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Palliative care has made great strides in improving the lives of people living with serious illness, with an empirical premise for increasing quality, and sometimes quantity of life. Yet in some cases, there exist gaps that impede the ability of palliative care clinicians to truly advocate, procure, and provide the comprehensive services needed for patients, family caregivers, and communities, particularly in the contexts of caring for marginalized populations and working in under-resourced practice settings. The end-of-life doula role has emerged over the last decade and the availability of trained doulas in the community has burgeoned. An end-of-life doula is a nonmedical, holistic support person who provides education, guidance, emotional, spiritual, and practical support to persons and families navigating serious and terminal illness, ideally early in the disease process, throughout the time surrounding death, and during bereavement. A pervasive Western culture of avoiding the subject of death means that we, as a society, often do not know or remember how to navigate the journey of end of life in a way that is caring, compassionate, skilled, holistic, and centered on the needs and worldview of the dying one. The ten tips provided here can guide palliative care clinicians to leverage collaboration with trusted, community-based end-of-life doulas to ensure comprehensive and people-centered palliative care.

Hormones regulate behavior either through activational effects that facilitate the acute expression of specific behaviors or through organizational effects that shape the development of the nervous system thereby altering adult behavior. Much research has implicated the neuropeptide oxytocin (OXT) in acute modulation of various aspects of social behaviors across vertebrate species, and OXT signaling is associated with the developmental social deficits observed in autism spectrum disorders (ASDs); however, little is known about the role of OXT in the neurodevelopment of the social brain. We show that perturbation of OXT neurons during early zebrafish development led to a loss of dopaminergic neurons, associated with visual processing and reward, and blunted the neuronal response to social stimuli in the adult brain. Ultimately, adult fish whose OXT neurons were ablated in early life, displayed altered functional connectivity within social decision-making brain nuclei both in naive state and in response to social stimulus and became less social. We propose that OXT neurons have an organizational role, namely, to shape forebrain neuroarchitecture during development and to acquire an affiliative response toward conspecifics. SIGNIFICANCE STATEMENT Social behavior is developed over the lifetime of an organism and the neuropeptide oxytocin (OXT) modulates social behaviors across vertebrate species, and is associated with neuro-developmental social deficits such as autism. However, whether OXT plays a role in the developmental maturation of neural systems that are necessary for social behavior remains poorly explored. We show that proper behavioral and neural response to social stimuli depends on a developmental process orchestrated by OXT neurons. Animals whose OXT system is ablated in early life show blunted neuronal and behavioral responses to social stimuli as well as wide ranging disruptions in the functional connectivity of the social brain. We provide a window into the mechanisms underlying OXT-dependent developmental processes that implement adult sociality., (Copyright © 2021 the authors.)

Recent evidence suggests neurogenesis is on-going throughout life but the relevance of these findings for neurodegenerative disorders such as Parkinson's disease (PD) is poorly understood. Biallelic PINK1 mutations cause early onset, Mendelian inherited PD. We studied the effect of PINK1 deficiency on adult neurogenesis of dopaminergic (DA) neurons in two complementary model systems. Zebrafish are a widely-used model to study neurogenesis in development and through adulthood. Using EdU analyses and lineage-tracing studies, we first demonstrate that a subset of ascending DA neurons and adjacent local-projecting DA neurons are each generated into adulthood in wild type zebrafish at a rate that decreases with age. Pink1-deficiency impedes DA neurogenesis in these populations, most significantly in early adult life. Pink1 already exerts an early effect on Th1 + progenitor cells rather than on differentiated DA neurons only. In addition, we investigate the effect of PINK1 deficiency in a human isogenic organoid model. Global neuronal differentiation in PINK1-deficient organoids and isogenic controls is similar, but PINK1-deficient organoids display impeded DA neurogenesis. The observation of impaired adult dopaminergic neurogenesis in Pink1 deficiency in two complementing model systems may have significant consequences for future therapeutic approaches in human PD patients with biallelic PINK1 mutations.

Oxytocin-like peptides have been implicated in the regulation of a wide range of social behaviors across taxa. On the other hand, the social environment, which is composed of conspecifics that may vary in their genotypes, also influences social behavior, creating the possibility for indirect genetic effects. Here, we used a zebrafish oxytocin receptor knockout line to investigate how the genotypic composition of the social environment (G s ) interacts with the oxytocin genotype of the focal individual (G i ) in the regulation of its social behavior. For this purpose, we have raised wild-type or knock-out zebrafish in either wild-type or knock-out shoals and tested different components of social behavior in adults. G i xG s effects were detected in some behaviors, highlighting the need to control for G i xG s effects when interpreting results of experiments using genetically modified animals, since the genotypic composition of the social environment can either rescue or promote phenotypes associated with specific genes., Competing Interests: DR, AN, MT, SA, JB, GL, RO No competing interests declared, (© 2020, Ribeiro et al.)

