Transcriptional regulators with broad expression in the zebrafish spinal cord.

BACKGROUND: The spinal cord is a crucial part of the vertebrate CNS, controlling movements and receiving and processing sensory information from the trunk and limbs. However, there is much we do not know about how this essential organ develops. Here, we describe expression of 21 transcription factors and one transcriptional regulator in zebrafish spinal cord. RESULTS: We analyzed the expression of aurkb, foxb1a, foxb1b, her8a, homeza, ivns1abpb, mybl2b, myt1a, nr2f1b, onecut1, sall1a, sall3a, sall3b, sall4, sox2, sox19b, sp8b, tsc22d1, wdhd1, zfhx3b, znf804a, and znf1032 in wild-type and MIB E3 ubiquitin protein ligase 1 zebrafish embryos. While all of these genes are broadly expressed in spinal cord, they have distinct expression patterns from one another. Some are predominantly expressed in progenitor domains, and others in subsets of post-mitotic cells. Given the conservation of spinal cord development, and the transcription factors and transcriptional regulators that orchestrate it, we expect that these genes will have similar spinal cord expression patterns in other vertebrates, including mammals and humans. CONCLUSIONS: Our data identify 22 different transcriptional regulators that are strong candidates for playing different roles in spinal cord development. For several of these genes, this is the first published description of their spinal cord expression.

Transcriptional Regulators with Broad Expression in the Zebrafish Spinal Cord.

Background: The spinal cord is a crucial part of the vertebrate CNS, controlling movements and receiving and processing sensory information from the trunk and limbs. However, there is much we do not know about how this essential organ develops. Here, we describe expression of 21 transcription factors and one transcriptional regulator in zebrafish spinal cord. Results: We analyzed the expression of aurkb, foxb1a, foxb1b, her8a, homeza, ivns1abpb, mybl2b, myt1a, nr2f1b, onecut1, sall1a, sall3a, sall3b, sall4, sox2, sox19b, sp8b, tsc22d1, wdhd1, zfhx3b, znf804a, and znf1032 in wild-type and MIB E3 ubiquitin protein ligase 1 zebrafish embryos. While all of these genes are broadly expressed in spinal cord, they have distinct expression patterns from one another. Some are predominantly expressed in progenitor domains, and others in subsets of post-mitotic cells. Given the conservation of spinal cord development, and the transcription factors and transcriptional regulators that orchestrate it, we expect that these genes will have similar spinal cord expression patterns in other vertebrates, including mammals and humans. Conclusions: Our data identify 22 different transcriptional regulators that are strong candidates for playing different roles in spinal cord development. For several of these genes, this is the first published description of their spinal cord expression.

Molecular analyses of zebrafish V0v spinal interneurons and identification of transcriptional regulators downstream of Evx1 and Evx2 in these cells.

BACKGROUND: V0v spinal interneurons are highly conserved, glutamatergic, commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However, we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. METHODS: To identify candidate members of V0v gene regulatory networks, we FAC-sorted wild-type and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression-profiled them using microarrays and single cell RNA-seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 double mutants and wild-type siblings. RESULTS: Our data reveal two molecularly distinct subtypes of zebrafish V0v spinal interneurons at 48 h and suggest that, by this stage of development, evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons, or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons, plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly, we show that Hmx2/3a, repress dI2 interneuron expression of skor1a and nefma, two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. CONCLUSIONS: This study identifies two molecularly distinct subsets of zebrafish V0v spinal interneurons, as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of these cells. Our data further suggest that in the absence of both Evx1 and Evx2, V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then, later, start to express markers of distinct types of inhibitory spinal interneurons, or motoneurons. Taken together, our findings significantly increase our knowledge of V0v and spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type.

Molecular Analyses of V0v Spinal Interneurons and Identification of Transcriptional Regulators Downstream of Evx1 and Evx2 in these Cells.

Background: V0v spinal interneurons are highly conserved, glutamatergic, commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However, we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. Methods: To identify candidate members of V0v gene regulatory networks, we FAC-sorted WT and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression-profiled them using microarrays and single cell RNA-seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 double mutants and wild-type siblings. Results: Our data reveal two molecularly distinct subtypes of V0v spinal interneurons at 48 h and suggest that, by this stage of development, evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons, or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons, plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly, we show that Hmx2/3a, repress dI2 interneuronal expression of skor1a and nefma, two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression. Conclusions: This study identifies two molecularly distinct subsets of V0v spinal interneurons, as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of these cells. Our data further suggest that in the absence of both Evx1 and Evx2, V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then, later, start to express markers of distinct types of inhibitory spinal interneurons, or motoneurons. Taken together, our findings significantly increase our knowledge of V0v and spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type.

