LIM-homeodomain Transcription Factor Islet1 Research Articles

The Roles of Histamine Receptor 1 (hrh1) in Neurotransmitter System Regulation, Behavior, and Neurogenesis in Zebrafish

Histamine receptors mediate important physiological processes and take part in the pathophysiology of different brain disorders. Histamine receptor 1 (HRH1) is involved in the development of neurotransmitter systems, and its role in neurogenesis has been proposed. Altered HRH1 binding and expression have been detected in the brains of patients with schizophrenia, depression, and autism. Our goal was to assess the role of hrh1 in zebrafish development and neurotransmitter system regulation through the characterization of hrh1−/− fish generated by the CRISPR/Cas9 system. Quantitative PCR, in situ hybridization, and immunocytochemistry were used to study neurotransmitter systems and genes essential for brain development. Additionally, we wanted to reveal the role of this histamine receptor in larval and adult fish behavior using several quantitative behavioral methods including locomotion, thigmotaxis, dark flash and startle response, novel tank diving, and shoaling behavior. Hrh1−/− larvae displayed normal behavior in comparison with hrh1+/+ siblings. Interestingly, a transient abnormal expression of important neurodevelopmental markers was evident in these larvae, as well as a reduction in the number of tyrosine hydroxylase 1 (Th1)–positive cells, th1 mRNA, and hypocretin (hcrt)-positive cells. These abnormalities were not detected in adulthood. In summary, we verified that zebrafish lacking hrh1 present deficits in the dopaminergic and hypocretin systems during early development, but those are compensated by the time fish reach adulthood. However, impaired sociability and anxious-like behavior, along with downregulation of choline O-acetyltransferase a and LIM homeodomain transcription factor Islet1, were displayed by adult fish.

ISL1 regulates lung branching morphogenesis via Shh signaling pathway

Lung branching morphogenesis relies on a complex coordination of multiple signaling pathways and transcription factors. Here, we found that ablation of the LIM homeodomain transcription factor Islet1 (Isl1) in lung epithelium resulted in defective branching morphogenesis and incomplete formation of five lobes. A reduction in mesenchymal cell proliferation was observed in Isl1ShhCre lungs. There was no difference in apoptosis between the wild-type (ShhCre) and Isl1ShhCre embryos. RNA-Seq and in situ hybridization analysis showed that Shh, Ptch1, Sox9, Irx1, Irx2, Tbx2, and Tbx3 were downregulated in the lungs of Isl1ShhCre embryos. ChIP assay implied the Shh gene served as a direct target of ISL1, since the transcription factor ISL1 could bind to the Shh epithelial enhancer sequence (MACS1). Also, activation of the Hedgehog pathway via ectopic gene expression rescued the defects caused by Isl1 ablation, confirming the genetic integration of Hedgehog signaling. In conclusion, our works suggest that epithelial Isl1 regulates lung branching morphogenesis through administrating the Shh signaling mediated epithelial-mesenchymal communications.

The islet-expressed Lhx1 transcription factor interacts with Islet-1 and contributes to glucose homeostasis.

The LIM-homeodomain (LIM-HD) transcription factor Islet-1 (Isl1) interacts with the LIM domain-binding protein 1 (Ldb1) coregulator to control expression of key pancreatic β-cell genes. However, Ldb1 also has Isl1-independent effects, supporting that another LIM-HD factor interacts with Ldb1 to impact β-cell development and/or function. LIM homeobox 1 (Lhx1) is an Isl1-related LIM-HD transcription factor that appears to be expressed in the developing mouse pancreas and in adult islets. However, roles for this factor in the pancreas are unknown. This study aimed to determine Lhx1 interactions and elucidate gene regulatory and physiological roles in the pancreas. Co-immunoprecipitation using β-cell extracts demonstrated an interaction between Lhx1 and Isl1, and thus we hypothesized that Lhx1 and Isl1 regulate similar target genes. To test this, we employed siRNA-mediated Lhx1 knockdown in β-cell lines and discovered reduced Glp1R mRNA. Chromatin immunoprecipitation revealed Lhx1 occupancy at a domain also known to be occupied by Isl1 and Ldb1. Through development of a pancreas-wide knockout mouse model ( Lhx1∆Panc), we demonstrate that aged Lhx1∆Panc mice have elevated fasting blood glucose levels, altered intraperitoneal and oral glucose tolerance, and significantly upregulated glucagon, somatostatin, pancreatic polypeptide, MafB, and Arx islet mRNAs. Additionally, Lhx1∆Panc mice exhibit significantly reduced Glp1R, an mRNA encoding the insulinotropic receptor for glucagon-like peptide 1 along with a concomitant dampened Glp1 response and mild glucose intolerance in mice challenged with oral glucose. These data are the first to reveal that the Lhx1 transcription factor contributes to normal glucose homeostasis and Glp1 responses.