Social living animals need to recognize the presence of conspecifics in the environment in order to engage in adaptive social interactions. Social cues can be detected through different sensory modalities, including vision. Two main visual features can convey information about the presence of conspecifics: body form and biological motion (BM). Given the role that oxytocin plays in social behavior regulation across vertebrates, particularly in the salience and reward values of social stimuli, we hypothesized that it may also be involved in the modulation of perceptual mechanisms for conspecific detection. Here, using videoplaybacks, we assessed the role of conspecific form and BM in zebrafish social affiliation, and how oxytocin regulates the perception of these cues. We demonstrated that while each visual cue is important for social attraction, BM promotes a higher fish engagement than the static conspecific form alone. Moreover, using a mutant line for one of the two oxytocin receptors, we show that oxytocin signaling is involved in the regulation of BM detection but not conspecific form recognition. In summary, our results indicate that, apart from oxytocin role in the regulation of social behaviors through its effect on higher-order cognitive mechanisms, it may regulate social behavior by modulating very basic perceptual mechanisms underlying the detection of socially-relevant cues.

The neurohypophysis (NH), located at the posterior lobe of the pituitary, is a major neuroendocrine tissue, which mediates osmotic balance, blood pressure, reproduction, and lactation by means of releasing the neurohormones oxytocin (OXT) and arginine-vasopressin (AVP) from the brain into the peripheral blood circulation. The major cellular components of the NH are hypothalamic axonal termini, fenestrated endothelia and pituicytes, the resident astroglia. However, despite the physiological importance of the NH, the exact molecular signature defining neurohypophyseal cell types and in particular the pituicytes, remains unclear. Using single-cell RNA sequencing (scRNA-Seq), we captured seven distinct cell types in the NH and intermediate lobe (IL) of adult male mouse. We revealed novel pituicyte markers showing higher specificity than previously reported. Bioinformatics analysis demonstrated that pituicyte is an astrocytic cell type whose transcriptome resembles that of tanycyte. Single molecule in situ hybridization revealed spatial organization of the major cell types implying intercellular communications. We present a comprehensive molecular and cellular characterization of neurohypophyseal cell types serving as a valuable resource for further functional research., (Copyright © 2020 Chen et al.)

To maintain body homeostasis, endocrine systems must detect and integrate blood-borne peripheral signals. This is mediated by fenestrae, specialized permeable pores in the endothelial membrane. Plasmalemma vesicle-associated protein (Plvap) is located in the fenestral diaphragm and is thought to play a role in the passage of proteins through the fenestrae. However, this suggested function has yet to be demonstrated directly. We studied the development of fenestrated capillaries in the hypophysis, a major neuroendocrine interface between the blood and brain. Using a transgenic biosensor to visualize the vascular excretion of the genetically tagged plasma protein DBP-EGFP, we show that the developmental acquisition of vascular permeability coincides with differential expression of zebrafish plvap orthologs in the hypophysis versus brain. Ultrastructural analysis revealed that plvapb mutants display deficiencies in fenestral diaphragms and increased density of hypophyseal fenestrae. Measurements of DBP-EGFP extravasation in plvapb mutants provided direct proof that Plvap limits the rate of blood-borne protein passage through fenestrated endothelia. We present the regulatory role of Plvap in the development of blood-borne protein detection machinery at a neuroendocrine interface through which hormones are released to the general circulation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The regulation of neuropeptide level at the site of release is essential for proper neurophysiological functions. We focused on a prominent neuropeptide, oxytocin (OXT) in the zebrafish as an in vivo model to visualize and quantify OXT content at the resolution of a single synapse. We found that OXT-loaded synapses were enriched with polymerized actin. Perturbation of actin filaments by either cytochalasin-D or conditional Cofilin expression resulted in decreased synaptic OXT levels. Genetic loss of robo2 or slit3 displayed decreased synaptic OXT content and robo2 mutants displayed reduced mobility of the actin probe Lifeact-EGFP in OXT synapses. Using a novel transgenic reporter allowing real-time monitoring of OXT-loaded vesicles, we show that robo2 mutants display slower rate of vesicles accumulation. OXT-specific expression of dominant-negative Cdc42, which is a key regulator of actin dynamics and a downstream effector of Robo2, led to a dose-dependent increase in OXT content in WT, and a dampened effect in robo2 mutants. Our results link Slit3-Robo2-Cdc42, which controls local actin dynamics, with the maintenance of synaptic neuropeptide levels., Competing Interests: SA, JB, MG, LG, RR, TD, ES, GL No competing interests declared, (© 2019, Anbalagan et al.)