Analyses of binding partners and functional domains for the developmentally essential protein Hmx3a/HMX3.

HMX3 is a homeodomain protein with essential roles in CNS and ear development. Homeodomains are DNA-binding domains and hence homeodomain-containing proteins are usually assumed to be transcription factors. However, intriguingly, our recent data suggest that zebrafish Hmx3a may not require its homeodomain to function, raising the important question of what molecular interactions mediate its effects. To investigate this, we performed a yeast two-hybrid screen and identified 539 potential binding partners of mouse HMX3. Using co-immunoprecipitation, we tested whether a prioritized subset of these interactions are conserved in zebrafish and found that Tle3b, Azin1b, Prmt2, Hmgb1a, and Hmgn3 bind Hmx3a. Next, we tested whether these proteins bind the products of four distinct hmx3a mutant alleles that all lack the homeodomain. Embryos homozygous for two of these alleles develop abnormally and die, whereas zebrafish homozygous for the other two alleles are viable. We found that all four mutations abrogate binding to Prmt2 and Tle3b, whereas Azin1b binding was preserved in all cases. Interestingly, Hmgb1a and Hmgn3 had more affinity for products of the viable mutant alleles. These data shed light on how HMX3/Hmx3a might function at a molecular level and identify new targets for future study in these vital developmental processes.

Guanosine tetraphosphate (ppGpp) accumulation inhibits chloroplast gene expression and promotes super grana formation in the moss Physcomitrium (Physcomitrella) patens.

The nucleotides guanosine tetraphosphate and pentaphosphate (or (p)ppGpp) are implicated in the regulation of chloroplast function in plants. (p)ppGpp signalling is best understood in the model vascular plant Arabidopsis thaliana in which it acts to regulate plastid gene expression to influence photosynthesis, plant development and immunity. However, little information is known about the conservation or diversity of (p)ppGpp signalling in other land plants. We studied the function of ppGpp in the moss Physcomitrium (previously Physcomitrella) patens using an inducible system for triggering ppGpp accumulation. We used this approach to investigate the effects of ppGpp on chloroplast function, photosynthesis and growth. We demonstrate that ppGpp accumulation causes a dramatic drop in photosynthetic capacity by inhibiting chloroplast gene expression. This was accompanied by the unexpected reorganisation of the thylakoid system into super grana. Surprisingly, these changes did not affect gametophore growth, suggesting that bryophytes and vascular plants may have different tolerances to defects in photosynthesis. Our findings point to the existence of both highly conserved and more specific targets of (p)ppGpp signalling in the land plants that may reflect different growth strategies.

Evolution of lbx spinal cord expression and function.

Ladybird homeobox (Lbx) transcription factors have crucial functions in muscle and nervous system development in many animals. Amniotes have two Lbx genes, but only Lbx1 is expressed in spinal cord. In contrast, teleosts have three lbx genes and we show here that zebrafish lbx1a, lbx1b, and lbx2 are expressed by distinct spinal cell types, and that lbx1a is expressed in dI4, dI5, and dI6 interneurons, as in amniotes. Our data examining lbx expression in Scyliorhinus canicula and Xenopus tropicalis suggest that the spinal interneuron expression of zebrafish lbx1a is ancestral, whereas lbx1b has acquired a new expression pattern in spinal cord progenitor cells. lbx2 spinal expression was probably acquired in the ray-finned lineage, as this gene is not expressed in the spinal cords of either amniotes or S. canicula. We also show that the spinal function of zebrafish lbx1a is conserved with mouse Lbx1. In zebrafish lbx1a mutants, there is a reduction in the number of inhibitory spinal interneurons and an increase in the number of excitatory spinal interneurons, similar to mouse Lbx1 mutants. Interestingly, the number of inhibitory spinal interneurons is also reduced in lbx1b mutants, although in this case the number of excitatory interneurons is not increased. lbx1a;lbx1b double mutants have a similar spinal interneuron phenotype to lbx1a single mutants. Taken together these data suggest that lbx1b and lbx1a may be required in succession for correct specification of dI4 and dI6 spinal interneurons, although only lbx1a is required for suppression of excitatory fates in these cells.

Hmx3a Has Essential Functions in Zebrafish Spinal Cord, Ear and Lateral Line Development.

