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Adult Neurogenesis in Injury-Induced Self-Repair: Use It or Lose It

One of the most hotly pursued topics in neuroscience and therapeutic medicine is the use of stem cells in the adult brain. Growing in parallel to this emerging field is the recognition that the adult brain is indeed capable of generating new cells. While neurogenesis was understood to be restricted to a few areas, recent studies suggest that damage to the adult brain can trigger neurogenesis even in regions outside of these specific areas. This finding raises the possibility that neurons born in response to perturbation in the brain may be involved in the recovery of function in the damaged adult brain. The key is understanding how to cultivate these newborn cells, because they do not remain viable if they are not accepted into the damaged network of interconnected neurons which support specific functions. From a birth site, undifferentiated precursor cells or neurons undergo migration and differentiation. Many factors influence the safe journey of migrating cells and their survival after maturation at their destination. This review will present evidence from ring dove studies that an activity-dependent mechanism underlies the survival of adult newborn neurons and establishment of their functionality. This evidence includes: [1] unique electrophysiological properties or specific connectivity associated with various type of neurons involved in ring dove coo behavior and reproductive function, [2] emergence of electrophysiological properties and specific projection neurons emanating from newborn neurons after hypothalamic lesion, and finally [3] collective behavioral analyses of social stimulations suggesting that sensorimotor events contribute to the integration of new neurons and reinstatement of function.

INTRODUCTION
Advances in neuroscience have provided an understanding of how sensory events contribute to brain and bodily functions. The brain is endowed with unique sets of sensory and perceptual attributes that filter external stimuli to facilitate the stability and maintenance of the living organism. As interest in the study of aging has surged, particularly regarding the decline of bodily and brain functions, the old maxim of "use it or lose it" has been touted as a guiding principle for medical treatment and health advice [1]. The science behind this maxim, however, is still in its infancy. Some advances have been made in the study of peripheral systems. For example, animal studies have elucidated the physiological processes of healing after usage versus resting of injured limbs [2][3][4][5]. Findings that exercise significantly increases synovial fluid in osteoarthritisaffected horses [6,7] and that the role of synovial fluid is to reduce friction between the articular cartilage of synovial joints during movement in animals and ISSN 2213-6304/16/17/$35.00 © 2016/2017 -IOS Press and the authors. All rights reserved humans [8] support the seemingly counterintuitive recommendation of exercise as a first-line treatment for managing arthritic joint pain in humans. Most studies, however, are correlational in nature, and in particular, studies validating the causal relationship between usage of neural circuits or cognitive function and the retention or recovery of function are far and few between.
In this paper, we will present how the same principle of usage operates in the cultivation of newborn neurons in the adult brain. Brain cells born (neurogenesis) after birth are conserved across species from crustaceans to higher vertebrates [9,10]. In phylogenetically more recent species including humans [10][11][12][13] neurogenesis appears to be limited to specific brain regions. The dentate gyrus of hippocampus and subventricular zone (SVZ) of olfactory bulb are two of the regions which are most characteristic of, and accepted as, neurogenic niches: that is, regions that normally incorporate new neurons throughout adulthood [14][15][16]. Functional neurogenesis exists in other less known regions, most notably, the dietresponsive neurogenic niche in median eminence regions [17][18][19]. Equally important are reports of low level regenerative activity in the neocortex, amygdala, and striatum of primates, the CA1 region of the hippocampus and in the nigra substantia of mice, and in striatal dopaminergic neurons [20][21][22][23], although some of these have been controversial [16,24]. Interestingly, non-neurogenic precursor cells from outside of the dentate gyrus can differentiate into neurons specific to the region when transplanted into the neurogenic dentate gyrus [16,25]. This finding suggests the feasibility that non-neurogenic regions can recruit new neurons in the presence of the following two permissive factors.
(1) Neurogenic promoting factors. Selective evolutionary pressure in mammals may have restricted neurogenic activity to a few brain regions for specific normal functions. However, various form of growth factors appear to enable the brain to retain some capacity to support and incorporate new neurons. For example, in mice and rats, infusion of EGF (epidermal growth factor) after stroke promotes SVZ proliferation and migration to the damaged striatum [26,27]. In rats intraventricular infusion of EGF and albumen or FGF2 (fibroblast growth fac-tor2) after large forebrain ischemia resulted in the regeneration of striatal interneuron and hippocampal CA1pyramidal neurons, and reversed some functional deficit [28][29][30]. For a review of neurogenesis promoting factors, see Sohur et al. [16].
Regenerative activity in the non-or-low neurogenic regions can therefore be elevated by transforming the region into "permissive microenvironment" 1 with the aid of various growth hormones.
(2) Cell death-induced chemical signals. The brain's reaction to damage includes chemical signals released by cell death which may serve to guide precursor cells from other brain regions; in particular, from neurogenic niches to the damaged region. Experimentally induced stroke in rats can trigger proliferation in SVZ and migration to non-neurogenic target sites [26][27][28][29]. Similarly, non-neurogenic brain regions such as the mouse neocortex, rat striatum, dove hypothalamus, and other regions can be induced to recruit neurons born from neurogenic regions following assault, [22,31,32]. Although the functionality of induced-new neurons following cell death has been documented in the normally neurogenic adult hippocampus and song nuclei HVC [10,33,34], with the exception of hippocampal pyramidal neurons [30], a rigorous elucidation of the self-repair process (compensatory neurogenesis) in non-neurogenic brain regions has yet to be accomplished [35].
In birds, research interest in adult neurogenesis is closely related to understanding the birdsong system. Seasonal changes in adult neurogenesis in the HVC (high vocal center: a telencephalic structure for song production) region of canaries provides a rationale for linking seasonal song learning to seasonal changes in neurogenic activity. Supportive evidence can be found in zebra finch studies which show that the selective killing of HVC neurons which send projection to area RA (the nucleus robustus archistriatum) responsible for song output will trigger neuronal recruitment. The rate of induced neurogenesis and the recovery of the birds' ability to produce song coincide with new projections from HVC and area RA [37]. These findings have led to the notion that adult neurogenesis is limited to the song system of song birds [38]. In truth, adult neurogenesis is evolutionarily conserved for a variety of functions [17]. We chose the ring dove for the study of adult neurogenesis, because the hypothalamic system in the adult ring dove is non-neurogenic, as described further below. As a consequence, results may be generalized to other non-neurogenic regions. Furthermore, comparative analyses between birds and mammals can provide useful knowledge at a structural level.
For example, the precursor cells in the subventricular zone in rodents proliferate and migrate to the olfactory bulb, but in birds, migrate to a wider regions [10], importantly, similar mechanisms of cell death induced compensatory neurogenesis in non-neurogenic regions are active in both species. Comparative analyses at cellular and behavioral levels will clarify the applicability of various animal models for brain repair.

RING DOVE MODEL FOR ADULT NEUROGENESIS AND RECOVERY OF FUNCTION
The focus of our experimental series specifically addressed whether adult neurogenesis participates in the recovery of functionality after injury to the adult brain. Attempts to demonstrate functionality of adult newborn neurons beyond the hippocampus are difficult; the challenge is identifying a verifiable model of function and behavior that can be measured and used to assess newborn neurons in the adult system.
We chose the ventral medial hypothalamus (VMH) 2 of the ring dove as our model based on three selection criteria: [1] the specific physiology and behavior of the species is quantifiable and repeatable; [2] the basic neural circuitry of the physiological and behavioral events to be studied has been documented; and [3] the brain region to be studied is not normally neurogenic in the adult brain 3 . This last feature will allow us to generalize our findings to other brain regions that are not normally neurogenic. Importantly, although the VMH region is not neurogenic [38], it generates new neurons in the adult brain in response to cell loss after lesion [32]. Moreover, the neural mediation of key courtship behavior, the subject of these studies, is centrally congregated in the hypothalamus. This enables us to interpret neural events in the hypothalamus that are associated with functional changes attributable solely to this region and not eclipsed by the potential involvement of other intact regions of the brain.