The hypothalamo-neurohypophyseal system (HNS) regulates homeostasis through the passage of neurohormones and blood-borne proteins via permeable blood capillaries that lack the blood-brain barrier (BBB). Why neurohypophyseal capillaries become permeable while the neighboring vasculature of the brain forms BBB remains unclear. We show that pituicytes, the resident astroglial cells of the neurohypophysis, express genes that are associated with BBB breakdown during neuroinflammation. Pituicyte-enriched factors provide a local microenvironment that instructs a permeable neurovascular conduit. Thus, genetic and pharmacological perturbations of Vegfa and Tgfβ3 affected HNS vascular morphogenesis and permeability and impaired the expression of the fenestral marker plvap. The anti-inflammatory agent dexamethasone decreased HNS permeability and downregulated the pituicyte-specific cyp26b gene, encoding a retinoic acid catabolic enzyme. Inhibition of Cyp26b activity led to upregulation of tight junction protein Claudin-5 and decreased permeability. We conclude that pituicyte-derived factors regulate the "decision" of endothelial cells to adopt a permeable endothelial fate instead of forming a BBB., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Presynaptic cGMP-gated ion (CNG) channels positively or negatively modulate neurotransmitter secretion as well as the strength of synaptic transmission. Zebrafish cGMP-gated ion channel, CNGA2a (a.k.a. CNGA5), was previously reported to be specifically enriched in synaptic terminals of zebrafish oxytocin (OXT) neurons. This conclusion was based on immunoreactivity of a monoclonal antibody (mAb) clone L55/54, which was directed against the carboxy terminal tail of the CNGA2a. To study the role of CNGA2a in oxytocin neurons function, we generated zebrafish mutants of cnga2a, cnga2b and oxt genes using clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing. We show that mAb L55/54 specifically recognizes CNGA2a protein when expressed in heterologous cell culture system. Surprisingly, anti-CNGA2a immunoreactivity was not eliminated following knockout of either cnga2a, cnga2b or both. However, knockout of oxt resulted in total loss of anti-CNGA2a mAb immunoreactivity despite the lack of sequence and structural similarities between OXT and CNGA2a proteins. Our results provide a noteworthy lesson of differences in antibody immunoreactivity, which could only be revealed using specific genetic tools.

Proper response to stress and social stimuli depends on orchestrated development of hypothalamic neuronal circuits. Here we address the effects of the developmental transcription factor orthopedia (Otp) on hypothalamic development and function. We show that developmental mutations in the zebrafish paralogous gene otpa but not otpb affect both stress response and social preference. These behavioral phenotypes were associated with developmental alterations in oxytocinergic (OXT) neurons. Thus, otpa and otpb differentially regulate neuropeptide switching in a newly identified subset of OXT neurons that co-express the corticotropin-releasing hormone (CRH). Single-cell analysis revealed that these neurons project mostly to the hindbrain and spinal cord. Ablation of this neuronal subset specifically reduced adult social preference without affecting stress behavior, thereby uncoupling the contribution of a specific OXT cluster to social behavior from the general otpa -/- deficits. Our findings reveal a new role for Otp in controlling developmental neuropeptide balance in a discrete OXT circuit whose disrupted development affects social behavior., Competing Interests: The authors declare that no competing interests exist.

The zebrafish has become a model of choice in fundamental and applied life sciences and is widely used in various fields of biomedical research as a human disease model for cancer, metabolic and neurodegenerative diseases, and regenerative medicine. The transparency of the zebrafish embryo allows real-time visualization of the development and morphogenesis of practically all of its tissues and organs. Zebrafish are amenable to genetic manipulation, for which innovative genetic and molecular techniques are constantly being introduced. These include the study of gene function and regulation using gene knockdown, knockout and knock-in, as well as transgenesis and tissue-specific genetic perturbations. Complementing this genetic toolbox, the zebrafish exhibits measurable behavioral and hormonal responses already at the larval stages, providing a viable vertebrate animal model for high-throughput drug screening and chemical genetics. With the available tools of the genomic era and the abundance of disease-associated human genes yet to be explored, the zebrafish model is becoming the preferred choice in many studies. Its advantages and potential are being increasingly recognized within the Israeli scientific community, and its use as a model system for basic and applied science has expanded in Israel in recent years. Since the first zebrafish-focused laboratory was introduced at Tel Aviv University 16 years ago, seven more zebrafish-centric research groups have been established, along with more than two dozen academic research groups and three bio-medical companies that are now utilizing this model.

