Role of hypoxia-inducible factor 1-alpha (HIF-1α) in the regulation of hypoxia-induced hemoglobin synthesis in cladocerans

다국어 초록 (Multilingual Abstract)

Aquatic organisms have evolved sophisticated molecular mechanisms to survive in low-oxygen (hypoxic) environments. A master regulator of this response is the transcription factor Hypoxia-Inducible Factor 1-alpha (HIF-1α). While its role is well-established in vertebrates, the precise mechanisms by which HIF-1α regulates adaptation in aquatic invertebrates, particularly the regulation of oxygen-carrying proteins like hemoglobin (Hb), remain less understood. This dissertation investigates the pivotal role of HIF-1α in mediating hypoxia-induced Hb synthesis in cladoceran crustaceans, a key zooplankton group in freshwater and brackish ecosystems.
Using the model species Daphnia magna and Diaphanosoma celebensis, this study first characterized the physiological and molecular responses to hypoxia, establishing a strong correlation between HIF-1α activation and the upregulation of Hb expression. To functionally validate this link, CRISPR/Cas9-mediated knockout of the HIF-1α gene was performed in D. magna. Comparative analyses revealed that HIF-1α deficient mutants exhibited significantly increased susceptibility to hypoxia and, critically, were unable to mount the characteristic Hb response observed in wild-type individuals under hypoxic stress. Transcriptomic and pathway analyses further demonstrated that HIF-1α governs a network of genes essential for hypoxic adaptation, including those involved in anaerobic metabolism and oxidative stress mitigation. Additionally, the study explored how this regulatory pathway is affected by co-stressors, such as nanoplastics, providing insights into adaptation under environmentally realistic, complex scenarios.
Collectively, these findings provide direct genetic evidence that HIF-1α is a key regulator of hypoxia-induced Hb synthesis and a critical component of the adaptive strategy in cladocerans. This research elucidates a fundamental molecular mechanism of survival in an ecologically important group of invertebrates and offers a deeper understanding of how aquatic ecosystems may respond to the growing threat of global deoxygenation.

국문 초록 (Abstract)

수생 생물은 저산소 환경에서 생존하기 위해 정교한 분자 메커니즘을 진화시켜 왔다. 이 반응의 주요 조절자는 전사 인자인 Hypoxia-Inducible 1-alpha (HIF-1α)다. 이 인자의 역할은 척추동물에서 잘 확립되어 있지만, HIF-1α가 수생 무척추동물에서 적응을 조절하는 정확한 메커니즘, 특히 헤모글로빈과 같은 산소 운반 단백질의 조절에 대해서는 아직 잘 이해되지 않고 있다. 이 논문은 담수 및 기수 생태계의 주요 동물성 플랑크톤 그룹인 Cladoceran 갑각류에서 저산소증에 의해 유도된 헤모글로빈 합성을 매개하는 HIF-1α의 중추적인 역할을 조사할 것이다.
이 연구는 모델 종 Daphnia magna와 Diapanosoma celebensis를 사용하여 저산소증에 대한 생리적 및 분자적 반응을 먼저 특성화하여 HIF-1α 활성화와 헤모글로빈 발현의 상향 조절 사이에 강한 상관관계를 확립했다. 이 연결을 기능적으로 검증하기 위해 D. magna에서 HIF-1α 유전자의 CRISPR/Cas9 매개 녹아웃을 수행했다. 비교 분석 결과, HIF-1α 결핍 돌연변이체는 저산소증에 대한 감수성이 현저히 증가했으며, 특히 저산소 스트레스 하에서 wild-type 개체에서 관찰되는 특징적인 헤모글로빈 반응을 억제할 수 없는 것으로 나타났다. 전사체 및 경로 분석을 통해 HIF-1α가 혐기성 대사 및 산화 스트레스 완화에 관여하는 유전자를 포함한 저산소 적응에 필수적인 유전자 네트워크를 조절한다는 것을 추가로 입증했다. 또한, 이 연구는 나노플라스틱과 같은 공동 스트레스 요인에 의해 이 조절 경로가 어떻게 영향을 받는지 탐구하여 환경적으로 현실적이고 복잡한 시나리오에서의 적응에 대한 통찰력을 제공한다.
종합적으로, 이러한 발견들은 HIF-1α가 저산소증에 의해 유도된 헤모글로빈 합성의 주요 조절인자이자 클라도세란들의 적응 전략의 중요한 구성 요소라는 직접적인 유전적 증거를 제공한다. 이 연구는 생태적으로 중요한 무척추동물 그룹에서 생존의 기본적인 분자 메커니즘을 설명하고, 수생 생태계가 증가하는 전 세계 탈산소화 위협에 어떻게 대응할 수 있는지에 대한 더 깊은 이해를 제공할 것이다.

