An erythroid to myeloid cell fate conversion is elicited by LSD1 inactivation

Histone H3 lysine 4 methylation (H3K4Me) is most often associated with chromatin activation, and removing H3K4 methyl groups has been shown to be coincident with gene repression. H3K4Me demethylase KDM1a/LSD1 is a therapeutic target for multiple diseases, including for the potential treatment of b-globinopathies (sickle cell disease and b-thalassemia) since it is a component of g-globin repressor complexes, and LSD1 inactivation leads to robust induction of the fetal globin genes. The effects of LSD1 inhibition in definitive erythropoiesis are not well characterized, so we examined the consequences of conditional inactivation of Lsd1 in adult red blood cells using a new Gata1creERT2 BAC transgene. Erythroid-specific loss of Lsd1 activity in mice led to a block in erythroid progenitor differentiation and to the expansion of GMP-like cells, converting hematopoietic differentiation potential from an erythroid to a myeloid fate. The analogous phenotype was also observed in human hematopoietic stem and progenitor cells (HSPC), coincident with induction of myeloid transcription factors (e.g. PU.1 and CEBPa). Finally, blocking the activity of transcription factors PU.1 or RUNX1 at the same time as LSD1 inhibition rescued myeloid lineage conversion to an erythroid phenotype. These data show that LSD1 promotes erythropoiesis by repressing myeloid cell fate in adult erythroid progenitors, and that inhibition of the myeloid differentiation pathway reverses the lineage switch induced by LSD1 inactivation.

The Drosophila gene smoke alarm regulates nociceptor-epidermis interactions and thermal nociception behavior

The detection and processing of noxious sensory input depends on the proper growth and function of nociceptor sensory neurons in the peripheral nervous system. In Drosophila melanogaster, the class IV (cIV) multidendritic dendritic arborization (md-da) neurons detect noxious stimuli through their highly branched dendrites that innervate the epidermis of the larval body wall. Here, we describe requirements of a previously uncharacterized gene named smoke alarm (smal), a discoidin domain receptor, in cIV md-da dendrite morphogenesis and nociception behavior. We find that smal mutant larvae exhibit thermal hyperalgesia that is fully rescued with a BAC transgene containing smal. Consistent with this phenotype, a smal reporter gene was expressed in nociceptors and other peripheral sensory neurons. Smal::GFP protein localized to punctate structures throughout the cIV md-da neurons. We further show that smal loss-of-function results in reduced nociceptor dendrite branching. Interestingly, mammalian homologues of smal act as collagen receptors, and we find that smal mutant dendrites showed an increase in epidermal cell ensheathment relative to animals that are wild type for smal. Based on this phenotype we propose that Smal protein function is required for attachment of dendrites to the extracellular matrix (ECM) and the loss of activity results in thermal hyperalgesia.

The epigenetic eraser LSD1 lies at the apex of a reversible erythroid to myeloid cell fate decision

H3K4Me demethylase KDM1a/LSD1 is a therapeutic target for multiple diseases, including the β-globinopathies (sickle cell disease and β-thalassemia) since its inactivation has been shown to lead to robust induction of the fetal globin genes. Here we examined the consequences of conditional inactivation of Lsd1 in adult red blood cells using a new Gata1creERT2 BAC transgene. Loss of Lsd1 activity in mice blocked erythroid differentiation and expanded GMP-like cells, converting hematopoietic differentiation potential from an erythroid to a myeloid fate. The analogous phenotype was also observed in human HSPC, coincident with induction of myeloid transcription factors (e.g. PU.1 and CEBPα). Finally, blocking the activity of myeloid transcription factors PU.1 or RUNX1 at the same time as LSD1 reverted myeloid lineage conversion to an erythroid phenotype. The data show that LSD1 promotes erythropoiesis by repressing myeloid cell fate, and that inhibition of myeloid differentiation reverses the lineage switch caused by LSD1 inactivation.

Repeat length increases disease penetrance and severity in C9orf72 ALS/FTD BAC transgenic mice

C9orf72 ALS/FTD patients show remarkable clinical heterogeneity, but the complex biology of the repeat expansion mutation has limited our understanding of the disease. BAC transgenic mice were used to better understand the molecular mechanisms and repeat length effects of C9orf72 ALS/FTD. Genetic analyses of these mice demonstrate that the BAC transgene and not integration site effects cause ALS/FTD phenotypes. Transcriptomic changes in cell proliferation, inflammation and neuronal pathways are found late in disease and alternative splicing changes provide early molecular markers that worsen with disease progression. Isogenic sublines of mice with 800, 500 or 50 G4C2 repeats generated from the single-copy C9–500 line show longer repeats result in earlier onset, increased disease penetrance, and increased levels of RNA foci and dipeptide RAN protein aggregates. These data demonstrate G4C2 repeat length is an important driver of disease and identify alternative splicing changes as early biomarkers of C9orf72 ALS/FTD.