Alternative splicing of the precursor mRNA encoding for the neuropeptide receptor PAC1/ADCYAP1R1 generates multiple protein products that exhibit pleiotropic activities. Recent studies in mammals and zebrafish have implicated some of these splice isoforms in control of both cellular and body homeostasis. Here, we review the regulation of PAC1 splice variants and their underlying signal transduction and physiological processes in the nervous system.

Regulation of corticotropin-releasing hormone (CRH) activity is critical for the animal's adaptation to stressful challenges, and its dysregulation is associated with psychiatric disorders in humans. However, the molecular mechanism underlying this transcriptional response to stress is not well understood. Using various stress paradigms in mouse and zebrafish, we show that the hypothalamic transcription factor Orthopedia modulates the expression of CRH as well as the splicing factor Ataxin 2-Binding Protein-1 (A2BP1/Rbfox-1). We further show that the G protein coupled receptor PAC1, which is a known A2BP1/Rbfox-1 splicing target and an important mediator of CRH activity, is alternatively spliced in response to a stressful challenge. The generation of PAC1-hop messenger RNA isoform by alternative splicing is required for termination of CRH transcription, normal activation of the hypothalamic-pituitary-adrenal axis and adaptive anxiety-like behavior. Our study identifies an evolutionarily conserved biochemical pathway that modulates the neuronal adaptation to stress through transcriptional activation and alternative splicing., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The hypothalamo-neurohypophyseal system (HNS) is the neurovascular structure through which the hypothalamic neuropeptides oxytocin and arginine-vasopressin exit the brain into the bloodstream, where they go on to affect peripheral physiology. Here, we investigate the molecular cues that regulate the neurovascular contact between hypothalamic axons and neurohypophyseal capillaries of the zebrafish. We developed a transgenic system in which both hypothalamic axons and neurohypophyseal vasculature can be analyzed in vivo. We identified the cellular organization of the zebrafish HNS as well as the dynamic processes that contribute to formation of the HNS neurovascular interface. We show that formation of this interface is regulated during development by local release of oxytocin, which affects endothelial morphogenesis. This cell communication process is essential for the establishment of a tight axovasal interface between the neurons and blood vessels of the HNS. We present a unique example of axons affecting endothelial morphogenesis through secretion of a neuropeptide., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We have explored the effects of robust neural plate patterning signals, such as canonical Wnt, on the differentiation and configuration of neuronal subtypes in the zebrafish diencephalon at single-cell resolution. Surprisingly, perturbation of Wnt signaling did not have an overall effect on the specification of diencephalic fates, but selectively affected the number of dopaminergic (DA) neurons. We identified the DA progenitor zone in the diencephalic anlage of the neural plate using a two-photon-based uncaging method and showed that the number of non-DA neurons derived from this progenitor zone is not altered by Wnt attenuation. Using birthdating analysis, we determined the timing of the last cell division of DA progenitors and revealed that the change in DA cell number following Wnt inhibition is not due to changes in cell cycle exit kinetics. Conditional inhibition of Wnt and of cell proliferation demonstrated that Wnt restricts the number of DA progenitors during a window of plasticity, which occurs at primary neurogenesis. Finally, we demonstrated that Wnt8b is a modulator of DA cell number that acts through the Fz8a (Fzd8a) receptor and its downstream effector Lef1, and which requires the activity of the Fezl (Fezf2) transcription factor for this process. Our data show that the differential response of distinct neuronal populations to the Wnt signal is not a simple interpretation of their relative anteroposterior position. This study also shows, for the first time, that diencephalic DA population size is modulated inside the neural plate much earlier than expected, concomitant with Wnt-mediated regional patterning events.

In the developing hypothalamus, a variety of neurons are generated adjacent to each other in a highly coordinated, but poorly understood process. A critical question that remains unanswered is how coordinated development of multiple neuronal types is achieved in this relatively narrow anatomical region. We focus on dopaminergic (DA) and oxytocinergic (OT) neurons as a paradigm for development of two prominent hypothalamic cell types. We report that the development of DA and OT-like neurons in the zebrafish is orchestrated by two novel pathways that regulate the expression of the homeodomain-containing protein Orthopedia (Otp), a key determinant of hypothalamic neural differentiation. Genetic analysis showed that the G-protein-coupled receptor PAC1 and the zinc finger-containing transcription factor Fezl act upstream to Otp. In vivo and in vitro experiments demonstrated that Fezl and PAC1 regulate Otp at the transcriptional and the post-transcriptional levels, respectively. Our data reveal a new genetic network controlling the specification of hypothalamic neurons in vertebrates, and places Otp as a critical determinant underlying Fezl- and PAC1-mediated differentiation.