On the Evolution of the Cardiac Pacemaker.

The rhythmic contraction of the heart is initiated and controlled by an intrinsic pacemaker system. Cardiac contractions commence at very early embryonic stages and coordination remains crucial for survival. The underlying molecular mechanisms of pacemaker cell development and function are still not fully understood. Heart form and function show high evolutionary conservation. Even in simple contractile cardiac tubes in primitive invertebrates, cardiac function is controlled by intrinsic, autonomous pacemaker cells. Understanding the evolutionary origin and development of cardiac pacemaker cells will help us outline the important pathways and factors involved. Key patterning factors, such as the homeodomain transcription factors Nkx2.5 and Shox2, and the LIM-homeodomain transcription factor Islet-1, components of the T-box (Tbx), and bone morphogenic protein (Bmp) families are well conserved. Here we compare the dominant pacemaking systems in various organisms with respect to the underlying molecular regulation. Comparative analysis of the pathways involved in patterning the pacemaker domain in an evolutionary context might help us outline a common fundamental pacemaker cell gene programme. Special focus is given to pacemaker development in zebrafish, an extensively used model for vertebrate development. Finally, we conclude with a summary of highly conserved key factors in pacemaker cell development and function.

Pax2-Islet1 Transgenic Mice Are Hyperactive and Have Altered Cerebellar Foliation

The programming of cell fate by transcription factors requires precise regulation of their time and level of expression. The LIM-homeodomain transcription factor Islet1 (Isl1) is involved in cell-fate specification of motor neurons, and it may play a similar role in the inner ear. In order to study its role in the regulation of vestibulo-motor development, we investigated a transgenic mouse expressing Isl1 under the Pax2 promoter control (Tg+/−). The transgenic mice show altered level, time, and place of expression of Isl1 but are viable. However, Tg+/− mice exhibit hyperactivity, including circling behavior, and progressive age-related decline in hearing, which has been reported previously. Here, we describe the molecular and morphological changes in the cerebellum and vestibular system that may cause the hyperactivity of Tg+/− mice. The transgene altered the formation of folia in the cerebellum, the distribution of calretinin labeled unipolar brush cells, and reduced the size of the cerebellum, inferior colliculus, and saccule. Age-related progressive reduction of calbindin expression was detected in Purkinje cells in the transgenic cerebella. The hyperactivity of Tg+/− mice is reduced upon the administration of picrotoxin, a non-competitive channel blocker for the γ-aminobutyric acid (GABA) receptor chloride channels. This suggests that the overexpression of Isl1 significantly affects the functions of GABAergic neurons. We demonstrate that the overexpression of Isl1 affects the development and function of the cerebello-vestibular system, resulting in hyperactivity.

The role of Islet-1 in cell specification, differentiation, and maintenance of phenotypes in the vertebrate neural retina.

