A perspective on notions of spirituality, democracy, social cohesion and public theology
03 Sept. 2014

Rothney S Tshaka email@example.com
Department of Philosophy, Practical and Systematic Theology
University of South Africa
South Africa

University of South Africa
0003Muckleneuk, PretoriaSouth Africa

A perspective on notions of spirituality, democracy, social cohesion and public theology
03 Sept. 2014EAAE47ADC49247203FA742805ABFED6910.4102/ve.v35i3.1336Received: 19 Feb. 2014 Accepted: 21 May 2014
This article explores the notions of spirituality, democracy, social cohesion and public theology briefly.Whilst these notions seem unrelated to each other, the article finds that they are indeed very much related especially in a conversation pertaining to the role of theology in democratic South Africa today.It argues that these notions are particularly important if one seeks the unity of a dislocated people.The word dislocation here refers to more than merely those who were dispossessed of their wealth and land, but speaks especially also to a spiritual dislocation which happened as a result of that material dislocation.Black liberation theology which has always been public must be seen to incorporate issues of the spiritual in its reflections.All this, it is argued, has pertinent repercussions for social cohesion in South Africa today.

Introduction

In this article, the notions of spirituality, democracy, social cohesion and public theology are explored, albeit briefly.The article strives to argue that all these notions are intrinsically linked, although it might not seem inevitable at first.The notion of spirituality, it is argued, is of particular importance, simply because this notion appears to have been pushed into the background of theological discourse particularly during the era of the church's struggle against apartheid.It argues that spirituality remains particularly important today as many people -especially those still on the underside of history -continue to strive to make sense of their relationship with their Creator.

It is also argued that speaking properly of spirituality reveals that there is no separation of the spiritual from the material, but that there is in fact unity between the spiritual and the material.Therefore the misconception of spirituality as that which concerns itself only with the spiritual and the world, and that which will come into force once this material world has vanished, is chastised as false perception of the notion especially when viewed from within African Christian circles.The article questions a theology that has engendered this false perception of the notion.It then follows to explore the notion of democracy and exposing it as a notion not without its own flaws.Several questions relating to the notion of social cohesion are asked especially given the existing huge gap between those who have and those who exist on the margins of society.

Lastly it engages the notion of public theology and, whilst it does not discourage talk on public theology, it does remain suspicious of attempts that are aimed at replacing black liberation theology with the notion of public theology, which appears to be neutral for those who falsely associate black liberation theology with militancy and disorder.

A perspective on spirituality

Let us engage this task by exploring the notion of spirituality.We start with this notion first for no particular reason other than the fact that it is listed first in the title of this article.The notion in question, in my view, has not received the needed attention at least within the ambit of academic theology 1 in South Africa.This is particularly also the case within black theological circles.There are of course several reasons why spirituality was pushed, perhaps unintentionally, into the background of academic theological discourse in South Africa.One reason that one could think of seems to be the assumption inherent in the notion that spirituality lacks passion and militancy in a context where the imbibed position of black docility had become entrenched.

1.By academic theology here I refer especially to those institutions of higher learning where theology was taught as an academic discipline.This takes into account the fact that black liberation theology during the apartheid years was strongly discouraged and never considered a subject worthy of study at these predominantly white institutions.It is for this very reason that refers to black liberation theology as a guerrilla enterprise -by this I argue that since it was never welcomed in academic circles, it had to find ways of penetrating other black circles through means of ministers' caucuses and workshops.

The frustration caused by mainline church traditions in South Africa not doing enough to fight apartheid and yet continuing to encourage a spirituality that seems detached from the socioeconomic, cultural and political situations, has been a subject for conversation amongst many black theologians in South Africa for years now.Kumalo refers to a conversation between Itumeleng Mosala and his mentor, Gabriel Setiloane, which prompted the latter to suggest that he was bewitched by the Christian faith and could therefore not simply shed it like a snake would shed its skin (Kumalo 2006:1f.; author's own emphasis).