HYPOTHALAMIC SYSTEMS AND REPRODUCTIVE FUNCTION AND BEHAVIOR
The ring dove is a monogamous species that pair bonds for life. The elaborate interactive courtship behavior between the partners, guided by "state reading" from each partner, helps to coordinate a predictable sequence culminating in egg-laying and rearing the young [39]. The courtship commences with male bow cooing (solicitation) to the female, followed by male nest cooing with oblique posture at the potential nest site. A critical milestone occurs when the female joins the male and together they perform duet nest cooing. This duet is followed by the female's solo nest coo, at which point the male leaves the site to hunt for nest materials and brings them back. Together the pair constructs the nest. The female continues cooing until she lays a clutch of two eggs, and then the male and female take turns incubating until the eggs hatch. Together, the pair raises their two squabs to 21 days of age. Adequate lighting is required for maintaining reproductive physiology, and lighting interacts with environmental factors in initiating courtship behavior which culminates in successful breeding [40]. The predictable sequence of these behavioral events during the breeding cycle provides us with a measurable reference from which to compare the nature and quantity of loss and reinstatement of functions after regeneration of new neurons. As it becomes clear in the following sections, the different types of neurons responsible for initiation of nest coo courtship behavior and reproduction are all conveniently located in the hypothalamus. These features make ring dove breeding behavior particularly suitable to the study of the role of adult born neurons in the recovery of function after brain injury.
Bilateral lesion in the ventral medial portion of the hypothalamus (VMH) abolishes the courtship behavior, specifically the nest coo behavior in both the male and female [41,42]. Could destruction of some neurons in the VMH account for loss of courtship behavior and breeding cycle after electrolytic lesion in the region? We have previously identified three types of neurons in this region that are most related to breeding behavior: [1] Acoustic neurons, [2] Projection neurons, [3] GnRH (gonadotrophic release hormone) neurons. These are described further below.
(1) Acoustic neurons (coo-sensitive neurons). The nucleus ovoidalis (avian thalamic auditory relay) and its shell region project to the ventral hypothalamus [43] and transmit auditory cues, including coo signals to the VMH [44]. Recordings of neurons in the VMH receiving these inputs led us to identify neurons sensitive to gender-specific nest coo stimulation [45]. In females, the neurons sensitive to female nest coo stimulation (FC specific neurons), give a signature firing pattern when activated (Fig. 1); no other stimulation, including male nest coo or female nest coo played backward, can activate these FC specific neurons (Fig. 2).
To our delight, we noted a chain reaction of these FC specific neuronal firings, culminating in activation of the hypothalamus-pituitary -ovarian axis (HPO). Activation of FC specific neurons resulted in a threefold increase of luteinizing hormone (LH) output in the anterior pituitary of reproductively inexperienced females. This mechanism explains a behavioral observation made in 1986 that female coo promotes ovarian growth [46] and the female's persistent solo coos coincide with the exponential rise of LH prior to laying a clutch of two eggs [47,48].
(2) Projection neurons. These neurons extend from VMH to the midbrain vocal control pathway, the pathway responsible for production of nest cooing [49].
(3) GnRH (gonadotrophic release hormone) neurons. These neurons control the hypothalamuspituitary-ovarian axis, and are mostly scattered in the preoptic area (POA, a poorly defined area with no clear border in the ring dove) just underneath the septomesencephalic tract (TSM) of anterior hypothalamus [50].
When electrolytic lesion is applied to the VMH region, the anterior hypothalamic region is invari- ably included [49,51,52 ] consequently, GnRH neurons are affected. Destruction of these three neuron types (acoustic neurons, projection neurons and GnRH neurons) disrupts the sequence of courtship behavior and ultimately egg-laying. The loss of cooresponsive acoustic neurons deprives a bird of its ability (a) to respond to a partner's nest-coo and her own nest coo, (b) to utter its own nest-coo through projection neurons, and importantly for females, (c) to activate FC specific neurons which in turn trigger GnRH neurons to activate the HPO axis, resulting in elevated LH output for oviposition and ensuing egg-laying.
Given that VMH lesion triggers adult neurogenesis, precursor cells migrate from subventricular zone to VMH [32], we contemplated the possible role of adult neurogenesis in the recovery of behavior. In the 2006 and 2007 studies, we observed that recovery of nest coo behavior is possible after a recuperation period (8 weeks) during which time the lesioned birds were returned to their bonded mates [49,51]. Only later in this series of studies did we realize that the nature of this housing significantly impacts the rate of recovery, a topic we will return to later.

SPECIFIC ELECTRICAL FIRING PATTERNS FROM NEWBORN NEURONS
To better understand the neural events underlying the loss of function and eventual recovery in doves, we conducted a series of experiments in which we monitored the electrical and cellular changes in the region for 14 weeks after electrolytic lesion [52]. Most birds in this study were returned to stock cages after surgery. Immediately following lesion, the region was silent without any sign of electrical activity for up to 36 days, and then in a gradual, stepwise manner, electrical signal reappeared. The signal was initially just a blip, then a phasic pattern lacking the complete form of a discharge. Normal forms of spontaneous discharge did not appear reliably until after 13-14 weeks (Fig. 3).
During the first 50 days after lesion, newborn cells (cells labeled with a marker for detection of proliferating cells: BrdU+ cells), dominated the region and electrical signals from the region were without characteristic discharge. Newborn neurons (proliferating cells labeled with a marker for neuronal specific nuclear protein: BrdU+/NeuN+ cells), gradually appeared at 60-70 days, a time when the number of abnormal spontaneous firing units in the region increased, of which about 1% responded to coo stimulation. Specific firing patterns evoked in response to a female's coo stimulation appeared at about the same time as the appearance of newborn neurons in the region. This coincidental timing suggests that the characteristic female coo-sensitive firing pattern (FCspecific pattern) likely originates from the newborn neurons. To test if this is the case, the position of the recording electrode was marked by iontophoresis of potamine sky blue dye (PSB). The dye at the tip of single unit recording micropipettes was dispensed at the end of each positive recording session. If cooresponsive new neurons give rise to the characteristic FC-specific firing pattern, only coo-responsive new neurons should show PSB labeling. There were indeed triple-labeled cells (BrdU+/NeuN+/ PSB+), suggesting that some newborn neurons had assumed the characteristic function of coo responsive auditory units [52]. It is worth emphasizing that our criteria for functional recovery was not limited to identifying the characteristic spontaneous firing pattern; we provide further evidence that the characteristic firing pattern is invoked only by specific coo (female coo) stimulation (Fig. 4).
We found that some of the newborn neurons send direct projections to the nucleus intercolliculus of the midbrain that controls vocal output in birds (Fig. 5). Reinstatement of this pathway before behavioral recovery may explain the parallel recovery of nest coo performance [49,51]. Finally, blocking the newborn cells blocks the recovery of nest coo behavior, a finding which supports the causal relationship between newborn neurons and recovery of lost functions [51].

ACTIVITY-DEPENDENT ADULT NEUROGENESIS AND RECOVERY OF BEHAVIOR
With the basic recovery timeline established, we next examined external (environmental and social) factors that might intervene in the process of recovery. The first question we addressed is whether housing conditions during recuperation affect the outcome of behavioral recovery. Our initial study of this question illustrated the advantage of (post-lesion males) being housed with the same mate (female) compared to being housed in solitude [49]. We then addressed whether the "healing" affect of a mate is specific to the female gender [51]. Finally, experiments were designed to identify the characteristics of "healing" promoting females. The most recent study on this topic highlights the principle of activity-dependent effect [53]. Simply put, the effect of an external factor (environmental or social) on recovery is contingent upon whether the external factor is involved in stimulating (i.e. engaging) newborn neurons during various stages of their incorporation into the specific circuitry responsible for a behavior. In our case, when newborn neurons, located in a previously damaged network of coo-specific responsive neurons, are activated or engaged, their synaptic connections to the circuit are presumably consolidated, which in turn leads to the survival of the newly arrived neurons and development of a functional network supporting nest coo behavior.
In studies completed in 2006-2007, we showed that lesioned males do not recover well when placed alone in the cage, nor if caged with another male. Lesioned males housed with female mates consistently fare better [49][50][51]. The data up to then suggested that the nature of the cage mate was the deciding factor, but it remained unclear which characteristics of this stimulus accounted for the differential effect. To address this question, we compared the effects of housing the lesioned male with their female mate or with a new female. In the latter situation, we introduced a new female every three days to block formation of bonding. We suspected that the bonding status of a female might have played a role in the earlier studies where the experimental males were always returned to their mates after surgery. Traditionally, a bird's bonding status to a mate is defined upon completing one successful breeding cycle. We recently established that the bond status (at least in the ring dove) can be reliably assessed by expression of the ZENK gene which encodes a zinc-finger-containing transcriptional regulator and can be rapidly activated in the brain [54]. This enables us to determine a dove's bond status without relying exclusively on breeding history. Zenk data was therefore used to determine the bonding status of female mates and new females in this study.
Pair bonding evolved as a reproductive strategy to ensure reproductive success. We predicted that the male housed with its female mate would recover better since bonding is critical for this species to maintain viable breeding cycles, and therefore the female mate would harbor higher value. Contrary to our prediction, the results showed that the male housed with a new female every three days recovered significantly faster, and more importantly, these males recovered within 4 weeks compared to the 8 weeks we had observed in earlier studies. Analysis of behavioral data for both males and females revealed the previously unsuspected role of male behavior in the differential pace of recovery. When exposed to a new female, the male significantly engaged or attempted to engage a new female more than their pair-bonded female, even though there was no difference in the level of female behavior between the groups to account for the male behavior; in fact, the bonded female tended to exhibit a higher level of courtship behavior at week 3 after her mate was returned from surgery. In other words, by attempting to court a new female by nest coo, the male engages in activity-dependent neural synaptic events, thereby successfully completing recruitment of functional newborn neurons.
In a subsequent study, we sought to replicate the findings and using a generalized linear mixed model, we analyzed the level of nest coo and newborn neurons at weeks 4 and 8 between bonded and nonbonded groups. This study would allow us to validate or discredit the notion that rapid behavioral recovery is made possible through the earlier appearance of newborn neurons, which we had long hypothesized as the underlying mechanism for behavioral recovery. The results replicated the earlier study that the males housed with new females recovered nest coo levels sooner (week 4). Specifically, males housed with new females exhibited higher levels of newborn neurons and nest coo behavior at week 4 than when housed with their female mates [53]. This differential performance persisted until week 8. The finding strengthens our conclusion that the males reinstate nest coo behavior sooner and better when exposed to a novel stimulus (a new female) during recuperative period. The new females' "healing power" lies in their ability to invoke the male's biological temptation to court or engage fresh females, thereby physiologically engaging the circuitry containing the newly arrived neurons, and consolidating their connection to the circuitry and survival of the new neurons. This hypothesis is consistent with numerous studies indicating the role of excitatory inputs in the form of electrical activities in developmental processes [55][56][57].