Trabajo financiado por: Ministerio de Ciencia y Tecnologia. Proyecto BFU2007-67540 Junta de Extremadura. Becas PRI06A195 y GR10152

Abstract 16013: Direct Nkx2-5 Transcriptional Repression of Isl1 Controls Cardiomyocyte Subtype Identity

During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the LIM-homeodomain transcription factor Islet-1 (Isl1) and the homeobox protein Nkx2-5. Here, we show that a direct repression of Isl1 transcription by Nkx2-5 is necessary for proper specification and maturation of ventricular and atrial chamber-specific myocardial lineages. Overexpression of Nkx2-5 in mouse embryonic stem cells (ESCs) delayed specification of cardiac progenitors and inhibited expression of Isl1 and its downstream targets in the Isl1+ precursor population. These effects were partially rescued by Isl1 overexpression. Embryos deficient for Nkx2-5 in the Isl1+ lineage failed to downregulate Isl1 protein in cardiomyocytes of the heart tube (Figure 1A). We demonstrated that Nkx2-5 directly binds to an Isl1 gene enhancer and represses the transcriptional activity of Isl1. Furthermore, we showed that overexpression of Isl1 does not prevent cardiac differentiation of ESCs and in Xenopus laevis embryos. Instead, Isl1 overexpression in ESCs leads to enhanced specification of cardiac progenitors, earlier cardiac differentiation, and increased number of cardiomyocytes (Figure 1B). Functional and molecular analysis of Isl1-overexpressing cardiomyocytes revealed higher beating frequencies in both ESC-derived contracting areas and Xenopus Isl1-gain-of-function hearts (Figure 1C), which was associated with upregulation of nodal-specific genes and downregulation of transcripts of working myocardium. Our findings provide an Isl1/Nkx2-5-mediated mechanism that coordinately regulates the specification of cardiac progenitors towards the different myocardial lineages and ensures proper acquisition of myocyte subtype-identity (Figure 1D).

An Org-1-Tup transcriptional cascade reveals different types of alary muscles connecting internal organs in Drosophila.

The T-box transcription factor Tbx1 and the LIM-homeodomain transcription factor Islet1 are key components in regulatory circuits that generate myogenic and cardiogenic lineage diversity in chordates. We show here that Org-1 and Tup, the Drosophila orthologs of Tbx1 and Islet1, are co-expressed and required for formation of the heart-associated alary muscles (AMs) in the abdomen. The same holds true for lineage-related muscles in the thorax that have not been described previously, which we name thoracic alary-related muscles (TARMs). Lineage analyses identified the progenitor cell for each AM and TARM. Three-dimensional high-resolution analyses indicate that AMs and TARMs connect the exoskeleton to the aorta/heart and to different regions of the midgut, respectively, and surround-specific tracheal branches, pointing to an architectural role in the internal anatomy of the larva. Org-1 controls tup expression in the AM/TARM lineage by direct binding to two regulatory sites within an AM/TARM-specific cis-regulatory module, tupAME. The contributions of Org-1 and Tup to the specification of Drosophila AMs and TARMs provide new insights into the transcriptional control of Drosophila larval muscle diversification and highlight new parallels with gene regulatory networks involved in the specification of cardiopharyngeal mesodermal derivatives in chordates.

Spinal neurons require Islet1 for subtype-specific differentiation of electrical excitability.

BackgroundIn the spinal cord, stereotypic patterns of transcription factor expression uniquely identify neuronal subtypes. These transcription factors function combinatorially to regulate gene expression. Consequently, a single transcription factor may regulate divergent development programs by participation in different combinatorial codes. One such factor, the LIM-homeodomain transcription factor Islet1, is expressed in the vertebrate spinal cord. In mouse, chick and zebrafish, motor and sensory neurons require Islet1 for specification of biochemical and morphological signatures. Little is known, however, about the role that Islet1 might play for development of electrical membrane properties in vertebrates. Here we test for a role of Islet1 in differentiation of excitable membrane properties of zebrafish spinal neurons.ResultsWe focus our studies on the role of Islet1 in two populations of early born zebrafish spinal neurons: ventral caudal primary motor neurons (CaPs) and dorsal sensory Rohon-Beard cells (RBs). We take advantage of transgenic lines that express green fluorescent protein (GFP) to identify CaPs, RBs and several classes of interneurons for electrophysiological study. Upon knock-down of Islet1, cells occupying CaP-like and RB-like positions continue to express GFP. With respect to voltage-dependent currents, CaP-like and RB-like neurons have novel repertoires that distinguish them from control CaPs and RBs, and, in some respects, resemble those of neighboring interneurons. The action potentials fired by CaP-like and RB-like neurons also have significantly different properties compared to those elicited from control CaPs and RBs.ConclusionsOverall, our findings suggest that, for both ventral motor and dorsal sensory neurons, Islet1 directs differentiation programs that ultimately specify electrical membrane as well as morphological properties that act together to sculpt neuron identity.

