Neurons derived from patients with bipolar disorder divide into intrinsically different sub-populations of neurons, predicting the patients' responsiveness to lithium.

Bipolar disorder (BD) is a progressive psychiatric disorder with more than 3% prevalence worldwide. Affected individuals experience recurrent episodes of depression and mania, disrupting normal life and increasing the risk of suicide greatly. The complexity and genetic heterogeneity of psychiatric disorders have challenged the development of animal and cellular models. We recently reported that hippocampal dentate gyrus (DG) neurons differentiated from induced pluripotent stem cell (iPSC)-derived fibroblasts of BD patients are electrophysiologically hyperexcitable. Here we used iPSCs derived from Epstein-Barr virus-immortalized B-lymphocytes to verify that the hyperexcitability of DG-like neurons is reproduced in this different cohort of patients and cells. Lymphocytes are readily available for research with a large number of banked lines with associated patient clinical description. We used whole-cell patch-clamp recordings of over 460 neurons to characterize neurons derived from control individuals and BD patients. Extensive functional analysis showed that intrinsic cell parameters are very different between the two groups of BD neurons, those derived from lithium (Li)-responsive (LR) patients and those derived from Li-non-responsive (NR) patients, which led us to partition our BD neurons into two sub-populations of cells and suggested two different subdisorders. Training a Naïve Bayes classifier with the electrophysiological features of patients whose responses to Li are known allows for accurate classification with more than 92% success rate for a new patient whose response to Li is unknown. Despite their very different functional profiles, both populations of neurons share a large, fast after-hyperpolarization (AHP). We therefore suggest that the large, fast AHP is a key feature of BD and a main contributor to the fast, sustained spiking abilities of BD neurons. Confirming our previous report with fibroblast-derived DG neurons, chronic Li treatment reduced the hyperexcitability in the lymphoblast-derived LR group but not in the NR group, strengthening the validity and utility of this new human cellular model of BD.

Rhomboid and Star facilitate presentation and processing of the Drosophila TGF-alpha homolog Spitz.

Activation of the Drosophila epidermal growth factor receptor (DER) by the transmembrane ligand, Spitz (Spi), requires two additional transmembrane proteins, Rhomboid and Star. Genetic evidence suggests that Rhomboid and Star facilitate DER signaling by processing membrane-bound Spi (mSpi) to an active, soluble form. To test this model, we use an assay based on Xenopus animal cap explants in which Spi activation of DER is Rhomboid and Star dependent. We show that Spi is on the cell surface but is kept in an inactive state by its cytoplasmic and transmembrane domains; Rhomboid and Star relieve this inhibition, allowing Spi to signal. We show further that Spi is likely to be cleaved within its transmembrane domain. However, a mutant form of mSpi that is not cleaved still signals to DER in a Rhomboid and Star-dependent manner. These results suggest strongly that Rhomboid and Star act primarily to present an active form of Spi to DER, leading secondarily to the processing of Spi into a secreted form.

Expression of Pax-3 in the lateral neural plate is dependent on a Wnt-mediated signal from posterior nonaxial mesoderm.

During early patterning of the vertebrate neuraxis, the expression of the paired-domain transcription factor Pax-3 is induced in the lateral portions of the posterior neural plate via posteriorizing signals emanating from the late organizer and posterior nonaxial mesoderm. Using a dominant-negative approach, we show in explant assays that Pax-3 inductive activities from the organizer do not depend on FGF, retinoic acid, or XWnt-8, either alone or in combination, suggesting that the organizer may produce an unknown posteriorizing factor. However, Pax-3 inductive signals from posterior nonaxial mesoderm are Wnt-dependent. We show that Pax-3 expression in the lateral neural plate expands in XWnt-8-injected embryos and is blocked by dominant-negative XWnt-8. Similarly, we show that the homeodomain transcription factor Msx-1, which like Pax-3 is an early marker of the lateral neural plate, is induced by posterior nonaxial mesoderm and blocked by dominant-negative XWnt-8. Finally, we show that Rohon-Beard primary neurons, a cell type that develops within the lateral neural plate, are also blocked in vivo by dominant-negative Xwnt-8. Together these data support a model in which patterning of the lateral neural plate by Wnt-mediated signals is an early event that establishes a posteriolateral domain, marked by Pax-3 and Msx-1 expression, from which Rohon-Beard cells and neural crest will subsequently arise.