RATE OF RECOVERY
The conclusion that male courtship behavior after brain damage can be restored sooner than 8 weeks prompted us to revisit our older data, specifically our 2004 work in which we recorded normally evoked discharges of newborn neurons of birds kept for up to 78-99 days. We detected seemingly incompatible results between this earlier study and the later study. The later study consistently suggests that 8 weeks time is sufficient to see behavioral recovery, whereas results of the 2004 study suggest that normal neuronal discharge related to LH output in response to female nest coo stimulation does not begin until around 60 days and more typically beginning around 78-99 days [52].
Two factors could contribute to the apparent inconsistency. Firstly, behavioral recovery measured in the studies suggesting an 8-week recovery timeline was limited to measurement of courtship behavior, and did not include egg-laying behavior. It is plausible that the recovery of egg-laying behavior does not appear until 78-99 days after brain lesion. Secondly, in the 2004 study, the majority of birds were returned to stock or solitude cages after brain surgery [52], in other words, these birds were not exposed to positive, facilitative external factors as in the later studies in which birds were returned to cages containing female mates. In essence, the speed of recovery of function depends to a degree on the nature of the function.

ACTIVITY-DEPENDENT SYNAPTOGENESIS IN LEARNING AND MEMORY
The "use it or lose it" principle is evident in the hippocampus, a region which has been widely studied in recent years. Progenitor cells in the subgrandular zone of the detate gyrus migrate into the granular layer and mature rapidly, extending axons to the CA3 region of the hippocampus. Several studies in rodents have shown how environmental and behavioral factors can modulate neurogenesis and affect hippocampal-dependent learning and memory [58][59][60][61]. One study did cast some doubt on the role of neurogenesis in hippocampaldependent memory [62], possibly because not all hippocampal-dependent learning tasks engage in the hippocampal circuitries exclusively. The fact remains that more hippocampal neurons survive after certain hippocampal-dependent tasks are learned compared to when non-hippocampal tasks are learned [60,62,63].
The finding that young newly generated neurons show a low threshold for induction of synaptic formation in mice [64] provides one tantalizing explanation for what transpires during the coding process of the recruitment of new neurons into the circuit. While the factors responsible for circuit integration and survival are likely to be complex and numerous, our systematic ring dove studies described above have identified the activity-dependent synaptogenesis as one viable underlying mechanism for the successful recruitment of functional newborn cells. Activitydependent synaptic event can also be activated by sensory input during development. For example, in song birds, before a song is learned, tutor song exposure drives pre-motor spiking activity within HVC (a sensorimotor interface for song). This process is suppressed by an inhibition mechanism once the song is learned [65]. Similar mechanisms may also apply to vision-mediated motor learning in adult born young neurons. In the ring dove, female exposure before new neurons establish firm synaptic connections in the nest-coo network may have driven network activity within VMH sensorimotor neurons for nest coo behavior. We speculate that compared to the bonded (familiar) female, the mere presence of a fresh (new) female is a more potent stimulus in driving new neurons arriving at the nest-coo network, thereby facilitating synaptogenesis. Future study should be able to tease apart the relative contributions of sensory and motor engagement. Whether by sensory or motor engagement, crucial to our hypothesis is the role of network activity in driving synaptic consolidation in the recruitment and survival of new neurons. Recent studies of molecular mechanisms provide further support for an activity-dependent mechanism. Activity-dependent transcription in the intracellular signaling of neurons, e.g., response to stimuli creates a new class of RNA (eRNA), thought to regulate synaptic development [66,67].

CONCLUSION
Aging adults generally take longer to learn and remember new tasks, in part because the encoding process (solidifying use of the circuit) takes longer to initiate and to complete. The popular advice for aging adults to be more mindful and to exercise the brain with mental activities is presumably to engage the related circuit more intensely. As we have shown there is scientific data from multiple species to support this recommendation.
In summary, the "use it or lose it" concept is strikingly evident in our analysis of the functionality of the nervous system in the adult ring dove. This phenomenon continues throughout the life span of a living animal and may be most critical for an aging population when a sedentary lifestyle compounds the vicious cycle of declining use and declining functionality.

ACKNOWLEDGMENTS
This work was supported by the Rutgers Research Fund. I would like to thank John Franchak for technical assistant.


extfieldsofstudy: Psychology Medicine

s2fieldsofstudy: Biology Psychology

text:
BDNF Val66Met Polymorphism Is Associated with Self-Reported Empathy

Empathy is an important driver of human social behaviors and presents genetic roots that have been studied in neuroimaging using the intermediate phenotype approach. Notably, the Val66Met polymorphism of the Brain-derived neurotrophic factor (BDNF) gene has been identified as a potential target in neuroimaging studies based on its influence on emotion perception and social cognition, but its impact on self-reported empathy has never been documented. Using a neurogenetic approach, we investigated the association between the BDNF Val66Met polymorphism and self-reported empathy (Davis’ Interpersonal Reactivity Index; IRI) in a sample of 110 young adults. Our results indicate that the BDNF genotype is significantly associated with the linear combination of the four facets of the IRI, one of the most widely used self-reported empathy questionnaire. Crucially, the effect of BDNF Val66Met goes beyond the variance explained by two polymorphisms of the oxytocin transporter gene previously associated with empathy and its neural underpinnings (OXTR rs53576 and rs2254298). These results represent the first evidence suggesting a link between the BDNF gene and self-reported empathy and warrant further studies of this polymorphism due to its potential clinical significance.

