A mouse infection model and long-term lymphatic endothelium co-culture system to evaluate drugs against adult Brugia malayi

The development of new drugs targeting adult-stage lymphatic filarial nematodes is hindered by the lack of a robust long-term in vitro culture model. Testing potential direct-acting and anti-Wolbachia therapeutic candidates against adult lymphatic filariae in vitro requires their propagation via chronic infection of gerbils. We evaluated Brugia malayi parasite burden data from male Mongolian gerbils compared with two immune-deficient mouse strains highly susceptible to B. malayi: CB.17 Severe-Combined Immmuno-Deficient (SCID) and interleukin-4 receptor alpha, interleukin-5 double knockout (IL-4Rα-/-IL-5-/-) mice. Adult worms generated in IL-4Rα-/-IL-5-/- mice were tested with different feeder cells (human embryonic kidney cells, human adult dermal lymphatic endothelial cells and human THP-1 monocyte differentiated macrophages) and comparative cell-free conditions to optimise and validate a long-term in vitro culture system. Cultured parasites were compared against those isolated from mice using motility scoring, metabolic viability assay (MTT), ex vivo microfilariae release assay and Wolbachia content by qPCR. A selected culture system was validated as a drug screen using reference anti-Wolbachia (doxycycline, ABBV-4083 / flubentylosin) or direct-acting compounds (flubendazole, suramin). BALB/c IL-4Rα-/-IL-5-/- or CB.17 SCID mice were superior to Mongolian gerbils in generating adult worms and supporting in vivo persistence for periods of up to 52 weeks. Adult females retrieved from BALB/c IL-4Rα-/-IL-5-/- mice could be cultured for up to 21 days in the presence of a lymphatic endothelial cell co-culture system with comparable motility, metabolic activity and Wolbachia titres to those maintained in vivo. Drug studies confirmed significant Wolbachia depletions or direct macrofilaricidal activities could be discerned when female B. malayi were cultured for 14 days. We therefore demonstrate a novel methodology to generate adult B. malayi in vivo and accurately evaluate drug efficacy ex vivo which may be adopted for drug screening with the dual benefit of reducing overall animal use and improving anti-filarial drug development.

Introduction
Lymphatic filariaisis (LF), caused by the vector-borne parasitic nematodes Wuchereria bancrofti, Brugia malayi and Brugia timori, presents a significant global health burden with an estimated 51 million people currently infected in tropical regions of the world [1].
Clinically, LF manifests as elephantiasis and hydrocele in approximately 40 million individuals, making it a leading cause of global disability [2]. Lymphatic filariasis is prioritised for elimination as a public health problem by 2030. The current elimination strategy relies on annual mass drug administration (MDA) of albendazole alone, or in combination with either or both ivermectin and diethylcarbamazine, which target the transmissible stage of the disease, the microfilariae (mf). In Africa, in regions of overlapping onchocerciasis and/or loiasis endemicity, delivery of albendazole-and ivermectin-based MDA drug regimens for up to 6 years with high population coverage is required to block transmission of infection. Currently, MDA is still needed in 49 countries, with 13 countries having not fully scaled up their elimination programmes, and five countries yet to commence MDA [3].
Development of a safe and effective short-course curative (macrofilaricidal) drug or, alternatively, a drug which permanently sterilises female adult lymphatic filariae, would be an important new tool to dock the long-tail of filariasis elimination, particularly in transmission 'hot-spots' and hard-to-reach areas currently without MDA delivery, or where existing strategies are failing [4].
Novel macrofilaricidal drug candidates, targeting nematode molecules or the intracellular filarial symbiont, Wolbachia, are identified by screening chemical compound libraries using a variety of in vitro assays. Screening drug activity against adult stage lymphatic filariae in particular is hampered due to low throughput and short life-span in vitro which jeopardises accurate evaluations of drug efficacy in culture due to decline in viability. To increase throughput and extend culture periods, anti-filarial or anti-Wolbachia in vitro drug screens which utilise more accessible and/or abundant life cycle stages; namely the mf [5,6] or infectious L3 stage [7] and other, surrogate, non-LF filarial parasites such as Litomosoides sigmodontis [8], L. loa [9], Mansonella perstans [10] or Onchocerca spp. [11,12] have been validated and implemented. Whilst useful for initial screening, a caveat to these systems is that anti-filarial or anti-Wolbachia activities may not translate to the target adult stages of lymphatic filariae due to variation in stage-or species-specific expression of nematode drug targets or relative yields and cell division rates of Wolbachia symbionts.
A further impediment to macrofilaricidal drug discovery is that there is no established system of maintaining filarial life cycles outside of an animal host. Whilst there is no suitable permissive rodent model of Wuchereria bancrofti or Onchocerca volvulus, the Mongolian Gerbil, Meriones unguiculatus, is the standard laboratory model of Brugia malayi infection, and the closely related animal filariae, Brugia pahangi [13,14]. Since the 1970s, Gerbils have been utilised to provide all life cycle stages for Brugia spp. for drug screening in vitro as well as onward therapeutic evaluations in vivo. Productivity in terms of numbers of adults and mf produced is highly variable, declines with age of infection and is generally low yielding, especially for B. malayi. More recently, Severe-Combined ImmunoDeficient (SCID) mice, deficient in adaptive immunity, have been appraised as long-term fully susceptible hosts for B. malayi and the human filarial pathogen, Loa loa, and have been subsequently validated as drug screening models [15][16][17]. Brugia malayi infection success and variability in adult yields are more consistent in SCID mice compared with gerbils [18]. We have recently identified that adaptive immune control of B. malayi in mice is dependent on interleukin (IL)-4 receptor-alpha (Rα) macrophage activation mediating recruitment of a vigorous tissue eosinophilia [19]. By ablating gene function of IL-4Rα and the eosinophil growth factor, IL-5, full development of B. malayi in mice is also possible [20].
Here, we compare two immuno-deficient mouse strains; CB.17 SCID and BALB/c IL-4Rα -/-IL-5 -/-, for long-term adult B. malayi parasite generation in comparison to the standard M. unguiculatis gerbil model. We utilised adult male and female B. malayi generated in the best performing rodent line to optimise a mammalian cell co-culture system for long-term maintenance over a time-course of 4 weeks. A primary human lymphatic endothelial cell coculture with adult female B. malayi was selected and initially validated as a drug screening system against macrofilaricidal and Wolbachia depleting drugs.

Ethics statement
All animal experiments were approved by the ethical committees of the University of Liverpool and Liverpool School of Tropical Medicine (LSTM) and conducted under Home Office Animals (Scientific Procedures) Act 1986 (UK) requirements. Animals had free access to food and water throughout the duration of studies, checked daily for welfare and weighed weekly.

Brugia malayi parasite maintenance
The life cycle of B. malayi (Bm) was maintained in Aedes aegypti mosquitoes and Mongolian gerbils. For Bm larvae (BmL3), microfilariae (mf) were collected from gerbils via catheterisation. Gerbils were anaesthetised with isoflurane and subjected to peritoneal washes with RPMI 1640 medium (ThermoFisher Scientific) to harvest mf. Microfilariae were then purified using PD10 size exclusion columns (Amersham), enumerated by microscopy and mixed with human blood to a final concentration of 20,000 mf/ml. Microfilariae were fed to female A. aegypti mosquitoes through an artificial membrane feeder heated to 37˚C (Hemotek). Blood fed mosquitoes were reared for 14 days with daily sugar-water feeding to allow development to BmL3 stage. At day 14, BmL3 were collected from infected mosquitoes by stunning at 4˚C, then crushed and concentrated using a Baermann's apparatus and RPMI medium.

B. malayi experimental infections
For BmL3 infection, male Mongolian gerbils, aged 4-6 months, were injected via the intraperitoneal route, with 400 highly motile BmL3. Alternatively, male IL-4Rα -/-IL-5 -/mice or male CB.17 SCID mice, both aged 6-8 weeks, were injected with 150 BmL3. Animals were left for periods between 12-and 52-weeks post-infection to allow infections to proceed to the chronic adult stage.

Adult B. malayi implantation surgeries
Brugia malayi adults were collected from infected donor CB.17 SCID mice via peritoneal lavage post-mortem. Parasites were then separated into male and female, washed with preheated, sterile RPMI (Sigma) and collected into groups of 10 female parasites for implantation (total n = 6-8). CB.17 SCID mice were then placed under surgical anaesthesia using isofluorane and received a subcutaneous injection of buprenorphine prior to implantation of female parasites into the peritoneal cavity. Implantation was achieved by making a small incision into the skin and abdominal cavity in the upper right quadrant and inserting parasites into the lower abdominal quadrant using a glass pipette to ensure all parasites were maintained in the cavity. The incisions were then re-sutured after implant and animals were re-housed as before and monitored closely.

In vitro culture of Brugia malayi adults
For parasite cultivation, adult B. malayi were isolated from the peritoneal cavities of BALB/c IL-4Rα -/-/ IL-5 -/mice via peritoneal lavage post-mortem. Parasites were washed with prewarmed, sterile RPMI and separated into males and females, with worms of the highest motility and similar lengths selected for culture.
Initial culture studies evaluated the survival of female and male B. malayi on different endothelial cell monolayers; lymphatic human microvascular endothelial cells derived from human dermis (HMVECdly; LEC; Lonza), human embryonic kidney cells (HEK293; European Collection of Authenticated Cell Cultures; ECACC), and their recommended cellular media; endothelial basal medium plus supplements (EGM-2 MV; Lonza), and Dulbecco's Modified Eagle Medium (DMEM; Sigma) for LECs and HEKs, respectively, to function as relative cell-free controls conditions in order to determine optimum culture conditions and longevity of culture systems. Individual cell lines were plated and allowed to form confluent monolayers in 6-well plates (Corning). At this point, parasites were added at a density of 2 adults per well with the addition of 6 ml of corresponding cell media containing 10% FBS, 2% penicillin-streptomycin and 2% amphotericin B. All cultured parasites were compared against parasites of the same age, freshly isolated from mice, to ensure cultured parasites were compared against an appropriate in vivo control. Culture media was replenished every 3 days and cells were replenished every week. Parasite motility was analysed every day using a 4-scale scoring system devised by Rao and Weil [21]. Parasites, both in vitro and in vivo, were taken for viability (MTT) and Wolbachia load analysis (qPCR) at the time points indicated in the corresponding results section.