For those who were engaged in an alternative theological hermeneutic, the black church could only come to its black self realisation once it departed from a theology that seemed to justify the subservient position and role of blacks especially during the apartheid epoch (Mosala 1989).

That is one way of looking at spirituality as a phenomenon.

In fact spirituality proper cannot be understood unless one recognises its inherent ambivalence.There is of course another dimension to this phenomenon, and that relates to its elasticity and significance particularly for those on the margins of society.It was none other than James Cone who helped me to understand the endurance of the black soul in a context of harsh oppression.In his book, The spirituals and the blues, Cone reminds us of how black slaves could somewhat understand their positions of oppression by identifying particularly with the oppressed people in the Bible (Cone 1992:53ff.).

About this ambivalence of spirituality, Cone (1992:54) writes, 'to create the future in the "extreme situation" of American slavery was very difficult for black slaves.It meant accepting the burden and the risk of the not yet.' Cone (1992) continues:

actually when they encountered the divine presence and promise, as revealed in the event of Jesus' (death and resurrection, they knew that there was only one possibility for authentic human existence.And that was to live in freedom for the future.(p.21)

Logic therefore becomes immaterial in such a situation.

In the South African situation, we realise that a theology that seems to have penetrated black communities is the very theology that intentionally does not link structural injustices, for instance, with the spirituality of its adherents.This perception was underscored once again when I was recently invited to address a conference of the Christian Youth Movement (CYM) of the Uniting Reformed Church in Southern Africa (URCSA) in the Brits circuit. 2

In that address I argued for the importance of having a teaching ministry for our church.That ministry, in my opinion, would entail providing opportunities for our congregants to be educated as we minister to them.To begin to see the relationship between the material and the spiritual 2.The Christian Youth Movement is the youth wing of the URCSA.Like many Christian youth movements in the country, it remains an essential organization, especially in a context of a great divide between the opulent and the extremely poor.Its mission is informed by Christian principles which are enshrined in its constitution.What seems to be valued most is the song and preaching which characterises their meetings, yet these invariably are removed from the structural problems created by apartheid and seem to temporarily escape from the real material issues of the day.

and to eventually come to the realisation that poverty is in fact not God ordained and that it is not by God's design that most that are poor happen to be black.More significantly, I argued, context and historicity play vital roles as we strive towards that teaching ministry.

I referred to the Easter celebrations which had become one of the key Christian events in our church calendar and reminded my audience of the disgusting objective of crucifixion which was essentially aimed at silencing those who dared to challenge delinquent hegemonies in their societies. 3I was very surprised, even disturbed, to realise that the perspective that I gave challenged the so-called spiritual perspective of the crucifixion which for them had nothing at all to do with politics.This as we will observe later is nothing strange and uniquely South African.

The American condition of slavery is another context in which we see the ambivalence of spirituality -its elasticity and its irrationality to the logical being (Cone 1992:23f.).To return to the South African context, one sees a similar functioning of spirituality as pointed out earlier.Our conception of spirituality is clearly informed by the lived experiences of the very people who embrace a particular form of spirituality.

In other words, there are clearly diverse factors that then suggest that contrary to what academic theology purports, there are in fact various spiritualities even within the different racialised groupings.When some people for instance speak of a Reformed spirituality, invariably they conjure up a form of life as was observed in the Geneva of Calvin (Tshaka 2009:294).This spirituality in my view has no consideration for those who were later converted to this faith.This was also because of attempts to make faith sophisticated as per the Reformed understanding of faith.Faith as an activity especially of the mind features nowhere more prominently than within the Protestant tradition.It was Wells and Quash (2010) who had this to say of the Christian faith:

the white man's largely cerebral religion was hardly touching the depths of his African soul; he was being redeemed from sins he did not believe he had committed; he was given answers to questions he had not asked.(p. 195) In the South African context, the ambivalence of spirituality remains similar to the context of slaves in the United States of America (USA).Here, like in the USA past and present, one sees the duality which is exemplified in exploitative labour during the week and church on Sunday.Just like the spirituals of which Cone speaks, so do hymns play significant roles in expressing the need to live in freedom for the future (Cone 1992:22).