Introduction
Social interactions are tightly linked to survival, so much so that the complexity of social environments has been proposed as one of the main factors driving the increasing size of primates' brains across evolution [1]. Accordingly, an impressive amount of data indicates specific neural processes for social perception and social cognition that are highly conserved across species [2]. Empathy, defined as the capacity to understand and share the actions, intentions and emotions of others [3,4], is one of these processes that has been shown to be deeply rooted in the neurobiology [5] and the evolution of the human brain [2]. For example, a twin study found that in 24 and 36 month-old infants, genetic factors accounted respectively for 34% and 47% of the variance in empathy [6]. However, current evidence from the scientific literature does not fully detail the specific genetic variants contributing to the observed interindividual variability in empathy.
Empathy is conceptualized as a multilayered construct where higher order social cognitive processes rely partly on social perception to allow the understanding of others (see [7]). Accordingly, empathy often begins with the perception of social stimuli that can trigger perception-action coupling (Preston & De Waal,[8]; also referred to as shared representations: Decety and Jackson [3]) and even emotional contagion, a process thought to be evolutionarily old as it has been observed in non-primate species such as mice [9]. In humans, a wide network of structures that includes the amygdala nuclei has been shown to be crucial for the perception of socially and emotionally salient stimuli (see [5,10]). This network feeds sensory information to cortical structures that have been shown to support the processing of higher order empathic processes in the brain (see [3,5]). These processes are typically divided in two broad categories: affective and cognitive components of empathy. On the one hand, the affective component of empathy allows individuals to automatically form shared representations of the feelings of others while preserving the self-other distinction, which differentiate this component of empathy from emotional contagion [3,8]. This process is thought to be phylogenetically older than the cognitive components of empathy (see [7]) and to rely on cortical regions such as the anterior cingulate cortex, the insula [11][12][13][14] and the inferior frontal gyrus [15,16]. On the other hand, the cognitive component of empathy is conceptualized as the capacity to consciously adopt the perspective of others and requires perspective taking as well as mentalizing [3,17]. The cognitive component of empathy has been shown to rely on the ventromedial prefrontal cortex [16], the superior temporal cortex, precuneous and the temporo-parietal junction [14].
The cognitive and affective components of empathy are often assessed using self-reported measures such as the Interpersonal Reactivity Index (IRI) [18]. The IRI is one of the most commonly used measure of self-reported empathy and decomposes empathy into four constructs [18]. The affective component is measured by the Empathic Concern (i.e., the 'other-oriented' feelings of sympathy and concerned for others) and the Personal Distress (i.e., the 'self-oriented' feeling of anxiety and discomfort in interpersonal settings) subscales, while the cognitive component is measured by the Perspective Taking (i.e., the spontaneous tendency to adopt the psychological perspective of others) and the Fantasy (i.e., the tendency to identify imaginatively with fictitious characters) subscales.
Pioneer neurogenetic studies have investigated the source of the genetic variability of empathy by examining the effect of specific genetic variants on self-reported empathy [19] and intermediate processes in the brain, such as emotion perception [20]. So far, the oxytocin receptor (OXTR) gene is probably the most studied gene with respect to social cognition and social behaviors. For instance, single-nucleotide polymorphisms (SNP) of the OXTR genes have been associated with social cognition in adults [19,21], children [22] as well as in schizophrenic individuals [23]. Amongst these findings, only two SNPs of the OXTR gene, rs53576 and rs2254298, have been associated both with self-reported empathy and functional neuroimaging tasks of emotion perception [20,24]. Accordingly, Rodrigues and colleagues (2009) were the first to report an association between a genetic variant and self-reported empathy by showing that A allele carriers of the rs53576 polymorphism reported less dispositional empathy on the IRI, as well as less accurate judgement of facial expressions. Furthermore, Tost and colleagues (2010) showed that A allele carriers of the rs53576 SNP presented decreased amygdala reactivity during the observation of angry and fearful faces, as well as a greater functional coupling between the amygdala and the hypothalamus during this task. Similarly, A allele carriers of the rs2254298 were also shown to present less self-reported empathy on the total IRI score [21], smaller amygdala volumes [25], as well as "deficient deactivation" of the dorsal anterior cingulate cortex during emotion perception [24]. This convergence of evidences strongly suggests a role of both polymorphisms in the neurobiology of social perception as well as in selfreported empathy. Interestingly, both SNPs are located in the third intron, a non-coding region of the gene, and their functional effects still remain unknown.
To further investigate the genetic factors affecting empathy, a likely genetic candidate is the Brain-derived neurotrophic factor (BDNF) Val66Met polymorphism (196 A/G; rs6265), a genetic variant extensively studied for its role in intermediate phenotypes of social perception as well as in psychopathologies associated with social cognitive deficits such as schizophrenia (see [26]). Notably, using similar experimental tasks of social perception as the ones used to study the OXTR SNPs, the BDNF Val66Met variant has been associated with amygdala reactivity to emotional stimuli in healthy [27] and anxious individuals (Lau et al., 2010). More precisely, Met allele carriers (ValMet / MetMet) were reported to present more reactivity than Val/Val participants to emotional stimuli. Furthermore, Met allele carriers have also been shown to present greater attention biases to social threats, as well as increased connectivity between the amygdala and the ventromedial prefrontal cortex [28]. This polymorphism has been linked to three effects in BDNF signalling: (1) a decreased concentration of pro-BDNF in dendrites (2) a decreased level of pro-BDNF in the secretory granules and (3) a decreased secretion of the molecule in the synaptic cleft [29][30][31].
Given the crucial role of the amygdala nuclei and the ventromedial prefrontal cortex in social perception and empathy, it is possible that the BDNF Val66Met might also play an important role in self-reported empathy. Therefore, we hypothesized that this increased reactivity of social perceptive processes in Met allele carriers might lead to altered self-reported empathy.
To test this hypothesis, we investigated the effect of the BDNF Val66Met polymorphism on one of the most commonly used self-reported measure of empathy, the IRI [18]. Furthermore, we tested whether this SNP could have a distinct influence on self-reported empathy beyond the effect of the two SNPs of the OXTR gene previously associated with the IRI [19,21] and to intermediate phenotypes of emotion perception [20,24]. Also, we investigated the interaction effect of rs53576 and rs2254298 on self-reported empathy as the influence of these polymorphisms have until now only been considered separately. We hypothesized that Met participants of the BDNF Val66Met polymorphism would report less self-reported empathy than Val/Val participants and that the A allele of both OXTR SNPs would be associated with less selfreported empathy.

Materials and Methods
Participants and Procedure 113 young adults (57 females; mean age = 24.14; SD = 4.44) were recruited using the University Laval electronic newsletter. The sample size was determined using effect sizes from previous studies that documented the effect of the rs53576 and the rs2254298 on self-reported empathy. Cohen's d were computed and averaged from all published articles that reported a significant effect of either SNPs on self-reported empathy [19,21,23,32,33]. One article was excluded from the analysis since insufficient information was reported [33]. The result indicated an average Cohen's d of 0.6, which is considered to be a medium size effect according to Cohen's guidelines [34]. A prospective Power analysis achieved using G Ã Power 3 [35] indicated that a sample size of 110 participants was sufficient to obtain a power of .7 with our statistical model (i.e., MANOVA). The power level was set at .7, which represents a 30% risk of not detecting an effect of genotypes (i.e., making a type II error). Participants were right-handed, self-reported no diagnosis of psychopathologies or neurological conditions and reported not taking any prescription medication. Only self-reported Caucasians of European ancestry were included to avoid population stratification artefacts, as this is the most accessible population in Quebec City. Inform consent was obtained in writing. Three participants were excluded from further analyses because they provided too little saliva for genetic analyses. The study was approved by the local ethics committee (i.e., Institut de réadaptation en déficience physique de Québec).

Genotyping procedure
Saliva samples were collected using the Oragene DNA self-collection kit (DNAGenotek). DNA was extracted using the Blood & cell culture DNA maxi kit (Qiagen) and concentration evaluated by fluorescence (Qubit). Some 50 nanograms of DNA was used to amplify the region of the BDNF Val66Met polymorphism using a real-time polymerase chained reaction cycler (Ligthcycler 480, Roche) and a TaqMan 5' nuclease assay kit (Life Technologies). PCR was made using 0.5 ul of 10X PCR MasterMix (Roche), 0.25 ul 40X TaqMan assay, 50 ng DNA in a final volume of 5 uL. PCR run included a denaturing step of 10 min-95°C followed by 35 PCR cycles (1 min 95°C, 1 min 55°C, 1 min 72°C).

Empathy Questionnaire
The Interpersonal Reactivity Index (IRI; [18]) is a 28-item self-reported scale assessing empathy comprising four subscales: Perspective Taking, Empathic Concern, Fantasy and Personal Distress. Each subscale includes 7 items rated on a 5-point Likert scales (1 = Does not describe me well; 5 = Describes me well) and were scored using criteria developed by Davis [18]. The test re-test validity of the IRI subscales has been reported to range from .63 to .71 [36]. In our sample, all subscales presented acceptable internal consistency (Cronbach's alpha: Perspective Taking = .70; Fantasy = .82; Empathic concern = .69; Personal Distress = .76). Data were analyzed using IBM SPSS Statistics version: 20.

Data Analysis
To determine the effect of BDNF Val66Met on self-reported empathy, a MANOVA was conducted on the four IRI subscales as dependant variables with the BDNF Val66Met, the OXTR rs53576 and the OXTR rs2254298 as independent variables, as well as the interaction between the two OXTR SNPs. Significant main effects and interactions were further decomposed using separate stepwise discriminant analyses to determine individual contribution of subscales to discriminate genotypes (see [37]). Each discriminant analysis was conducted by considering as covariates the genotypes present in the MANOVA but not considered in this particular discriminant analysis. For instance, to determine the contribution of each IRI subscale to discriminate the BDNF genotype, individual scores on the IRI subscales were adjusted for the effect of the two OXTR SNPs and their interaction. Adjusted IRI scores were obtained by taking the non-standardised residuals of the regression between each IRI subscales and covariates (genotypes), which is similar to the procedure used to control for covariates in analyses of covariance [38,39]. To evaluate the interaction between the OXTR polymorphisms, we determined if the adjusted IRI subscales could discriminate the four groups created by the interaction of OXTR polymorphisms in the discriminant analysis.

Results
The genotype distribution of the Val66Met, rs53576 and rs2254298 are presented in Table 1. None of the distribution differed significantly from the expected numbers calculated on the basis of observed allele frequencies according to the Hardy-Weinberg equilibrium (p > 0.05).
Participants in the Met and Val/Val groups did not differ on age (t (108) = -.679; p = .50) and males and females were equally distributed between groups (χ2 (1, N = 110) = 3.379; p = .07) (see Table 2). Furthermore, on both OXTR SNPs, participants in the AA/AG and the GG groups did not differ on age (rs53576: t (108) = 1.68; p = .10; rs2254298: t (108) = -.729; p = .47) and the sex of participants was equally distributed between genetic groups (rs53576: χ2 (1, N = 110) = 0.324; p = .56; rs2254298: χ2 (1, N = 110) = 1.01; p = .32) (see Table 2). For each polymorphism, as achieved in most previous studies, homozygous carriers of the mutant alleles were pooled with heterozygous carriers to allow sufficient statistical power for comparisons. The BDNF genotype presented no association with either the rs53576 (ϕ = .062 p = .52) or the rs2254298 (ϕ = .069 p = .47) polymorphisms, but the two OXTRs SNPs presented a weak negative association (ϕ = -.267 p = .005), which represent a certain linkage disequilibrium These results indicates that participants of one of the two groups on the first SNP were more likely to be in the other group on the second SNP. This linkage disequilibrium is expected since both SNPs are located in the same intron and should not influence the results of the MANOVA for two reasons: first, the postulates of MANOVA allow independent variables to be correlated and second, the association between two independent variables do not indicate that they will interact in their effect on another dependant variable.
To further study how individual IRI subscales are associated with the BDNF Val66Met and to the interaction of the OXTR SNPs, we introduced the IRI subscales as predictors in two separate stepwise discriminant analysis to discriminate (1) BDNF Val66Met polymorphism and (2) the four groups created by the interaction of the OXTR SNPs (see Procedure). The results indicated that a significant function discriminates the BDNF polymorphisms using the Perspective Taking, Fantasy and Empathic Concern subscales when the effect of the SNPs of the OXTR on individual subscales are considered (Λ = .910, χ2 (3) = 10.07, p = .018; Canonical R 2 = .09). The correlations between the subscales and the discriminant function indicate that the Perspective Taking (r = -.58) and the Fantasy subscale (r = .42) make significant unique contribution to the prediction of the BDNF Val66Met genotype while the Empathic Concern (r = .28) and the Personal Distress (r = .19) subscales do not present a significant association with this prediction. Specifically, the Met