3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) viability assessments
To assess parasite viability quantitatively, parasites were removed from culture and washed in pre-warmed phosphate buffered saline (PBS; Gibco). Individual parasites were then transferred into separate wells of a 96-well plate and incubated with 0.5 mg/ml MTT (Sigma) for 2 hours at 37˚C. Following incubation, parasites were washed in PBS, and incubated for 1 hour with 100% Dimethyl Sulfoxide (DMSO; Sigma) to solubilise the blue formazan product. The plate was then analysed using a fluorescent plate reader set at 450nm, including a primary shake step. Data were expressed as a percentage change in optical density readings from the in vivo control median.

qPCR for Wolbachia quantification
DNA extractions were performed on individual parasites using a Qiagen QIAmp DNA mini kit in accordance with the manufacturer's instructions. Quantification of Bm wsp copy numbers was performed using qPCR as previously described [15].

Anti-Wolbachia drug screen validation
To assess the in vitro culture system's ability to evaluate anti-Wolbachia drug activity, a drug challenge was carried out using the 'gold-standard' reference drug, doxycycline (DOX; Sigma Aldrich). Flubentylosin (ABBV-4083, also referred to as 'tylosin A microfilaricide'; 'TylAMac' [24, 25]), a novel oral tylosin macrolide derivative compound proven to have superior in vitro and in vivo anti-Wolbachia activities compared with DOX, was synthesised as described [25] and assessed in comparison to DOX. Doxycycline was solubilised in ddH 2 O and prepared to a concentration of 5 μM in EGM-2 MV medium, whilst the same concentration of flubentylosin was solubilised in 100% DMSO and diluted in EGM-2 MV medium, equating to a final percentage of 0.05% DMSO per well. Drugs were replenished at each medium change. An equivalent percentage of DMSO used in the drug groups was added to vehicle control groups. Parasites were scored daily for motility and survival, with treatment end points at 7 and 14 days. An additional wash-out group, which entailed drug treatment for 7 days followed by a 7-day washout period was included in the study to evaluate any Wolbachia recrudescence. Quantitative PCR analysis, as previous, was employed at endpoints to evaluate Wolbachia reductions in response to treatment.

Direct-acting macrofilaricide drug screen validation
To evaluate the system's functionality in determining direct acting macrofilaricide activity, Flubendazole (FBZ) and Suramin (SUR) (both Merck) were used as reference drugs. Compounds were prepared at 10 μM in DMSO. The equivalent DMSO concentration was added to control wells. Parasites were maintained for 14 days in culture with daily motility scoring and an end-point MTT readout.

Statistics
Data were tested for normal distribution using D'Agostino & Pearson omnibus normality tests. Data that passed normality tests were analyzed by one-way ANOVA with Tukey's multiple comparisons tests. Data significantly different from a normal distribution were analyzed using Kruskal-Wallis with Dunn's multiple comparisons tests. Significance was defined at alpha <0.05 and analyzed using GraphPad Prism v7.0h. Survival curves were compared using Mantel-Cox log-rank tests.

Results
Severe-combined and IL-4Rα -/-IL-5 -/immunodeficient mice are superior animal models for the production and long-term maintenance of adult B. malayi compared with M. unguiculatus.

Human lymphatic endothelium monolayers extend life-span of B. malayi adults in culture
Initial experimentation was conducted to determine lifespans of male and female B. malayi adult parasites in culture following isolations from IL-4Rα -/-IL-5 -/mice. Mammalian cell monolayers of human embryonic kidney cells (HEK-293) or human adult dermal lymphatic microvascular endothelial cells (referred to as lymphatic endothelial cells; LEC) were used to test whether their inclusion extended adult B. malayi survival, motility and metabolic activity to levels comparable to typically recorded within adult worms of the same age freshly isolated from rodent models. Cell co-cultures were compared with corresponding cell growth media only (Dulbecco's Modified Eagles Medium + 10% foetal calf serum; DMEM+10%FCS or endothelial growth medium 2 microvascular, EGM-2MV, Fig 2). By day 14, females cultured on LEC monolayers displayed high rates of survival (46/48 surviving, 96%, Fig 2A). Other culture conditions showed lower survival by day 14 (HEK = 38/48, 79%, DMEM = 36/48, 75%, EGM-2MV = 32/48, 67%) although survival was not significantly different between culture conditions (Fig 2A). Average (median) motility of surviving female B. malayi worms in culture declined from day 6 (HEK) or day 12 (DMEM and EGM-2MV) whereas average motility of females cultured on LECs reflected the vigorous motile phenotype typical of freshly isolated worms ( Fig 2B). When evaluated at day 14, average motility was significantly higher compared with other culture conditions (Kruskal-Wallis with Dunn's comparison post-hoc test, P = 0.0006, Fig 2C). By comparing the metabolic activity of cultured B. malayi females against those freshly recovered from mice, female worms cultured on LEC monolayers displayed, on average, a non-significant 15% reduction in MTT reductase activity (Fig 2D). In comparison, the metabolic activity of female B. malayi cultures under other cell-culture conditions exhibited a 58-83% reduction in metabolic activity. The decline in metabolic activity in female B. malayi cultured on HEK cells was significant compared to female B. malayi cultured on LECs (P = 0.0098, Kruskal-Wallis with Dunn's comparisons test Fig 2D).

A transwell co-culture system further improves female B. malayi survival in culture
In attempts to extend culture longevity of fully viable B. malayi females for periods �14 days, a lymphatic / leukocyte co-culture trans-well system was tested. These used a LEC monolayer in EGM-MV, as deemed the best performing culture (Fig 2), with the addition of a LEC or THP-1 human monocyte-derived macrophage (Mϕ) monolayer 6-well 0.4 μm pore transwell insert. THP-1 Mϕ were either seeded in a non-polarised state or conditioned with recombinant (r) IFN-γ or rIL-4/rIL-13, termed Mϕ(naïve), Mϕ(IFN-γ) or Mϕ(IL-4/13) (Fig 3A). Control LEC cultures (LEC monolayers) supported 100% survival (12/12 female B. malayi) for >16 days in culture (Fig 3B). Beyond this point, survival decreased slightly, with 92% (11/12) female B. malayi surviving by the 21-day end-point. Similar survival was apparent with LEC+Mϕ(IFN-γ) or LEC+Mϕ(IL-4/13) inserts, whereby 100% survival was maintained for 15 and 16 days (12/12 female B. malayi), before declining to 67% (8/12) and 83% (10/12), respectively, by day 21. Full survival was achieved for the duration of the 21-day culture period in LEC+Mϕ(naïve) or LEC +LEC insert co-cultures, a significant improvement on the other culture conditions tested (P = 0.0027 Mantel-Cox log-rank test). Full motility was sustained for the 21-day duration with the LEC+Mϕ(naïve) and LEC+LEC insert groups, whilst other groups demonstrated significant declines in motility during the culture period (P<0.0001, Kruskal-Wallis with Dunn's comparison test Fig 3C and 3D). Analysis of metabolic activity in cultured worms at the day 21 endpoint, compared against in vivo recovered female B. malayi, determined an 80% median reduction in metabolic activity of B. malayi cultured on LEC monolayers and a 69% reduction in female worms cultured in the presence of LEC+Mϕ(IFNγ) insert (Fig 3E). Metabolic activity was maintained to a level more representative of in vivo isolated worms in LEC+Mϕ(naïve), LEC+Mϕ(IL-4/13) and LEC+LEC insert co-cultures after 21-day incubations (22%, 26% and 24% reductions in metabolic activity, respectively). The improvement in metabolic activity supported by LEC+Mϕ(naïve) inserts was significant compared with LEC monolayers (P = 0.0132, Kruskal-Wallis with Dunn's comparisons post-hoc test).

in LEC bilayers
Microfilariae release by female B. malayi into the lower chamber medium of LEC bilayers were assessed after 3, 7 or 14 days of culture (Fig 4A), in which mf production declined with time in culture. At day 3, a median of 2850 mf had been released per female B. malayi. This reduced to 300 mf per female at day 7 and 0 mf per female by day 14 (P<0.0001, Kruskal Wallis test). Wolbachia yields within female B. malayi after 1, 2 or 3 weeks in LEC+LEC insert co-cultures were compared with those from parasites immediately retrieved from IL-4Rα -/-IL-5 -/mice ( Fig  4B). After one to two weeks in culture, Wolbachia loads were significantly lower, on average by 55%, than in vivo controls (both 1.4 vs 3.13x10 7 median wsp copies per worm) and by three weeks, levels had declined by 74.4% (0.8 vs 3.12x10 7 median wsp copies per worm (P<0.005, Kruskal-Wallis with Dunn's comparisons post-hoc test).

Reductions in metabolic activity and Wolbachia content in female B. malayi after 2-week cultures in LEC bilayers reflect cessation of mf production
A 21-day culture period with a 6-well 0.4 μm pore transwell, LEC co-culture 'bilayer' had been defined as sufficient to maintain female B.malayi survival at 100% with full motility. However, metabolic activity, whilst not significantly different from in vivo isolated females, was reduced on average by 25% after two weeks in culture and Wolbachia content had declined significantly. As mf production was not sustained beyond 7 days in vitro (Fig 4A), the cessation of embryogenesis may have impacted on measures of overall metabolic activity and Wolbachia content, compared with freshly isolated, gravid female worms. To more accurately interpret whether these measures reflected a real decline in viability or cessation of mf production, a sample of 62 adult female worms were isolated from CB.17 SCID mouse infections, and approximately equal batches were either processed immediately for metabolic activity and Wolbachia molecular assays, set up into culture within LEC bilayers, or implanted into naïve CB.17 SCID recipients (Fig 4C). After removal of male worms and 2-week in vivo implantation, female worm mf production had ceased. This was confirmed by overnight culture release assays. Although ex-implanted female worms were fully motile, their total metabolic activity had reduced by 17% when compared against freshly isolated female worms from mixed-sex infections (0.55 vs 0.66 median O.D. Fig 4D). Median Wolbachia titres within female B. malayi ex-implanted worms were 37% significantly lower than those recovered from in vivo mixed-sex controls (2.0 vs 3.3x10 7 ; P<0.05, Kruskal-Wallis with Dunn's comparisons post-hoc test, Fig 4E). Reductions in metabolic activity and Wolbachia production in female-only explants after two weeks were similar to levels in B. malayi cultured on LEC bilayers, which displayed an average 24% decline in metabolic activity (median 0.5 O.D.), and a 45% reduction in Wolbachia content (1.8x10 7 vs 3.3x10 7 ). Levels of metabolic activity and Wolbachia were adjudged not to vary significantly between female ex-implanted worms and cultured parasites.