Dual role for Islet-1 in promoting striatonigral and repressing striatopallidal genetic programs to specify striatonigral cell identity

Striatal projection neurons comprise two populations of striatonigral and striatopallidal neurons. These two neuronal populations play distinct roles in controlling movement-related functions in the basal ganglia circuits. An important issue is how striatal progenitors are developmentally specified into these two distinct neuronal populations. In the present study, we characterized the function of Islet-1 (Isl1), a LIM-homeodomain transcription factor, in striatal development. Genetic fate mapping showed that Isl1(+) progeny specifically developed into a subpopulation of striatonigral neurons that transiently expressed Isl1. In Nestin-Cre;Isl1(f/f) KO mouse brain, differentiation of striatonigral neurons was defective, as evidenced by decreased expression of striatonigral-enriched genes, including substance P, prodynorphin, solute carrier family 35, member D3 (Slc35d3), and PlexinD1. Striatonigral axonal projections were also impaired, and abnormal apoptosis was observed in Isl1 KO striatum. It was of particular interest that striatopallidal-enriched genes, including dopamine D2 receptor (Drd2), proenkephalin, A2A adenosine receptor (A2aR) and G protein-coupled receptor 6 (Gpr6), were concomitantly up-regulated in Isl1 mutant striatum, suggesting derepression of striatopallidal genes in striatonigral neurons in the absence of Isl1. The suppression of striatopallidal genes by Isl1 was further examined by overexpression of Isl1 in the striatum of Drd2-EGFP transgenic mice using in utero electroporation. Ectopic Isl1 expression was sufficient to repress Drd2-EGFP signals in striatopallidal neurons. Taken together, our study suggests that Isl1 specifies the cell fate of striatonigral neurons not only by orchestrating survival, differentiation, and axonal projections of striatonigral neurons but also by suppressing striatopallidal-enriched genes. The dual action of developmental control by Isl1 in promoting appropriate striatonigral but repressing inappropriate striatopallidal genetic profiles may ensure sharpening of the striatonigral identity during development.

The LIM homeobox geneIsl1is required for the correct development of the striatonigral pathway in the mouse

The mammalian striatum controls the output of the basal ganglia via two distinct efferent pathways, the direct (i.e., striatonigral) and the indirect (i.e., striatopallidal) pathways. The LIM homeodomain transcription factor Islet1 (Isl1) is expressed in a subpopulation of striatal progenitors; however, its specific role in striatal development remains unknown. Our genetic fate-mapping results show that Isl1-expressing progenitors give rise to striatal neurons belonging to the striatonigral pathway. Conditional inactivation of Isl1 in the telencephalon resulted in a smaller striatum with fewer striatonigral neurons and reduced projections to the substantia nigra. Additionally, conditional inactivation in the ventral forebrain (including both the telencephalon and diencephalon) revealed a unique role for Isl1 in diencephalic cells bordering the internal capsule for the normal development of the striatonigral pathway involving PlexinD1-Semaphorin 3e (Sema3e) signaling. Finally, Isl1 conditional mutants displayed a hyperlocomotion phenotype, and their locomotor response to psychostimulants was significantly blunted, indicating that the alterations in basal ganglia circuitry contribute to these mutant behaviors.

