Dlk1 is a negative regulator of emerging hematopoietic stem and progenitor cells.

The first mouse adult-repopulating hematopoietic stem cells emerge in the aorta-gonad-mesonephros region at embryonic day (E) 10.5. Their numbers in this region increase thereafter and begin to decline at E12.5, thus pointing to the possible existence of both positive and negative regulators of emerging hematopoietic stem cells. Our recent expression analysis of the aorta-gonad-mesonephros region showed that the Delta-like homologue 1 (Dlk1) gene is up-regulated in the region of the aorta-gonad-mesonephros where hematopoietic stem cells are preferentially located. To analyze its function, we studied Dlk1 expression in wild-type and hematopoietic stem cell-deficient embryos and determined hematopoietic stem and progenitor cell activity in Dlk1 knockout and overexpressing mice. Its role in hematopoietic support was studied in co-culture experiments using stromal cell lines that express varying levels of Dlk1. We show here that Dlk1 is expressed in the smooth muscle layer of the dorsal aorta and the ventral sub-aortic mesenchyme, where its expression is dependent on the hematopoietic transcription factor Runx1. We further demonstrate that Dlk1 has a negative impact on hematopoietic stem and progenitor cell activity in the aorta-gonad-mesonephros region in vivo, which is recapitulated in co-cultures of hematopoietic stem cells on stromal cells that express varying levels of Dlk1. This negative effect of Dlk1 on hematopoietic stem and progenitor cell activity requires the membrane-bound form of the protein and cannot be recapitulated by soluble Dlk1. Together, these data suggest that Dlk1 expression by cells of the aorta-gonad-mesonephros hematopoietic microenvironment limits hematopoietic stem cell expansion and is, to our knowledge, the first description of such a negative regulator in this tissue.

The effects of verbalisation on cognitive performance in schizophrenia: a pilot study using tasks from the Delis Kaplan Executive Function System.

Cognitive impairment is common in schizophrenia, and has adverse effects on functional outcome. Cognitive remediation strategies in which people with schizophrenia speak aloud (verbalise) during task performance have demonstrated some success in improving performance on the Wisconsin Card Sorting Test. This study extends previous research by assessing whether verbalisation also improves performance on tasks selected from the Delis-Kaplan Executive Function System (D-KEFS). Twenty two subjects with schizophrenia participated in the study. We used a within subjects design to compare performance on the D-KEFS Tower Test and Trail Making Test when participants (a) produced concurrent verbalisation, or (b) remained silent. Results demonstrated selective benefits of verbalisation on a neuropsychological task requiring multiple executive functions (number-letter switching task), while performance on tasks requiring simpler single-component cognitive functions (visual scanning and motor speed tasks) was adversely affected. The effects of verbalisation on the cognitive task performance of patients with schizophrenia differ depending on the nature of the task. Benefits are seen in tests of executive skills but performance worsens in single component cognitive tasks. When developing cognitive remediation strategies for people with schizophrenia, consideration should be given to the nature and cognitive demands of each task before recommending verbalisation strategies.

The nature of imagery processes underlying food cravings.

OBJECTIVE: The study used a working memory approach to examine the nature of the imagery processes underlying food cravings. DESIGN AND METHOD: A sample of 60 dieters and 60 non-dieters were asked to imagine either a food induction or a holiday induction scenario. Participants then performed 18 trials of either a visual imagery task (loading the visuo-spatial sketch pad) or auditory imagery task (loading the phonological loop). Food craving was measured before and after the induction scenario, and then after every 6 trials of the imagery task. RESULTS: Craving intensity increased following instructions to imagine the food (but not holiday) induction scenario, especially for dieters. As predicted, the visual imagery task was superior to the auditory imagery task in reducing the level of food craving. DISCUSSION: The results confirmed the imaginal basis of food cravings. Specifically, they demonstrated that the imagery processes involved in food cravings are predominantly visual in nature. Hence concurrent tasks that load the visuospatial sketch pad of working memory can be used to reduce food cravings. The findings have potential application in the treatment of craving episodes in clinical populations.

Generation of murine stromal cell lines: models for the microenvironment of the embryonic mouse aorta-gonads-mesonephros region.

We describe a method to derive cell lines and clones from cells of the murine midgestation aorta-gonads-mesonephros (AGM) microenvironment. We start from subdissected AGM regions in "explant-" or "single-cell suspension"-type cultures from embryos transgenic for tsA58, a temperature-sensitive mutant of the SV40 T antigen gene. The number of cells in such cultures initially expand, but in most cases, this expansion phase is followed by a stable or even decline in cell number. After this so-called crisis phase, cell proliferation is noticeable in more than 90% of the cultures. Stromal cell clones can be isolated from these cultures, some of which have been cultured for more than 50 population doublings. These stromal cell clones are valuable tools for the study of the regulation of hematopoietic stem and progenitor cells in the midgestation mouse embryo.

Cell-cell contact and anatomical compatibility in stromal cell-mediated HSC support during development.