Validation of B. malayi female LEC bilayer co-cultures to screen anti-Wolbachia drugs
Doxycycline (DOX), and the anti-Wolbachia clinical candidate, flubentylosin (ABBV-4083), a novel tylosin analogue, were used to validate LEC bilayers as a screening system to evaluate anti-Wolbachia drug efficacies. Female B. malayi in LEC bilayers were dosed with drug at 5 μM for either 7-days or 14-days before evaluation of efficacy. In certain cultures, drugs were removed following 7-day exposure, parasites washed, and cultures extended for a further 7-day period. Due to significant decline in Wolbachia content with time in culture due to loss of mf production (Fig 4), vehicle treated cultures were ran simultaneously and sampled at 7 or 14 days as matched controls (Fig 5A). Negligible changes in survival were observed throughout the study duration ( Fig 5A). Whilst there was some decline in average motility between days 7-14 in certain treatment groups, this was also observed in vehicle treated cultures, thus motility was not significantly influenced by DOX or flubentylosin drug exposure (Fig 5B and 5C). After 7-day treatment, both treatments significantly reduced Wolbachia levels. Doxycycline depleted Wolbachia on average by 77%, whilst treatment with flubentylosin reduced levels by 85% median depletion (5.26x10 6 and 3.47x10 6 median Wolbachia load per female vs 2.3x10 7 vehicle control, P<0.0001, Kruskal-Wallis with Dunn's comparison post-hoc test, Fig 5D). A further 7 days of DOX treatment maintained a significant 73% decrease in Wolbachia load (3.89x10 6 vs 1.44x10 7 median levels, P<0.0001, Kruskal-Wallis with Dunn's comparison posthoc test, Fig 5D). Removal of DOX at day 7 followed by a 7-day washout initiated a recrudescence of Wolbachia within the cultured female B. malayi worms, resulting in a 57% non-significant reduction of Wolbachia (Fig 5E). Contrastingly, 7-days post-removal of flubentylosin, Wolbachia recrudescence was not apparent, with a significant median Wolbachia reduction of 79% in comparison to the vehicle control group (3.02x10 6 vs 1.44x10 7 median Wolbachia loads, P<0.001, Kruskal-Wallis with Dunn's comparison post-hoc test, Fig 5E).

Validation of B. malayi female LEC bilayer co-cultures to screen macrofilaricidal drugs
Flubendazole (FBZ) and suramin (SUR), at fixed doses of 10 μM, were used to assess the functionality of the LEC bilayer co-culture system in evaluating direct macrofilaricidal activity ( Fig  6A). Vehicle control treated female B. malayi retained 100% survival and motility throughout the course of the study, whereas survival declined from 12 days of treatment in both FBZ and SUR treatment groups with 12% surviving after 14 days of SUR treatment and 0% survival with FBZ treatment (P = 0.0019 & 0.0003, respectively, Log-rank Mantel Cox test; Fig 6B). Motility significantly decreased in both drug treatment groups, reducing to average scores of 0 or 1 by day 14 for FBZ and SUR, respectively (P = 0.0006, Kruskal-Wallis with Dunn's comparison post-hoc test, Fig 6C and 6D). Quantitative metabolic activity assessment readouts at day 14 revealed significantly lower metabolic activity (72%) in FBZ-treated worms, which also had significantly lower activity in comparison to SUR treated worms, where 28% non-significant reduction compared to the vehicle control was observed (P<0.0001, Kruskal-Wallis with Dunn's comparison post-hoc test, Fig 6E).

