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Regulation of blood cell transdifferentiation by oxygen sensing neurons

Sean Corcoran, Anjeli Mase, Yousuf Hashmi, Debra Ouyang, Jordan Augsburger, Thea Jacobs, Katelyn Kukar, Katja Brückner

Preprint posted on May 28, 2020 https://www.biorxiv.org/content/10.1101/2020.04.22.056622v3

Protean plasmatocytes: how sensory neurons instruct blood cells to change their fate

Selected by Sophia Friesen

Background:

The process of cell differentiation, by which unspecialized cells can gain functions ranging from conduction of electrical impulses to production of stomach acid, is classically modeled as one-way only. Generally speaking, less differentiated cells develop through a series of progressively more limited stages, eventually arriving at their final differentiated state. However, in some cases, fully differentiated cells can turn into other specialized cell types, a process called transdifferentiation. In C. elegans, for instance, a differentiated hindgut cell ultimately leaves its epithelial layer and develops into a motor neuron [1], and regeneration of the adult newt eye requires iris cells to turn into lens cells [2].

In vitro, blood cells seem particularly adept at changing from one type to the other – experimental manipulations of transcription factor expression in vertebrates can convert T cells to natural killer cells [3], red blood cell precursors to platelet precursors [4], or B cells into macrophages [5].

Blood cell transdifferentiation also occurs in vivo in Drosophila. One of the three blood cell types found in Drosophila larvae can transdifferentiate into two other blood cell types, under conditions of immune stress or even just normal development [6][7]. Transdifferentiation could potentially allow organisms to adapt their development to the environment, but it’s important that this process is regulated to achieve the appropriate balance of cell types. Here, the authors ask what regulates blood cell transdifferentiation in vivo, and uncover a surprising role for a group of oxygen-sensing peripheral neurons that form a niche for developing blood cells.

 

Main findings:

Drosophila have several kinds of differentiated blood cells, including plasmatocytes, which are specialized to engulf and destroy pathogens, as well as the poetically named crystal cells, which store and secrete enzymes that defend against infection [8]. Previously, live imaging of developing blood cells had suggested that differentiated plasmatocytes could turn directly into crystal cells [7].

To confirm that some crystal cells originated from plasmatocytes, the authors performed a creative experiment that relied on the ability of plasmatocytes to engulf large particles. The authors injected fluorescent beads into a developing larva and saw that plasmatocytes rapidly engulfed the beads. But later, as blood cells continued to develop, the authors started to observe crystal cells containing the beads. Neither crystal cells nor undifferentiated progenitor cells are able to uptake the beads, so the only explanation for bead-containing crystal cells is that they used to be plasmatocytes. The majority of crystal cells eventually became labeled by beads, indicating that most of them arose through transdifferentiation.

What controls the conversion of plasmatocytes to crystal cells? Intriguingly, the authors suspected that this process might be regulated by the peripheral nervous system. While the canonical role of sensory neurons is to detect information about the environment and pass it along to the rest of the nervous system, they also cluster with blood cell progenitors and directly support their growth [9]. Crystal cells accumulate in especially large numbers around the sensory cone neurons, a cluster of oxygen-sensing neurons that help larvae detect low-oxygen conditions and avoid suffocation [10]; this close association prompted the authors to ask whether these neurons also regulate blood cell transdifferentiation.

The authors used genetic tools to either overactivate the sensory cone neurons or kill them outright. Increased sensory cone activity increased crystal cell number, and destruction of the sensory cone neurons reduced crystal cell number, demonstrating that these specific neurons regulate blood cell transdifferentiation. Finally, the authors showed a direct role for oxygen sensing in this process by raising larvae in low-oxygen conditions and observing fewer crystal cells. Blocking the ability of sensory cone neurons to sense oxygen by knocking down part of the oxygen-sensing signaling pathway also reduced crystal cell numbers, confirming that the oxygen-sensing function of sensory cone neurons regulates conversion between blood cell types.