Discussion
Our results provide the first evidence of an association between the BDNF Val66Met polymorphism and self-reported empathy. Specifically, the Met group membership is predicted by less Perspective Taking and more Fantasy when the effects of the targeted OXTR SNPs are considered. Furthermore, our results indicate that the rs53576 and rs2254298 SNPs of the OXTR may interact to confer their effect on self-reported empathy. More precisely, our results indicate that less Personal Distress is associated with participants presenting the A alleles on both OXTR SNPs when the effect of the BDNF Val66Met is considered. By uncovering dissociable effects of the BDNF Val66Met and the OXTR SNPs, our results make a significant contribution to our understanding of the genetic factors affecting self-reported empathy. The fact that less Perspective Taking and more Fantasy predict the Met group membership indicates a differential effect of the BDNF Val66Met on the two subscales. In fact, even though the two subscales are often regrouped under the construct of Cognitive empathy, they describe types of social interactions that differ in terms of the level of personal involvement they require. On the one hand, the Fantasy construct mainly represents the tendency to identify with fiction characters, which does not necessitate personal involvement. On the other hand, Perspective Taking conceptualizes the cognitive aspects of empathy in the context of actual social interactions requiring a high level of personal involvement. There are indeed evidences that offline (i.e., inferential representation of a social situation requiring no direct involvement) and online (i.e., direct involvement in a social situation) social cognitive processes recruit distinct regions of the ventromedian prefrontal cortex [40]. In fact, several results support the hypothesis that different social cognitive processes are recruited in situations where individuals are directly in interaction with others and when they are simply observing others (for a review, see [41]). One possibility is that the ValVal genotype may favour the understanding of others in the context of direct social interactions, which requires personal involvement, as opposed to the ValMet / MetMet genotypes that may favour the capacity to adopt the point of view of fiction characters in situations that do not require personal involvement.
Interestingly, the biological functionality of the BDNF Val66Met can provide hypotheses regarding the discrepancy of its effect on the Perspective Taking and the Fantasy subscales. The BDNF Val66Met has indeed been reported to influence experience-dependant plasticity [29][30][31], a process that might affect differently the online and offline processing of social information. For instance, Perspective Taking during online social interactions might require rapid plasticity to coordinate social actions and to facilitate the integration of multiple social cues while offline social processing might not rely as heavily on this plasticity. For instance, offline social processing such as reading a book, daydreaming and fantasizing do not require the coordination of social responses, and the relevant information can often be integrated much more slowly then typical online social interactions. It is even possible to imagine that too much plasticity might generate interference in situations where less plasticity would be required. Future studies could test these hypotheses by investigating the propensity for the cognitive component of empathy in online and offline social interactions in order to further determine the role of the BDNF Val66Met polymorphism in these social cognitive processes.
Furthermore, if online and offline social interactions present distinct neural and cognitive processes, do they also present distinct levels of heritability? In fact, some evidences indicate that genetic variants might be responsible for 34% to 47% of the variance of empathy [6], but little is known about the heritability of its individual components. If online and offline social interactions indeed serve different purposes with different evolutionary advantages like some authors have proposed [41], it is possible that they may also be influenced by different genetic factors and have different levels of heritability. For instance, reflecting on social interactions from an observer's point of view (offline social interactions) might allow to elaborate and finetune general rules of social interactions in order to guide future behaviour which cannot be easily achieved during online social interactions that are emotionally involving and that might have direct consequences for the individual's survival. Interestingly, our results do not seem to support this hypothesis since we observed that one genetic factor, the BDNF Val66Met, accounted for part of the variability of both these constructs. As our results do not allow the determination of the heritability of these components of empathy, future studies are needed to investigate this specifically.
Our results also build on the existing literature linking the OXTR rs53576 and the OXTR rs2254298 to self-reported empathy and highlight the necessity for a multivariate approach. It is in fact possible that genetic variants such as the rs53576 [20] and the rs2254298 [24] acting on the neurobiological level, may have to simultaneously interfere with the perception of emotional stimuli to affect Personal Distress, an higher-order cognitive construct related to the reactivity to emotion perception. Therefore, using multivariate approaches will most likely further improve our capacity to determine the many genetic factors contributing to self-reported empathy and more importantly these approaches could also help bridge the gap between intermediate neural phenotypes and behavioural measurements of psychopathologies.
The fact that BDNF Val66Met is associated with empathy also opens new research avenues for the study of psychopathologies involving social-cognitive deficits, such as autism or schizophrenia. For instance, schizophrenia has been associated both with decreased self-reported Perspective Taking [42] and with the BDNF Val66Met polymorphism (see [26]). Our results provide new evidences suggesting a link between Perspective Taking and the BDNF Val66Met polymorphism, which may partly explain why schizophrenia was associated independently with Perspective Taking and the BDNF Val66Met polymorphism. Interestingly, the rs2254298 polymorphism has already been associated with the affective component of empathy in schizophrenic patients (see [23]) and studying the role of the BDNF Val66Met on Perspective Taking could further improve our understanding of the genetic factors contributing to social cognitive difficulties in this psychopathology.
One must although keep in mind that these results should be replicated in independent studies to further establish their validity. While our approach aimed at controlling for confounding genetic factors, replication remains one of the most effective ways to establish the validity of these results, since every study that use random sampling of the population can unknowingly report false positive.
In conclusion, this study provides results indicating for the first time a link between the BDNF Val66Met polymorphism and self-reported empathy that goes beyond the previously reported effects of the rs53576 and rs2254298 SNPs. The results also further builds on the previous literature suggesting a link between the rs53576, the rs2254298 and self-reported empathy and highlights the importance of studying gene X gene interactions to better understand the genetic factors contributing to interindividual variability in self-reported empathy.
Supporting Information S1 Data. Data underlying the findings reported in the manuscript. (XLSX) Philip L. Jackson was supported by a New Investigator Award from the CIHR and a Chercheur-Boursier Senior Award from the FRQS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contributions
Conceived and designed the experiments: VTD SH YCC PLJ. Performed the experiments: VTD AB AL MR. Analyzed the data: VTD SH AB AL MR YCC PLJ. Contributed reagents/ materials/analysis tools: VTD YCC PLJ. Wrote the paper: VTD SH AB AL MR YCC PLJ.


extfieldsofstudy: Chemistry Medicine

text:
Microtubule-Mediated Misregulation of Junctophilin-2 Underlies T-Tubule Disruptions and Calcium Mishandling in mdx Mice

Summary Cardiac myocytes from the mdx mouse, the mouse model of Duchenne muscular dystrophy, exhibit t-tubule disarray and increased calcium sparks, but a unifying molecular mechanism has not been elucidated. Recently, improper trafficking of junctophilin (JPH)-2 on an altered microtubule network caused t-tubule derangements and calcium mishandling in a pressure-overload heart failure model. Mdx cardiac myocytes have microtubule abnormalities, but how this may affect JPH-2, t-tubules, and calcium handling has not been established. Here, we investigated the hypothesis that an inverse relationship between microtubules and JPH-2 underlies t-tubule disruptions and calcium mishandling in mdx cardiac myocytes. Confocal microscopy revealed t-tubule disorganization in mdx cardiac myocytes. Quantitative Western blot analysis demonstrated JPH-2 was decreased by 75% and showed an inverse hyperbolic relationship with α- and β-tubulin, the individual components of microtubules, in mdx hearts. Colchicine-induced microtubule depolymerization normalized JPH-2 protein levels and localization, corrected t-tubule architecture, and reduced calcium sparks. In summary, these results suggest microtubule-mediated misregulation of JPH-2 causes t-tubule derangements and altered calcium handling in mdx cardiac myocytes.