Physical lineage tracing of cells contributing to the chicken cardiac conduction system

Purpose: Knowledge about the developmental origin of the cardiac conduction system (CCS) is important to understand the pathophysiology of arrhythmias. During early development, the heart consists of two bilateral plates of splanchnic mesoderm, which fuse to form the primary heart tube (PHT) derived from the first heart field. A large number of cells at the venous pole of the PHT constitute a different cardiac progenitor population, called the second heart field (SHF). This population of cells expresses the LIM-homeodomain transcription factor Islet-1 (Isl-1). The contribution of this SHF population to the developing CCS remains the subject of discussion. We explored the feasibility of in ovo physical lineage tracing in chicken embryos, to perform long-term follow-up of SHF-derived cells contributing to the CCS. Methods and results: Labeling was achieved by injecting a solution of two fluorescent dyes (DiI/5-TAMRA, Invitrogen) at HH stages 14-17, using a programmable microinjector (IM-300 Narishige, Japan). Right-sided labeling of the splanchnic mesoderm and the right cardinal vein was performed. Early embryos were sacrificed and analyzed 1-3 hours after injection (HH stages 14-17), and sectioned serially. Sections were stained for the myocardial marker cTnI, for Nkx2.5 which was expressed in both myocardium and mesenchyme and for Isl-1. Fluorescent labeling was found in the second heart field mesenchyme surrounding the sinus venosus and the right cardinal vein. In older stages of development (up to HH26), labeling of the developing sinus node and venous valves was found. Conclusions: Our results show that it is feasible to label the SHF and elements of the CCS in ovo, which makes it possible to trace the fate of these cells over a longer period of time. Follow-up after HH stage 26 is needed to determine whether the contributions of the SHF are not only found in the sinus node but also in the atrioventricular node, common bundle and bundle branches Future plans include earlier labeling experiments as well as contribution of the left-sided SHF and determination of the differentiation capacities of these SHF derived cells.

The LIM-Homeodomain transcription factor Islet-1 is required for the development of sympathetic neurons and adrenal chromaffin cells

Islet-1 is a LIM-Homeodomain transcription factor with important functions for the development of distinct neuronal and non-neuronal cell populations. We show here that Islet-1 acts genetically downstream of Phox2B in cells of the sympathoadrenal cell lineage and that the development of sympathetic neurons and chromaffin cells is impaired in mouse embryos with a conditional deletion of Islet-1 controlled by the wnt1 promotor. Islet-1 is not essential for the initial differentiation of sympathoadrenal cells, as indicated by the correct expression of pan-neuronal and catecholaminergic subtype specific genes in primary sympathetic ganglia of Islet-1 deficient mouse embryos. However, our data indicate that the subsequent survival of sympathetic neuron precursors and their differentiation towards TrkA expressing neurons depends on Islet-1 function. In contrast to spinal sensory neurons, sympathetic neurons of Islet-1 deficient mice did not display ectopic expression of genes normally present in the CNS. In Islet-1 deficient mouse embryos the numbers of chromaffin cells were only mildly reduced, in contrast to that of sympathetic neurons, but the initiation of the adrenaline synthesizing enzyme PNMT was abrogated and the expression level of chromogranin A was diminished. Microarray analysis revealed that developing chromaffin cells of Islet-1 deficient mice displayed normal expression levels of TH, DBH and the transcription factors Phox2B, Mash-1, Hand2, Gata3 and Insm1, but the expression levels of the transcription factors Gata2 and Hand1, and AP-2ß were significantly reduced. Together our data indicate that Islet-1 is not essentially required for the initial differentiation of sympathoadrenal cells, but has an important function for the correct subsequent development of sympathetic neurons and chromaffin cells.