Nucleolar segregation is observed under some physiological conditions of transcriptional arrest. This process can be mimicked by transcriptional arrest after actinomycin D treatment leading to the segregation of nucleolar components and the formation of unique structures termed nucleolar caps surrounding a central body. These nucleolar caps have been proposed to arise from the segregation of nucleolar components. We show that contrary to prevailing notion, a group of nucleoplasmic proteins, mostly RNA binding proteins, relocalized from the nucleoplasm to a specific nucleolar cap during transcriptional inhibition. For instance, an exclusively nucleoplasmic protein, the splicing factor PSF, localized to nucleolar caps under these conditions. This structure also contained pre-rRNA transcripts, but other caps contained either nucleolar proteins, PML, or Cajal body proteins and in addition nucleolar or Cajal body RNAs. In contrast to the capping of the nucleoplasmic components, nucleolar granular component proteins dispersed into the nucleoplasm, although at least two (p14/ARF and MRP RNA) were retained in the central body. The nucleolar caps are dynamic structures as determined using photobleaching and require energy for their formation. These findings demonstrate that the process of nucleolar segregation and capping involves energy-dependent repositioning of nuclear proteins and RNAs and emphasize the dynamic characteristics of nuclear domain formation in response to cellular stress.

Transcriptional repression of E-cadherin, characteristic of epithelial to mesenchymal transition, is often found also during tumor cell invasion. At metastases, migratory fibroblasts sometimes revert to an epithelial phenotype, by a process involving regulation of the E-cadherin-beta-catenin complex. We investigated the molecular basis of this regulation, using human colon cancer cells with aberrantly activated beta-catenin signaling. Sparse cultures mimicked invasive tumor cells, displaying low levels of E-cadherin due to transcriptional repression of E-cadherin by Slug. Slug was induced by beta-catenin signaling and, independently, by ERK. Dense cultures resembled a differentiated epithelium with high levels of E-cadherin and beta-catenin in adherens junctions. In such cells, beta-catenin signaling, ErbB-1/2 levels, and ERK activation were reduced and Slug was undetectable. Disruption of E-cadherin-mediated contacts resulted in nuclear localization and signaling by beta-catenin, induction of Slug and inhibition of E-cadherin transcription, without changes in ErbB-1/2 and ERK activation. This autoregulation of E-cadherin by cell-cell adhesion involving Slug, beta-catenin and ERK could be important in tumorigenesis.

During animal development, regions of the embryo become committed to position-specific identities, which are determined by spatially restricted expression of Hox/homeotic genes. This expression pattern is initially established by the activity of the segmentation genes and is subsequently maintained during the proliferative stage through the action of transcription factors encoded by the trithorax (trx) and Polycomb (Pc) groups of genes. trithorax (trx)and ash1 (absent, small, or homeotic 1) are members of the Drosophila trx group. Their products are associated with chromosomes and are believed to activate transcription of target genes through chromatin remodeling. Recently, we reported molecular studies indicating that TRX and ASH1 proteins act in concert to bind simultaneously to response elements located at close proximity within the same set of target genes. Extension of these and other studies to mammalian systems required identification and cloning of the mammalian homologue of ash1 (the mammalian homologue of trx, ALL-1, was previously cloned). We have identified a human expressed sequence tag (EST) clone with similarity to the SET domain of Drosophila ASH1, and used it to clone the human gene. huASH1 resides at chromosomal band 1q21. The gene is expressed in multiple tissues as an approximately 10.5-kb transcript and encodes a protein of 2962 residues. The protein contains a SET domain, a PHD finger, four AT hooks, and a region with homology to the bromodomain. The last region is not present in Drosophila ASH1, and as such might confer to the human protein a unique additional function. Using several anti-huASH1 Ab for immunostaining of cultured cells, we found that the protein is distributed in intranuclear speckles, and unexpectedly also in intercellular junctions. Double-immunofluorescence labeling of huASH1 and several junctional proteins localized the huASH1 protein into tight junctions. The significance of huASH1 dual location is discussed. In particular, we consider the possibility that translocation of the protein between the junctional membrane and the nucleus may be involved in adhesion-mediated signaling.

Recent reports have shown that estradiol increases the hypophagic effect of exogenous cholecystokinin-octapeptide (CCK). CCK is known to increase the expression of Fos, a marker of neuronal activation, in specific medullary and hypothalamic nuclei. The present experiment tested the hypothesis that as estradiol enhances that behavioral effects of CCK, there is a parallel amplification of CCK-induced Fos expression. Instead, estradiol pretreatment reduced the level of CCK-induced Fos expression in the nucleus of the solitary tract (NTS) and in the medial parvocellular region of the hypothalamic paraventricular nucleus (PVN). The number of Fos-containing cells in the supraoptic nucleus and other regions of the PVN were not affected by estradiol pretreatment. The results are consistent with the hypothesis that the NTS and/or the PVN may mediate the estrogen-induced increased sensitivity to peripheral inhibitory signals in the control of food intake.

