77
humans due to a specific mutation, they do not serve as good models of CDAs. Cdan1 knockout mice responsible for CDA I die at an early embryonic stage (E6.5) suggesting that the gene is important for early development of the mice (R Renella et al. 2011). The information about Cdin1 knockout mice (another CDA I-associated gene) was not available in the literature but the International Mouse Phenotyping Consortium (IMPC) has Cdin1em1(IMPC)J homozygous mice available in their dataset and they were described lethal at embryonic day 9.5. Sec23b knockout mice, responsible for CDA II die shortly after birth that don’t show any signs of anemia but degeneration in the salivary glands and pancreas (Tao et al. 2012). Conditional knockout mice of erythropoietic cell specific Sec23b are viable but show no signs of anemia. Kif23 heterozygous or homozygous mutant mice failed to show any symptoms of dyserythropoiesis unlike the erythropoietic precursor cells of humans. In Klf1 knockout mice, homozygous mutation is lethal in the uterus having severe anemia like beta-thalassemia (Nuez et al. 1995; Parkins, Sharpe, and Orkin 1995). However, Klf1-/- mice results in embryonic lethality and balance of globin chains could not help in the rescue of the mice (Yang et al. 1995). Gata1-/- knockout mice are lethal at an early embryonic age causing severe defects in primitive and definitive erythropoiesis (Fujiwara et al. 1996).
The phenotypes expressed in the Tmcc2-/- mice closely resemble the hallmarks of a rare genetic disorder named congenital dyserythropoietic anemia (CDA). Due to differences in the expression of CDA mutations among humans and mice, our novel knockout mouse model would serve a good model for CDA to further characterize the molecular pathway of terminal erythropoiesis and enucleation.
Relevance of Tmcc2 knockout mice
The results presented in this thesis addressed the study of the physiological role of Tmcc2 with the use of a constitutive knockout mouse model. Studying what happens when a gene of interest is absent in vivo has been one of the most successful approaches to address the function of a knocked-out gene and provide information about the biochemical, developmental, physical, and behavioral roles of the protein encoded by the gene. In this thesis I describe the phenotype of the Tmcc2-/- mice that were generated to get an indication of what role TMCC2 plays in hair cells of inner ear. Humans share the majority of genes with mice and in most cases the functions of human and mouse orthologs overlap to a large extent.
For this reason, mice are frequently used to find the functions of human genes and to study human diseases.
78
Constant efforts are currently been taken to establish the function of human genes through mouse model by publicly available or published data via International Mouse Phenotyping Consortium (IMPC) and Knockout Mouse Program (KOMP). There are 20-30 thousand genes in mice and Tmcc2-/- mouse was not one of the available lines when we started the project and it is still not one of the phenotyped lines.
I made a rough count of gene knockouts generated by targeted mutagenesis, excluding chemically induced lines from random ENU mutagenesis projects or gene traps available through the International Mouse Strain Resource (IMSR) web page. Taking all available forms: live and archived mice, sperm and ovaries from all sources in the world but exclude ES cells than there are roughly 59 thousand lines in approximately 11 thousand unique genes.
This means that we are between 1/3 and half way towards the goal of knocking out every gene in the mouse genome. Then there are all the possibilities of different mutations in the same gene leading to different phenotypes. Phenotyping of these available lines is far less advanced. Despite having knockouts for so many genes there are thousands of key pieces missing from this puzzle and we know very little about the physiological function of the genes we have knocked out. Therefore, such simple knockout projects as the one described in this thesis are still very much needed.
After establishing the absence of TMCC2 in hair cells of the knockout mice, I looked for other phenotypes in different organs and based on the study of Ludwig et al. where they found a blood-specific isoform of TMCC2 which is expressed during the process of erythropoiesis (Ludwig et al. 2019). Later, I looked closely at blood, bone marrow and organs like spleen and liver where definitive erythropoiesis takes place in utero and right after birth, to decipher the function of TMCC2 in erythropoiesis.