Expression of Pax-3 is initiated in the early neural plate by posteriorizing signals produced by the organizer and by posterior non-axial mesoderm.

Pax-3 is a paired-type homeobox gene that is specifically expressed in the dorsal and posterior neural tube. We have investigated inductive interactions that initiate Pax-3 transcript expression in the early neural plate. We present several lines of evidence that support a model where Pax-3 expression is initiated by signals that posteriorize the neuraxis, and then secondarily restricted dorsally in response to dorsal-ventral patterning signals. First, in chick and Xenopus gastrulae the onset of Pax-3 expression occurs in regions fated to become posterior CNS. Second, Hensen's node and posterior non-axial mesoderm which underlies the neural plate induce Pax-3 expression when combined with presumptive anterior neural plate explants. In contrast, presumptive anterior neural plate explants are not competent to express Pax-3 in response to dorsalizing signals from epidermal-ectoderm. Third, in a heterospecies explant recombinant assay with Xenopus animal caps (ectoderm) as a responding tissue, late, but not early, Hensen's node induces Pax-3 expression. Chick posterior non-axial mesoderm also induces Pax-3, provided that the animal caps are neuralized by treatment with noggin. Finally we show that the putative posteriorizing factors, retinoic acid and bFGF, induce Pax-3 in neuralized animal caps. However, blocking experiments with a dominant-inhibitory FGF receptor and a dominant-inhibitory retinoic acid receptor suggest that Pax-3 inductive activities arising from Hensen's node and posterior non-axial mesoderm do not strictly depend on FGF or retinoic acid.

Regulation of vertebrate neural cell fate by transcription factors.

Evidence that region- and cell-type-specific transcription factors regulate morphogenesis and differentiation of the vertebrate nervous system comes from numerous studies, including descriptions of discrete patterns of expression during neural development and analysis of mutant phenotypes. Recently published works provide insights into the roles of vertebrate transcription factors in regulating the generation of neural precursors, regionalization of the nervous system, and subsequent differentiation of specific cell types within these regions. For instance, misexpression studies in Xenopus embryos show that the newly isolated basic helix-loop-helix protein NeuroD is able to promote neurogenesis, whereas analysis of mouse embryos mutant for the homeobox gene En-1 demonstrates that this transcription factor is required for proper development of the midbrain-hindbrain region. A recent study in chick shows that the combinatorial expression of Islet-1, Lim-1, and two other LIM homeobox genes, Islet-2 and Lim-3, defines subclasses of motor neurons in the spinal cord, supporting a model where combinatorial repertoires of transcription factors may act to generate diverse cell types.

Hairless promotes stable commitment to the sensory organ precursor cell fate by negatively regulating the activity of the Notch signaling pathway.

In Drosophila imaginal discs, the function of the Hairless (H) gene is required at multiple steps during the development of adult sensory organs. Here we report the results of a series of experiments designed to investigate the in vivo role of H in sensory organ precursor (SOP) cell specification. We show that the proneural cluster pattern of proneural gene expression and of transcriptional activation by proneural proteins is established normally in the absence of H activity. By contrast, single cells with the high levels of achaete, scabrous, and neuralized expression characteristic of SOPs almost always fail to appear in H mutant proneural clusters. These results indicate that H is required for a relatively late step in the development of the proneural cluster, namely, the stable commitment of a single cell to the SOP cell fate. We also show that expression of an activated form of the Notch receptor leads to bristle loss with the same cellular basis--failure of SOP determination--as loss of H function and that simultaneous overexpression of H suppresses this effect. Finally, we demonstrate by epistasis experiments that the failure of stable commitment to the SOP fate in H null mutants requires the activity of the genes of the Enhancer of split complex, including groucho. Our results indicate that H promotes SOP determination by antagonizing the activity of the Notch pathway in this cell, thereby protecting it from inhibitory signaling by its neighbors in the proneural cluster. We propose a simple threshold model in which the principal role of H in SOP specification is to translate a quantitative difference in the activity of the Notch pathway (in the SOP versus the non-SOP cells) into a stable binary cell fate decision.