SUMMARY
Cardiac myocytes from the mdx mouse, the mouse model of Duchenne muscular dystrophy, exhibit t-tubule disarray and increased calcium sparks, but a unifying molecular mechanism has not been elucidated. Recently, improper trafficking of junctophilin (JPH)-2 on an altered microtubule network caused t-tubule derangements and calcium mishandling in a pressure-overload heart failure model. Mdx cardiac myocytes have microtubule abnormalities, but how this may affect JPH-2, t-tubules, and calcium handling has not been established. Here, we investigated the hypothesis that an inverse relationship between microtubules and JPH-2 underlies t-tubule disruptions and calcium mishandling in mdx cardiac myocytes. Confocal microscopy revealed t-tubule disorganization in mdx cardiac myocytes. Quantitative Western blot analysis demonstrated JPH-2 was decreased by 75% and showed an inverse hyperbolic relationship with aand b-tubulin, the individual components of microtubules, in mdx hearts. Colchicine-induced microtubule depolymerization normalized JPH-2 protein levels and localization, corrected t-tubule architecture, and reduced calcium sparks. In summary, these results suggest microtubule-  (1). Heart failure is the cause of death for 20% to 25% of DMD patients (2); therefore research aimed at understanding the molecular and cellular phenotypes underlying the cardiomyopathy of DMD has been conducted. Altered calcium homeostasis marked by increased calcium sparks (3)(4)(5) and t-tubule disarray were documented in cardiac myocytes from the dystrophin-deficient mdx mouse (6,7), but a unifying molecular mechanism has not been identified.
Junctophilin (JPH)-2, the protein that links the plasma membrane of t-tubules to the ryanodine receptor, is essential for proper t-tubule structure and function (8). Recently, it was reported that JPH-2 mislocalization due to abnormalities in microtubule cytoskeleton caused pathological t-tubule remodeling and abnormal calcium handling in the pressure overload-induced heart failure model (9). However, the relationship between microtubules and JPH-2 in other models of heart failure or cardiomyopathy has not been analyzed. Because previous studies documented microtubule derangements in mdx cardiac myocytes (5,10), we tested the hypothesis that microtubule alterations cause JPH-2 misregulation and result in t-tubule disruptions and calcium mishandling in mdx mice.
Finally, to investigate the translational aspects of our hypothesis, we examined the cardiomyopathy of mdx mice via echocardiography and isoproterenol stress tests as previous studies showed mildly reduced systolic function (11)(12)(13)(14) and excessive mortality with isoproterenol administration (10,15,16) Figures 2C and 2D).
When the relationships between aand b-tubulin and JPH-2 in older mice was analyzed, we observed a significant inverse hyperbolic relationships (r 2 ¼ 0.95 Abbreviations as in Figure 1.
Prins et al.
These data suggested increased microtubule content was associated with reduced JPH-2 protein levels.  2 AU) ( Figures 3F and 3G).  Although we were able to document restoration of t-tubule organization with colchicine treatment, we did not observe organ-level cardiac improvements in mdx mice. One explanation is that t-tubule correc- Finally, our results add to several other publications that document a crucial role for JPH-2 in t-tubule integrity. The first link between JPH-2 and t-tubules was shown when knockout of JPH-2 resulted in embryonic lethality due to lack and cardiac contractility and improper t-tubule formation (27). Several other studies document that alterations of JPH-2 via miR-24 (28,29), RNA interference (21,22,30), or calpain-mediated protein cleavage (23) cause t-tubule derangements and rescue of JPH-2 protein levels or localization via transgenic overexpression (17), inhibition of miR-24 (28)


extfieldsofstudy: Biology Medicine

text:
Hyphal ontogeny in Neurospora crassa: a model organism for all seasons

Filamentous fungi have proven to be a better-suited model system than unicellular yeasts in analyses of cellular processes such as polarized growth, exocytosis, endocytosis, and cytoskeleton-based organelle traffic. For example, the filamentous fungus Neurospora crassa develops a variety of cellular forms. Studying the molecular basis of these forms has led to a better, yet incipient, understanding of polarized growth. Polarity factors as well as Rho GTPases, septins, and a localized delivery of vesicles are the central elements described so far that participate in the shift from isotropic to polarized growth. The growth of the cell wall by apical biosynthesis and remodeling of polysaccharide components is a key process in hyphal morphogenesis. The coordinated action of motor proteins and Rab GTPases mediates the vesicular journey along the hyphae toward the apex, where the exocyst mediates vesicle fusion with the plasma membrane. Cytoplasmic microtubules and actin microfilaments serve as tracks for the transport of vesicular carriers as well as organelles in the tubular cell, contributing to polarization. In addition to exocytosis, endocytosis is required to set and maintain the apical polarity of the cell. Here, we summarize some of the most recent breakthroughs in hyphal morphogenesis and apical growth in N. crassa and the emerging questions that we believe should be addressed.

Abstract
Filamentous fungi have proven to be a better-suited model system than unicellular yeasts in analyses of cellular processes such as polarized growth, exocytosis, endocytosis, and cytoskeleton-based organelle traffic. For example, the filamentous fungus develops a variety of Neurospora crassa cellular forms. Studying the molecular basis of these forms has led to a better, yet incipient, understanding of polarized growth. Polarity factors as well as Rho GTPases, septins, and a localized delivery of vesicles are the central elements described so far that participate in the shift from isotropic to polarized growth. The growth of the cell wall by apical biosynthesis and remodeling of polysaccharide components is a key process in hyphal morphogenesis. The coordinated action of motor proteins and Rab GTPases mediates the vesicular journey along the hyphae toward the apex, where the exocyst mediates vesicle fusion with the plasma membrane. Cytoplasmic microtubules and actin microfilaments serve as tracks for the transport of vesicular carriers as well as organelles in the tubular cell, contributing to polarization. In addition to exocytosis, endocytosis is required to set and maintain the apical polarity of the cell. Here, we summarize some of the most recent breakthroughs in hyphal morphogenesis and apical growth in and the emerging questions that N. crassa we believe should be addressed. Polarity is the key feature in all living cells and allows the asymmetrical transport of components to precise cellular sites. Filamentous fungi present one of the most extreme cases of polarized tip growth, among the different cell types that also extend apically (for example, pollen tubes, neurons, and root hairs). Fungal tubular cells, so-called hyphae, extend by continuous expansion of their apices through a complex mechanism that involves the coordinated directional supply of material needed for plasma membrane (PM) and cell wall growth. This process, hyphal morphogenesis, has increasingly been in the spotlight of fungal biology research in recent years.

Referee Status:
Neurospora crassa, "the orange bread mold" discovered in French bakeries in the 19th century 1 , has been at the vanguard of biochemistry, genetics, and biological research for nearly a century. Several reviews have highlighted the historical contribution of N. crassa to many areas of biology, including pioneering work on DNA silencing and circadian rhythms 2,3 . The genetic basis of the transition from single spherical conidia (asexual spore) to a large network of filamentous tubular hyphae 4 has been at the forefront of fungal biological research, elucidating both fungal morphogenesis and polarized growth. Most research aimed at identifying key players in hyphal morphogenesis before the availability of genome data involved forward genetics screens-bottom-up approaches-in which randomly generated mutants were analyzed to discover the function of a gene. This in fact was the basis for the leading work of the Nobel laureates George Beadle and Edward Tatum that established the relationship between genetics and biochemistry 5 . When this strategy was followed, many N. crassa morphological mutants were generated 6 .
The increasing availability of sequenced genomes made it possible to apply reverse genetics screens-top-down directed approachesto silence or mutate specific genes and evaluate the resulting phenotypes. Sequencing of the N. crassa genome 7,8 , along with other key developments (expression plasmids for protein tagging 9,10 , recipient strains deficient in non-homologous end joining 11 , and knockout cassettes for all annotated open reading frames 12,13 ), revolutionized the field of fungal biology and quickly accelerated the number of studies on N. crassa, which set out to analyze the distribution, dynamics, and function of subcellular components 14 . Needless to say, this led to significant advances in our understanding of fungal morphogenesis.
Research in genetics, biochemistry, and more recently "omics" conducted on N. crassa clearly contributed to many important insights into how fungal hyphae are shaped. This review focuses on the most recent findings on key subcellular structures determining hyphal ontogenesis in N. crassa.