The Tmcc2-/- mouse model has benefitted by providing a model for novel form of congenital dyserythropoietic anemia (CDA). The characteristics of the knockout mouse model show features of CDAs type II and type III along with some distinctive characteristics.
The classification of different types of CDAs are easier based on the morphological abnormalities; however, there is lack of suitable model in order to understand cellular and molecular mechanism of dyserythropoiesis. The mouse models available to date die during gestation or soon after birth. Surprisingly, some of the mouse models specifically engineered to replicate human CDA causing mutations do not develop CDA. This difference among the human and mice mutations in erythropoiesis indicate interspecies differences. There are three
79
models currently useful:the Gata1neoΔHS and the conditional RhoA and mDia2 (Diaph3) knockouts. Gata1neoΔHS is the first model replicating XLTDA, a part of naturally occurring CDA while conditional RhoA and mDia2 (Diaph3) knockout mimics different features of CDA type 3 like the presence of binucleated and multinucleated erythroblasts because of defect in cytokinesis (Konstantinidis et al. 2015; Mei et al. 2016). The complete knockout mice model of these genes is lethal. RhoAfl/fl gene when inactivated with erythroid specific EpoR-creTg/+ will eventually lead to death of the fetus (E16.5) but it is still possible to study the defects of fetal, definitive erythropoiesis. The function of RhoA in adult erythropoiesis could be studied by inducing inactivation with the Mx1-cre but later needs irradiation in mice using hematopoietic stem cell transplantation experiment which results in neutrophils, monocytes, and platelets (PLT) along with erythroid cells reconstitution (X. Zhou et al.
2013). RhoA and DIAPH3 genes have not been associated with CDA-causing mutations in humans. Hence, the status of these mouse lines has yet to be determined either faithful models of human CDA or just useful tools that replicates some aspects of CDA type 3.
Functional relevance of TMCC2 in hearing
Hearing impairment is the most frequent type of sensory deficit with 466 million people all around the world has mild to profound hearing loss (Www.Who.Int/Health-Topics/Hearing-Loss#tab=tab_2, n.d.). The latest estimates and projections from the World Health Organization are even more alarming. Close to 2.5 billion people are expected to experience some hearing loss out of which 700 million will require hearing rehabilitation (WHO 2021). In general, half of deafness cases are genetic with 70% of them caused by non-syndromic and 30% by non-syndromic mutations. There are hundreds of known genetic syndromes leading to hearing loss (Petit et al. 2019) and around 124 loci known for non-syndromic hearing loss in humans (hereditaryhearingloss.org). The research on genetic basis of non-syndromic hearing loss started with the whole-genome linkage analysis of large and clinically well characterized families, providing a certain approach for mapping critical chromosomal intervals (Botstein and Risch 2003). Another approach was used for characterizing autosomal recessive loci using homozygosity mapping of long stretches of homozygous genotypes in consanguineous families and homogenous populations with clinically similar features (T. B. Friedman et al. 1995; Schraders et al. 2012). Large chromosomal regions comprising several mega bases and hundreds of genes are further mapped by positional cloning strategies, such as bacterial artificial chromosome (BAC)-mediated cloning, using a priori hypotheses for selection and sequencing of candidate genes
80
based on literature and expression data or using a pre-existing mouse model of deafness for selection of orthologous human candidate genes to sequence (A. Wang 1998; Mburu et al.
2003; Naz 2004; Zwaenepoel et al. 2002; Robertson et al. 1998; von Ameln et al. 2012; X.
Liu et al. 2010; Kurima et al. 2002). Candidate genes in the locus were sequenced sequentially based on an arbitrary rank to pinpoint the mutation. The first reported SNHL locus mapping was a seven-year long journey that led to the identification of the POU3F4 gene to chromosome Xq via linkage analysis in 1988 (Wallis 1988; de Kok et al. 1995). This work was valued at the time as these loci were mapped when human molecular genetics was still beginning to develop and physical genomic maps contained serious errors that reduced the power of linkage analysis (Snoeckx 2004; He et al. 2011). Many classical NSHL candidate genes were identified in a similar way. The quick development of NGS technology has clearly resulted in faster and more accurate detection of variants for all Mendelian disorders, including SNHL. The scope of NGS approaches ranged from small targeted gene panels to whole exome sequencing (WES) or whole genome sequencing (WGS). In parallel to approaches that start with SNHL patients, mouse models contributed significantly to the discovery and characterization of mutations which causes hearing loss. Many different analyses that are impossible in human patients can be performed in mice, including genetic manipulation, breeding strategies that help to map mutations, physiological studies of dissected tissue, and the analysis of large numbers of genetically identical individuals of a certain genotype.