Discussion
The discovery and development of new macrofilaricidal drugs for the prioritised filarial nematode NTDs: B. malayi, B. timori, O. volvulus and W. bancrofti, is hampered by a lack of longterm in vitro culture models to robustly test compounds for their effectiveness against the target adult life-cycle stage. This is reflected by deficits in culturing systems to reliably support the full development of human adult filariae from insect-derived, third-stage infective larvae in vitro, and a paucity of permissive laboratory models to propagate large quantities of male and female adult worms in vivo. 'Two-dimensional' (2D) cultures with human serum and/or monolayers of various mammalian cell lines are sufficient to support the initial growth of larvae through the third and potentially fourth cuticle moults for B. malayi [26], O. volvulus [27] or W. bancrofti [28]. In these studies, whilst certain adult internal organ morphological features can be microscopically determined, growth is severely retarded, with length typically �20-fold stunted compared to typical in vivo adult parasites. Moreover, sexual maturity and reproduction is not evidenced in these culture experiments. In one exceptional study, the technical feasibility of growing physiologically relevant, sexually mature adult B. malayi over 100 days, using cultures supplemented with a single-source of human serum has been demonstrated [29]. However, these data have not been reproduced using similar culture conditions, which is perhaps explained by variation in growth factors essential for filarial development in different sources of human serum [30]. More complex, 2D multi-cell, insert co-culture systems and 3D skin organoid culture scaffolds have been tested for O. volvulus which support 'preadult' stages surviving in culture for months, yet these in vitro generated filariae still display a severely stunted phenotype (typically <1mm) not dissimilar to growth achieved when culturing long-term on monkey kidney cell (LL-CMK2) monolayers [11,31].
With no reliable way of propagating physiologically relevant filarial adult stages from larvae, the only viable approach available for drug screening in vitro is to isolate adult filarial worms from animals that are either naturally, or experimentally infected with filariasis. For onchocerciasis, several closely related cattle Onchocerca species can be utilised. The female intra-nodular niche is unsuitable for ex vivo culture and only by careful collagenase digestion of excised nodules can culture of female stages be supported long-term [32]. Due to their greater abundance within nodule tissues and their natural migratory behaviour between nodules, male Onchocerca spp. are more readily isolated and can survive for prolonged periods of several months, when cultured on LL-CMK2 monolayers [32]. This system, using the cattle parasite, O. gutturosa, has become adopted as the gold-standard in vitro screen to evaluate nematodicidal or anti-Wolbachia drug candidates [33][34][35][36]. However, limitations of the screen are throughput via sourcing infected cattle, mainly within Sub-Saharan Africa, and differential macrofilaricidal susceptibilities between Onchocerca and lymphatic filariae (as determined for drugs such as moxidectin and albendazole) [37-40]. Incidence of differential macrofilaricidal activities between male and female worms is another caveat to reliance on this screening system alone [41]. Excised B. malayi derived from experimentally infected Meriones unguiculatus (Mongolian gerbils) exhibit rapid loss of viability in serum-supplemented media which is reflected by upregulation of a number of stress-response genes over the first five days in culture [42]. A recent study utilising image-based motion analysis demonstrated decline in female B. malayi PLOS NEGLECTED TROPICAL DISEASES motility (a late-stage indicator of in vitro fitness) begins at 5-7 days in optimised serum supplemented medium culture [43]. Due to this rapid demise in vitro, moderate automated throughput screening utilising motility phenotype as a readout of Brugia female macrofilaricidal activity has been limited to a three-day exposure window [44]. This limits hit-compound identification to more rapid-acting agents with drug compounds exerting more gradual killing effects being over-looked. Further, titrations to determine minimum effective concentrations of hit-compounds may generate under-estimates of true efficacy following longer-exposures or washout time-frames. Finally, compounds synergising with artefactual Brugia physiology induced by sub-optimal culturing may risk driving identification of false-positive hit compounds.
For these reasons, in this study we wished to advance a more robust and long-term culture model of lymphatic filariasis for drug screening applications. Our aims were to 1) improve in vivo propagation by examining whether yields of adult B. malayi could be increased with inbred immunodeficient mouse strains, 2) assess lymphatic endothelium / leukocyte 2D coculture systems to improve the longevity of adult worms in culture, and 3) evaluate reference macrofilaricidal drug responses in long-term culture.
Through an extensive meta-analysis of 32 independent sub-periodic human strain B. malayi infection experiments undertaken in our laboratory between 2012-2018, including untreated or vehicle control data derived from published in vivo preclinical model development, drug screening or immunological investigations [6, 15, 18-20, 23, 25, 45-49], we determined that immunodeficient mouse strains serve as improved models for propagations of adults. Whilst we have previously described reduced variability of adult worm burden and less frequent occurrence of infection failures in CB.17 SCID mice compared with gerbils [18], we extend our conclusions here that CB.17 SCID mice afford a >3-fold more productive source of adult infections per unit inoculation. Improvements in yields become more pronounced with chronicity of infection with >10-fold increases in adult burdens in >25-week old infections. In addition, we determine that an additional selective immunodeficient mouse model (BALB/c IL-4Rα -/-/IL-5 -/-) could persistently yield greater burdens of adult B. malayi compared with gerbils, similar to productivity in CB.17 SCID mice. This long-term susceptibility reflects the profound loss of eosinophil-specific mediated immunity in these mice, controlled by IL-4 receptor and IL-5, as detailed in our prior investigations [20]. Increased susceptibility to adult filarial infections in IL-4R -/-/IL-5 -/mice has also been determined for the related filarial parasites, L. loa and S. sigmodontis [50,51]. In both mouse models, we noted an increased bias toward female B. malayi infections which might indicate residual immune-mediated or other host-dependent factors influencing male survival in mice. We also noted that initial mf accruement within the peritoneum in the first few months of patency, normalised per adult female worm, was most abundant in gerbils, with CB.17 SCID mice being intermediate and IL-4R -/-/IL-5 -/mice displaying significantly reduced mf burdens. Whilst this might appear paradoxical given the increased adult burdens in mice, raised mf burdens in gerbils may reflect more balanced sex ratios in this model. In addition, other adaptive immune-mechanisms distinct from the IL-4 receptor and IL-5 signalling axis and lacking in gerbils and CB.17 SCID mice may exert immune-pressure on mf survival in IL-4R -/-/IL-5 -/mice. In contrast to developing larvae, B. malayi mf activate IFN-γ producing lymphocytes in stage-specific BALB/c mouse experimental infections [52]. Our data indicate that CB.17 SCID or BALB/c IL-4R -/-/ IL-5 -/mice offer superior alternatives to gerbils specifically where a large demand for mature female B. malayi in ex vivo research is required, such as drug screening. In our experience, the reduced costs to produce BALB/c IL-4R -/-/IL-5 -/mice in-house and more prolonged infection courses without incurring animal welfare issues compared with CB.17 SCID mice make this research model most suitable to propagate adult female B. malayi.
From our initial culture studies, we determined that co-cultures with human primary LEC monolayers extended the period of full motility and survival of mature female B. malayi to 14 days. Further analysis of metabolic activity confirmed that female adult B. malayi displayed only minor reductions compared with freshly isolated parasites from BALB/c IL-4R -/-/IL-5 -/mice. This extended, fully viable life-span in culture was specific to the presence of LEC cells in culture as neither full endothelial growth medium nor human embryonic kidney cells could emulate the extended survival time. Our data is consistent with a recent publication demonstrating benefit of co-culture with a mouse endothelial cell line monolayer in augmenting survival of mf and adult L. sigmodontis [8]. Because in this study conditioned medium derived from endothelial cell cultures supported full survival of adult worms for 15 days, a hypothesis was proposed that endothelial cell-restricted macromolecules and metabolites released into culture provide a necessary source of nutrition to support in vitro adult worm fitness [8]. An alternative hypothesis proffered is that host cell monolayers may modify oxygen content in the liquid phase which favours filarial nematode survival [30]. Whilst LEC monolayers also extended the male B. malayi life-span in vitro, males cultured declined in viability from 12 days, and metabolic activity, motility, and percentage survival was severely reduced by day 14. We conclude that, unlike Onchocerca spp., it is more challenging to extend male vs female lymphatic filarial life-spans in vitro. Whilst the reasons for this are not clear, we speculate the smaller size and rapid motility of male B. malayi may make them more vulnerable to depletion of inherent energy stores which they are unable to sustainably replenish in culture.
Upon selection of a favourable monolayer and B. malayi adult-stage, we wished to test whether a mixture of leukocytes and lymphatic endothelium could emulate the lymphatic / lymphoid parasitic niche of the lymphatic system to further prolong longevity in culture. We selected a monocytic-Mϕ human THP-1 cell line to test as representative of abundant circulating or patrolling monocytes within the circulatory / lymphatic systems as well as major resident populations within afferent lymphatic-lymph node junctions [53]. Thus, we hypothesised monocyte-macrophage lineage cells would naturally occur in intimate and frequent contact with lymphatic-dwelling filariae in vivo. Further, because lymphocytes are absent, yet peritoneal Mϕ constitute as much as 50% of the white blood cells found within the long-term permissive CB.17 SCID B. malayi infection model, we speculated that this leukocytic cell may provide a source of nutritional factors suitable for supporting long-term parasite survival in the absence of a strong eosinophil larvicidal/adulticidal immune effector response. Finally, in support of this hypothesis of the Mϕ as a 'feeder cell', in earlier studies, we observed improved survival of B. malayi larvae when cultured with peritoneal Mϕ [54]. Because LF patients display a spectrum of T cell activation states that can influence the activation and metabolic status of Mϕ via differential cytokine production [54][55][56], we artificially polarised THP-1 Mϕ toward a classical (M1) or alternative (M2) phenotype with pre-exposure of recombinant IFNγ or IL-4/IL-13 [23]. We determined that non-polarised Mϕ in an upper chamber with LEC mono-layers in contact with filariae in the lower chamber improved full female B. malayi survival and motility for 3-weeks and maintained metabolic activity at approximately 75% of in vivo levels, indicating that these cells may provide an additional source of soluble factors to prolong adult lymphatic filarial survival ex vivo. Mϕ conditioned with IL-4/13 also appeared somewhat beneficial. Contrastingly, IFNγ conditioned Mϕ were marginally detrimental in B. malayi female in vitro fitness at 3 weeks, compared with LEC monolayers. This perhaps reflects production of soluble released mediators by classically-activated Mϕ, such as nitric oxide, which are toxic to adult B. malayi in culture [57,58]. Surprisingly, the inclusion of a further LEC mono-layer in the upper chamber re-capitulated full survival, motility and sustained metabolic activity observed with non-polarised Mϕ+LEC cocultures.
Given the simplicity of working with a single versus a double cell system, we opted to take forward the LEC bi-layer system for further validation as a drug screening system, including for anti-Wolbachia compound assessments. However, the transwell co-culture system could be of benefit in future applications to study other immune and non-immune cell types. We determined that Wolbachia content significantly declined with time in culture. Wolbachia are found both in somatic tissues of the hypodermis and the female germline, where populations expand within all embryonic stages [59,60]. Female embryogenesis ceases in the absence of male mating in vitro (as evidenced by loss of mf production by 14 days in culture). By implanting single sex female worms into CB.17 SCID mice for periods matching those in culture (and thus blocking fertilisation in vivo) before estimating total Wolbachia content, we deduced that Wolbachia decline reflected a natural loss of embryogenesis and mf production. Further, the minor impact in metabolic activity of cultured B. malayi within LEC bi-layer cultures were similar to reductions following cessation of the embryogenesis pathway in vivo; a biological pathway of high metabolic demand. Thus, we conclude that notwithstanding loss of mf gestation, our in vitro LEC bi-layer system supports fully viable female B. malayi in culture for three weeks.
We subsequently demonstrated the advantage of being able to sustain fully viable B. malayi females for prolonged periods in vitro to accurately discriminate efficacy of anti-filarial drugs using physiologically relevant fixed dose concentrations (5-10 μM). Over a two-week period, the system was able to discern the inferior efficacy of the slower-acting anti-Wolbachia agent, doxycycline, versus the more potent novel tylosin analogue, flubentylosin, over a one-week exposure interval, as previously determined in animal infection models [25,45,47,61]. Following doxycycline drug removal at one week, Wolbachia levels recrudesced by two weeks, a phenomenon consistently observed when using sub-optimal dosing of tetracyclines or rifamycins to treat Brugia or Litomosoides in vivo [25,45,47,61]. Observing Wolbachia re-expansion within female B. malayi following drug removal is confirmation of the in vitro health of the Wolbachia-filarial symbiosis in our culture system. Thus, we have confidence that this culture system could be applied in the future to evaluate anti-Wolbachia candidates with putatively faster kinetics of symbiont depletion compared with registered antibiotics and to investigate mode-of-action and target identification studies. LEC bi-layer maintenance of female B. malayi was also successfully validated as an effective model in determining the long-term impact of the direct-acting nematodicidal agents, flubendazole or suramin. The pharmacology of flubendazole has been recently better fully characterised, whereby sustained release of injectable formulations for several weeks are necessary to exert significant macrofilaricidal activities against Brugia and Onchocerca filarial parasites in preclinical infection models [48,62]. Conversely, it has been challenging to emulate flubendazole sustained exposure and macrofilaricidal activity in vitro or with mixed in vitro / in vivo B. malayi systems due to limitations in culture period [63,64]. Our findings demonstrate that periods of two weeks in culture are necessary to determine macrofilaricidal activity of this benzimidazole drug at physiologically relevant dose concentrations. Therefore, our culture screening system might be useful to elucidate the pharmacodynamics of other members of the benzimidazole class against B. malayi, including albendazole [39] and oxfendazole [33] in deployment or in clinical development as alternative treatment strategies for lymphatic filariasis and onchocerciasis, respectively.
In conclusion, we have established an improved method for both in vivo propagations and in vitro maintenance of female B. malayi. We have demonstrated the utility of this approach for improving accuracy of assessing efficacy of anti-Wolbachia and direct-acting nematodicidal agents. The culture system could be deployed to more accurately evaluate the potential translation of 'hit' compounds against lymphatic filariasis following chronic drug exposures and to compare efficacy against Onchocerca spp. We recognise that the complexity of a 2-3 week-long LEC bi-layer culture system in current large well format (6-well) is unlikely to be adopted as a screen with moderate-throughput capacity. Miniaturisation into 24-48 well plates may be possible but will require onward careful evaluation of female B. malayi in vitro fitness over multiple weeks. As an alternative, this culture system may be incorporated as a secondary confirmatory screen to robustly validate initial hits identified in moderate screening systems and aid rational selection of compounds for advancement into in vivo preclinical testing. In this way, the screen may filter out false hit compounds that are synergising with stress responses in sub-optimal in vitro systems or compounds with differential active against surrogate life-cycle stages such as filarial mf. Similarly, LEC bi-layers can be utilised to determine efficacies that manifest more gradually at physiologically achievable dose concentrations prior to selection for animal testing. As an example application, we have utilised B. malayi female LEC bi-layers as a secondary screen following hit-identification against mf in the discovery of dihydrobenzoxazepinone compounds with broad-spectrum anthelmintic activities [65]. Further opportunities for innovation include incorporation of this long-term co-culture model with human hepatic organoids via a microfluidics physiological system ('organ-on-a-chip') to more accurately model drug metabolism and in vivo pharmacokinetics of late-stage candidate anti-filarial drugs. As such, the novel methodologies described herein, if adopted, should reduce overall animal use, lower costs and improve decision making toward expediting new curative drugs for filarial NTDS.


Notch Signaling in HSC Emergence: When, Why and How

The hematopoietic stem cell (HSC) sustains blood homeostasis throughout life in vertebrates. During embryonic development, HSCs emerge from the aorta-gonads and mesonephros (AGM) region along with hematopoietic progenitors within hematopoietic clusters which are found in the dorsal aorta, the main arterial vessel. Notch signaling, which is essential for arterial specification of the aorta, is also crucial in hematopoietic development and HSC activity. In this review, we will present and discuss the evidence that we have for Notch activity in hematopoietic cell fate specification and the crosstalk with the endothelial and arterial lineage. The core hematopoietic program is conserved across vertebrates and here we review studies conducted using different models of vertebrate hematopoiesis, including zebrafish, mouse and in vitro differentiated Embryonic stem cells. To fulfill the goal of engineering HSCs in vitro, we need to understand the molecular processes that modulate Notch signaling during HSC emergence in a temporal and spatial context. Here, we review relevant contributions from different model systems that are required to specify precursors of HSC and HSC activity through Notch interactions at different stages of development.