 

Why I picked this paper:

I love that this paper challenges two common simplifying assumptions about development: that cells follow a linear path from undifferentiated to terminally differentiated, and that the peripheral nervous system only communicates with other neurons. This work is a fascinating reminder that biology is always more complex and interconnected than we expect it to be.

I also thought that the authors’ lineage-tracing experiment, using fluorescent beads to track cells with the current or historical ability to do phagocytosis, was a really interesting and creative way to show that one blood cell type can turn into another. I’m used to thinking about forms of lineage tracing that rely on differential gene expression, but using cells’ different abilities to uptake a reporter is a novel idea for me.

 

Questions for the authors:

  1. Since the oxygen-sensing Gycs are activated under low oxygen conditions [Morton 04], how do you explain the fact that Gyc knockdown and hypoxia lead to the same reduction in crystal cell numbers? Wouldn’t you expect those two experiments to produce opposite results?
  2. Do you think there’s an adaptive advantage to plasmatocyte-to-crystal cell transdifferentiation being under the control of oxygen levels?
  3. What do you think the intermediate state between plasmatocytes and crystal cells looks like? Do plasmatocytes de-differentiate before turning into crystal cells?

References:

  1. Jarriault S, Schwab Y, Greenwald I. A Caenorhabditis elegans model for epithelial-neuronal transdifferentiation. PNAS USA 105(10): 3790-3795 (2008). https://doi.org/10.1073/pnas.0712159105
  2. Hayashi T, Mizuno N, Ueda Y, Okamoto M, Kondoh H. FGF2 triggers iris-derived lens regeneration in newt eye. Mech Dev 121: 519-526 (2004). https://doi.org/10.1016/j.mod.2004.04.010
  3. Li P, Burke S, Wang J, Chen X, Ortiz M, Lee SC, Lu D, Campos L, Goulding D, Ng BL, Dougan G, Huntly B, Gottgens B, Jenkins NA, Copeland NG, Colucci F, Liu P. Reprogramming of T cells to natural killer-like cells upon Bcl11b deletion. Science 329(5987): 85-89 (2010). https://doi.org/10.1126/science.1188063
  4. Siripin D, Kheolamai P, Yaowalak UP, Supokawej A, Wattanapanitch M, Klincumhom N, Laowtammathron C, Issaragrisil S. Transdifferentiation of erythroblasts to megakaryocytes using FLII and ERG transcription factors. Thromb Haemost 114: 593-602 (2015).
  5. Di Tullio A, Vu Manh TP, Schubert A, Castellano G, Mansson R, Graf T. CCAAT/enhancer binding protein alpha (C/EBP(alpha))-induced transdifferentiation of pre-B cells into macrophages involves no overt retrodifferentiation. PNAS USA 108(41): 17016-17021 (2011). https://doi.org/10.1073/pnas.1112169108
  6. Markus R, Laurinyecz B, Kurucz E, Honti V, Bajusz I, Sipos B, Somogyi K, Kronhamn J, Hultmark D, Ando I. Sessile hemocytes as a hematopoietic compartment in Drosophila melanogaster. PNAS USA 106(12): 4805-4809 (2009). https://doi.org/10.1073/pnas.0801766106
  7. Leitao AB, Sucena E. Drosophila sessile hemocyte clusters are true hematopoietic tissues that regulate larval blood cell differentiation. Elife 4 (2015). https://doi.org/10.7554/eLife.06166.001
  8. Binggeli O, Neyen C, Poidevin M, Lemaitre B. Prophenoloxidase activation is required for survival to microbial infections in PLOS Pathogens 10(5): e1004067 (2014). https://doi.org/10.1371/journal.ppat.1004067
  9. Makhijani K, Alexander B, Tanaka T, Rulifson E, Brückner K. The peripheral nervous system supports blood cell homing and survival in the Drosophila Development 138: 5379-5391 (2011). https://doi.org/10.1242/dev.067322
  10. Morton DB. Behavioral responses to hypoxia and hyperoxia in Drosophila larvae: molecular and neuronal sensors. Fly 5(2): 119-125 (2011). https://doi.org/10.4161/fly.5.2.14284

Tags: blood cell, differentiation, fly, hemocyte, transdifferentiation

Posted on: 3rd June 2020 , updated on: 8th June 2020

doi: https://doi.org/10.1242/prelights.21530

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  • Author's response

    Katja Brückner shared

    1. Since the oxygen-sensing Gycs are activated under low oxygen conditions [Morton 04], how do you explain the fact that Gyc knockdown and hypoxia lead to the same reduction in crystal cell numbers? Wouldn’t you expect those two experiments to produce opposite results?

