zebrafish ventral spinal cord at these stages, even in the absence of Hh signalling. Consistent with this, ectopic expression of Gli3R represses ventral progenitor domain expression at these early stages and knocking down Gli repressor activity rescues later expression. We investigated whether retinoic acid (RA) signalling specifies ventral spinal neurons in the absence of Hh signalling. The results suggest that RA is required for the correct number of many different spinal neurons to form. This is probably mediated, in part, by an effect on cell proliferation. However, V0v, V1 and V2 cells are still present, even in the absence of both Hh and RA signalling. We demonstrate that Gli1 has a Hh-independent role in specifying most of the remaining motoneurons and V3 domain cells in embryos that lack Hh signalling, but removal of Gli1 activity does not affect more dorsal neurons.

Polycystic kidney disease (PKD) proteins are trans-membrane proteins that have crucial roles in many aspects of vertebrate development and physiology, including the development of many organs as well as left–right patterning and taste. They can be divided into structurally-distinct PKD1-like and PKD2-like proteins and usually one PKD1-like protein forms a heteromeric polycystin complex with a PKD2-like protein. For example, PKD1 forms a complex with PKD2 and mutations in either of these proteins cause Autosomal Dominant Polycystic Kidney Disease (ADPKD), which is the most frequent potentially-lethal single-gene disorder in humans. Here, we identify the complete family of pkd genes in zebrafish and other teleosts. We describe the genomic locations and sequences of all seven genes: pkd1, pkd1b, pkd1l1, pkd1l2a, pkd1l2b, pkd2, and pkd2l1. pkd1l2a/pkd1l2b are likely to be ohnologs of pkd1l2, preserved from the whole genome duplication that occurred at the base of the teleosts. However, in contrast to mammals and cartilaginous and holostei fish, teleosts lack pkd2l2, and pkdrej genes, suggesting that these have been lost in the teleost lineage. In addition, teleost, and holostei fish have only a partial pkd1l3 sequence, suggesting that this gene may be in the process of being lost in the ray-finned fish lineage. We also provide the first comprehensive description of the expression of zebrafish pkd genes during development. In most structures we detect expression of one pkd1-like gene and one pkd2-like gene, consistent with these genes encoding a heteromeric protein complex. For example, we found that pkd2 and pkd1l1 are expressed in Kupffer's vesicle and pkd1 and pkd2 are expressed in the developing pronephros. In the spinal cord, we show that pkd1l2a and pkd2l1 are co-expressed in KA cells. We also identify potential co-expression of pkd1b and pkd2 in the floor-plate. Interestingly, and in contrast to mouse, we observe expression of all seven pkd genes in regions that may correspond to taste receptors. Taken together, these results provide a crucial catalog of pkd genes in an important model system for elucidating cell and developmental processes and modeling human diseases and the most comprehensive analysis of embryonic pkd gene expression in any vertebrate.