Introduction to HSC Development
Hematopoietic stem cells (HSCs) sustain the adult blood system by generating mature blood cells of all lineages through multi-potent progenitors of intermediate stages [1]. During embryogenesis, the hematopoietic system is established through several waves starting from Embryonic day (E) 7.5. In mouse, the earliest blood cells are produced in the blood islands of the yolk sac (extra embryonic) which continue to distribute hematopoietic cells with erythro-myeloid lineage potential by E8.5 and multipotent hematopoietic cells also with lymphoid lineage potential at later stages [2][3][4]. Cells originating from the early waves of hematopoiesis also include tissue resident macrophages that infiltrate various organs and fulfil tissue-specific and niche-specific functions, including functions during HSC development [5,6]. However, the first HSCs with hematopoietic reconstitution capacity are detected from E10.5 onwards within the embryo (intra-embryonically). They are particularly enriched in the trunk of the embryo where the aorta, gonads and the mesonephros meet (AGM). The hematopoietic stem and progenitor cells (HSPC) accumulate as Intraaortic hematopoietic clusters (IAHC) in the dorsal aorta (DA) [7][8][9][10][11]. Although nascent HSCs have been associated to other sites (umbilical cord, placenta, head [12][13][14][15]), blood emergence is closely associated with a specialized endothelial cell population, termed hemogenic endothelium (HE), that trans differentiate to blood by losing their endothelial identity and gaining hematopoietic potential. Over the years, several studies have conclusively demonstrated this endothelial-to-hematopoietic transition (EHT) by in vivo imaging of different animal models, as well as in vitro differentiation to blood from Embryonic Stem (ES) cells [16][17][18][19][20][21][22]. HE cells can be identified by the co-expression of endothelial marker gene expression such as CD31, CDH5, ACE and CD44 and key hematopoietic transcription factors, including Runx1, Gfi1 and Gata2 [23][24][25][26][27][28][29][30]. The earliest transcription factors detected 2 of 19 in the HE, Runx1 and Gata2, are downstream of Notch signaling, [31,32] and later the expression of Gfi1 is detected in a discrete subset of Runx1 positive cells that are restricted to the HE and IAHC, while runx1 expression extends to the subaortic mesenchyme [30]. Several other surface markers and transcription factors have been described to enrich HSC activity, including Sca1, Gpr56, CD27 and CD201 (PROCR) [33] [34][35][36][37]. Once the EHT process is completed, the cells proliferate and recruit other cells [29,38] forming IAHC that appear associated to the ventral wall of the dorsal aorta starting between the embryonic days E10.25-E12 in the mouse (week 4-5 in human embryo). Although HE and IAHC can be observed on both the ventral and the dorsal side of the aorta within this time window, only the IAHC associated with the ventral side contain transplantable HSCs [27,39]. This has been mainly attributed to pathways, including BMP, hedgehog and Notch signaling that are polarized to the ventral domain [39]. The emerging HSPCs then migrate to the fetal liver for maturation and expansion [40]. The sites of HSC emergence and their migration between hematopoietic niches are very well conserved in vertebrates [41]. In addition, in the zebrafish, HSPCs emerge from the dorsal aorta of the trunk. However, unlike in the mouse model, the early erythroid-myeloid progenitors and the emergence of progenitors with HSC properties occur within a shared spatial and temporal manner [42]. At least in the mouse, transplantation assays performed at different time points of HSPC emergence, early (pre)-HSC can readily contribute to the blood system of neonates, but not directly to the adult system [43]. This potency is only evident in HSCs that are older than E11.5. Even then, only a very small fraction of these HSPCs are functional HSCs [25][26][27]44], with the majority being blood progenitors. Therefore, although there is consensus regarding the site of HSC emergence. It is unclear whether HPCs and HSC share the same HE precursors, or if in fact, the HE is a heterogeneous cell population with different capacities. Moreover, further clarity is required in understanding which molecular pathways are unique to HSC emergence or shared with HPCs. Adding further to the complexity, EHT and HSPC emergence occurs at a developmental stage when angiogenesis is in progress and vascular identity (arterial versus venous) is being established. Therefore, it is highly plausible that both these processes share common signaling pathways to some extent.
Importantly, arterial fate acquisition and HSC emergence are dependent on Notch activity, but its specific requirement in HE and EHT is only now to emerge. There is some evidence suggesting that the arterial fate can be uncoupled from HE, but also evidence arguing that arterial specification of the aorta is a prerequisite for HE specification and subsequent EHT of HSPC/HSCs. Currently, these questions remain open. In this review, we will discuss and highlight the current studies that can give us clues on the requirement of Notch activity for HE and specifically, HSC emergence in mouse and zebrafish since these two model organisms have contributed the most to our understanding of embryonic hematopoiesis.

The Basics of Notch Signaling
The Notch signaling pathway controls cell fate decisions during embryonic development and is highly conserved in metazoan [45]. In the classical model, Notch signaling is established through cell-cell contact. Adjacent cells express the Notch receptor and/or ligand on their cell surface. Upon interaction, the receptor is activated and results in the nuclear translocation of the active form of the Notch signaling molecule (Figure 1). In mammals, there are four Notch receptors (Notch1-4) and five ligands: there are three Delta ligands (Dll1, Dll3 and Dll4) and two Jagged ligands (Jag1 and Jag2), but in zebrafish some of these receptors and ligands have undergone duplications and therefore have two isoforms [42]. Typically, upon ligand binding (Delta or Jagged), the Notch receptor undergoes three protease cleavages thereby releasing the Notch Intracellular Domain (NICD), which then translocates into the nucleus. In the nucleus, the NICD forms a complex with its coregulator, RBPJ (Recombination signal-Binding Protein for Ig Kappa J region) and recruits co-activators such as MAML (Mastermind-Like) to its gene targets. The best characterized NICD-RBPJ complex targets are the transcriptional repressors genes of the Hes/Her family in vertebrates ( Figure 1) [46]. These Hes/Her transcription factors can repress genes driving cell specification, cell differentiation and cell cycle arrest [46]. Hes/Her can also form a negative feedback loop and repress Notch ligand expression in a particular cell. This first receiver can now send out a signal and inhibit the neighboring cell through the remaining ligands on its cell surface ( Figure 2). This lateral inhibition mechanism results in one cell that is unique in a homogenous cell population. This cell can be destined to acquire a distinctive fate, by inducing Notch activation and repressing this specific fate in the neighboring cells ( Figure 2) [47][48][49]. However, the reverse mechanism can be used to specify a small group of cells with the identical fate. Here, an adjacent cell expresses the Notch ligand upon Notch activation which then further spreads this (positive) feedback loop (lateral induction) to the next neighboring cell [50,51]. Subsequently, both the interacting cells and a small group of cells within a population acquire the same fate ( Figure 2). On the contrary, Notch receptors and ligands can also form cis interactions that inhibit or activate Notch signaling. In these instances, the receptor and the ligand are present on the surface of the same cell, form a complex and thereby mask this cell from further Notch activity [52][53][54]. In vertebrates, different combinations of Notch receptor and ligand can be expressed in a cell population and their spatiotemporal abundance contributes further to the complexity of Notch signaling. Interactions of different ligands with the same receptor can also trigger distinct responses. For example, during angiogenesis, JAG1 and DLL4 drive different outcomes in the control of cell fate decisions. Similarly, Dll1 and Dll4 induce different Notch activation dynamics resulting in opposing gene programs and cell fates [55]. Finally, Fringe glycotransferases (Radical, Lunatic and Manic) can modify the Notch receptor and alter the affinity between Notch receptors and their ligands and further fine tune the cellular Notch activity [56].

Release of the Transcriptionally Active NICD
The Notch receptor is cleaved at multiple sites and stages before its NICD is released into the nucleus. Initially, the Notch receptor is post-translationally cleaved at the S1 site whilst in the trans-Golgi network by a Furin-like protease, which results in a heterodimer that is held together by Ca 2+ -dependent ionic bonds and can be integrated into the cell membrane. Here, it is important to note, that this first cleavage exposes the negative regulatory region (NRR) at the base of the extracellular domain (ECD) and this is critical in preventing Notch activation in the absence of the correct signals. When a receptorligand interaction occurs, conformational changes in the NRR allow access to the ADAM proteins to the S2 cleavage site that is also based in the ECD of the Notch receptor [57][58][59]. Once the S2 cleavage occurs, further conformational changes expose the S3 cleavage site on the intracellular domain of the receptor to the γ-secretase complex that is comprised of multiple protein subunits including Nicastrin, Anterior pharynx defective-1, Presenilin enhancer-2 and the catalytically active subunit PRESENILIN. This complex is present at the plasma membrane and localizes to early/late endosomes and in lysosomes. Notch receptor and γ-secretase co-localization to the endocytic compartment is critical to Notch activation. During endocytosis, CATHRIN is recruited to the plasma membrane and

Release of the Transcriptionally Active NICD
The Notch receptor is cleaved at multiple sites and stages before its NICD is released into the nucleus. Initially, the Notch receptor is post-translationally cleaved at the S1 site whilst in the trans-Golgi network by a Furin-like protease, which results in a heterodimer that is held together by Ca 2+ -dependent ionic bonds and can be integrated into the cell membrane. Here, it is important to note, that this first cleavage exposes the negative regulatory region (NRR) at the base of the extracellular domain (ECD) and this is critical in preventing Notch activation in the absence of the correct signals. When a receptor-ligand interaction occurs, conformational changes in the NRR allow access to the ADAM proteins to the S2 cleavage site that is also based in the ECD of the Notch receptor [57][58][59]. Once the S2 cleavage occurs, further conformational changes expose the S3 cleavage site on the intracellular domain of the receptor to the γ-secretase complex that is comprised of multiple protein subunits including Nicastrin, Anterior pharynx defective-1, Presenilin enhancer-2 and the catalytically active subunit PRESENILIN. This complex is present at the plasma membrane and localizes to early/late endosomes and in lysosomes. Notch receptor and γ-secretase co-localization to the endocytic compartment is critical to Notch activation. During endocytosis, CATHRIN is recruited to the plasma membrane and adheres to lipid-or protein-binding domains at the membrane with the help of adaptor proteins. These adaptors help to form a curved vesicle called CLATHRIN-coated pits. These pits invaginate with the help of bending-proteins, such as EPSIN and form vesicles that eventually bud off from the membrane. The vesicle is then uncoated and can then fuse with other intracellular structures such as endosomes and lysosomes [60,61]. The S3 cleavage occurs within these intracellular vesicles and releases the NICD and allows Notch signaling to be initiated.

The Role of Notch Ligand in Activating Notch Signaling
Notch activation is not only dependent on receptor-ligand interactions, but also on the ECD dissociation from the receptor and its trans-endocytosis into the ligand-expressing cell [62,63]. It has been suggested that this pulling force is necessary for conformational changes in the NRR region and for S2 cleavage by ADAM family members, ADAM10 and ADAM17/TACE, after which the ECD is free to be trans-endocytosed [63].