In an age of secularisation, to speak about spirituality and to link that to the public domain can be seen as being highly

3.The celebration of the Easter weekend is one of the key celebrations on the Christian calendar, especially in black South Africa.This is evidenced by the great rush to the Moria Mountain in Limpopo which is the headquarters for the Zion Christian Church (ZCC).The same is true within the mainline churches.My church, the URCSA, also values this weekend particularly and vigils are observed throughout the weekend.With no theological training, ordinary people relate the happenings of Easter to their own concrete situations in a spiritual manner.

controversial, in fact even discouraging.This is so for the following reason: according to Charles Taylor (2007) in his book, A secular age, he writes:

[A] certain awe still surrounds reason as a critical power, capable of liberating us from illusions and blind forces of instinct, as well as the phantasies bred of our fear and narrowness and pusillanimity.The nearest thing to fullness lies in this power of reason and it is entirely ours, developed if it is through our own, often heroic action.(p.9)

In listening to the input of Itumeleng Mosala, who was invited to the conversation on spirituality, social cohesion and public theology at the University of Pretoria, 4 I personally had hoped that he would speak and elaborate a little bit more to the audience about the matter of methodology on the matter, because it is a fact that for black liberation theology to be where it is today in South Africa -that is alive and kicking, and not irrelevant and dead as some people would want to believe -this theological hermeneutic had to engage in an intensive exercise of methodological restlessness.One only has to read the first two chapters of Mosala's magnum opus to see this fact (Mosala 1989).This was because black liberation theology had to defend itself to the dominant theological hegemony, that it was in fact genuine theological reflection, especially when it insisted on the lived experiences of those on the margins as crucial to its reflections.Methodology was considered significant, yet it needed to take the lived experiences of black people very seriously.These lived experiences, we contend, are not fantasies bred by the fear and narrowness of black people.

I believe that we shall have to insist on putting epistemologies and methodologies that were traditionally perceived as moribund on the table as we embark on this project.

The work of Vuyani Vellem on Imvuselelo is of particular importance if we are to tap into the spiritualities in which black people are raised (Vellem 2009).Again these Imvuselelo [revivals] speak to an ambivalent spirituality; one that defies human logic and reason.If the Trinity which is Western in all its conceptions is something that some of us agree is beyond human comprehension, it then is only right that we must admit that the spiritual realm is much bigger than our human logic permits.And so we must park this point and ask questions such as: Whose spirituality are we talking about?Who dictates the spirituality which must engender social cohesion?And whether those of us in the public domain recognise the many lived experiences which influence our spiritualities?

This then reminds me of a significant point made by Antonio Gramsci according to Forgacs (cited in Forgacs 2000).He argues essentially that from time to time when some from the masses progress, they then seem to lose bits of their organic being (Forgacs 2000:305).This is very important especially for those in the middle class.I am of the view that because of this progress, the majority have developed a love-hate relationship with the black middle class.They love them because they represent hope, the exit from squalor and poverty, yet at the same time they hate them because this class claims to speak on the behalf of the majority who still remain on the underside of history and therefore have no idea of the continued challenges faced by the majority.

A perspective on democracy

Having said all the above, I now come to the notion of democracy.My current research interests include, amongst others, a research project of looking at Malcolm X's 1964 speech titled 'The Ballot or the Bullet' (Tshaka 2012:882-898).

In this project I try to compare this watershed speech with the current South African context.Looking at how the ballot, which for us transpired in 1994, is currently being threatened by the bullets of service delivery and the still huge economic discrepancy between whites and blacks in this country.So what was significant for Malcolm is also significant for us in South Africa: a functional democracy.Stellenbosch economist Sampie Terreblanche argues that the notion of democracy is not as sacrosanct as we would like to believe (Tshaka & Makofane 2010:532-546).