Islet1 is a direct transcriptional target of the homeodomain transcription factor Shox2 and rescues the Shox2-mediated bradycardia

The heart’s rhythm is initiated and regulated by a group of specialized cells in the sinoatrial node (SAN), the primary pacemaker of the heart. Abnormalities in the development of the SAN can result in irregular heart rates (arrhythmias). Although several of the critical genes important for SAN formation have been identified, our understanding of the transcriptional network controlling SAN development remains at a relatively early stage. The homeodomain transcription factor Shox2 is involved in the specification and patterning of the SAN. While the Shox2 knockout in mice results in embryonic lethality due to severe cardiac defects including improper SAN development, Shox2 knockdown in zebrafish causes a reduced heart rate (bradycardia). In order to gain deeper insight into molecular pathways involving Shox2, we compared gene expression levels in right atria of wildtype and Shox2 −/− hearts using microarray experiments and identified the LIM homeodomain transcription factor Islet1 (Isl1) as one of its putative target genes. The downregulation of Isl1 expression in Shox2 −/− hearts was confirmed and the affected region narrowed down to the SAN by whole-mount in situ hybridization. Using luciferase reporter assays and EMSA studies, we identified two specific SHOX2 binding sites within intron 2 of the ISL1 locus. We also provide functional evidence for Isl1 as a transcriptional target of Shox2 by rescuing the Shox2-mediated bradycardia phenotype with Isl1 using zebrafish as a model system. Our findings demonstrate a novel epistatic relationship between Shox2 and Isl1 in the heart with important developmental consequences for SAN formation and heart beat.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-013-0339-z) contains supplementary material, which is available to authorized users.

Islet-1 Immunoreactivity in the Developing Retina ofXenopus laevis

The LIM-homeodomain transcription factor Islet1 (Isl1) has been widely used as a marker of neuronal differentiation in the developing visual system of different classes of vertebrates, including mammals, birds, reptiles, and fish. In the present study, we analyzed the spatial and temporal distribution of Isl1-immunoreactive cells during Xenopus laevis retinal development and its relation to the formation of the retinal layers, and in combination with different markers of cell differentiation. The earliest Isl1 expression appeared at St29-30 in the cell nuclei of sparse differentiating neuroblasts located in the vitreal surface of the undifferentiated retina. At St35-36, abundant Isl1-positive cells accumulated at the vitreal surface of the neuroepithelium. As development proceeded and through the postmetamorphic juveniles, Isl1 expression was identified in subpopulations of ganglion cells and in subsets of amacrine, bipolar, and horizontal cells. These data together suggest a possible role for Isl1 in the early differentiation and maintenance of different retinal cell types, and Isl1 can serve as a specific molecular marker for the study of retinal cell specification in X. laevis.

Islet1-expressing cardiac progenitor cells: a comparison across species

Adult mammalian cardiac stem cells express the LIM-homeodomain transcription factor Islet1 (Isl1). They are considered remnants of Isl1-positive embryonic cardiac progenitor cells. During amniote heart development, Isl1-positive progenitor cells give rise mainly to the outflow tract, the right ventricle, and parts of the atria. This led to the hypothesis that the development of the right ventricle of the amniote heart depends on the recruitment of additional cells to the primary heart tube. The region from which these additional, Isl1-positive cells originate is called second heart field, as opposed to the first heart field whose cells form the primary heart tube. Here, we review the available data about Isl1 in different species, demonstrating that Isl1 is an important component of the core transcription factor network driving early cardiogenesis in animals of the two clades, deuterostomes, and protostomes. The data support the view of a single cardiac progenitor cell population that includes Isl1-expressing cells and which differentiates into the various cardiac lineages during embryonic development in vertebrates but not in other phyla of the animal kingdom.Electronic supplementary materialThe online version of this article (doi:10.1007/s00427-012-0400-1) contains supplementary material, which is available to authorized users.