Notch Ligand Independent NICD Activation
As an alternative, Notch endocytosis has been suggested not only as necessary for its activation, but also in order to decrease the level of Notch signal by reducing its expression on the cell surface. Notch can indeed be marked for degradation via ubiquitination by E3-ligases such as AIP4/ITCH [64] or Nedd4 [65,66]. It is then endocytosed via NUMB which recruits the AP2-clathrin adaptor-complex [67,68]. Finally, Notch activity can also occur independent of ligand interaction. DELTEX, a E3 ubiquitin ligase, can facilitate the processing of the Notch receptor in the endosomes and thereby release the NICD in the absence of a ligand [69,70].
In conclusion, complete processing of Notch requires multiple cleavages, classically through interaction with a ligand and internalization of the receptor, where it then becomes cleaved and fully activated in the endosome. Each of these steps requires the participation of several proteins and they all together fine tune the final NICD activity.

Notch Activity during Zebrafish Angiogenesis
Fate-mapping studies in zebrafish of mesodermal progenitors labeled at midblastula or gastrula stages indicate that at least some of the putative hemangioblast are bipotential and capable of giving rise to cells of both lineages, although the majority of labeled cells give rise to only one lineage [71][72][73]. Nevertheless, the early angioblasts assemble via migration to the midline of the trunk into two axial vessels: the dorsal aorta and the posterior cardinal vein (PCV), found just below the DA. Interestingly, fate-mapping studies in the zebrafish indicate that angioblasts become restricted to either an arterial or venous fate before they start to migrate to the trunk midline to form the DA and PCV [74]. Hedgehog (Hh) is a morphogen known to regulate epithelial/mesenchymal interactions during embryonic development and sits at the apex of the signaling cascade that leads to arterial and venous identity [74,75]. In zebrafish, its secreted from the endoderm during gastrulation and later from the notochord and forms a decreasing gradient to induce the expression of two families of angiogenic cytokines, vascular endothelial growth factor-1 (vegf ) from the somites and angiopoietins-1 and -2 (Ang1/Ang2) [76]. Vegf-A is produced by multiple cell types, including somites and smooth muscle cells and regulates differentiation, proliferation and survival of ECs [77,78]. In angioblasts that have arterial identity, Vegf interacts with the Vegf receptor Flk1/Vegfr2 and Np1 complex to induce the activation of the Notch and Erk signaling pathways [79,80]. In contrast, in venous angioblasts, chicken ovalbumin upstream promoter transcription factor II (Coup-TFII) acts to suppress the Notch and Erk pathways and thereby repress arterial fate induction [74,80]. The Notch pathway activation is required for imperative for arterial specification and arterial marker expression, including Ephrinb2, Notch1, Notch4, Dll4 and the Notch downstream target gene Hey2 (a hairy/enhancer-ofsplit-related basic helix-loop-helix transcription factor) [81]. The venous angioblast express COUP-TFII and the B4 ephrin receptor (EphB4) [80,82].

Notch Activity Contribution to Zebrafish HSPC Emergence from the Dorsal Aorta
Analysis of the mind bomb mutant in zebrafish embryos has demonstrated the requirement of Notch signaling in HSPC emergence from the dorsal aorta. Mind bomb is an E3 ubiquitin ligase, which is essential for endocytosis of Notch ligands upon Notch receptor and ligand interaction [83]. The mutants display a complete absence of transcripts for the HSC marker genes cmyb and runx1 in the ventral floor of the dorsal aorta at 36 h post fertilization (hpf), although the erythro-myeloid progenitor (EMP) generation is unaffected [32,84]. Accordingly, overexpression of the Notch intracellular domain resulted in an expansion of cmyb and runx1 at 36 hpf that reaches into the venous endothelium, but notably, without increasing efnB2a expression into the vein [32]. Studies have also addressed the requirement for Notch signaling in HSPC emergence by generating morphants for Notch receptors and ligands. The two zebrafish Notch ligands dlc and dld are expressed from the somitic tissue around 17.5 hpf, just after the axial vessels are beginning to segregate. Both ligands are dependent on Wnt16 expression from the somites and critical for proper blood emergence [85]. Enforced expression of Nicd at 14 hpf could rescue cmyb expression at 36 hpf in these morphants. However, later induction failed to induce cmyb expression along the dorsal aorta [85]. The authors reason that the critical phase WNT16 (WNT family member 16) mediated Notch signaling required for HSC specification occurs between 15-17 hpf and not thereafter. A later study from the same group identified the adhesion molecules Jam1a and Jam2a as the critical contributors to this dlc and dld ligand function. jam1a and jam2a are expressed on early vascular progenitors and are activated through Notch by the dlc and dld ligand presenting somites [86]. When this interaction is missing, arterial specification is unaffected, but HE is lost [86]. In order to identify if any of the Notch signaling molecules were affected by Jam1a loss, they sampled all the aortic Notch receptor and ligand genes (notch1a, notch1b, notch3, dlc and dll4), but detected no changes. In yet a different study, the same group discovered notch3 receptor expression in the somites and on the vascular progenitors at early time points. By using morpholino mediated knock down approaches, they show that Notch3 is molecularly situated downstream of dlc and dld [87] that is important for HE specification, but dispensable for arterial fate determination. Only a second wave of Notch activity which is driven by the aortic expression of Notch1a and Notch1b is crucial for both arterial identity and most importantly, HSPC formation [87]. Which Notch receptors the zebrafish Dlc and Dld interact with and which downstream targets they mediate is currently, not known. At least within the dorsal aorta, where the two Notch isoforms notch1a and notch1b are expressed, the ligand Jag1a seem to play a dominant role in HSPC emergence, as in the mouse. Morphants for Jag1a display an established arterial fate but have compromised HE and HSPC formation ( [88]. Finally, time lapse imaging on a Notch responsive transgenic zebrafish line illustrates Notch activity in HE and cells undergoing EHT which decreases as they leave the DA [89].
Altogether, Notch activity is pivotal for HSPC emergence from the DA of the zebrafish model. Two waves of Notch activity seem required. At least in the zebrafish model, firstly, Notch3 from the somites interacts with the ligands Dlc and Dld in the vascular progenitors. Epistatic analyses demonstrate that Notch3 function lies downstream of Wnt16, which is required for HSC specification through its regulation of two Notch ligands, dlc and dld [87]. Once the vascular progenitors segregate according to their arterial/venous fate, Notch activation in aortic cells through Notch1a/Notch1b and Jag1a is essential. Together, they provide the indispensable Notch activity levels for HE specification and HSPC emergence.

Downstream Targets of Notch Activation in Zebrafish HSPC
Genome duplication within the teleost lineage has given rise two Gata2 paralogs in zebrafish, gata2a and gata2b. Gene expression analysis demonstrates a distinct pattern of gata2a and gata2b expression in zebrafish [90]. Gata2a is expressed throughout the endothelium, but Gata2b is restricted to the HE subpopulation of the DA. Expression of Gata2b begins in the vascular cord during posterior lateral mesoderm migration and is initiated in a subpopulation of fli1a+ (early vascular progenitors) cells [90]. This study on genetic morphants demonstrates that Notch1a and Notch1b are required for gata2b expression in the aorta, but not gata2a. Furthermore, gata2b morphants lose runx1/cmyb expression in the trunk, but not the expression of EfnB2a [90] with the study further highlighting how two isoforms can be employed to direct different cell fates. Finally, another direct Notch target, Hey2 is demonstrated to act upstream of Notch activation in the dorsal aorta in a zebrafish model [81]. Hey2 expression is first detected in the early angioblast when the formation of the axial vessels and independent of Notch signaling, as the hey2 expression persists in Rbpj morphants and mind bomb mutants [81]. Intriguingly, Hey2 morphants do not express the receptor notch1b or notch5 in the dorsal aorta that are important for Notch signaling in arterial cells, but the Notch ligand dll4 is readily detected, suggesting that initiation of dll4 expression is not dependent on Notch activation through notch1b (or notch5). Consequently, the arterial gene efnb2a is not expressed (due to lack of Notch1b), flt4 is not downregulated (venous gene that is usually silenced by Notch activity) and runx1+ hematopoietic cells do not emerge from the DA angioblast cord. However, the lack of hematopoietic progenitors can be rescued by enforced expression of NICD in Hey2 morphants, suggesting that Hey2 indeed acts upstream of Notch activity induced by Notch1b in the aorta [81]. In summary, in zebrafish, the Notch target hey2 seems to mark the arterial and HE primed endothelial cells from an early precursor stage already. During the dorsal aorta segregation from the venous endothelium, Notch activity is resumed for arterial identity and hematopoietic commitment. Here, Notch signaling mainly fine tunes the expression and cross regulation of the transcription factors gata2.

Notch during Early Angiogenic Cord Formation
The mouse angiogenic development is less well studied compared to the zebrafish model since assessment of early embryos is limited and ex vivo culture is not established. Nevertheless, the early events of vasculogenesis and angiogenesis are conserved to a high degree within vertebrates. The existence of an hemangioblast precursor has also been postulated in the mouse embryo as well as in the zebrafish, but its existence is both rare and under debate [91][92][93]. Fate mapping studies lead to the discovery that the endothelium and blood cells are already specified at the pre-gastrula epiblast stage in adjacent, but independent regions [92,94]. Like in the zebrafish, defects in both the hematopoietic and angioblastic lineages are observed in embryos lacking VEGF-R2/Flk1 [95], but unlike in the zebrafish, in mouse embryos lacking the transcription factor Scl/Tal-1, mainly the HSPC production is affected and secondary capillary sprouting from blood vessels are perturbed [96][97][98]. Additionally, Indian hedgehog, one of the three mammalian hedgehog isoforms, signals from the extraembryonic endoderm to promote vascular and hematopoietic cells [99]. Notably, mouse embryos lacking hedgehog signaling experience regional defects in vasculogenesis, affecting the anterior (but not posterior) region of the dorsal aorta and to the vessels of the yolk sac. This phenotype can be reversed with VEGF treatment [100,101]. On the contrary, over-activation of Hh signaling increases the expression of the Notch ligand DLL4 in the vascular cells. The secreted growth factor VEGF-A is found in multiple isoforms in mammals and signals through multiple receptor tyrosine kinases, including VEGFR2/FLK1, FMS-like tyrosine 1 (VEGFR1/FLT1), FLT4 (VEGFR3) and NEUROPILIN (NP)1 and NP2, all of which are abundantly expressed in the vascular system. Downstream of this cascade, the vascular cord segregates based on its identity.