Hematopoietic stem cells (HSCs) are able to generate the wide variety of blood cells found in the adult and are maintained in the bone marrow (BM) stromal microenvironment. In the aorta-gonads-mesonephros (AGM), which autonomously generates the first HSCs, the stromal microenvironment is largely uncharacterized. We have previously made an extensive panel of stromal clones from AGM subregions and have found that clones from the urogenital ridges (UG) provide the most potent support for adult BM HSCs. However, it is unknown to what extent the stroma from this developmentally and anatomically distinct microenvironment can support HSCs from other regions of the embryo, such as yolk sac. Moreover, it is unknown whether cell-cell contact is necessary in this microenvironment. Here, we show that the HSCs from the embryonic aorta are the most potently supported HSCs in UG stromal clone co-cultures and that contact is required for the maintenance and expansion of embryo-derived HSCs.

The role of apoptosis in the development of AGM hematopoietic stem cells revealed by Bcl-2 overexpression.

Apoptosis is an essential process in embryonic tissue remodeling and adult tissue homeostasis. Within the adult hematopoietic system, it allows for tight regulation of hematopoietic cell subsets. Previously, it was shown that B-cell leukemia 2 (Bcl-2) overexpression in the adult increases the viability and activity of hematopoietic cells under normal and/or stressful conditions. However, a role for apoptosis in the embryonic hematopoietic system has not yet been established. Since the first hematopoietic stem cells (HSCs) are generated within the aortagonad-mesonephros (AGM; an actively remodeling tissue) region beginning at embryonic day 10.5, we examined this tissue for expression of apoptosis-related genes and ongoing apoptosis. Here, we show expression of several proapoptotic and antiapoptotic genes in the AGM. We also generated transgenic mice overexpressing Bcl-2 under the control of the transcriptional regulatory elements of the HSC marker stem cell antigen-1 (Sca-1), to test for the role of cell survival in the regulation of AGM HSCs. We provide evidence for increased numbers and viability of Sca-1(+) cells in the AGM and subdissected midgestation aortas, the site where HSCs are localized. Most important, our in vivo transplantation data show that Bcl-2 overexpression increases AGM and fetal liver HSC activity, strongly suggesting that apoptosis plays a role in HSC development.

Embryonal subregion-derived stromal cell lines from novel temperature-sensitive SV40 T antigen transgenic mice support hematopoiesis.

Throughout life, the hematopoietic system requires a supportive microenvironment that allows for the maintenance and differentiation of hematopoietic stem cells (HSC). To understand the cellular interactions and molecules that provide these functions, investigators have previously established stromal cell lines from the late gestational stage and adult murine hematopoietic microenvironments. However, the stromal cell microenvironment that supports the emergence, expansion and maintenance of HSCs during mid-gestational stages has been largely unexplored. Since several tissues within the mouse embryo are known to harbor HSCs (i.e. aortagonads-mesonephros, yolk sac, liver), we generated numerous stromal cell clones from these mid-gestational sites. Owing to the limited cell numbers, isolations were performed with tissues from transgenic embryos containing the ts SV40 Tag gene (tsA58) under the transcriptional control of constitutive and ubiquitously expressing promoters. We report here that the growth and cloning efficiency of embryonic cells (with the exception of the aorta) is increased in the presence of the tsA58 transgene. Furthermore, our results show that the large panel of stromal clones isolated from the different embryonal subregions exhibit heterogeneity in their ability to promote murine and human hematopoietic differentiation. Despite our findings of heterogeneity in hematopoietic growth factor gene expression profiles, high-level expression of some factors may influence hematopoietic differentiation. Interestingly, a few of these stromal clones express a recently described chordin-like protein, which is an inhibitor of bone morphogenic proteins and is preferentially expressed in cells of the mesenchymal lineage.

Stromal cell lines from mouse aorta-gonads-mesonephros subregions are potent supporters of hematopoietic stem cell activity.

The aorta-gonads-mesonephros (AGM) region autonomously generates the first adult repopulating hematopoietic stem cells (HSCs) in the mouse embryo. HSC activity is initially localized to the dorsal aorta and mesenchyme (AM) and vitelline and umbilical arteries. Thereafter, HSC activity is found in the urogenital ridges (UGs), yolk sac, and liver. As increasing numbers of HSCs are generated, it is thought that these sites provide supportive microenvironments in which HSCs are harbored until the bone marrow microenvironment is established. However, little is known about the supportive cells within these midgestational sites, and particularly which microenvironment is most supportive for HSC growth and maintenance. Thus, to better understand the cells and molecules involved in hematopoietic support in the midgestation embryo, more than 100 stromal cell lines and clones were established from these sites. Numerous stromal clones were found to maintain hematopoietic progenitors and HSCs to a similar degree as, or better than, previously described murine stromal lines. Both the AM and UG subregions of the AGM produced many supportive clones, with the most highly HSC-supportive clone being derived from the UGs. Interestingly, the liver at this stage yielded only few supportive stromal clones. These results strongly suggest that during midgestation, not only the AM but also the UG subregion provides a potent microenvironment for growth and maintenance of the first HSCs.