A scholar who helped me to understand this ambivalence of democracy even better is Naom Chomsky.In one of his numerous books titled, Media control: The spectacular achievements of propaganda, he speaks of a spectator democracy (Chomsky 2011:22).Chomsky recalls Walter Lippmann who argued that the media's manufacturing of consent is necessary because the common interest eludes public opinion entirely and can only be understood and managed by a specialised class of responsible men who are smart enough to figure things out.

Having established that class, Lippmann (cited in Chomsky 2011) then argues that this class has to pit itself against the majority, which he calls 'the bewildered herd'.In other words, for him the smart people have to protect themselves from the trampling and roar of the bewildered herd (Chomsky 2011:22).Seen in this way, what has therefore happened in Marikana 5 was nothing but the actions of a bewildered herd.This picture is not only observed in Marikana, but in all the townships where we are seeing the spiralling service-delivery protests in this country.So, contrary to Mosala likening the protesters at Marikana to a child who never thinks that a parent will exert maximum force, this specialised class, because they are protecting their interests, exert maximum force against the bewildered herd. 6

There are therefore two functions in a democracy, according to Chomsky.The specialised class, the responsible men, 5.Marikana is a place located in the North West province of South Africa and home to the platinum mines owned by London Mines (Lonmin).It shot to fame when police opened fire, killing more than 30 miners who were striking for better wages.The killing of civilians seems to have become a familiar phenomenon in recent South African past.In the same year, 2012, Andries Tatane was shot dead by police as he participated in a service-delivery protest in the Free State.

6.This reasoning became apparent as Mosala tried to explain the Marikana situation.This is not surprising to me as this one-time very reputable black scholar has joined the Tenderpreneurship which seems to have taken South Africa by storm.

carry out the executive function, which means they do the thinking and planning and understand the common interest.Then there is the bewildered herd, and they have a function in democracy as well (Chomsky 2011:23).Their function in democracy is to be spectators.However, since it is a democracy, occasionally they are allowed to lend their weight to one or other member of the specialised classand this happens during voting time when you will see politicians again speaking the language of the people on the ground and that are seen actually entering the shanty towns to assure the masses that they have their interest at heart.This attitude was not made clearer than at the African National Congress (ANC)'s 100-year birthday celebration when the deputy president of the ANC, Kgalema Motlanthe made a toast and said they were drinking sparkling wine on behalf of the masses (Du Plessis 2013).

The picture painted so far raises significant questions for social cohesion.In a country fraught with contradictions, it is perhaps significant to ask a few critical questions relating to the subject of social cohesion.We must, I believe, ask: Social cohesion for what?Why is it necessary?For what purpose must this be encouraged?To what does such a call for social cohesion want to respond?Responding faithfully to these questions suggests that we see a common problem and we agree on the causes of the problem.Social cohesion in my opinion, in the context of present-day South Africa, is a means towards a particular end.

Tavis Smiley and Cornel West prefer to speak of a covenant with black America (cited in Smiley 2006).They realise that there are particular problems that seem to be specifically ganging up more so on black communities than they do on white communities in present-day America.We in South Africa have been branded by the world as the miracle child and this branding forces us to act accordingly by all means necessary.We have at best become the most insincere nation in terms of what our true feelings really are.We have compressed our feelings and seem astounded when pockets of racial eruptions occur in this country.Most of our problems are as a direct result of a legal racialised past that we share.This is not unique to us, but refers to darker people across the globe.

Critical theology in this country has always insisted on recognising how we have been racialised and how that process of racialisation impacts on our theological reflections.

In other words, therefore, critical theological reflections on issues such as the black theology of liberation and others have always been public theological discourses.This brings us to the notion of public theology which seems to have been lifted up as the newest, most relevant theological reflection today in South Africa and elsewhere.Public theology has become the buzz word in South Africa; it has become a buzz word in some Princeton circles as well as in some circles in Brazil.A book edited by Joel Carpenter of Calvin College with the title, Walking together: Christian thinking and public life in South Africa is dedicated to three South African public theologians, John De Gruchy, Nico Koopman and Tinyiko Maluleke (Carpenter 2012:i).