Hyphal morphogenesis
N. crassa exhibits a variety of cell morphologies corresponding to different developmental stages. The morphogenetic changes initiate when a conidium begins to grow isotropically during the first hours of hydration; soon after, the symmetry is broken, growth becomes polarized, and the resulting germ tube continues extending by apical polarized growth until it becomes a fully mature hypha. Further branching from subapical compartments generates new hyphal tips capable of fusing with each other and generating a mycelium 6,15-18 . Some remarkable differences in growth and intracellular organization have been described between germ tubes and mature vegetative hyphae in N. crassa. The most prominent characteristic of the apex in mature hyphae of N. crassa is the Spitzenkörper (Spk), a conspicuous accumulation of vesicles, ribosomes, actin microfilaments, and amorphous material of undefined nature 19 . During the early stages of development, no Spk can be perceived at the germling apex 20 and this is most likely because of the insufficient number of tip-directed secretory vesicles 21 . In addition, organelle distribution is disorganized in germlings and cytoplasmic microtubules (MTs) are less abundant, shorter, and differently distributed compared with mature hyphae 20 . A widely accepted fungal morphogenesis model proposed that the Spk behaves as a vesicle supply center (VSC) 22 . According to this model, the forward advancement of the VSC and the concomitant release of vesicles would generate an ideal hypha 23 . An alteration of the number of released vesicles, the rate of advancement of the VSC, or a sustained displacement of its central position would generate several cell shapes, including branches and meandering hyphae 24 . Growth of N. crassa in confined microfluidic structures, which mimic some of the characteristics in the natural environment, has allowed analysis of the thigmotropic response of individual hyphae and tip growth to changes in the environment 25 and has enabled long-term tracking to monitor, in real time, fluorescent reporters of molecular mechanisms such as circadian rhythms 26 . In germlings with structures associated with cell fusion, so-called conidial anastomosis tubes (CATs), the displacement of activated GTPase clusters initiates repositioning of the apical secretory vesicle delivery machinery in response to chemotropic cues, offering an explanation of how directional tip growth is accomplished in cell types that lack a Spk 27 .
Prior to symmetry breakage of an N. crassa spore, there is accumulation and localized activation of the small Rho GTPase CDC-42 and its guanidine exchange factor (GEF) CDC-24 27,28 . Once a polarized germ tube has emerged, a second RHO GTPase, RAC, is recruited at the incipient tip forming a crescent 28 . CDC-42 and RAC regulate the negative chemotropism exhibited during germ tube development and the positive chemotropism observed during CAT formation and cell fusion 27 . In mature hyphae, CDC-42, CDC-24, and RAC are localized at the apical dome 28 ( Figure 1A). The homologues of the polarisome components BUD-6 and SPA-2 are required for the maintenance of apical growth and cell morphology of N. crassa germlings and hyphae but not for cell symmetry breakage during conidial germination 28,29 . SPA-2 accumulates at apices of hyphae adopting a fan shape, whose base co-localizes partially with the Spk core 30 . BUD-6 accumulates at the apical PM, excluding the very tip. In addition, the formin BNI-1 (an F-actin polymerization protein), another polarisome putative component, has a distribution similar to that of BUD-6 but also co-localizes with the Spk 29 ( Figure 1A). Polarisome proteins are possibly involved in the thigmotropic response of N. crassa by mediating the reorientation of actin microfilaments, thereby regulating the vectorial flow of vesicles to the tip 31 . It has been proposed that to convey information from the sensing apex to the transcriptional machinery that is located several micrometers behind the tip (11 μm in Aspergillus nidulans, 12 μm in N. crassa, and 22 μm in Ustilago maydis 32-34 ), a signal is able to travel in the opposite direction from growing tips to the nuclei 33 .

Building the cell wall
Fungal hyphae are surrounded by a wall made of polysaccharides and glycoproteins, which helps the cell withstand internal pressure and serves as an external barrier against environmental stresses. It is therefore essential for the optimal development and survival of fungi. In N. crassa, the cell wall comprises an inner layer of chitin (9%) and β-1,3-glucans (87%) microfibrils, embedded in a matrix-like material composed of galactomannans, mixed traces of β-1,4-glucans, glucuronic acid, and glycoproteins 35-39 . These elements are enzymatically cross-linked to form a three-dimensional network.
α-1,3-glucans have been described as an important component of the fungal cell wall 45,46 , but in N. crassa these polysaccharides have been detected in macroconidia only 47 . Additional proteomic analysis indicated compositional differences between cell walls of conidia and hyphae, suggesting differential regulation of polysaccharide biosynthesis through development 48 . Mannan and galactomannan are found as glycoconjugates on cell walls. The α-1,6-mannosyltransferase OCH-1 and the α-1,6-mannanases DFG-5 and DCW-1 are required for the efficient covalent incorporation of glycosylated proteins into the cell wall 37,44 . Many of these proteins are found attached to the PM by a glycosylphosphatidylinositol (GPI) anchor, an indispensable post-translational modification for optimal cell wall function in N. crassa 49 .
A few decades ago, the unitary model of cell wall growth proposed that cell wall construction during apical extension requires a delicate balance of exocytic vesicles carrying lytic enzymes for cell wall softening as well as simultaneous secretion of vesicles transporting synthesizing enzymes 50 . Subsequently, the steady-state model introduced the concept of plastic cell wall material extruded to the apex as individual chains, which gradually become crosslinked at the subapex by glycosyltransferases, resulting in the hardening of the cell wall 51 . The role of remodeling enzymes such as chitinases, glucanases, or glycosyltransferases has recently been addressed in N. crassa. To elucidate the role of glycosyl hydrolase (GH) family 18 in N. crassa growth, deletion strains for the corresponding genes were phenotypically characterized. The lack of GPI-anchored chitinase CHIT-1 led to a decrease in growth rate, suggesting a potential role of chitinases in hyphal extension 52 . Subcellular distribution analysis of chitinases has not been carried out in N. crassa. However, recently, two GPI-anchored β-1,3-endoglucanases, BGT-1 and BGT-2, with predicted glycosyltransferase activity (GH-17) were reported at cell wall remodeling sites (that is, hyphal apices and septa) during N. crassa vegetative development 53 . These observations and previous attempts to reconcile the above-mentioned models 54 led to a new, integrated model of cell wall assembly. Remodeling enzymes, such as β-1,3-endoglucanases with glycosyltransferase activity that are anchored to the PM, hydrolyze long chains of β-1,3-glucans (at the tip) and transfer the cleaved residues to other chains. The new amenable free ends generated can be further cross-linked with chitin or glycoproteins, thus contributing to the construction of new cell wall 53 (Figure 2). Nonetheless, further analysis of the cellular distribution of different cell wall remodeling enzymes is needed to attain a more comprehensive understanding of the process.

The apical machinery
Decades after the discovery of the Spk in Coprinus fixed cells using iron-hematoxylin staining and in living hyphae of Polystictus versicolor by phase-contrast microscopy 55,56 , the pivotal role of the Spk in maintaining apical growth and determining hyphal growth direction and morphology was confirmed 19 . Hence, it was predicted that the vesicles that comprise the Spk carry cell wall biosynthetic enzymes 50,57 . This hypothesis has been proven correct in the past few years. In N. crassa, all CHSs (CHS-1 to CHS-7) accumulate at the core of the Spk 58-60 , whereas FKS-1, GS-1, and RGF-1 (RHO-1 GEF) accumulate at the periphery of the Spk 61-63 . The localization pattern of CHS and the GSC in the inner and outer regions of the Spk corresponds to the microvesicular (chitosomes) and macrovesicular regions of the Spk ( Figure 1B and Figure 3), revealed by transmission electron microscopy in N. crassa and by electron tomography in A. nidulans 64,65 . Small RAB GTPases mediate the traffic of vesicles. In N. crassa, YPT-1 Rab1 was detected in the Spk microvesicular core, whereas SEC-4 Rab8 and YPT-31 Rab11 occupied the Spk macrovesicular peripheral layer, implying different regulatory pathways for each type of vesicle 66 ( Figure 1B). In A. nidulans, RabC Rab6 and RabO Rab1 were also found at the Spk, although their localization could not be correlated to a specific population of vesicles 67,68 . Recent evidence demonstrated that the A. nidulans GS-1 homologue GsaA also accumulates at the outer region of the Spk 69 . Remarkably, the flippases DnfA and DnfB, which regulate phospholipid asymmetry and presumably membrane bending, occupy the outer and inner regions of the Spk in A. nidulans, respectively, a finding that supports differences in the nature of the Spk secretory vesicles 69 . In contrast, Chs6, a class VII CHS in the maize pathogen basidiomycete U. maydis, is transported in vesicles carrying Mcs1, a class V CHS with an N-terminal myosin motor domain similar to that of myosin-5 70 . Also, Gsc1, the U. maydis homologue of Fks1, is delivered in Mcs1-containing vesicles. U. maydis, however, does not display an Spk in any of its yeast or hyphal forms and has growth rates several orders of magnitude lower than those of N. crassa.
The short-distance delivery of vesicles from the Spk to the cell surface requires the orchestrated action of the exocyst complex, which tethers vesicles to the PM before their soluble N-ethylmaleimide sensitive factor attachment protein receptor-dependent fusion at specific PM sites. In N. crassa, the exocyst consists of eight proteins that seem to be essential for viability, except for SEC-5, whose loss resulted in impaired secretion and aberrant morphology 71 . SEC-3 accumulates between the Spk outer layer and the PM; SEC-5, SEC-6, SEC-8, SEC-10, and SEC-15 accumulate as a surface crescent in the apical PM; and EXO-70 and EXO-84 accumulate primarily in the Spk outer layer 71 ( Figure 1B). Thus, in N. crassa, all exocyst components were excluded from the microvesicular region, leaving an open question about the secretion mechanism followed by chitosomes from the Spk to the cell surface. In the rice pathogen Magnaporthe oryzae, the exocyst proteins Sec5 and Exo70 participate in an alternate exocytic pathway for cytoplasmic effector proteins destined to be delivered to the host cells during biotrophic invasion of rice 72 .