Mouse genetics played a crucial role in the unravelling of the development and the functioning of the mammalian auditory system (Brown, Hardisty-Hughes, and Mburu 2008;
Kikkawa et al. 2012). With the use of forward-genetic screens, novel hearing loss loci were identified in mice and genes essential for the function of hearing were discovered (Habiby Kermany et al. 2006; M. Schwander et al. 2007; Acevedo-Arozena et al. 2008; Hardisty-Hughes, Parker, and Brown 2010). Utilising gene-driven approaches, mouse models for hearing loss continue to grow in number helping to understand the molecular mechanisms and physiological bases of hearing impairment in mammals, the disease onset and progression and the pathological changes happening in the inner ear. Slowly with time, the research on hearing in mice got advanced in last two decades as described but still, there are a lot of unanswered questions in the field. In order to answer some of the questions, we generated Tmcc2 knockout mouse model and tested different molecular markers important for the development and maintenance of the hair cells.
81
TMCC2 is a novel hair cell marker expressed specifically in the hair cell body of both OHCs and IHCs in the inner ear. It was an interesting candidate for a novel deafness gene due to a hair cell specific expression, but our observations of the Tmcc2 knockout mice did not uphold this notion. It is still possible that certain point mutations in humans could lead to deafness, particularly age related hearing loss that takes much longer than a lifespan of a mouse to develop, and for this possibility our results regarding the expression and subcellular localization of TMCC2 are valuable. It is also possible that the mice have some specific electrophysiological defects that could be studied in a more specialized setting. TMCC2 is among a handful of proteins postulated to have a function in vesicular protein transport that have been localized in hair cells. Mutations in genes might result in profound disturbance of the hair bundle growth, splayed or missing stereocilia or kinocilia and in some cases to altered bundle polarity. The phenotypes mentioned below are a good indication of a potential problem with the transport of the components of the stereociliary links and can be easily detected by fluorescent confocal microscopy. The importance of TMCC2 in hearing is yet to be determined, but the expression of CDH23 appears normal in the Tmcc2-/- mice. CDH23 is a transmembrane protein playing a key role in the development of the hair bundle as the component of transient lateral links as well as tip-links. All Cdh23 mutations known in mice cause a recessive phenotype. The loss of function alleles of the Cdh23v class show profound hearing loss from birth, severe stereocilia disorganization and abnormal shaker/waltzer behavior (Palma et al. 2001; Di Palma, Pellegrino, and Noben-Trauth 2001; Wada et al. 2001;
Wilson et al. 2001; Yonezawa et al. 2006). Mutant mice with missense mutations in the extracellular cadherin repeats, Cdh23elong, Cdh23salsa, Cdh23jera present a phenotype of early onset hearing loss with no vestibular dysfunction. In contrast to the Cdh23v alleles, these are hypomorphic mutations where the development of the stereocilia is normal but progressive loss is observed in the tip links of the bundles (S. Liu et al. 2012; Manji et al. 2011; M.
Schwander et al. 2009). Since CDH23 was still localized normally in the stereocilia of Tmcc2-/- mice and these mice do not turn deaf with time it is clear that despite the hair cell specific expression and a confirmed role in protein vesicular transport, TMCC2 is not critical for the functional expression of at least one key hair bundle protein.