The ALL-1 gene was discovered by virtue of its involvement in human acute leukemia. Its Drosophila homolog trithorax (trx) is a member of the trx-Polycomb gene family, which maintains correct spatial expression of the Antennapedia and bithorax complexes during embryogenesis. The C-terminal SET domain of ALL-1 and TRITHORAX (TRX) is a 150-aa motif, highly conserved during evolution. We performed yeast two hybrid screening of Drosophila cDNA library and detected interaction between a TRX polypeptide spanning SET and the SNR1 protein. SNR1 is a product of snr1, which is classified as a trx group gene. We found parallel interaction in yeast between the SET domain of ALL-1 and the human homolog of SNR1, INI1 (hSNF5). These results were confirmed by in vitro binding studies and by demonstrating coimmunoprecipitation of the proteins from cultured cells and/or transgenic flies. Epitope-tagged SNR1 was detected at discrete sites on larval salivary gland polytene chromosomes, and these sites colocalized with around one-half of TRX binding sites. Because SNR1 and INI1 are constituents of the SWI/SNF complex, which acts to remodel chromatin and consequently to activate transcription, the interactions we observed suggest a mechanism by which the SWI/SNF complex is recruited to ALL-1/trx targets through physical interactions between the C-terminal domains of ALL-1 and TRX and INI1/SNR1.

ALL1, the human homologue of Drosophila trithorax, is directly involved in human acute leukemias associated with abnormalities at 11q23. Using the differential display method, we isolated a gene that is down-regulated in All1 double-knockout mouse embryonic stem (ES) cells. The gene, designated ARP1 (also termed RIEG, Ptx2, or Otlx2), is a member of a family of homeotic genes containing a short motif shared with several homeobox genes. Using a bacterially synthesized All1 polypeptide encompassing the AT-hook motifs, we identified a 0.5-kb ARP1 DNA fragment that preferentially bound to the polypeptide. Within this DNA, a region of approximately 100 bp was protected by the polypeptide from digestion with ExoIII and DNase I. Whole-mount in situ hybridization to early mouse embryos of 9.5-10.5 days indicated a complex pattern of Arp1 expression spatially overlapping with the expression of All1. Although the ARP1 gene is expressed strongly in bone marrow cells, no transcripts were detected in six leukemia cell lines with 11q23 translocations. These results suggest that ARP1 is up-regulated by the All1 protein, possibly through direct interaction with an upstream DNA sequence of the former. The results are also consistent with the suggestion that ALL1 chimeric proteins resulting from 11q23 abnormalities act in a dominant negative fashion.

The ALL-1 gene positioned at 11q23 is directly involved in human acute leukemia either through a variety of chromosome translocations or by partial tandem duplications. ALL-1 is the human homologue of Drosophila trithorax which plays a critical role in maintaining proper spatial and temporal expression of the Antennapedia-bithorax homeotic genes determining the fruit fly's body pattern. Utilizing specific antibodies, we found that the ALL-1 protein distributes in cultured cells in a nuclear punctate pattern. Several chimeric ALL-1 proteins encoded by products of the chromosome translocations and expressed in transfected cells showed similar speckles. Dissection of the ALL-1 protein identified within its approximately 1,100 N-terminal residues three polypeptides directing nuclear localization and at least two main domains conferring distribution in dots. The latter spanned two short sequences conserved with TRITHORAX. Enforced nuclear expression of other domains of ALL-1, such as the PHD (zinc) fingers and the SET motif, resulted in uniform nonpunctate patterns. This indicates that positioning of the ALL-1 protein in subnuclear structures is mediated via interactions of ALL-1 N-terminal elements. We suggest that the speckles represent protein complexes which contain multiple copies of the ALL-1 protein and are positioned at ALL-1 target sites on the chromatin. Therefore, the role of the N-terminal portion of ALL-1 is to direct the protein to its target genes.

Receptor dimerization is ubiquitous to the action of all receptor tyrosine kinases, and in the case of dimeric ligands, such as the stem cell factor (SCF), it was attributed to ligand bivalency. However, by using a dimerization-inhibitory monoclonal antibody to the SCF receptor, we confined a putative dimerization site to the nonstandard fourth immunoglobulin-like domain of the receptor. Deletion of this domain not only abolished ligand-induced dimerization and completely inhibited signal transduction, but also provided insights into the mechanism of the coupling of ligand binding to dimer formation. These results identify an intrinsic receptor dimerization site and suggest that similar sites may exist in other receptors.