Notch Signaling in Arterial Specification
Surprisingly, endothelial cells of the dorsal aorta lack expression of most arterial markers, including ephrinB2-LacZ, Cx40, Hey2, Nrp1, Notch1, Notch4, or Jag1 before E8. 25 and only show weak expression of Cx37 and Hey1. Dll4 was the only robustly expressed (arterial) marker at this stage [102]. Just after this period, the embryo acquires blood circulation. Blood flow itself is essential to maintain of arterial identity since mouse embryos lacking the cardiac sodium-calcium ion exchanger Ncx1 do not develop blood flow, which results in inhibition of endothelial Notch activation and expression of EphrinB2 in the DA [103]. Finally, the Forkhead box c proteins Foxc1 and 2 can also induce DLL4 expression in the arterial endothelium. Notably, the promoter of the DLL4 gene harbors Foxc-binding sites and, thus, foxc appears to positively regulate Notch signaling by activating the Dll4 promoter during arterial specification [104]. Arterial, but not venous vessels co-express Notch1, Notch4, Jag1, Jag2 and Dll4 [31], whereas venous endothelium is identified by the expression of Coup-TFII and EphB4 like in zebrafish. However, the individual requirements of these receptors and ligands are still unclear.

Genetic Knockout Mice of Notch Signaling and Aorta Specification
Notch1 knockout mice show a reduction in the radius of axial vessels, cannot properly remodel the vasculature and die during embryonic development, while deletion of Notch4 has no major effect in vessel formation. However, Notch1/Notch4 double mutants show a more severe phenotype in abnormal axial vessel development than the Notch1 mutants alone, suggesting that the two genes are at least partially functionally overlapping [105]. Similarly, mice lacking even one copy of the Notch ligand Dll4 exhibit severe vascular defects [106]. The role of JAG1 and JAG2 in this context is more elusive. While JAG2 deletion has no impact on angiogenic development and HSPC formation, Jag1 knockout mice die between embryonic day E10.5-E11.5 due to aberrant angiogenesis in the yolk sac and embryonic vasculature, although the aorta is formed and expresses the key marker EfnB2 and CD44 [107,108]. During sprouting angiogenesis, the process whereby blood vessels create secondary capillaries, tip cell selection is controlled by an antagonistic role for DLL4 and JAG1 and fine balance of these two ligands [48].

Notch Activity Requirement for HSC Emergence in the Mouse Model
There is multiple evidence from different vertebrate animal models that indicate the requirement for Notch activity in HSC emergence during embryonic development. Data has been collected from different Notch loss-of-function mutants, but the interpretation of the results is not always straightforward.

Notch Receptor Mutants
Experiments with embryonic chimeras demonstrated that Notch1-deficient cells failed to contribute to hematopoiesis after E15.5 of development, indicating that Notch1 is a cell autonomously needed for definitive hematopoietic development [109,110]. Curiously, only the definitive wave is affected since the early yolk sac hematopoiesis is preserved in these embryos. This is the most elegant demonstration of the Notch requirement for definitive hematopoiesis, although the presence of HSCs or HE in AGM was not specifically tested and no conclusions can be drawn as to the temporal requirement of Notch1 in this process. Studies in both Notch1 and Rbpj knockout embryos have confirmed the lack of HSCs or pre-HSCs at early stages of AGM development, though arterial development is also affected [109,110]. In contrast, Notch2 knockouts show no obvious hematopoietic defects [110,111] and Notch3 and Notch4 knockouts are viable, without obvious defects in HSC generation [112,113].

Notch Target Genes
In both mice and zebrafish, Notch signaling functions genetically upstream of the key and most nascent hematopoietic transcription factor Runx1 [114,115]. Runx1 expression occurs in the dorsal aorta and the mesenchyme below [11,29]. Although Notch signaling activates several genes that are important for HSC formation, including Hes1, Hey2 and Gata2, there is no evidence for direct regulation of Runx1 by Notch. Instead, Notch contributes to the oscillatory expression of Hes1 and Gata2 and the latter drives Runx1 expression as part of a transcriptional complex with further unknown factors [46,116]. Accordingly, the hematopoietic defect of Notch signaling deficiency can be rescued by the induction of Runx1 [117]. Notch signaling itself participates in the transcriptional regulation of several Notch receptors and ligands by positive or negative feedback loop. Mutant RBPJ embryos which lack any Notch transcriptional activity show a specific reduction in the expression level of Jag1 and Jag2 in the AGM aortic cells, suggesting that Notch activity in the aorta can control the expression of these ligands [118]. Strikingly, Jag1 deficient AGMs specify the aortic endothelium, but there is no IAHC formation [119]. However, Jag1 knockout AGM does possess cKIT along the dorsal aorta [120]. They retain high expression of endothelial genes, do not gain CD45 expression and show reduced CFU-S activity [120]. The Jag1 knockout aorta also almost completely lacks Runx1 expression and has no Gata2 expression [119]. Next to Gata2 activation, Notch signaling also initiates the expression of the transcriptional repressor Hes1 in the dorsal aorta. Curiously, Hes1/Hes5 double knockout AGM (Hes5 compensates for Hes1 loss), present a striking phenotype, whereby IAHC formation occurs, but the cells within the IAHC have no stem cell activity upon transplantation into recipients. Hes1/5 mutant AGMs have increased levels of Gata2 (since the negative feedback loop is compromised) and this increase can be reduced to WT levels with the compound DAPT upon which the HSPC activity is partially restored [116]. How the initiation of Jag1 expression fits in this Notch mediated control of HSC emergence is currently unknown, although the HSC loss in Jag1 mutants can be overcome with ectopic Gata2 expression.

Repressors of Notch Activity
The Sox transcription factor Sox17 can directly counteract Notch1 expression by binding to its promoter or the promoter of Dll4 and Notch1 and thereby modulate Notch activity [121]. Genetic loss of either Sox17 or Notch1 during EHT results in increased production of hematopoietic cells due to loss of Sox17-mediated repression of runx1 and gata2 [121]. Studies using DAPT during in vitro culture of AGMs from mouse and chicken have also described an increase in hematopoietic progenitor numbers [121,122]. At least in the mouse study, the authors determined that this increase in number was due to proliferation of the progenitors and not EHT as assessed by BrdU labeling [121]. However, the increase in EHT can be abolished by increased Notch signaling [121].
Altogether, the findings on Notch participation in HSC emergence suggest that it is essential for the formation of HSC activity. The ligand JAG1 is crucial for HSC generation, while the right activation of the downstream targets, especially Hes1 is necessary for HSC activity. Hes1/5 double mutants have plenty of IAHC, but no HSC activity.

Notch Activity during In Vitro Differentiation of Embryonic Stem Cell to Blood
Mouse Embryonic stem (mES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage embryos and have the capability to differentiate into progeny of the different germ layers in culture [123,124]. It provides a powerful model system for studying mammalian development. More recently, human ES (hES) cell cultures have also been established although they differ in their properties. Notably, mES cells do not differentiate to trophectoderm in culture, but hES cells can be induced with BMP4 upon which they will give rise to cells that display characteristics of the trophoblast lineage [125]. Under appropriate culture conditions, mostly by removing the stem cell maintenance factor LIF, mES cells can generate cells in vitro that express the hematopoietic/vascular marker Flk-1 [126], together with mesodermal gene Brachyury [127] in some specific conditions. These cells can give rise to vascular smooth muscle (VSM) cells, in addition to hematopoietic and endothelial progeny. Based on these observations, it was postulated that these cells are equivalent to the yolk sac hemangioblast and, to date, there is no standardized protocol available to derive multi-lineage, long term repopulating HSC from them [128,129]. Like during embryonic development, key transcription factors leading to vascular and hematopoietic transition, including Scl/Tal1 and Runx1, have been also found to be essential during ES cell differentiation to blood [130,131]. Interestingly, Scl-/-deficiency in endothelial cells leads to a growth deficiency in monolayer cultures, but can be partially reverted by culturing them in 3-dimensional aggregates [131]. However, like their embryonic counterpart, blood gener-ation is completely diminished and only the smooth vascular muscle is retained [131]. More recently, efforts have been made to induce the generation of hematopoietic cells beyond the yolk sack stage, i.e., lymphoid cells and transplantable HSC. Co-culture with Notch ligands and culture media with defined cytokines have improved these efforts, but has not been accomplished yet [132,133]. Several studies have therefore focused on understanding and resolving the need for Notch activity during in vitro differentiation. The repertoire of Notch signaling molecules was assessed during the time course of human ES cell differentiation and was mainly found to be expressing the Notch receptor Notch4 and the ligands Dll4 and Jag2 [134]. Interestingly, they found the ligand Dll4 to be expressed at higher levels on endothelial fated cells and the expression level was declined upon transition to the hematopoietic fate [134]. Several studies have explored the Notch pathway involvement during hematopoietic differentiation of mouse and human ES. Notch activation increases the frequency of CD45+ blood cells [126,[134][135][136], while NOTCH inhibition with DAPT decreases the percentage of CD45+ cells in cultures of hES derived CD34 + CD73 − CD43 − progenitors [137]. NOTCH activation in hPSC cultures is predominantly mediated through the NOTCH ligand, DLL4, expressed by endothelial cells [134]. More recent studies have focused on whether arterial specification is a prerequisite for enhanced lymphoid and HSC (like) fate. Human ES cultured in chemically defined conditions and isolated HE cells were either exposed to DLL4 to activate Notch, or DAPT to inhibit Notch during EHT. After this Notch manipulation, hematopoietic cells were assessed for the number of colonies and type of blood cells they could generate [138]. In this process, the authors discovered that NOTCH activation (through DLL1 exposure) in hPSC-derived immature HE progenitors lead to the formation of CD144 + CD43 − CD73 − DLL4 + Runx1 + hemogenic arterial-like endothelial cells, which requires NOTCH activation to undergo EHT and produce definitive lympho-myeloid and erythroid [138]. This is in contrast with the previous report in which they specifically found that HE cells were DLL4 − and distinguished from the arterial precursors [137]. Strikingly, DLL4 + HE could only produce blood cells when induced with OP9-DLL4, but not if they were exposed to DLL1 [138].
In summary, HE activity was associated with DLL4 + cells in studies employing ES differentiation. They further highlight that Notch manipulation, especially its activation during HE to EHT can enhance blood progenitor production.