The kinocilium in mammals plays an important role in the morphogenesis of hair cells and its localization on the surface of the cell is one of the prominent manifestations of planar cell polarity (PCP) that is essential for hearing. The development of kinocilia in mouse hair cells takes place around E15, later on they follow the basal body and shift to the non-neural
82
part of the cell. In the same time, around E17 the stereocilia keep on growing and form the three to five rows of varying heights arranged in a staircase-like V-shaped structure known as the hair bundle (Williams et al. 2017; D. Wang and Zhou 2021). The generation of kinocilium precedes the appearance of stereocilia. Hence, kinocilia are essential for maturation and proper arrangement of hair cells. Kinocilia degenerate once mice start hearing at about P8 and later disappears completely around P12 (Leibovici et al. 2005). The deletion of a PCP gene Par3 affects the orientation of hair bundles and the positioning of the kinocilium within the hair bundle (Landin Malt et al. 2019). Other PCP proteins are also involved in the positioning of kinocilia, so a potential defect in the intracellular transport of these proteins should lead to a PCP phenotype. We have visualized kinocilia in Tmcc2-/- mice using an anti-tubulin antibody and observed that the appearance and the localization of kinocilia is normal.
In the course of experiments aimed at determining the subcellular localization of TMCC2 in hair cells we noticed that in early postnatal hair cells the TMCC2 signal in the apical domain of the cell resembles the staining for MYO6 and MYO7A, two members of the myosin family that are known to play a fundamental role in hair cell physiology. The MYO6 is localized to the cuticular plate in mammalian hair cells near the apical cell surface of the stereocilia. The MYO6 functions as a membrane anchor surrounding individual stereocilia (Hasson et al. 1997). In Snell’s waltzer mouse (a Myo6 mutant), the development of stereocilia is normal but immediately after the birth the stereocilia fuse together (Tim Self et al. 1999; Avraham et al. 1995). In the absence of MYO6, the membrane leads to the formation of giant stereocilia which later degenerate, hence it is hypothesized that MYO6 has a function to anchor the plasma membrane around individual stereocilia (Tim Self et al. 1999;
Hasson et al. 1997). The function of anchor is unique to MYO6 as it has a backward movement along actin filaments (towards the minus end) (Wells et al. 1999).
Shaker-1 homozygotes (Myo7ash1/sh1) display characteristic circling, head-tossing and hyperactivity resulting from impaired vestibular system. Cochlear defects of neuroepithelial-type result in progressive degeneration of the organ of Corti (Gibson et al. 1995). In hair cells MYO7A is expressed almost along the whole length of stereocilia, in the cytoplasm, the pericuticular necklace and cuticular plate (Hasson et al. 1997). I report that the expression and the localization of MYO7A in the Tmcc2-/- mice are normal. Consequently, we saw no signs of hair bundle degeneration.
83
Finally, the knockout mice can hear and show no signs of balance disorders, making it very unlikely that TMCC2 is required for basic hair cell functions. Perhaps TMCC2 simply does not have an important role in hair cells. Alternatively, the loss of TMCC2 might be compensated by the presence of its close paralog (TMCC3). I determined that the expression of TMCC3 protein in the Tmcc2-/- hair cells is much stronger than in wild-type controls.
Taking into account the fact that the expression of the Tmcc3 mRNA in utricular hair cells of the balance organ may be increased upon experimental induction of heat shock to the level that makes this gene one of the top 243 heat shock enriched signature genes (Ryals et al.
2018), it is plausible that TMCC3 has some stress-response function that can counteract the consequences of the loss of TMCC2. Only a double knockout mouse line with both these proteins missing from hair cells can conclusively resolve this issue. Nevertheless, future research in the field of targeted vesicular protein transport towards the apical surface of hair cells as well as the clearance of these proteins from the plasma membrane may benefit from the knowledge that TMCC2 and possibly TMCC3 are present in the apical domain of early postnatal hair cells.
Additionally, by the use of confocal super resolution microscopy I have uncovered that the TMCC2 immunofluorescence signal in hair cells is localized at or extremely close to the plasma membrane. Both outer hair cells, that are known to have a system of ER cisterns below the plasma membrane as well as inner hair cells that do not have such cisterns. This modest observation may have important implications for the research on the TMCC family of proteins in general, as so far these proteins were described as residents of the intracellular membrane compartment, not the plasma membrane (Hopkins, Sáinz-Fuertes, and Lovestone 2011; Hopkins 2013).