For us to understand the conversation on public theology in South Africa, we need to have an understanding of the centre for theological inquiry in Princeton.Storrar's (2007) article titled 'A Kairos moment for public theology', 7 is noteworthy and has received interesting feedback from the likes of Maluleke.For Storrar doing public theology suggests participation in the public sphere.He employs Habermas's understanding of the public sphere, which is the domain where citizens gather to consider issues of general interest (Storrar 2011:23-43).He names what he believes to be the four tasks for public theology; creating an inclusive public sphere; welcoming the stranger; expressing anger and resisting privatisation (Storrar 2011:23-43).Whilst all these issues which for him constitute public theology sound acceptable and undoubtedly noble, Storrar appears to be a bit naive in as far as he does not engage the question of power which lies at the heart of those who make available such space for genuine public theology to thrive.

In a way, according to Storrar, liberation theologies are not necessarily public theologies but rather expressions of public anger (Storrar 2011:36).They are necessary therefore only insofar as they struggle for inclusion in the public sphere.Storrar's understanding of public theology is highly problematic if it assumes that the questions that frame the public are predetermined (Storrar 2011:23-46).In fact it is my view that there can never be one public theology, but in fact several public theological discourses.The fact that I do not reserve some time to deal with the question of power and who dictates the terms for discourse blinds us from seeing the questions that frames the public spheres of those on the underside of history.

In fact if we follow Storrar (2011:23-46) blindly in this direction, we will fall into the same trap that black theology of liberation fell into when it began to obsess about a methodology in an attempt to become more acceptable to mainline theology.The choice for neutral language to explain the real structural problems of South Africa has resulted in an obfuscation which is manifested in the many societal challenges we are facing in present-day South Africa.Since in the minds of many a black theology of liberation wrongly conjures up a militant figure, this theological reflection is to be rejected and to be replaced with a more civilized reflection such as public theology, for instance.The question of interlocution which remains central to practitioners of black theology of liberation is ignored all together.

An African Christology remains relevant today simply because it is not by chance that the majority of the world's 7.The Global Network for Public Theology (GNPT) is an initiative borne from the vision of the Director of the Centre for Theological Inquiry (CTI) at Princeton, Dr William Storrar.Dr Nico Koopman, Dean of the Faculty of Theology at Stellenbosch was its first president from the inception of the network in 2007 till 2008.On its official website, the network explains part of her mission as follows: 'The GNPT seeks, through interaction and collaboration, to be a genuinely global network, where the global and the local come together.Despite our differences, the Christian faith challenges us to engage theologically with public life in all its aspects, political, economic, legal and social.We are concerned with the expression of public theology, how it is conceived in particular contexts, and how it is applied' (Haire 2008(Haire -2011)).

people who happen to be poor and marginal are Africans and black.The existential questions that concern these people are no doubt different from those who happen to be non-black and European.Because of this reality, it cannot be denied that African Christians' relation and imagination about God is different.

Again it seems to me to be an attempt at suggesting a theology that is safer for the fragile South African context.Black theology of liberation is not particularly considered relevant and seems to be made out as being too militant and angry.I have no issues with the notion of public theology until it wants to give a false impression that it brings something new to the public debate that black liberation theology has not done yet.From the history of black liberation theology in this country it is clear that what the practitioners of this hermeneutic were busy with was public all along.I am going to end on this note and hope that what I said gives us enough to engage with each other as we put hands and feet to this timely and very relevant project.

