Deletion of a conserved Gata2 enhancer impairs haemogenic endothelium programming and adult Zebrafish haematopoiesis

Gata2 is a key transcription factor required to generate Haematopoietic Stem and Progenitor Cells (HSPCs) from haemogenic endothelium (HE); misexpression of Gata2 leads to haematopoietic disorders. Here we deleted a conserved enhancer (i4 enhancer) driving pan-endothelial expression of the zebrafish gata2a and showed that Gata2a is required for HE programming by regulating expression of runx1 and of the second Gata2 orthologue, gata2b. By 5 days, homozygous gata2aΔi4/Δi4 larvae showed normal numbers of HSPCs, a recovery mediated by Notch signalling driving gata2b and runx1 expression in HE. However, gata2aΔi4/Δi4 adults showed oedema, susceptibility to infections and marrow hypo-cellularity, consistent with bone marrow failure found in GATA2 deficiency syndromes. Thus, gata2a expression driven by the i4 enhancer is required for correct HE programming in embryos and maintenance of steady-state haematopoietic stem cell output in the adult. These enhancer mutants will be useful in exploring further the pathophysiology of GATA2-related deficiencies in vivo

Desma ve Desmb Knockout Zebra Balığı Modellerinde Desmin İfadesinin İncelenmesi

Desmin, a basic intermediate filament protein specific to muscle tissue, facilitates the transmission of force during muscle contraction and relaxation by forming a large network structure that allows the myofibrils to connect to the nucleus, sarcolemma and intracellular organelles. In animal models used to understand the underlying pathophysiology of DES-related diseases, it is necessary to analyze tissues in which orthologous genes are expressed. In the zebrafish, which has many advantages in modeling muscle diseases, there are two DES orthologous gene (desma and desmb). In our preliminary studies conducted by our group with in situ hybridization experiments in wild-type AB zebrafish, it has been shown that the desma and desmb transcripts have similar skeletal muscle expression pattern until 70th hour after fertilization; but after 70 hours, it was observed that the desma was expressed predominantly in skeletal muscle, while desmb was expressed highly in the smooth muscle. In the thesis study, it was aimed to investigate mRNA and protein expressions of desma and desmb in wild type AB, desma KO (-/-) and desmb KO (-/-) zebrafish embriyos. In the quantitative real-time PCR study, 99% of desma mRNA expression in desma KO (-/-) fishes and 98% of desmb mRNA expression in desmb KO (-/-) fishes were found to decrease relative to wild type AB fish. Whole-mount in situ hybridization and whole-mount immunofluorescence staining studies have shown that desma in desmb KO (-/-) zebrafish is also expressed in the gut, additional to skeletal and cardiac muscle. However, it was observed that in the desma KO (-/-) fish, the desmb was mostly expressed in smooth muscle from the 70th hour after fertilization and that no expression occurs in skeletal muscle in the absence of desma.İÇİNDEKİLER ONAY SAYFASI iii YAYIMLAMA VE FİKRİ MÜLKİYET HAKLARI BEYANI iv ETİK BEYAN v TEŞEKKÜR vi ÖZET vii ABSTRACT viii İÇİNDEKİLER ix SİMGELER VE KISALTMALAR xii ŞEKİLLER xiii TABLOLAR xvi 1. GİRİŞ 1 1.1 İskelet Kası İşlevi ve Hastalıkları 1 1.2 Desmin ve Desminopatiler 1 1.3 Zebra Balığı ve Desmin 2 1.4 Amaç ve Kapsam 3 2. GENEL BİLGİLER 4 2.1 İskelet Kası Yapısı 4 2.2 Sarkomerik Hücre İskeleti 5 2.2.1 İskelet Kası Ara Filament Proteinleri 6 2.3 Desmin 7 2.3.1 Desminopatiler 9 2.4 Zebra Balığı (Danio rerio) 10 2.4.1 Zebra Balığında İskelet Kası Yapısı 11 2.4.2 Zebra Balığında İskelet