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In contrast to mammals, the brain of the adult zebrafish has a remarkable ability to regenerate. In mammals, injuries induce proliferation of astrocytes and oligodendrocyte progenitors contributing to the formation of a glial scar. We analyzed the proliferation of glial cells and microglia in response to stab injury in the adult zebrafish telencephalon: Radial glial markers were up-regulated at the ventricle and co-expressed the proliferation nuclear antigen (PCNA). Microglia and oligodendrocyte progenitors accumulated transiently at the site of lesion. However, we could not find evidence of permanent scar formation. Parenchymal proliferation was almost negligible in comparison to the increase in proliferation at the ventricular zone. This suggests that most of the cellular material for regeneration is derived from regions of constitutive neurogenesis. Remarkably, the proliferative response is almost completely restricted to the lesioned hemisphere indicating that signals inducing regeneration remain mainly confined within the lesioned half of the telencephalon.

Adult neurogenesis arises from niches that harbor neural stem cells (NSC). Although holding great promise for regenerative medicine, the identity of NSC remains elusive. In mammals, a key attribute of NSC is the expression of the filamentous proteins glial fibrillary acidic protein (GFAP) and NESTIN. To assess whether these two markers are relevant in the fish model, two transgenic zebrafish lines for gfap and nestin were generated. Analysis of adult brains showed that the fusion GFAP-green fluorescent protein closely mimics endogenous GFAP, while the nestin transgene recapitulates nestin at the ventricular zones. Cells expressing the two reporters display radial glial morphology, colocalize with the NSC marker Sox2, undergo proliferation, and are capable of self-renewal within the matrix of distinct thickness in the telencephalon. Together, these two transgenic lines reveal a conserved feature of putative NSC in the adult zebrafish brain and provide a means for the identification and manipulation of these cells in vivo.

Summary Acetylcholinesterase (AChE) hydrolyses acetylcholine (ACh) ensuring the fast clearance of released neurotransmitter at cholinergic synapses. Many studies led to the hypothesis that AChE and the closely related enzyme butyrylcholinesterase (BChE) may play other, non-hydrolytic roles during development.In this review, we compare data from in vivo studies performed on invertebrate and vertebrate genetic models. The loss of function of ache in these systems is responsible for the appearance of several phenotypes. In all aspects so far studied,thephenotypescanbeexplainedbyanexcessof the undegraded substrate, ACh, leading to misfunction and pathological alterations. Thus, the lack of AChE catalytic activity in the mutants appears to be solely responsible for the observed phenotypes. None of them appears to require the postulated adhesive or other nonhydrolytic functions of AChE. BioEssays 27:189–200, 2005. 2005 Wiley Periodicals, Inc.

In zebrafish, development of the ventral neural tube depends on the Nodal-related signal Cyclops (Cyc). One-day-old cyc mutant embryos lack the medial floor plate (MFP). We show here that cells expressing MFP marker genes differentiate gradually in cyc mutant embryos in a delayed manner during the second day of development. This late differentiation is restricted to the hindbrain and spinal cord and depends on an intact Hedgehog (Hh) signalling pathway. Cells expressing MFP marker genes in cyc mutant embryos appear to be derived from lateral floor plate (LFP) cells as they coexpress LFP and MFP marker genes. This finding suggests that the correct temporal development of the MFP is required for the distinction of LFP and MFP cells in wild-type embryos. © 2002 Wiley-Liss, Inc.

SummaryHomology searches between DNA sequences of evolu-tionarydistantspecies(phylogeneticfootprinting)offerafastdetectionmethodforregulatorysequences.Becauseof the small size of their genomes, tetraodontid speciessuch as the Japanese pufferfish and green spottedpufferfish have become attractive models for compara-tivegenomics.Adisadvantageofthetetraodontidspeciesis, however, that they cannot be bred and manipulatedroutinely under laboratory conditions, so these speciesare less attractive for developmental and genetic analy-sis. In contrast, an increasing arsenal of transgene tech-niques with the developmental model species zebrafishand medaka are being used for functional analysis of cisregulatory sequences. The main disadvantage is themuch larger genome. While comparison between manyloci proved the suitability of phylogenetic footprint-ing using fish and mammalian sequences, fast rate ofchange in enhancer structure and gene duplicationwithin teleosts may obscure detection of homologies.Here we discuss the contribution and potentials pro-vided by different teleost models for the detectionand functional analysis of conserved cis-regulatoryelements. BioEssays 24:564–572, 2002. 2002 Wiley Periodicals, Inc.IntroductionThe understanding of the cis-regulatory architecture of verte-brategenomesremainsamajorchallengeforthepostgenomicera. Since these cis-regulatory elements can be scatteredoverlargeregionsofnon-codingsequence,theirdetectioncanbe very time-consuming and laborious using conventionalfunctional assays. The rapidly increasing information on thenon-coding sequences of genomes allows researchers to useincreasingly sophisticated tools to detect regulatory regionsand motifs in large stretches of genomic DNA. For example,onemethodofdetectingnovelregulatorymotifsforexampleisthe alignment of non-coding sequences that are clustered byexpressionanalysisinyeast.