Spirituality and being intertwined with the material conditions of humans

The spiritual was always considered as being intertwined with the physical.This point was made poignantly clear by Steve Biko (1976) when he argued that:

We believed in one God, we had our own community of saints through whom we related to our God, and we did not find it compatible with our way of life to worship in isolation from the various aspects of our lives.Hence worship was not a specialized function that found expression once a week in a scheduled building, but rather it featured in our wars, in our beer drinking, our dances and our customs in general.Whenever Africans drank, they would first relate to God by giving a portion of their beer away as a token of thanks.When anything went wrong at home they would offer sacrifice to God to appease him and atone for their sins.There was no hell in our religion.We believed in the inherent goodness of man hence we took it for granted that all people at death joined the community of saints and therefore merited our respect.(p.42)

The problem, it would seem, comes up once we fall into the trap of separating the two, the spiritual from the material.My dismay with the CYM, when realising that any theological comment that challenges the hegemonies is considered political and departs from the spiritual task which is considered the core of Christian theology, reminded me of the challenge that is faced by academic theology even today.I have made mention of the perceptions of the spirituality that has become entrenched in the minds of many South African Christians (see for instance my reference to spirituality in this article as being perceived of as docile and lacking in militancy).More importantly has been the fact that a black theological hermeneutic which took the criticism of hegemonies as being central in Christian reflection has yet to transcend its position as a guerrilla enterprise (Tshaka 2010b:124-135).Whilst this hermeneutic might be known in academic theological circles, it seems especially ignored within church circles and in some instances even considered dangerous.

Elsewhere I have argued (Tshaka 2010a:51) that theological reflection is most at home within the realm of the church.I made this assertion contrary to attempts at theological reflections that seem to ignore and even discourage the context of the church.That context is the ambivalence that is created by a spirituality that inverts the cross from a symbol of harsh oppression to victory over the powers that are.It is this approach in my view which has kept the likes of James Cone in the church despite the harsh criticisms that they have of dominant theological hegemonies.In fact it is this approach which in my view resulted in the likes of Gabriel Setiloane to declare that they are bewitched by this faith (cited by Kumalo 2006:1).

Something is clearly amiss in the South African context.It is therefore not by chance that notions such as ethics, morality, et cetera, seem to take centre stage in South African public life today.That these issues and many others are debated in public life also happens during a time where service-delivery protests seem to be at their highest.Police brutality seems to become an everyday occurrence, with Africans from other parts of the continent particularly being on the receiving end of this ill treatment, not only from the police but some South Africans as well.The brutality displayed in the killings of ordinary people by thugs as well as the raping of the elderly and infants which grace our newspapers on an almost daily basis suggest that something is seriously wrong with this one-time miracle child of the world.Indeed the current South African situation can at best be described as a nervous condition.The need to link the spiritual to the material seems to be urgent at this stage.

Towards a covenant with black South Africa!

Moral regeneration which must have at its heart the intention to foster accountability of communities to their various constituencies must be a covenant especially with black South Africa.I say this simply because the black experience is still a matter that cannot and must not be ignored by serious political as well as theological leadership in South Africa.

If we agree that we have in this country a history that had encouraged black people to deny themselves and to imitate other cultures, then we must admit that there are things that black people will have to do for themselves and on their own terms in their communities.It means that in as much as we acknowledge the histories which had engendered differences amongst us a nation, as black people we need to begin to challenge our various leaderships to be more intentional in dealing with problems that seem to be uniquely ours.It seems very clear that this prediction applies very well to black people.I say this because black people and their communities remain divided because we are more concerned with looking at the issues that do not really seem to build us.We are led by envy and distrust for our fellow black people.A spirituality that has managed the oppressed to transcend adversity and to invert symbols of oppression into symbols of victory is the spirituality that is called for.More importantly this time, when making this call we need to realise the historicity which has brought us to where we are today.Our spirituality, we need to reckon, has never been one which makes us to distinguish between the spirit and the flesh.This has always been the point of the hermeneutics of black liberation theology.

Conclusion

The notions of spirituality, democracy, public theology and social cohesion are significant notions that call for diligent attention.Yet we are faced by the challenge of a divided country.Divided mainly along racial and class lines.