Kası Hastalıklarının Modellenmesi 12 2.4.3 Zebra Balığında Desmin 13 3. GEREÇ VE YÖNTEM 15 3.1 Zebra Balıkları ve Gereçler 15 3.1.1 Çalışmada İncelenen Zebra Balıkları 15 3.1.2 Zebra Balıklarının Bakımı ve İdamesi 16 3.1.3 DNA İzolasyonu 17 3.1.4 Primer Dizilerinin Tasarlanması 17 3.1.5 Polimeraz Zincir Reaksiyonu 17 3.1.6 DNA Agaroz Jel Elektroforezi 18 3.1.7 DNA Dizi Analizi 18 3.1.8 Zebra Balığı Embriyolarının Fiksasyonu 19 3.1.9 Total RNA İzolasyonu 19 3.1.10 cDNA Sentezi 19 3.1.11 Prob Sentezi 20 3.1.12 Tüm Örnek İn Sitü Hibridizasyon ve Dondurulmuş Kesitler 21 3.1.13 Kantitatif Gerçek Zamanlı Polimeraz Zincir Reaksiyonu 23 3.1.14 Tüm Örnek İmmünfloresan Boyama 23 3.2. Yöntemler 24 3.2.1 Zebra Balıklarının Bakımı ve İdamesi 24 3.2.2 Zebra Balığı Kuyruk Dokusundan DNA İzolasyonu 24 3.2.3 desma ve desmb İçin Yapılan PZR ve DNA Dizi Analizi Çalışmaları 25 3.2.4. Zebra Balığı Embriyolarının Fiksasyonu 29 3.2.5. Zebra Balığı Embriyolarından Total RNA İzolasyonu 30 3.2.6 cDNA Sentezi 32 3.2.7. İn Sitü Hibridizasyon Çalışmaları İçin Prob Sentezi 33 3.2.8 Tüm Örnek İn Sitü Hibridizasyon ve Dondurulmuş Kesitler 36 3.2.9. Kantitatif Gerçek Zamanlı Polimeraz Zincir Reaksiyonu 39 3.2.10. Tüm Örnek İmmünfloresan Boyama 41 4. BULGULAR 43 4.1 DNA Dizi Analizi ile desma KO (-/-) ve desmb KO (-/-) Zebra Balıklarının Belirlenmesi 43 4.2 desma ve desmb İfadesinin Kantitatif Gerçek Zamanlı PZR ile Analizi 48 4.3 Zebra Balığı Embriyolarında Tüm Örnek ve Dondurulmuş Kesitlerde İn Sitü Hibridizasyon 53 4.4 Zebra Balığı Embriyolarında Tüm Örnek İmmünfloresan Boyama 61 5. TARTIŞMA 65 6. SONUÇ VE ÖNERİLER 75 6.1 Sonuç 75 6.2 Öneriler 76 7. KAYNAKLAR 77 8. EKLER EK 1: Tez Çalışması ile İlgili Etik Kurul İzni. 82 EK 2: Vimentin İfadesinin kGZ-PZR Çalışması İle Analizi. 84 EK 3: Erişkin Zebra Balıklarının Kas Dokusundan Alınan Kesitlerde Gerçekleştirilen Hematoksilen Eozin Boyaması. 85 9. ÖZGEÇMİŞKas dokusuna özgül temel ara filament proteini olan desmin, miyofibrillerin çekirdek, sarkolemma ve hücre içi organellerle bağlantılarını sağlayan geniş bir ağ yapısı oluşturarak, kas kasılması ve gevşemesi sırasında güç aktarımını kolaylaştırmaktadır. Desmin geni (DES) ile ilişkili hastalıkların altında yatan patofizyolojinin anlaşılması için kullanılan hayvan modellerinde, ortolog genlerin ifade olduğu dokuların analiz edilmesi gerekmektedir. Kas hastalıklarını modellemede birçok avantajı bulunan zebra balığında, DES ortoloğu iki homolog gen (desma ve desmb) bulunmaktadır. Grubumuz tarafından, yabanıl tip AB zebra balıklarında in sitü hibridizasyon deneyleri ile gerçekleştirilen ön çalışmalarda, desma ve desmb transkriptlerinin döllenmeden sonra 70. saate kadar benzer iskelet kası yerleşimi gösterdiği; ancak 70. saatten sonra, desma ağırlıklı olarak iskelet kasında ifade olurken, desmb‘nin çoğunlukla düz kasta ifade edildiği gözlenmiştir. Tez çalışmasında, desma ve desmb’nin mRNA ve protein ifadelerinin, yabanıl tip AB, desma KO (-/-) ve desmb KO (-/-) zebra balıklarında karşılaştırılarak araştırılması amaçlanmıştır. Kantitatif gerçek zamanlı PZR çalışmasında, yabanıl tip AB balıklara göre, desma KO (-/-) balıklarda desma mRNA ifadesinin %99 ve desmb KO (-/-) balıklarda desmb mRNA ifadesinin %98 oranında göreceli olarak azaldığı bulunmuştur. Gerçekleştirilen tüm örnek in sitü hibridizasyon ve tüm örnek immünfloresan boyama çalışmalarında, desmb KO (-/-) zebra balıklarında desma’nın, iskelet kası ve kalp kasının yanında, bağırsakta da ifade olduğu saptanmıştır. Ancak, desma KO (-/-) balıklarda, desmb‘nin 70. saatten itibaren çoğunlukla düz kasta ifade olduğu ve desma yokluğunda iskelet kasında ifade olmadığı gözlenmiştir