Nodal signaling controls germ layer formation, left-right asymmetry, and patterning of the brain in the vertebrate embryo. Cellular responses to Nodal signals are complex and include changes in gene expression, cell morphology, and migratory behavior. Only little is known about the genes regulated by Nodal signaling. We designed a subtractive screening strategy by using a constitutively active Nodal receptor to identify putative target genes of Nodal signals in the early gastrula of zebrafish embryos. By quantitative analysis of macro-array hybridizations, 132 genes corresponding to 1.4% of genes on the entire macro-array were identified, which were enriched in the Nodal-induced probe pool. These genes encode components of signal transduction pathways, transcription regulators, proteins involved in protein metabolism but also cytoskeletal components and metabolic enzymes, suggesting dramatic changes of cell physiology in gastrula cells in response to Nodal signals. © 2001 Wiley-Liss, Inc.

The signalling molecule Sonic hedgehog is involved in a multitude of distinct patterning processes during vertebrate embryogenesis. In the nascent body axis of the zebrafish embryo, sonic hedgehog is co‐expressed with axial (HNF3β in mammals), a transcription regulator of the winged helix family. We show here that misexpression of axial leads to ectopic activation of sonic hedgehog expression in the zebrafish, suggesting that axial is a regulator of sonic hedgehog transcription. The sonic hedgehog gene was cloned from zebrafish and its promoter was characterized with respect to activation by axial . Expression of axial or rat HNF3β in HeLa cells results in activation of co‐transfected sonic hedgehog promoter–CAT fusion genes. This effect is mediated by two Axial (HNF3β) recognition sequences. We furthermore identified a retinoic acid response element (RARE) in the sonic hedgehog upstream region which can be bound by retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers in vitro and confers retinoic acid inducibility to the sonic hedgehog promoter in the HeLa cell system. Our results suggest that both Axial (HNF3β) and retinoic acid receptors are direct regulators of the sonic hedgehog gene.

A classical example for hormonal control of gene expression is the induction of the enzymes tyrosine aminotransferase (TAT) and tryptophan oxygenase (TO).1-3 These two enzymes are expressed exclusively in the parenchymal cells of the liver.4 The expression ofboth genes is affected by glucocorticoids.slO In nuclear run-on assays it could be shown that hormonal induction involves changes in the relative rates of transcription of these two genes. Apart from glucocorticoid induction, the activity of the TAT gene is also regulated by cAMP."-14 The accumulation ofTAT mRNA after cAMP stimulation is a consequence of transcriptional activation of the TAT gene. Combined dexamethasone and cAMP treatment leads to higher TAT mRNA concentrations than treatment with either inducer alone, which implies that dexamethasone and cAMP act by distinct mechanisms. IS In addition to hormonal regulation, both TAT and TO genes are under developmental control. Whereas TAT enzyme activity appears around birth, TO enzyme activity can be detected only 2 weeks later.16 The activity of the TAT gene is affected by two distinct genetic loci, which appear to operate in trans on the expression of the TAT gene. By genetic and biochemical analysis of several albino lethai mutants of the mouse, a control region required for expression and inducibility of several liver enzymes including TAT has been assigned to the region of the albino locus on chromosome 7 of the mouse. l7 The basal level of TAT mRNA is severely decreased, and the concentration of the mRNA is no longer inducible by glucocorticoids and cAMP, aIthough the structural gene for TAT is still present. Because the mouse TAT structural gene is located on chromosome 8,'9 the effect of this presumptive regulatory locus must operate in trans. Another locus, the so-called tissue-specific extinguisher-l (Tse-l) has been mapped to chromosome 11 of the mouse. When chromosome 11 of a fibroblast cell is

In order to define the mechanism of synergistic induction mediated by multiple glucocorticoid response elements (GRE), the affinity of the glucocorticoid receptor to a single or duplicated GRE was analyzed by gel retardation, nitrocellulose filter binding and by footprinting experiments. Direct measurement of the relative affinity and indirect determination by competition showed greater than 10-fold higher affinity of the glucocorticoid receptor to a duplicated GRE when compared to a single element. Maximal stability of the GRE-receptor complex was obtained using two closely spaced GREs positioned on the same side of the DNA helix. Increasing the distance or changing the helical position of the GREs considerably increased the off rate of the receptor. DNase I footprinting shows in addition to the protection of the GRE region, an altered pattern in the nonprotected intervening DNA indicating structural alteration of the DNA helix by the receptor bound to adjacent GREs.

Steroid responsive elements (SRE) have been mapped at variable positions relative to the transcription start site and are often adjacent to binding sites of transcription regulatory proteins. In order to define the role of these transcriptional control sequences in the induction process, we inserted the previously defined 15-bp glucocorticoid response element (GRE) or 15-bp estrogen response element (ERE) immediately upstream of the TATA box of the thymidine kinase promoter, deleting all distal promoter elements. Both ERE and GRE confer inducibility by the respective hormone to the truncated promoter. These data suggest that the steroid receptor protein, possibly in conjunction with the TATA box binding protein, is able to form an active transcription complex. In contrast, the GRE when inserted 351 bp upstream of the start site of transcription of the tyrosine aminotransferase gene (TAT) is not capable of mediating hormone inducibility. Inducibility can be attained at this position by either two GREs or a single GRE in combination with a CCAAT motif. A cluster of point mutations in the CCAAT box abolishes hormone inducibility, strongly suggesting a synergistic action between the glucocorticoid receptor and the factor recognizing the CCAAT motif. The CCAAT box can be replaced by a CACCC box, an NF I and an SP1 binding site, thus demonstrating that synergistic action is not restricted to the CCAAT box binding protein. These combinations of a GRE with different transcription factor binding sites show a pronounced cell-type-dependent glucocorticoid induction of expression.