Direct downstream targets of proneural activators in the imaginal disc include genes involved in lateral inhibitory signaling.

In Drosophila imaginal discs, the spatially restricted activities of the achaete (ac) and scute (sc) proteins, which are transcriptional activators of the basic-helix-loop-helix class, define proneural clusters (PNCs) of potential sensory organ precursor (SOP) cells. Here, we report the identification of several genes that are direct downstream targets of ac-sc activation, as judged by the following criteria. The genes are expressed in the PNCs of the wing imaginal disc in an ac-sc-dependent manner; the proximal promoter regions of all of these genes contain one or two high-affinity ac-sc binding sites, which define the novel consensus GCAGGTG(T/G)NNNYY; where tested, these binding sites are required in vivo for PNC expression of promoter-reporter fusion genes. Interestingly, these ac-sc target genes, including Bearded, Enhancer of split m7, Enhancer of split m8, and scabrous, are all known or believed to function in the selection of a single SOP from each PNC, a process mediated by inhibitory cell-cell interactions. Thus, one of the earliest steps in adult peripheral neurogenesis is the direct activation by proneural proteins of genes involved in restricting the expression of the SOP cell fate.

Dpbx, a new homeobox gene closely related to the human proto-oncogene pbx1 molecular structure and developmental expression.

Recently, a new class of homeodomain containing proteins, pbx1, pbx2, and pbx3 has been described. pbx proteins are most closely related to two yeast regulatory proteins, a1 and alpha 2. Here, we identify and characterize the pbx homolog in Drosophila, designated Dpbx. Dpbx is 95% identical to the pbx proteins within the homeodomain and, more remarkably, is 85% to 88% identical within a 201 amino acid region adjacent to the homeodomain. Cytologically, the Dpbx gene is located on the X chromosome at 14A. mRNA expression is both maternal and zygotic and occurs throughout the life cycle. Prior to full germband retraction, Dpbx is rather ubiquitously present and variations are minor. The most notable feature of Dpbx expression is that after germband retraction, high levels of Dpbx are observed in the anterior portion of the ventral nerve cord.

The Drosophila gene Hairless encodes a novel basic protein that controls alternative cell fates in adult sensory organ development.

The mechanosensory bristles of adult Drosophila are composed of four cells that, in most cases, are progeny of a single sensory organ precursor (SOP) cell. Two sister cells in this lineage, the trichogen and tormogen, produce the external shaft and socket of the bristle, respectively. Loss-of-function mutations of Hairless (H) confer two distinct mutant phenotypes on adult bristles. The bristle loss phenotype results from the failure to specify and/or execute the SOP cell fate; the double socket phenotype results from the transformation of the trichogen (shaft) cell into a second tormogen (socket) cell. We have found that the H gene encodes a novel basic protein with a predicted molecular mass of 109 kD. Basal levels of expression of a transgene (P[Hs-H]) in which the H protein-coding region is under the control of the Hsp70 promoter are sufficient to provide full rescue of H mutant phenotypes. Heat shock treatment of P[Hs-H] transgenic animals as late larvae and early pupae produces a tormogen-to-trichogen (double shaft) cell fate transformation, as well as bristle multiplication and loss phenotypes very similar to those caused by loss-of-function mutations in the neurogenic gene Notch. Our results indicate that the SOP cell fate requires H to antagonize the activity of the neurogenic group of genes and that the expression of distinct cell fates by the trichogen/tormogen sister cell pair depends on an asymmetry in their levels of H+ activity or in their thresholds for response to H.