From Basic Biology to Patient Mutational Spectra of GATA2 Haploinsufficiencies: What Are the Mechanisms, Hurdles, and Prospects of Genome Editing for Treatment

Inherited bone marrow failure syndromes (IBMFS) are monogenetic disorders that result in a reduction of mature blood cell formation and predisposition to leukemia. In children with myeloid leukemia the gene most often mutated is Gata binding protein 2 (GATA2) and 80% of patients with GATA2 mutations develop myeloid malignancy before the age of forty. Although GATA2 is established as one of the key regulators of embryonic and adult hematopoiesis, the mechanisms behind the leukemia predisposition in GATA2 haploinsufficiencies is ambiguous. The only curative treatment option currently available is allogeneic hematopoietic stem cell transplantation (allo-SCT). However, allo-SCT can only be applied at a relatively late stage of the disease as its applicability is compromised by treatment related morbidity and mortality (TRM). Alternatively, autologous hematopoietic stem cell transplantation (auto-SCT), which is associated with significantly less TRM, might become a treatment option if repaired hematopoietic stem cells would be available. Here we discuss the recent literature on leukemia predisposition syndromes caused by GATA2 mutations, current knowledge on the function of GATA2 in the hematopoietic system and advantages and pitfalls of potential treatment options provided by genome editing

Gata2-regulated Gfi1b expression controls endothelial programming during endothelial-to-hematopoietic transition

The first hematopoietic stem cells (HSCs) are formed through endothelial-to-hematopoietic transition (EHT) events during embryonic development. The transcription factor GATA2 is a crucial regulator of EHT and HSC function throughout life. Because GATA2 haploinsufficiency patients have inborn mutations, prenatal defects are likely to have an influence on disease development. In mice, Gata2 haploinsufficiency (Gata2+/-) reduces the number and the functionality of embryonic hematopoietic stem and progenitor cells (HSPCs) generated through EHT. However, the embryonic HSPC pool is heterogeneous and the mechanisms underlying this defect in Gata2+/- embryos are unclear. Here, we investigated whether Gata2 haploinsufficiency selectively affects a cellular subset undergoing EHT. We show that Gata2+/- HSPCs initiate but cannot fully activate hematopoietic programming during EHT. In addition, due to reduced activity of the endothelial repressor Gfi1b, Gata2+/- HSPCs cannot repress the endothelial identity to complete maturation. Finally, we show that hematopoietic-specific induction of gfi1b can restore HSC production in gata2b-null (gata2b-/-) zebrafish embryos. This study illustrates pivotal roles of Gata2 on the regulation of transcriptional network governing HSPC identity throughout EHT

Mutations In Anks6 Cause A Nephronophthisis-Like Phenotype With Esrd

Nephronophthisis (NPHP) is one of the most common genetic causes of CKD; however, the underlying genetic abnormalities have been established in <50% of patients. We performed genome-wide analysis followed by targeted resequencing in a Turkish consanguineous multiplex family and identified a canonic splice site mutation in ANKS6 associated with an NPHP-like phenotype. Furthermore, we identified four additional ANKS6 variants in a cohort of 56 unrelated patients diagnosed with CKD due to nephronophthisis, chronic GN, interstitial nephritis, or unknown etiology. Immunohistochemistry in human embryonic kidney tissue demonstrated that the expression patterns of ANKS6 change substantially during development. Furthermore, we detected increased levels of both total and active beta-catenin in precystic tubuli in Han:SPRD Cy/+ rats. Overall, these data indicate the importance of ANKS6 in human kidney development and suggest a mechanism by which mutations in ANKS6 may contribute to an NPHP-like phenotype in humans.WoSScopu

Knockout of zebrafish desmin genes does not cause skeletal muscle degeneration but alters calcium flux

Desmin is a muscle-specific intermediate filament protein that has fundamental role in muscle structure and force transmission. Whereas human desmin protein is encoded by a single gene, two desmin paralogs (desma and desmb) exist in zebrafish. Desma and desmb show differential spatiotemporal expression during zebrafish embryonic and larval development, being similarly expressed in skeletal muscle until hatching, after which expression of desmb shifts to gut smooth muscle. We generated knockout (KO) mutant lines carrying loss-of-function mutations for each gene by using CRISPR/Cas9. Mutants are viable and fertile, and lack obvious skeletal muscle, heart or intestinal defects. In contrast to morphants, knockout of each gene did not cause any overt muscular phenotype, but did alter calcium flux in myofibres. These results point to a possible compensation mechanism in these mutant lines generated by targeting nonsense mutations to the first coding exon