    Thank you for highlighting our manuscript in preLights and for the great questions. Indeed it seems a little paradoxical at first that Gyc88E silencing and hypoxia show similar phenotypes of reduced crystal cell numbers and diminished transdifferentiation, given that orthogonal approaches would provide consistent results. However, biochemical studies showed that Gyc88E is active under normoxia (Huang et al. Biochemistry 2007; Morton JBC 2004). In biochemical assays, Fe(II)- unligated Gyc-88E had an activity in the same order of magnitude as NO-stimulated ‘classical’ mammalian soluble guanylyl cyclases (Huang et al. Biochemistry 2007); binding of oxygen (O2) or nitric oxide (NO) to Gyc-88E reduced the activity by 3.2-, and 2-fold, respectively, which is a relatively mild reduction (Huang et al. Biochemistry 2007). In addition, it remains unclear whether O2 levels in the sensory cone neurons under normoxia fully reach the atmospheric oxygen concentration. Taken together, we expect significant activity of Gyc88E even under ambient oxygen conditions.

    Moreover, the time frame of hypoxia may make a big difference. Most studies examined Gyc88E under conditions of short term-hypoxia (in the range of minutes), measuring an increase in Gyc88E activity in vitro (Huang et al. Biochemistry 2007), in a heterologous cell system (Morton JBC 2004), and in a short-term behavioral escape response of larvae which suggested links of increased cGMP with the cyclic nucleotide gated channel CNGA (Vermehren-Schmaedick et al. Genetics 2010). However, this escape response is known to cease when hypoxic conditions are extended beyond 30min (Wingrove and O’Farrell Cell 1999), suggesting a switch in the regulation upon prolonged hypoxia. Likewise, we examine effects of long-term hypoxia. Since short-term hypoxia is not compatible with the slower process of blood cell transdifferentiation, we incubated larvae under hypoxic conditions for several hours. Long-term hypoxia may affect Gyc88E activity negatively through several possible mechanisms as is outlined below. It will be interesting to distinguish between these possibilities in the future.

    (1) Repression by hypoxia-induced NO elevation. Long-term hypoxia increases levels of NO (Wingrove and O’Farrell Cell 1999), a known inhibitor of Gyc88E activity (see above and Huang et al. Biochemistry 2007). Depending on the local concentration of NO in the sensory cones, this inhibition could compensate for enhanced activity due to reduced O2 levels, and could potentially be stronger than the inhibition by local oxygen concentrations in sensory cone neurons under normoxia. In support of this hypothesis, it is interesting to note that NO production is exceptionally high in the sensory cones of the larva (Wingrove and O’Farrell Cell 1999, Figure 3).

    (2) Mechanisms of long-term exhaustion. Long-term hypoxia could lead to refractoriness of the cellular response, perhaps due to changes in the activity of Gyc88E or potential associated or downstream signaling components.

    (3) Additional regulatory factors. Gyc88E might partner with regulatory factors in vivo, which could potentially change its activity profile toward oxygen. Biochemical studies tested only defined interactions of Gyc88E, and in some cases deleted the C-terminal domain of unknown function (Huang et al. Biochemistry 2007), which could be of regulatory importance in vivo. The authors therefore emphasized the need to examine actual Gyc88E activity in vivo (Huang et al. Biochemistry 2007). Protein interaction maps have predicted that a variety of other proteins interact with Gyc88E. Among these are heme-containing, potentially oxygen-sensitive cytoplasmic proteins that are prime candidates for regulators of Gyc88E function (Ding et al. Database 2020).