Focus on Molecules: Math5 and retinal ganglion cells

The LIM-homeodomain transcription factor Islet-1 (Isl1) has been widely used as a marker of different subtypes of neurons in the developing and mature retina of vertebrates. During retinal neurogenesis, early Isl1 expression is detected in the nuclei of neuroblasts that give rise to ganglion, amacrine, bipolar, and horizontal cells. In the mature retina, Isl1 expression is restricted to the nuclei of ganglion cells, cholinergic amacrine cells, ON-bipolar cells, and subpopulations of horizontal cells. Recent studies have explored the functional mechanisms of Isl1 during specification and differentiation of these retinal cell types. Thus, conditional inactivation of Isl1 in the developing mouse retina disrupts retinal function, and also results in optic nerve hypoplasia, marked reductions in mature ganglion, amacrine, and bipolar cells, and a substantial increase in horizontal cells. Furthermore, conditional knockout shows delayed ganglion cell axon growth, ganglion cell axon guidance error, and ganglion cell nerve fiber defasciculation. These data together suggest a possible role for Isl1 in the early differentiation and maintenance of different vertebrate retinal cell types. This review examines whether the expression pattern of Isl1 during vertebrate retinal development is conserved across vertebrate species, and discusses current understanding of the developmental functions of Isl1 in retinogenesis.

Isl1 Is required for multiple aspects of motor neuron development

The LIM homeodomain transcription factor Islet1 (Isl1) is expressed in multiple organs and plays essential roles during embryogenesis. Isl1 is required for the survival and specification of spinal cord motor neurons. Due to early embryonic lethality and loss of motor neurons, the role of Isl1 in other aspects of motor neuron development remains unclear. In this study, we generated Isl1 mutant mouse lines expressing graded doses of Isl1. Our study has revealed essential roles of Isl1 in multiple aspects of motor neuron development, including motor neuron cell body localization, motor column formation and axon growth. In addition, Isl1 is required for survival of cranial ganglia neurons.

Distinct Temporal Requirements for the Homeobox Gene Gsx2 in Specifying Striatal and Olfactory Bulb Neuronal Fates

The homeobox gene Gsx2 (formerly Gsh2) is known to be required for striatal and olfactory bulb neurogenesis; however, its specific role in the specification of these two neuronal subtypes remains unclear. To address this, we have employed a temporally regulated gain-of-function approach in transgenic mice and found that misexpression of Gsx2 at early stages of telencephalic neurogenesis favors the specification of striatal projection neuron identity over that of olfactory bulb interneurons. In contrast, delayed activation of the Gsx2 transgene until later stages exclusively promotes olfactory bulb interneuron identity. In a complementary approach, we have conditionally inactivated Gsx2 in a temporally progressive manner. Unlike germline Gsx2 mutants, which exhibit severe alterations in both striatal and olfactory bulb neurogenesis at birth, the conditional mutants exhibited defects restricted to olfactory bulb interneurons. These results demonstrate that Gsx2 specifies striatal projection neuron and olfactory bulb interneuron identity at distinct time points during telencephalic neurogenesis.

TheDrosophilahomolog of vertebrateIslet1is a key component in early cardiogenesis

In mouse, the LIM-homeodomain transcription factor Islet1 (Isl1) has been shown to demarcate a separate cardiac cell population that is essential for the formation of the right ventricle and the outflow tract of the heart. Whether Isl1 plays a crucial role in the early regulatory network of transcription factors that establishes a cardiac fate in mesodermal cells has not been fully resolved. We have analyzed the role of the Drosophila homolog of Isl1, tailup (tup), in cardiac specification and formation of the dorsal vessel. The early expression of Tup in the cardiac mesoderm suggests that Tup functions in cardiac specification. Indeed, tup mutants are characterized by a reduction of the essential early cardiac transcription factors Tin, Pnr and Dorsocross1-3 (Doc). Conversely, Tup expression depends on each of these cardiac factors, as well as on the early inductive signals Dpp and Wg. Genetic interactions show that tup cooperates with tin, pnr and Doc in heart cell specification. Germ layer-specific loss-of-function and rescue experiments reveal that Tup also functions in the ectoderm to regulate cardiogenesis and implicate the involvement of different LIM-domain-interacting proteins in the mesoderm and ectoderm. Gain-of-function analyses for tup and pnr suggest that a proper balance of these factors is also required for the specification of Eve-expressing pericardial cells. Since tup is required for proper cardiogenesis in an invertebrate organism, we believe it is appropriate to include tup/Isl1 in the core set of ancestral cardiac transcription factors that govern a cardiac fate.