Of filaments and nanomotors: connecting exocytosis and endocytosis
A complex network of proteins organized in a variety of forms such as tubules, patches, filaments, rings, and scaffolds composes the cytoskeleton in fungal cells. Collectively, cytoskeletal elements participate in shaping the hyphae, in the positioning and shuttling of vesicles and organelles, and in cellular division. The large cells of N. crassa constitute an excellent experimental system to study in great detail the distribution, dynamics, and function of the main cytoskeletal components: MTs, actin, associated proteins, molecular motors, and septins.
Immunolocalization studies of cryo-fixed N. crassa cells revealed long prominent cytoplasmic MTs distributed longitudinally along the axis of growth of hyphae 64 . Subsequently, live imaging by confocal laser scanning microscopy of N. crassa cells expressing green fluorescent protein (GFP)-tagged β-tubulin revealed with unprecedented detail the dynamics and three-dimensional organization of MTs 73 . Often an intertwined network of helical cytoplasmic MTs and individual MTs grouped at times into bundles was observed 73 . Although there is no direct evidence of vesicles moving along MTs, cumulative observations suggest tip-directed vesicle traffic via an MT-based mechanism until vesicles reach the Spk, where a switch to the actin cytoskeleton occurs 65 . MTs extend to the hyphal apex, either terminating at the vicinity of the Spk or traversing it and reaching the apical PM ( Figure 3). When the Spk is displaced from its most forward position and growth temporarily ceases, a rearrangement of the long MTs into short segments and bundles of MTs surrounding the retracted Spk is observed and this supports the association suggested between the Spk (or its constituents) and MTs 73 .
In N. crassa, both plus-ends and minus-ends are present at hyphal tips 74 , indicative of MT mixed polarity. The dynamic plus-ends of MTs undergo rapid polymerization and depolymerization, a phenomenon known as dynamic instability 74 . Analysis of MT dynamic instability in mature leading hyphae of N. crassa by total internal reflection fluorescence microscopy revealed very fast MT polymerization rates that depend on the role of molecular motors 74 . Lis1, an MT plus-end-binding protein, is involved in the regulation of the dynein/dynactin complex in humans 75 . In N. crassa, two paralogues of Lis1-LIS1-1 and LIS1-2-were identified in the hyphal apical dome as short filaments or comet-like structures decorating the terminal plus-ends of the MTs 76 , similar to the distribution of the MT plus-end-binding protein MTB-3, a homologue of EB1 77 . LIS1-1 localization was found to be dependent on the minus-end-directed MT-associated motor dynein and dynactin (p150Glued) 76,78 . Importantly, the dynein-dynactin complex has a role not only in minus-end-directed cargo traffic but also in the organization of the MTs in N. crassa 76 .
Within the superfamily of kinesins, 10 different kinesins have been reported for filamentous fungi 79 . In N. crassa, conventional kinesin-1 KIN-1 (NKIN) is believed to be responsible for vesicular transport and also nuclear, mitochondrial, and vacuolar migration 80 . However, an N. crassa strain expressing KIN-1-GFP displayed dispersed cytoplasmic fluorescence 81 . Microscopy approaches with a better temporal and spatial resolution may be needed to resolve the traffic of these motors. Alternatively, another unexplored kinesin could be responsible for vesicular traffic. Kinesin-3 NKIN2 co-localized with YPT-52 Rab5 , indicating its involvement in the transport of early endosomes, as has been previously observed, for kinesin-3 motors in U. maydis and A. nidulans [81][82][83] . The role of NKIN2 in early endosome transport was confirmed in Nkin2 deletion mutants, where endosomal movement was strongly reduced 81 . The shuttling of early endosomes in the opposite direction also depends on dynein 81,[84][85][86] . The role of early endosomes in apical growth is just starting to be disclosed. Early endosomes are much more than mere organelles originated from endocytosis. They are interconnected with other organelles of the secretory pathway and behave as another hub for sorting cargos (Golgi compartments have been traditionally considered the main sorting hub of the cell). For example, U. maydis and A. nidulans endosomes transport peroxisomes, endoplasmic reticulum, lipid droplets, and mRNAs 85,87,88 . N. crassa has a peroxisome-derived organelle, the Woronin body (WB), which reseals membrane lesions by occluding septa 89 . WBs are associated with the cell cortex and the cell apex via a Leashin tether that promotes WB inheritance and holds it in position until signals from cellular damage induce release, translocation to the septal pore, and membrane resealing 90 (Figure 3).
Motor protein rigor mutants, with a point mutation in the ATP-binding site, bind to the cytoskeletal element but cannot hydrolyze ATP, and this ATP-bound form is irreversibly locked to its cytoskeleton partner 91 . In N. crassa, NKIN2 rigor -GFP was found to associate preferentially with a subpopulation of straight detyrosinated MTs, as reported earlier for the corresponding A. nidulans orthologue UncA 83 . Although the role of post-translational modifications in MTs is not totally understood, it is possible that they serve as a traffic signal for specific organelles. Thus, the existence of different populations of MTs could ensure that the tip-directed transport of cargo along a subpopulation of MTs remains stable during mitosis, when most cytoplasmic MTs are disassembled 92 .
Though not absolutely indispensable for polarized growth, MTs and MT-associated molecular motors are important for Spk stability and hyphal morphogenesis 93 . Mutations or inhibitors leading to MT disorganization in N. crassa cause growth defects, distorted hyphal morphology, and erratic Spk movement while maintaining polarized growth 64, 78 . In contrast, actin is essential for hyphal polarity. Direct tagging of G-actin with fluorescent proteins has not been possible in N. crassa. To circumvent this, several F-actin-binding proteins, including fimbrin (FIM), tropomyosin (TPM-1), coronin (CRN-1), ARP-3, and Lifeact, a 17-amino-acid peptide derived from S. cerevisiae Abd120p, were fluorescently tagged and imaged by live microscopy 94-97 . The Lifeact reporter uncovered different subpopulations of actin macromolecular structures, including rings, actin patches, and actin microfilaments. FIM, ARP-3, and CRN-1 were found to be associated with patches arranged in a cortical ring at the hyphal subapical region (1 to 4 μm behind the growing tip). Although now it is widely accepted that this subapical collar of actin patches delimits the region of endocytosis, at the beginning of the 21st century it was doubted that endocytosis occurred in filamentous fungi 98 . Endocytosis and exocytosis are spatially coupled. Endocytosis at the subapical collar is thought to contribute to the recycling of apical receptors and cortical markers 98 since it has been proven necessary to establish polarity and to maintain apical growth 96,98-101 . TPM-1 was identified at the core of the Spk and at actin cables departing from the Spk toward the subapex. Lifeact elucidated all forms of actin. Whereas F-actin is thought to mediate short-range movements, the long cables observed might serve as tracks along which myosin motors transport secretory vesicles to and within the apex 94 (Figure 3). Although we focus on the hyphal apex in this review, it is noteworthy to point out that prior to the formation of the contractile actomyosin ring (CAR) at septation sites, a septal actomyosin tangle (SAT) was observed 102 .
Myosins are another superfamily of molecular motors that move along actin microfilaments. In N. crassa, MYO-1 is a component of the actin subapical collar and is thought to have a role in endocytosis 103 . MYO-2 is involved mainly in the formation of the SAT and CAR during cytokinesis 102,104 . MYO-5, found occupying all the Spk 61 ( Figure 1B), is thought to be involved in exocytosis and hyphal morphogenesis. The MYO-5 homologue in A. nidulans, MyoE, is needed for the accumulation of RabE Rab11 post-Golgi carriers at the tip 105 .
Finally, septins are cytoskeletal components with GTP and phosphoinositide-binding domains 106 . N. crassa septins CDC-3, CDC-10, CDC-11, CDC-12, and ASP-1 accumulate at or close to the PM in germinating conidia at sites of symmetry breakage ( Figure 1A) 107 . They are suggested to constrain and corral the polarity machinery to the apical PM, acting as a molecular boundary between the apex and the region behind 107 .

Future directions
There are several unresolved questions that need to be addressed to further develop a holistic view of N. crassa hyphal morphogenesis. We need to do the following: (a) identify the real triggers of polarity; (b) generate mathematical models to test whether the spatial segregation of the vesicles at the Spk provides any morphogenetic advantage; (c) elucidate the interaction, if any, between the MT and actin cytoskeletons; (d) investigate the role of the predicted hypothetical proteins encoded in the N. crassa genome in polarity establishment and hyphal morphogenesis; (e) discern whether endocytosis occurs at sites other than the subapical collar, where actin patches have also been found; (f) determine which enzymes are secreted by a non-conventional pathway depending on mRNA transport; and (g) investigate, within the hyphal apical compartment, which nuclei are responding to external signals sensed at the growing tip.
Something to take into consideration is that some of the findings obtained from research in N. crassa, while conserved among several fungal taxa, should not be extrapolated to all members of the fungal kingdom. There are innate differences at the subcellular level among fungal species that may reflect their evolutionary distance.