    (4) Differential downstream signaling. Gyc88E is expected to produce distinct levels of cGMP at different levels of oxygen (and/or NO) exposure. This quantitative variation could lead to quantitative or qualitative differences in downstream signaling and neuron activation, for example stimulating distinct sets of CNG channels (with different ion profiles), and/or protein kinase G (PKG) members.

    (5) Alternative functions of Gyc88E. While the guanylyl cyclase activity of Gyc88E is well established, it can never be ruled out that proteins could have alternative functions that might be triggered by certain conditions, such as long-term hypoxia. This may not be the most likely scenario, but remains a possibility to consider should other mechanisms be disproven.

    2. Do you think there’s an adaptive advantage to plasmatocyte-to-crystal cell transdifferentiation being under the control of oxygen levels?

    This is a good question and without any additional experiments we can only speculate. Indeed based on the transdifferentiation data we have, it looks like crystal cell production correlates with ample oxygen supply, and hypoxia seems to reduce the level of transdifferentiation (rather than simply triggering loss of crystal cells). Linking oxygen supply with crystal cell production could have a number of biological benefits:

    (1) It provides a mechanism that coordinates normal larval behavior and environmental conditions with crystal cell production. Larvae typically expose their caudal ends to the air, which allows air intake by the posterior spiracles of the tracheal system, and supports atmospheric oxygen monitoring by sensory cone neurons. As long as normal oxygen levels are detected, this signals that the larva progresses normally in its development and environmental conditions. Linking this detection with transdifferentiation ensures that adequate levels of crystal cells are produced throughout larval lifetime.

    (2) Generation of crystal cells, or the presence of phenoloxidases that are produced by crystal cells, could pose a disadvantage for the animal under hypoxic conditions. In fact, some evidence suggests that toxicity by crystal cells might be higher under hypoxia. The main function of crystal cells is the production of pro-phenoloxidases PPO1 and PPO2, which are cleaved by serine proteases to turn into active phenoloxidases. Phenoloxidases catalyze melanization reactions, which not only lead to the formation of melanin (required in barriers against microorganism invaders and wound scabs) but also generates reactive oxygen species (ROS) that defeat invading pathogens (Tang Fly 2009; Binggeli et al. PLoS Pathogens 2014). These reactive oxygen species might be toxic for the animal itself especially under conditions of hypoxia: It is known that acute hypoxia is associated with generation of ROS, for which hemocytes (the ones that are remaining) are at least one important source (Azad et al. Free Radic Biol Med. 2011 and refs therein). An imbalance between ROS production and antioxidant defenses leads to oxidative stress, injury, and death of the animal. In that study, overexpression of Hsp70 in all hemocytes reduced hypoxia-induced ROS production and increased animal survival (Azad et al. Free Radic Biol Med. 2011). These findings suggest that the intrinsic mechanism of downregulating ROS-producing crystal cells under hypoxia that we observe could have substantial benefits. It would be interesting to examine whether experimentally overriding this reduction in crystal cells and/or PPO production under hypoxia would increase oxidative stress, injury, and accelerate animal death.

    3. What do you think the intermediate state between plasmatocytes and crystal cells looks like? Do plasmatocytes de-differentiate before turning into crystal cells?

    Since our study mainly relies on fluorescent reporters, it leaves the possibility that a brief phase of de-differentiation in the transition from plasmatocytes to crystal cells may go unnoticed due to the relatively long half-life of fluorescent proteins. However we think this is unlikely, based on insights from recent single cell RNA-seq studies. For example, Tattikota and colleagues from the Perrimon lab performed Monocle3 pseudotime lineage analysis on scRNA-seq data from 3rd instar larval hemocytes, which revealed a continuum of intermediate states from plasmatocytes to the fully differentiated mature crystal cell type (Tattikota et al. eLife 2020). We therefore believe the process likely corresponds to a bona fide transdifferentiation, rather than reprogramming of plasmatocytes through a dedifferentiated state.

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