Functional relevance of TMCC2 in erythropoiesis
The role of the TMCC2 protein in erythropoiesis has been previously postulated based on genome wide expression studies and in vitro work but never addressed in vivo.
Comparison of gene expression in four distinct erythroid progenitor populations at successive erythropoietin-dependent stages of erythropoiesis identified 123 genes within 60 kb of single nucleotide polymorphisms (SNPs) that are found to be associated with blood cell traits (Merryweather-Clarke et al. 2011; Soranzo et al. 2009; Ganesh et al. 2009). Three genes out of 123 were considered differentially expressed at the most stringent level: transmembrane
84
coiled-coil domain family 2 (TMCC2), membrane-associated ring finger (C3HC4) 8 (MARCH8), and myeloblastosis oncogene (MYB).
A strong hint of a functional role of TMCC2 in erythropoiesis was its association with the mean corpuscular volume (MCV) in genome wide association studies (Soranzo et al.
2009; Thompson et al. 2010).
A previously unknown blood-specific isoform of human TMCC2 that expresses highly at the polychromatic to orthochromatic maturation stages (PolyE/OrthoE) was shown to be a regulator of terminal erythropoiesis. Severe defects in the proliferation and differentiation of RBCs were seen when TMCC2 was knocked down in vitro (Ludwig et al.
2019).
In a powerful single cell expression study TMCC2 turned out to be among 30 differentially expressed genes at the early-OrthoE stage when erythroid precursors undergo changes preparing them for enucleation (Huang et al. 2020). Taken together, these studies identify TMCC2 as a prime candidate for one of the key players in erythropoiesis and set the stage for the in vivo research described in this thesis. The phenotypic analysis of the Tmcc2 -/-mice comes at a time when these results are highly anticipated and needed to make further progress in the field. Even though some phenotype of disturbed erythropoiesis was to be expected it was not possible to predict whether the defect would be limited to slight alteration of erythrocyte properties or if it takes shape of severe, possibly life threatening anemia. What I found was that the severity of the phenotype was strongly dependent on age and that its presentation bears a striking similarity to a group of rare genetic disorders called congenital dyserythropoietic anemias.
Tmcc2 knockout mice bear striking resemblance to CDA II and CDA III, where the prominent morphological feature of CDA II are the double cell membranes observed in TEM microscopy and of CDA III is the presence of multinucleated erythroblasts. There are some unique features in Tmcc2 knockout mice that are not characteristic for CDA II or CDA III.
Tmcc2-/- mice have reduced body weight that we have analyzed systematically between the ages of three to five days, accompanied by a significantly lower number of RBCs, high fraction of nucleated erythrocytes and the presence of multinucleated erythroblasts in the blood of P5 mice. In adult Tmcc2-/- mice, we still observed nucleated erythroblasts in the peripheral blood, therefore the function of TMCC2 in erythropoiesis is not solely developmental but constitutive. The impact of the loss of TMCC2 on the RBC count varies
85
greatly between the young and the adult mice as early postnatal anemia gives way to slight erythrocytosis in the adults. However, the analysis of adult bone marrow exhibited that maturation of erythroblast is impeded, which suggests that in the knockout mice the process of erythropoiesis is hindered regardless of age. I propose that the age related improvement of the RBC number and size parameters could be due to the maturation of the bone marrow that leads to the controlled release of RBCs in the blood stream and the engagement of systemic compensatory mechanisms such as extramedullary erythropoiesis. The exact cellular and molecular mechanism of dyserythropoiesis in the Tmcc2 knockout mice will be a subject of future studies.