The mast cell growth factor (MGF) affects migration, proliferation and differentiation of erythroid and myeloid progenitor cells by binding to a transmembrane receptor tyrosine kinase encoded by the c-Kit proto-oncogene. By using MGF-dependent human myeloid cell lines (M-07e and TF-1), here we show that a Kit-related 100 kDa protein is associated with the cell but it undergoes release into the medium upon treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C. Immunological analysis with a series of antibodies to Kit indicated that the released protein (p100Kit) contains the whole glycosylated extracellular portion of the transmembrane Kit protein (p145Kit). The secreted protein retained the ability to specifically bind MGF. Moreover, p100Kit was able to block the mitogenic effect of MGF on cultured M-07e cells, suggesting that the soluble protein may function as a physiological antagonist of MGF.

Despite extensive research on the molecular mechanisms of signal transduction by growth factors and their oncogenic receptor tyrosine kinases, the physiological relevance of these pathways, especially in mammals, remains largely unknown. A unique exception is the Steel factor (SLF) and its c-kit-encoded receptor, because many natural germ line mutations of both the ligand and the receptor exist in mice. The protooncogene c-kit encodes a cell surface receptor that belongs to the immunoglobulin gene family and carries an intrinsic tyrosine kinase activity in its cytoplasmic portion. The precursor of the Kit ligand, SLF, is also a transmembrane protein that exists as a soluble factor as well as a cell surface protein. The interaction of Kit with SLF leads to receptor dimerization, kinase activation, and tyrosine phosphorylation of cytoplasmic proteins that contain Src homology 2 motifs. Various mutations in Kit and SLF result in a defective signaling pathway and underly the complex phenotypes of W and Sl mice, respectively. The early development of at least four cell lineages is affected. These are erythrocytes, melanocytes, germ cells, and mast cells. Correlation between the behavior of these lineages and specific mutations uncovered interesting physiological aspects of the mechanism of signal transduction by a polypeptide growth factor. These include the different degrees of severity of affected lineages, indications for distinct functions during early embryonic development and at late phases, the significance of synergy between a growth factor and lymphokines, the interaction between mutant and wild-type proteins in heterozygous animals, and the possibility that a surface-anchored ligand may act differently than a soluble factor. Predictably, the lessons learned with Kit and Sl mice will be widely relevant to other pairs of ligands and receptors that control the function of different cell lineages and physiological processes.

Binding of the Steel factor (SLF) to the product of the c-kit proto-oncogene stimulates the receptor's intrinsic tyrosine kinase that phosphorylates a set of cytoplasmic signaling molecules. Germ-line mutations in the genes that encode the receptor or the ligand result in remarkably similar phenotypes that affect melanogenesis, erythropoiesis and gametogenesis in mice. We concentrated on the initial events of the signal transduction pathway that underlies these processes. The extracellular portion of Kit is comprised of five immunoglobulin-(Ig)-like domains. Ligand binding to this domain induces rapid and extensive dimerization of the receptor molecules in a mechanism that involves monovalent binding of the dimeric ligand, followed by an increase in receptors' affinity and gradual stabilization of the dimers. It thus appears that Kit has at least two functions: ligand binding and ligand-induced receptor dimerization, in addition to the kinase activity. Both functions are independent of the transmembrane and cytoplasmic domains, as a recombinant soluble ectodomain retained high affinity to SLF and ligand-dependent dimerization. In order to correlate these functions with specific structures, we employed ligand-competitive monoclonal antibodies, soluble deletion mutants of the ectodomain and chimeric human-mouse Kit proteins. These approaches indicated that the N-terminal three Ig-like domains constitute the binding site, whose core is the second domain. Further experiments suggested that a putative dimerization site is distinct from the binding cleft and may be located on the fourth Ig-like domain.