Is Arterial Specification Necessary for HSPC/HSC Emergence?
The genetic program between endothelial and hematopoietic cell types and fate tracing studies are unequivocally supporting the common origin of both cell types. If the rare population of HE is already determined before the vascular progenitor migrates to the midline of the embryo to form the axial vessels, or only after the segregation of the vascular cells into arteries and veins, is under debate (Figure 3). Since HSCs are generated in the aortic niche of the AGM, it is likely that arterial specification is a pre-requirement for the hemogenic precursor of HSPCs (Figure 3). One of the key factors in defining this requirement is the Notch activity and the expression of ligands and target genes. This question has been addressed by different groups in several experimental models: ES cell differentiation, mouse embryos and zebrafish embryos.
In zebrafish morphants for Etsrp or Scl/Tal1 that have an earlier block in endothelial development (before the onset of angioblast migration) than Hh-, Vegf-or Hey2-depleted embryos could not be rescued for their runx1 deficiency through Nicd activation [81]. These findings suggest that early endothelial programming and arterial priming is an obligate prerequisite HSPC formation in the aorta. In order to address whether arterial requirement is a prerequisite for HE development, Bonkhofer et al. separated the dorsal aortic cells based on runx1 reporter gene expression levels and then were subjected to transcriptomics. They found arterial gene expression (Dll4) present in the Runx1+ fraction. Furthermore, they propose that Runx1 downregulates the arterial fate of HE cells as they undergo EHT [139].
in defining this requirement is the Notch activity and the expression of ligands and target genes. This question has been addressed by different groups in several experimental models: ES cell differentiation, mouse embryos and zebrafish embryos. In zebrafish morphants for Etsrp or Scl/Tal1 that have an earlier block in endothelial development (before the onset of angioblast migration) than Hh-, Vegf-or Hey2-depleted embryos could not be rescued for their runx1 deficiency through Nicd activation [81]. These findings suggest that early endothelial programming and arterial priming is an obligate prerequisite HSPC formation in the aorta. In order to address whether arterial requirement is a prerequisite for HE development, Bonkhofer et al. separated the dorsal aortic cells based on runx1 reporter gene expression levels and then were subjected to transcriptomics. They found arterial gene expression (Dll4) present in the Runx1+ fraction. Furthermore, they propose that Runx1 downregulates the arterial fate of HE cells as they undergo EHT [139].
To investigate whether arterial specification was mandatory for downstream HE/IAHC formation, the para-splanchnopleura (the precursor tissue of the AGM) from E9-9,5 mouse embryos that lack Efnb2 (the KO die by E11.5) was cultured ex vivo and examined for hematopoietic potential compared to wild type embryos. The analysis found that Efnb2 knock out para-splanchnopleura cannot generate blood ex vivo. Interestingly, the EfnB2 KO vasculature have comparable levels of the arterial-associated genes such as Notch4, Dll4 and Hey2, but abnormally low levels of hemangioblast/hematopoietic genes, including Scl/Tal-1, Runx1 and Foxc2 [140]. They hypothesize that the interaction between EFNB2 and EFNB4 during early angiogenesis is important for HE development [140]. These two reports provide some experimental evidence for a requirement of an arterial fate before a HSPC competent HE is established. Whether or not full arterial specification is needed remains unknown since all studies focus on very few (distinct) genes and expression, but do not assess the extent of arterialization of HE. Elegant experiments using two types of Notch activity reporter revealed that arterial cells experience high Notch activity during their ontology. In contrast, HE and IAHC have only been subject to low NOTCH activation throughout development [120]. Further To investigate whether arterial specification was mandatory for downstream HE/IAHC formation, the para-splanchnopleura (the precursor tissue of the AGM) from E9-9,5 mouse embryos that lack Efnb2 (the KO die by E11.5) was cultured ex vivo and examined for hematopoietic potential compared to wild type embryos. The analysis found that Efnb2 knock out para-splanchnopleura cannot generate blood ex vivo. Interestingly, the EfnB2 KO vasculature have comparable levels of the arterial-associated genes such as Notch4, Dll4 and Hey2, but abnormally low levels of hemangioblast/hematopoietic genes, including Scl/Tal-1, Runx1 and Foxc2 [140]. They hypothesize that the interaction between EFNB2 and EFNB4 during early angiogenesis is important for HE development [140]. These two reports provide some experimental evidence for a requirement of an arterial fate before a HSPC competent HE is established. Whether or not full arterial specification is needed remains unknown since all studies focus on very few (distinct) genes and expression, but do not assess the extent of arterialization of HE. Elegant experiments using two types of Notch activity reporter revealed that arterial cells experience high Notch activity during their ontology. In contrast, HE and IAHC have only been subject to low NOTCH activation throughout development [120]. Further supporting this notion, treatment of AGM explants of mouse and chicken (performed after E9.5 for mouse) with DAPT, increases hematopoietic output in culture [121,122]. The opposite was also detected in the chicken AGM explants. Here, more hematopoietic progenitors were observed with DAPT, when the colony forming units were assessed [122]. These experiments highlight the developmental time and duration sensitivity of Notch activity. Furthermore, mouse HSPC form clusters of cells that "hibernate" within the aorta and supposedly mature into HSCs. In the zebrafish model, this behavior is not present. Instead, the HSPC seems to emerge in Notch activated state and quickly join circulation without forming clusters of cells within the aorta (Figure 4) [89]. Thus, it is tempting to speculate that Notch activity or quality changes within this window of time. In line with this speculation, some cells in the IAHC gain NOTCH2 expression and Immunophenotyping of cKIT positive clusters for DLL4 expression shows a decrease in its level that is anti-correlated to the size of the cluster [38].
Notch activity. Furthermore, mouse HSPC form clusters of cells that "hibernate" within the aorta and supposedly mature into HSCs. In the zebrafish model, this behavior is not present. Instead, the HSPC seems to emerge in Notch activated state and quickly join circulation without forming clusters of cells within the aorta (Figure 4) [89]. Thus, it is tempting to speculate that Notch activity or quality changes within this window of time. In line with this speculation, some cells in the IAHC gain NOTCH2 expression and Immunophenotyping of cKIT positive clusters for DLL4 expression shows a decrease in its level that is anti-correlated to the size of the cluster [38]. Finally, plasticity of ES-derived hemogenic cells may be totally different from the embryonic precursors. One of the studies separated arterial endothelium from HSPC competent HE by using cell surface markers CD73 − CD184 − during hES differentiation to blood. When Day 8 Embryoid Bodies are separated based on CD34, CD73 and CD184, they find that arterial cells expressing EfnB2 are highly enriched in the CD73medCD184+ cells, but EHT competent HE cells are found in the CD34 + CD73 − CD184 − DLL4 − cell compartment based on hematopoietic marker [137].
On the other hand, a study overexpressing ETS1 or modulating MAPK/ERK signaling pathway at the mesodermal stage of hES differentiation to blood induced arterial type HE with DLL4 + CXCR4 +/− phenotype that dramatically enhanced the lymphoid potential of the HE by more than 100-fold [141]. It is more than likely that lymphoid potential is dependent on Notch activity and arterial fate of HE. Perhaps some specific Notch Finally, plasticity of ES-derived hemogenic cells may be totally different from the embryonic precursors. One of the studies separated arterial endothelium from HSPC competent HE by using cell surface markers CD73 − CD184 − during hES differentiation to blood. When Day 8 Embryoid Bodies are separated based on CD34, CD73 and CD184, they find that arterial cells expressing EfnB2 are highly enriched in the CD73medCD184+ cells, but EHT competent HE cells are found in the CD34 + CD73 − CD184 − DLL4 − cell compartment based on hematopoietic marker [137].
On the other hand, a study overexpressing ETS1 or modulating MAPK/ERK signaling pathway at the mesodermal stage of hES differentiation to blood induced arterial type HE with DLL4 + CXCR4 +/− phenotype that dramatically enhanced the lymphoid potential of the HE by more than 100-fold [141]. It is more than likely that lymphoid potential is dependent on Notch activity and arterial fate of HE. Perhaps some specific Notch activation in the HE/IAHC is needed to specify IAHC that can contribute to all lineages, including lymphoid cells.

How Much Notch Activity Is Needed for HSCs?
The studies presented and discussed in this review support a critical role for Notch signaling activity in HPC emergence and HSC activity. We are yet to determine if developing hematopoietic progenitors and HSCs in IAHC/aorta differ in their requirements of Notch activity (receptor-ligand interaction, or different downstream targets).
In this regard, we are still unable to properly translate these findings into in vitro settings and to generate transplantation competent HSCs. Reports where Notch signaling has been manipulated during hematopoietic development by either lowering the levels with DAPT or activating it by exposing the cells of interest to Notch ligands have not been able to accurately mimic the in vivo conditions. Hence, it is only plausible that we are still missing some vital knowledge or components about the process. The studies in search for a role in arterial specification as a requirement for HSPC emergence did not test the identified precursor cells for stem cell activity [134,[137][138][139]141]. During embryonic HSPC emergence from the dorsal aorta, a complex array of Notch ligands, receptors and direct downstream targets are involved. It is likely that different numbers of receptors, or ligands or combinations of receptors or ligands are unique to arterial or HE/IAHC cells. Clues for this difference can be found in the large quantity of single cell RNA sequencing data that is available for AGM derived hematopoiesis. Several Notch receptors, ligands and downstream targets including Notch1, Notch4, Dll4, Jag1, Hey2, Hey1 and Hes1 have been reported in the HE population. Here, we propose that Notch is required at several stages of HSPC formation. Early arterial cells express Notch target genes and downstream Jag1 expression is critical for HE/EHT and IAHC formation of all blood cells from the dorsal aorta. Notch activity also seems to dictate HSC activity. Here, it is essential that Hes1 and Gata2 are expressed in an oscillatory manner. Recent studies in the neuronal stem cell compartment studied the oscillation of Hes1 and its effect on stem cell activity [142]. Strikingly, they suggest that Notch1-induced Hes1 oscillation is a cue for cell proliferation and a transition from quiescent to active states of neuronal stem cells.
Importantly, the type of Notch activity changes over time. This might be the challenging aspect to reproduce in vitro by modulating all pathways (and cells) with inhibitors like DAPT/y-secretase that abolish all Notch activity, or by activating Notch activity with one type of ligand. The use of ligand or receptor specific inhibitors or activators would be ideal to assess the true impact of each Notch signaling molecule during hematopoietic/HSC development. As an example, the use of a blocking antibody directed against DLL4 during AGM hematopoiesis has provided great insight; blocking DLL4 during IAHC formation greatly enhances IAHC size and to a lesser extent HSC activity [38]. Finally, perhaps we will be able to decipher the molecular programs that are driven by Notch to specify HSCs by mining the vast number of single cell RNA sequencing data [29,33,37,[143][144][145][146][147][148][149]. In this case, we can circumvent modulation of Notch activity and directly induce the desired downstream signaling.