Transcription factors that contain a homeodomain DNA-binding domain have crucial functions in most aspects of cellular function and embryonic development in both animals and plants. Hmx proteins are a subfamily of NK homeodomain-containing proteins that have fundamental roles in development of sensory structures such as the eye and the ear. However, Hmx functions in spinal cord development have not been analyzed. Here, we show that zebrafish (Danio rerio) hmx2 and hmx3a are coexpressed in spinal dI2 and V1 interneurons, whereas hmx3b, hmx1, and hmx4 are not expressed in spinal cord. Using mutational analyses, we demonstrate that, in addition to its previously reported role in ear development, hmx3a is required for correct specification of a subset of spinal interneuron neurotransmitter phenotypes, as well as correct lateral line progression and survival to adulthood. Surprisingly, despite similar expression patterns of hmx2 and hmx3a during embryonic development, zebrafish hmx2 mutants are viable and have no obviously abnormal phenotypes in sensory structures or neurons that require hmx3a In addition, embryos homozygous for deletions of both hmx2 and hmx3a have identical phenotypes to severe hmx3a single mutants. However, mutating hmx2 in hypomorphic hmx3a mutants that usually develop normally, results in abnormal ear and lateral line phenotypes. This suggests that while hmx2 cannot compensate for loss of hmx3a, it does function in these developmental processes, although to a much lesser extent than hmx3a More surprisingly, our mutational analyses suggest that Hmx3a may not require its homeodomain DNA-binding domain for its roles in viability or embryonic development.

Anteroposterior patterning of the zebrafish ear through Fgf- and Hh-dependent regulation of hmx3a expression.

In the zebrafish, Fgf and Hh signalling assign anterior and posterior identity, respectively, to the poles of the developing ear. Mis-expression of fgf3 or inhibition of Hh signalling results in double-anterior ears, including ectopic expression of hmx3a. To understand how this double-anterior pattern is established, we characterised transcriptional responses in Fgf gain-of-signalling or Hh loss-of-signalling backgrounds. Mis-expression of fgf3 resulted in rapid expansion of anterior otic markers, refining over time to give the duplicated pattern. Response to Hh inhibition was very different: initial anteroposterior asymmetry was retained, with de novo duplicate expression domains appearing later. We show that Hmx3a is required for normal anterior otic patterning, and that otic patterning defects in hmx3a-/- mutants are a close phenocopy to those seen in fgf3-/- mutants. However, neither loss nor gain of hmx3a function was sufficient to generate full ear duplications. Using our data to infer a transcriptional regulatory network required for acquisition of otic anterior identity, we can recapitulate both the wild-type and the double-anterior pattern in a mathematical model.

Tal1, Gata2a, and Gata3 Have Distinct Functions in the Development of V2b and Cerebrospinal Fluid-Contacting KA Spinal Neurons.

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.

Using Zebrafish to Bring Hands-On Laboratory Experiences to Urban Classrooms.

Zebrafish are widely used as a model organism for research. Zebrafish embryos are also a useful resource for teaching students about vertebrate development. Here we describe a collaboration between two high school teachers and two university professors that used zebrafish to bring hands-on laboratory experiences to inner-city students, with the aim of increasing tangibility, and improving student understanding and retention, of several fundamental scientific concepts, such as the scientific method, cell division, mitosis, and Mendelian genetics. We describe and provide supporting material for each of the four laboratory modules that we developed. We also discuss the obstacles that we encountered and include suggestions of ways to overcome these. This collaboration provides an example of how high school teachers with very little zebrafish experience can gain the knowledge and confidence to develop and implement modules such as these in a relatively short period of time. Owing to the wide availability of zebrafish resources, these laboratories should provide a useful resource for other teachers who are interested in integrating more hands-on, inquiry-based investigations using live animals into their classes. We also hope to encourage other zebrafish researchers to collaborate with local teachers in similar projects.

Identification and Expression Analysis of the Complete Family of Zebrafish pkd Genes.

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.

Lmx1b is required for the glutamatergic fates of a subset of spinal cord neurons.