Repeat length increases disease penetrance and severity in C9orf72 ALS/FTD BAC transgenic mice

C9orf72 ALS/FTD patients show remarkable clinical heterogeneity, but the complex biology of the repeat expansion mutation has limited our understanding of the disease. BAC transgenic mice were used to better understand the molecular mechanisms and repeat length effects of C9orf72 ALS/FTD. Genetic analyses of these mice demonstrate that the BAC transgene and not integration site effects cause ALS/FTD phenotypes. Transcriptomic changes in cell proliferation, inflammation and neuronal pathways are found late in disease and alternative splicing changes provide early molecular markers that worsen with disease progression. Isogenic sublines of mice with 800, 500 or 50 G4C2 repeats generated from the single-copy C9-500 line show longer repeats result in earlier onset, increased disease penetrance, and increased levels of RNA foci and RAN aggregates. These data demonstrate G4C2 repeat length is an important driver of disease and identify alternative splicing changes as early biomarkers of C9orf72 ALS/FTD.

Abstract 020: Physiological Significance of Angiotensin AT 1A Receptors in Vasopressin-producing Cells of the Supraoptic Nucleus

Low-renin and salt-sensitive forms of hypertension are characterized by elevated activity of the brain renin-angiotensin system and secretion of arginine vasopressin (AVP). While angiotensin in the brain is a known stimulant of AVP secretion through its AT 1 receptor, the localization of relevant AT 1 receptors remains unclear. We tested whether AT 1A receptors localized to AVP-producing cells are important for AVP secretion. To examine AVP and AT 1A co-localization, mice expressing Cre-recombinase via the AVP gene (AVP-Cre) were bred with mice expressing a conditional red fluorescent ROSA-stop flox -tdTomato construct and GFP via an AT 1A BAC transgene. Dual-fluorescent cells were detected in supraoptic nuclei (SON) but not paraventricular nuclei. Mice lacking AT 1A specifically in AVP-producing cells (AT 1A AVP-KO ) were then generated by breeding AVP-Cre mice with mice harboring a conditional endogenous AT 1A gene. AT 1A AVP-KO mice exhibited normal serum (littermate n=13, 353±69 vs AT 1A AVP-KO n=7, 207±37 pg/mL, p=NS) and urine (n=26, 145±33 vs n=11, 170±54 pg/mL, p=NS) copeptin (the stable C-terminal fragment of AVP) as well as hematocrit (n=14, 46.3±0.7 vs n=7, 47.5±1.3 %, p=NS) despite increased serum osmolality (n=33, 324±1.3 vs n=19, 330±1.6 mOsm/kg, p<0.01), supporting a role for AT 1A in AVP-producing cells in modulating the osmotic control of AVP release. Systolic blood pressure (SBP) (n=18, 109±1.3 vs n=5, 107±1.2 mmHg), urine volume (n=27, 1.1±0.1 vs n=12, 0.9±0.2 mL/d), and fluid intake (n=27, 4.0±0.2 vs n=12, 3.9±0.2 mL/d) were all normal (p=NS) in AT 1A AVP-KO mice. Two-bottle choice between water and escalating concentrations of NaCl uncovered minor alterations in sodium intake behavior. Serum osmolality (n=22, 336±2 vs n=9, 333±3 mOsm/kg), SBP (n=23, +10.4±2.1 vs n=8, +12.9±2.0 mmHg), urine output (n=23, +12.7±0.8 vs n=9, +12.7±1.5 g/day), and fluid intake (n=23, +16.2±1.3 vs n=9, +14.8±2.5 mL/day) all increased normally (p=NS) in response to deoxycorticosterone acetate (DOCA)-salt treatment. Collectively these data support a role for AT 1A receptors, localized specifically to AVP-expressing cells of the SON, in the normal osmotic control of AVP secretion.

Spry2 regulates signalling dynamics and terminal bud branching behaviour during lung development

SummaryDevelopment of mammalian lung involves reiterative outgrowth and branching of an epithelial tube into the surrounding mesenchymal bed. Each coordinated growth and branching cycle is driven by reciprocal signalling between epithelial and adjacent mesenchymal cells. This signalling network includes FGF, SHH, BMP4 and other pathways. We have characterized lung defects in 36Pub mice carrying a deletion that removes an antagonist of FGF signalling, Spry2. Spry2 deficient mice show an enlarged cystic structure located in the terminus of each lobes. Our study shows that Spry2 deficient lungs have reduced lung branching and the cystic structure forms in the early lung development stage. Furthermore, mice carrying a targeted disruption of Spry2 fail to complement the lung phenotype characterized in 36Pub mice. A Spry2-BAC transgene rescues the defect. Interestingly, cystic structure growth is accompanied by the reduced and spatially disorganized expression of Fgf10 and elevated expression of Shh and Bmp4. Altered signalling balance due to the loss of Spry2 causes a delayed branch cycle and cystic growth. Our data underscores the importance of restricting cellular responsiveness to signalling and highlights the interplay between morphogenesis events and spatial localization of gene expression.