목차 (Table of Contents)

• Chapter 1. General introduction 1
• 1.1. The emergence of hypoxia and its impacts on aquatic ecosystem 1
• 1.2. The adverse impacts of hypoxia on aquatic invertebrates 5
• 1.2.1. Phenotypic responses 16
• 1.2.2. Oxidative stress-mediated responses 18
• 1.2.3. Metabolic responses 20
• 1.2.4. Immune responses 21
• 1.2.5. HIF-1 signaling pathway 23
• 1.2.6. Hemoglobin and hemocyanin 25
• Chapter 2. Oxidative stress-mediated deleterious effects of hypoxia in the brackish water flea Diaphanosoma celebensis 32
• 2.1. Introduction 32
• 2.2. Materials and methods 39
• 2.2.1. Animal culture 39
• 2.2.2. Hypoxia conditions 40
• 2.2.3. Determination of NOEC and LC50, and in vivo experiments 41
• 2.2.4. Measurement of reactive oxygen species, antioxidant enzymatic activity, and hemoglobin content 42
• 2.2.5. Total RNA extraction and quantitative real-time polymerase chain reaction 44
• 2.2.6. Western blotting 45
• 2.2.7. Statistical analysis 45
• 2.3. Results and Discussion 46
• 2.3.1. Acute toxicity and in vivo effects of Diaphanosoma celebensis in response to hypoxia 46
• 2.3.2. Oxidative stress, antioxidant enzymes, and hemoglobin content of Diaphanosoma celebensis in response to hypoxia 48
• 2.3.3. Fluorescent microscopy of reactive oxygen species of Diaphanosoma celebensis in response to time-dependent hypoxia 50
• 2.3.4. Activation of HIF-1α and MAPK signaling pathway proteins 53
• 2.4. Discussion 54
• Chapter 3. Combined exposure to hypoxia and nanoplastics leads to negative synergistic oxidative stress-mediated effects in the water flea Daphnia magna 63
• 3.1. Introduction 63
• 3.2. Materials and methods 67
• 3.2.1. Animal culture 67
• 3.2.2. Establishment of hypoxia and nanoplastic conditions 68
• 3.2.3. In vivo experiments 69
• 3.2.4. Detection of reactive oxygen species, antioxidant enzyme activity, and hemoglobin content 70
• 3.2.5. Total RNA extraction and quantitative real-time polymerase chain reaction 71
• 3.2.6. Differential gene expression and transcriptome analysis 72
• 3.2.7. Western blotting 73
• 3.2.8. Statistical analysis 74
• 3.3. Results and Discussion 75
• 3.3.1. Acute toxicity and in vivo effects of D. magna in response to hypoxia 75
• 3.3.2. Fluorescent microscopy and enzymatic activity of ROS in D. magna in time-dependent hypoxia 77
• 3.3.3. Enzymatic activities and transcriptional expression levels of antioxidant enzymes and hemoglobin 79
• 3.3.4. Differential gene expression and ingenuity pathway analysis 82
• 3.3.5. Western blot of HIF-1α, NF-κB, and mitogen-activated protein kinase pathway proteins 83
• 3.4. Discussion 87
• Chapter 4. Differential susceptibility to hypoxia in hypoxia-inducible factor 1-alpha (HIF-1α)-targeted freshwater water flea Daphnia magna mutants 98
• 4.1. Introduction 98
• 4.2. Materials and methods 102
• 4.2.1. Animal culture 102
• 4.2.2. CRISPR/Cas9-mediated gene targeting of hypoxia-inducible factor 1α gene 103
• 4.2.3. Establishment of hypoxia and determination of NOEC and LC50 105
• 4.2.4. Assessments of life-history traits 106
• 4.2.5. Western blotting of HIF-1α 106
• 4.2.6. RNA extraction of wild-type and mutant-type D. magna in response to hypoxia, and transcriptome analysis 107
• 4.2.7. Ingenuity pathway analysis of HIF-1αrelated genes in response to hypoxia 108
• 4.2.8. Statistical analysis 109
• 4.3. Results and Discussion 110
• 4.3.1. Generation of hypoxia-inducible factor 1a gene mutants by CRISPR/Cas9 110
• 4.3.2. Acute toxicity and in vivo effects of Daphnia magna in response to hypoxia 110
• 4.3.3. HIF-1α protein expression of wild-type and HIF-1α genetargeted mutants 114
• 4.3.4. Differential gene expression and gene ontology of the normoxia and hypoxia-exposed wild-type and HIF-1α genetargeted mutants 115
• 4.3.5. Ingenuity pathway analysis of HIF-1α generelated pathway in hypoxiaexposed wild-type and mutant Daphnia magna 117
• 4.4. Discussion 121
• Chapter 5. Conclusion 129
• REFERENCE 131
• ABSTRACT IN KOREAN 164