Hairless is required for the development of adult sensory organ precursor cells in Drosophila.

Reduction of the wild-type activity of the gene Hairless (H) results in two major phenotypic effects on the mechanosensory bristles of adult Drosophila. Bristles are either 'lost' (i.e. the shaft and socket fail to appear) or they exhibit a 'double socket' phenotype, in which the shaft is apparently transformed into a second socket. Analysis of the phenotypes conferred by a series of H mutant genotypes demonstrates (1) that different sensilla exhibit different patterns of response to decreasing levels of H+ function, and (2) that the 'bristle loss' phenotype results from greater loss of H+ function than the 'double socket' phenotype. The systematic study of H allelic combinations enabled us to identify genotypes that reliably produce specific mutant defects in particular positions on the bodies of adult flies. This permitted us to investigate the cellular development of sensilla in these same positions in larvae and pupae and thereby establish the developmental basis for the mutant phenotypes. We have found that H is required for at least two steps of adult sensillum development. In positions where 'double socket' microchaetes appear on the notum of H mutant flies, sensillum precursor cells are present in the developing pupa and divide normally, but their progeny adopt an aberrant spatial arrangement and fail to differentiate correctly. In regions of the notum exhibiting 'bristle loss' in adult H mutants, we were unable at the appropriate stages of development to detect sensillum-specific cell types, the precursor cell divisions that generate them, or the primary precursor cells themselves. Thus, the H 'bristle loss' phenotype appears to reflect a very early defect in sensillum development, namely the failure to specify and/or execute the sensory organ precursor cell fate. This finding indicates that H is one of a small number of identified genes for which the loss-of-function phenotype is the failure of sensillum precursor cell development.

Transcript and sequence analysis of a 5.1 kb contiguous fragment of Dictyostelium discoideum plasmid Ddp1 that contains the origin of replication and codes for several transcripts.

The sequence of a 5.1 kb contiguous fragment of the Dictyostelium plasmid Ddp1 is presented. This fragment contains three long open reading frames which correspond to the developmentally regulated and cAMP-inducible transcript d-5, the growth phase specific transcript g-1 and the three overlapping transcripts g-2, g-3 and d-4. The transcripts that originate from Ddp1 resemble chromosomally-encoded ones: they are products of RNA polymerase II, are polyadenylated and accumulate at different time points during Dictyostelium development. The presented nucleotide sequence encompasses a 2,033 bp HindIII fragment that had previously been shown to carry all the information necessary for extrachromosomal replication. None of the identified genes is completely contained within this HindIII fragment.

Human muscle glycogen synthase cDNA sequence: a negatively charged protein with an asymmetric charge distribution.

The cDNA for human muscle glycogen synthase encodes a protein of 737 amino acids. The primary structure of glycogen synthase is not related either to bacterial glycogen synthase or to any glycogen phosphorylase. All nine of the serines that are phosphorylated in the rabbit muscle enzyme in vivo are conserved in the human muscle sequence. The amino- and carboxyl-terminal fragments, which contain all the phosphorylation sites, are very negatively charged. Overall the unphosphorylated protein has a charge of -13, while the fully phosphorylated inactive protein has a net charge of -31. The importance of the asymmetrical charge distribution is discussed.

The watermelon mitochondrial URF-1 gene: evidence for a complex structure.

We have cloned and sequenced a fragment of watermelon mitochondrial DNA (mtDNA) which contains a gene homologous to mitochondrial URF-1 (Unidentified Reading Frame-1) of vertebrates, Drosophila yakuba and Aspergillus nidulans. URF-1 is thought to encode a component of the respiratory chain NADH dehydrogenase. Two coding regions in the watermelon gene are separated by approximately 1,450 bp of untranslatable DNA. These two exons encode the central portions of URF-1, and are highly conserved. We postulate that three additional exons, selected by their map location and amino acid homology to other URF-1 sequences, encode the remainder of the polypeptide. This is the first description of a plant mitochondrial gene with multiple introns.