The binding of the stem cell factor (SCF) to the c-kit-encoded receptor tyrosine kinase stimulates a variety of biochemical responses that culminate in cellular proliferation, migration, or survival. The extracellular domain of p145kit consists of five immunoglobulin-like domains. To confine the ligand binding site to this portion of the receptor we generated a panel of murine monoclonal antibodies (mAbs) to the Kit protein and identified two mAbs that efficiently displaced receptor-bound SCF and also inhibited proliferation of SCF-dependent human megakaryocytes. To map the epitopes of these mAbs we constructed and expressed soluble portions of the extracellular domain of Kit, which included either the two amino-terminal Ig-like domains (denoted Kit 1-2), three Ig-like domains (Kit 1-2-3), or the entire extracellular portion (Kit-X). All three recombinant proteins were recognized by the ligand inhibitory mAbs, suggesting that the SCF binding site resides in the amino-terminal half of the ecto-domain. Consistent with this conclusion, all of the soluble proteins inhibited SCF binding to Kit-expressing cells, and they also underwent specific covalent cross-linking to the radiolabeled ligand. However, whereas Kit 1-2-3 and Kit-X displayed comparable ligand affinities, deletion of the third Ig-like domain, in Kit 1-2, involved significant reduction in SCF binding. Hence, the binding site of SCF probably includes Ig-like domains 1 and 2, but structural determinants distal to this portion may also participate in ligand recognition.

Previously, we have demonstrated T-B cell interactions mediated by T cell recognition of immunoglobulin (Ig) peptide/major histocompatibility complex (MHC) class II complexes derived by the B cell processing of endogenously synthesized Ig molecules. In this report Ig-specific T-B cell interaction mediated by B cell presentation of idiotopes (Id) of anti-sIg antibodies to Id-specific T cell clones has been studied in Ig kappa-1-congenic rat strains. A panel of August (RT-1c; Ig kappa-1a) rat T helper clones specific for Id of syngeneic anti-Ig kappa-1b C3B9 monoclonal antibody (mAb) has been developed to study IdC3B9 presentation by Ig kappa-1b-bearing B cells from congenic August.1b (RT-1c; Ig kappa-1b) rats. Five of seven IdC3B9-specific T clones responded even at very low concentrations (100-200 pg/ml) of C3B9 mAb presented by Ig kappa-1b+ B cells. In contrast, the presentation of intact C3B9 mAb by nonspecific antigen-presenting cells (macrophages, Ig kappa-1a+ B cells, etc.) to IdC3B9-specific T cells was of low efficiency. The IdC3B9-specific T cell response to idiotopes of anti-Ig kappa-1b C3B9 mAb was found to be restricted by RT-1B molecule and required the processing of intact C3B9 molecule. IdC3B9 epitope recognized by C31 and C5 clones was mapped to the heavy chain of C3B9 mAb. Thus, B cells are able to present peptides related to the V region of anti-sIg Ab, i.e. Id peptide/MHC class II complexes, to Id-recognizing T cells. IdC3B9-presenting B cells are specifically activated both to proliferation and Ig production upon interaction with IdC3B9-specific T clones. Based on the results of our studies on B cell presentation of Ig epitopes to T cells a hypothetical model of Ig peptide-driven T-B cell interaction has been proposed.

In the preceding report (Eur. J. Immunol. 1989. 19: 1677) we have demonstrated that normal B cells, including small B cells, are capable of presenting Ig kappa-1b allotypic determinants of their endogeneously synthesized Ig+ to Ig kappa-1b-immune major histocompatibility complex (MHC) class II-restricted T cells. A panel of Ig kappa-1b allotype-specific T cell clones from August rats has been developed to study further the presentation of self surface Ig by B cells from Ig kappa-1-congeneic August.1b rats. All the clones studied were of the T helper/inducer phenotype (W3/25+,OX8-) and restricted by the RT-1Bc molecule. These clones responded both to the serum IgG(Ig kappa-1b) in the presence of irradiated spleen cells (SC) from August rats and to the Ig kappa-1b-bearing irradiated B cells from August.1b rats. SC presentation of secreted IgG was much less effective than B cell presentation of membrane Ig. Using CNBr cleavage of isolated C kappa (Ig kappa-1b) domain followed by high-performance liquid chromatography fractionation of the derived antigenic peptides, the kappa chain sequence between amino acids 176 and 214 has been identified as the T cell epitope recognized by all T cell clones in association with RT-1Bc. The fragment 176-214 of the Ig kappa-1b allotype differs from that of Ig kappa-1a allotype by three amino acid substitutions at positions 184, 185, 188. T cell recognition of pL kappa-1b(176-214) required no additional processing by the antigen-presenting cell: the efficient presentation of the peptide but not of intact IgG(Ig kappa-1b) by the paraformaldehyde-fixed SC was observed. These data provide clear-cut evidence for an absolute requirement of the processing of Ig molecules for T cell recognition to occur in our experimental system. Although the fixation of B cells from August.1b rats diminished their Ig kappa-1b-presenting ability, fixed Ig kappa-1b+ B cells were still able to induce Ig kappa-1b-specific T cell clone responses. Our results suggest that B cells can express the processed form of self-synthesized surface Ig in addition to intact surface Ig molecules. The former can be recognized by MHC-restricted T cell.