BACKGROUND: Alterations in neurotransmitter phenotypes of specific neurons can cause imbalances in excitation and inhibition in the central nervous system (CNS), leading to diseases. Therefore, the correct specification and maintenance of neurotransmitter phenotypes is vital. As with other neuronal properties, neurotransmitter phenotypes are often specified and maintained by particular transcription factors. However, the specific molecular mechanisms and transcription factors that regulate neurotransmitter phenotypes remain largely unknown. METHODS: In this paper we use single mutant, double mutant and transgenic zebrafish embryos to elucidate the functions of Lmx1ba and Lmx1bb in the regulation of spinal cord interneuron neurotransmitter phenotypes. RESULTS: We demonstrate that lmx1ba and lmx1bb are both expressed in zebrafish spinal cord and that lmx1bb is expressed by both V0v cells and dI5 cells. Our functional analyses demonstrate that these transcription factors are not required for neurotransmitter fate specification at early stages of development, but that in embryos with at least two lmx1ba and/or lmx1bb mutant alleles there is a reduced number of excitatory (glutamatergic) spinal interneurons at later stages of development. In contrast, there is no change in the numbers of V0v or dI5 cells. These data suggest that lmx1b-expressing spinal neurons still form normally, but at least a subset of them lose, or do not form, their normal excitatory fates. As the reduction in glutamatergic cells is only seen at later stages of development, Lmx1b is probably required either for the maintenance of glutamatergic fates or to specify glutamatergic phenotypes of a subset of later forming neurons. Using double labeling experiments, we also show that at least some of the cells that lose their normal glutamatergic phenotype are V0v cells. Finally, we also establish that Evx1 and Evx2, two transcription factors that are required for V0v cells to acquire their excitatory neurotransmitter phenotype, are also required for lmx1ba and lmx1bb expression in these cells, suggesting that Lmx1ba and Lmx1bb act downstream of Evx1 and Evx2 in V0v cells. CONCLUSIONS: Lmx1ba and Lmx1bb function at least partially redundantly in the spinal cord and three functional lmx1b alleles are required in zebrafish for correct numbers of excitatory spinal interneurons at later developmental stages. Taken together, our data significantly enhance our understanding of how spinal cord neurotransmitter fates are regulated.

Evx1 and Evx2 specify excitatory neurotransmitter fates and suppress inhibitory fates through a Pax2-independent mechanism.

BACKGROUND: For 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. METHODS: In this paper we use single and double mutant zebrafish embryos to identify the spinal cord functions of Evx1 and Evx2. RESULTS: We 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. CONCLUSIONS: Evx1 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.

Bmi-1 Regulates Extensive Erythroid Self-Renewal.

Red blood cells (RBCs), responsible for oxygen delivery and carbon dioxide exchange, are essential for our well-being. Alternative RBC sources are needed to meet the increased demand for RBC transfusions projected to occur as our population ages. We previously have discovered that erythroblasts derived from the early mouse embryo can self-renew extensively ex vivo for many months. To better understand the mechanisms regulating extensive erythroid self-renewal, global gene expression data sets from self-renewing and differentiating erythroblasts were analyzed and revealed the differential expression of Bmi-1. Bmi-1 overexpression conferred extensive self-renewal capacity upon adult bone-marrow-derived self-renewing erythroblasts, which normally have limited proliferative potential. Importantly, Bmi-1 transduction did not interfere with the ability of extensively self-renewing erythroblasts (ESREs) to terminally mature either in vitro or in vivo. Bmi-1-induced ESREs can serve to generate in vitro models of erythroid-intrinsic disorders and ultimately may serve as a source of cultured RBCs for transfusion therapy.

Extensively self-renewing erythroblasts derived from transgenic ß-yac mice is a novel model system for studying globin switching and erythroid maturation.

Globin gene regulation occurs in the context of a maturing erythroid cell, which is undergoing significant changes in chromatin structure and gene expression. There are few model systems available that facilitate studies of globin gene regulation in the context of erythroid maturation. Extensively self-renewing erythroblasts (ESREs) are a nontransformed model of erythroid maturation derived from murine fetal liver or yolk sac. Imaging flow cytometry and RNA-seq studies demonstrate that ESREs functionally and molecularly model erythroid maturation. To address the need for a model system that also recapitulates human globin switching, ESREs were derived from mice transgenic for the complete human ß-globin locus (ß-yac ESREs). ß-yac ESREs express ß-globin from the transgenic human locus, with minimal γ-globin expression. When treated with hydroxyurea or inhibitors to histone deacetylases, DNA methyltransferases, or the histone demethylase lysine specific demethylase 1 (LSD1), ß-Yac ESREs significantly increase their γ-globin expression, demonstrating their utility for studying agents that influence maturational globin switching. ß-yac ESREs were further used to characterize the secondary effects of LSD1 inhibition on erythroid maturation, with inhibition of LSD1 resulting in altered cell and nuclear size, prolonged Kit expression, and decreased rates of enucleation consistent with impaired maturation. Taken together, these studies demonstrate that ß-yac ESREs have significant utility for identifying modulators of maturational globin switching as well as for studying the broader role of those modulators in erythroid maturation.

Roles of Hedgehog pathway components and retinoic acid signalling in specifying zebrafish ventral spinal cord neurons.

In 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.

Evx1 is required for joint formation in zebrafish fin dermoskeleton.

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.

Immature erythroblasts with extensive ex vivo self-renewal capacity emerge from the early mammalian fetus.

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.