The findings described herein suggest that potential loss of function mutations of TMCC2 might result in a novel form of CDA in humans. There is no known pathogenic mutation in TMCC2 but single nucleotide polymorphisms (SNPs) are known to be located in this gene. A single nucleotide variant C of intronic rs61823972 is linked with the change in erythrocyte properties (Astle et al. 2016). Our observations provide a plausible explanation as to why some polymorphisms located in the TMCC2 gene are a subject of negative evolutionary pressure (Soranzo et al. 2009). As a more immediate practical consequence of my work, I postulate the inclusion of TMCC2 in hematological genetic testing panels used in diagnosing genetic forms of anemia. It is important to keep in mind, that the clinical picture of patients with anticipated TMCC2 mutations might differ from the phenotype of the Tmcc2 knockout mice, both because of the interspecies differences in the molecular and cellular mechanisms of erythropoiesis and because the patients may have only partial loss of function or even gain of function mutations that still lead to a haematological disorder.
Finally, based on my findings and the available literature, one can speculate in an attempt to deduce the possible cellular and molecular mechanism of TMCC2 function to guide future research plans. Let’s start with several simple facts. First, TMCC2 protein is an ER membrane protein recently shown to mediate vesicular transport and endosomal cargo sorting. Second, mutations in another vesicular transport protein (SEC23) lead to CDA II.
Third, there is a constant debate in the field on how the process of enucleation works and a growing body of evidence supports a crucial role of intracellular vesicle dynamics and transport in enucleation. Taking into account that abnormal abundance of mono- bi- and multinucleated RBCs in the bone marrow or circulation is a feature of different forms of CDA as well as a prominent phenotype of the Tmcc2-/- mice and that the retention of the nucleus can at least hypothetically be caused by a failure of preparatory steps that would
86
normally lead to enucleation or the enucleation itself it is interesting to consider the potential role that the vesicular transport proteins could play in enucleation. On the other hand, perhaps the multinucleated RBCs are formed because their precursors have undergone multiple nuclear divisions but failed to perform cytokinesis? If so, could a glitch in cytokinesis simultaneously explain the appearance of mononucleated RBCs or would it require invoking a malfunction in a separate cellular process? The existing literature offers some hints that could shed light on these issues.
Is enucleation more cytokinesis, vesicle transport or both?
Enucleation is one of the rate-limiting steps in the process of terminal erythropoiesis.
It is not entirely clear how enucleation happens, and there is evidence supporting two models:
asymmetric cytokinesis and vesicular trafficking.
One of the models for nuclear extrusion is asymmetric cytokinesis, where the nucleus is extruded from the cytoplasm in an active process that resembles cell division and involves the cytokinetic machinery. In this hypothesis an enucleating cell forms a contractile actomyosin ring, has a well-defined cleavage furrow, and completes the “division” by abscission of the expelled pyrenocyte (Barr and Gruneberg 2007). There are several studies that support asymmetric cytokinesis, which includes ultra-structural observations from the 1960s. For instance, pyrenocytes were surrounded by a thin rim of cytoplasm in an intact plasma membrane, which confirms that the separation of nucleus happens in a well-orchestrated process (Yoshida et al. 2005; Koury, Koury, and Bondurant 1989; Simpson and Kling 1967; Ehud Skutelsky and Danon 1967; E. Skutelsky and Danon 1970). Furthermore, there is a formation of a constricted surface on enucleating erythroblasts, which resembles a cleavage furrow (Ji, Jayapal, and Lodish 2008; Koury, Koury, and Bondurant 1989; E.
Skutelsky and Danon 1970; Repasky and Eckert 1981). In addition, cytochalasin D, an actin-depolymerizing agent that causes the disruption of actin filaments and inhibits actin polymerization, results in inhibition of enucleation in vitro (Kohara et al. 2019; Koury, Koury, and Bondurant 1989; Repasky and Eckert 1981). By the use of fluorescence microscopy, actin has been shown to accumulate in the constriction zone that exists between the nucleus and the cytoplasm (Koury, Koury, and Bondurant 1989; Wickrema et al. 1994;
Xue et al. 1997). RAC1 and RAC2 activity through mDIA2 GTPases contributes to the accumulation of actin in the constriction zone of murine primary erythroblasts (Ji, Jayapal, and Lodish 2008). These findings suggest a role of the actin cytoskeleton in the late stages of