Theology has a significant role to play.Yet honest theology is one which does not dismiss the past but uses it wisely to learn from it with the objective to avoid similar mistakes made in the past.We must admit that we are a racialised nation.To dismiss this is not helpful at all.To admit it and to inquire how to transcend this is what is of utmost importance.This article wants to contribute towards such a broader discourse.

4.Itumeleng Mosala was one of the key conversational partners invited to ponder on the questions of spirituality, democracy, Public Theology and social cohesion.His reflections were paramount especially in light of his own theological scholarship.This article flows from that conversation held at the University of Pretoria under the auspices of the Director for the Centre for Public Theology at the University of Pretoria, Dr Vuyani Vellem.

(Lynch 2009)e reflections were shared by myself at a congregation in the Eastern Cape organised by the public sector, training in collaboration with the Public Administration Leadership and Management Academy (PALAMA) (see especially the paper by Tshaka 2010c).I refer again to a quote from the WillieLynch (2009)letter.Lynch was the slave owner whose name became infamous for hanging black people, especially in the south of the USA in the 1700s.He was once invited to share his tactics with other slave owners about how to make a slave behave according to the expectations of his master.As he rode into Virginia he felt disturbed by the sight of black bodies hanging from the trees.He was disturbed simply because he thought these masters were messing precious cargo.This is what he(Lynch 2009) said as he shared his highly successful tactics on the banks of the Virginia River in 1712: on top of my list is age, but only because it starts with an 'a'.The second is color or shade.There is intelligence, size, sex, sizes of plantation, status of plantation, attitude of owners, whether the slaves live in a valley or on a hill .... Have fair hair, course hair, or is tall or short.Now that you have a list of differences ... I shall assure you that distrust is stronger than trust and envy, stronger than adulation, respect or admiration.The black slaves after receiving this indoctrination shall carry on and will become self refuelling and self generating for hundreds of years maybe thousands.(p.7ff.)

AcknowledgementsI want to acknowledge the continued support of my wife and partner, Mrs. Precia Galaletsang Tshaka.I also want to thank my three wonderful children who I believe are starting to understand my academic calling.Competing interestsThe author declares that he has no financial or personal relationship(s) that may have inappropriately influenced him in writing this article.
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Evidence for large domains of similarly expressed genes in the Drosophila genome

Paul T Spellman firstname.lastname@example.com
Department of Molecular and Cell Biology
Howard Hughes Medical Institute
University of California
94720-3400BerkeleyCAUSA

Gerald M Rubin
Department of Molecular and Cell Biology
Howard Hughes Medical Institute
University of California
94720-3400BerkeleyCAUSA

Evidence for large domains of similarly expressed genes in the Drosophila genome
Research article Correspondence: Paul T Spellman.
Background: Transcriptional regulation in eukaryotes generally operates at the level of individual genes. Regulation of sets of adjacent genes by mechanisms operating at the level of chromosomal domains has been demonstrated in a number of cases, but the fraction of genes in the genome subject to regulation at this level is unknown.Results: Drosophila gene-expression profiles that were determined from over 80 experimental conditions using high-density oligonucleotide microarrays were searched for groups of adjacent genes that show similar expression profiles. We found about 200 groups of adjacent and similarly expressed genes, each having between 10 and 30 members; together these groups account for over 20% of assayed genes. Each group covers between 20 and 200 kilobase pairs of genomic sequence, with a mean group size of about 100 kilobase pairs. Groups do not appear to show any correlation with polytene banding patterns or other known chromosomal structures, nor were genes within groups functionally related to one another.Conclusions:Groups of adjacent and co-regulated genes that are not otherwise functionally related in any obvious way can be identified by expression profiling in Drosophila. The mechanism underlying this phenomenon is not yet known.BackgroundThe regulation of gene expression is a fundamental process within every cell that often allows exquisite control over a genes activity (for review see[1]). Altering transcription rates is an effective strategy for regulating gene activity. It is well established that transcription of a given gene is dependent upon a promoter sequence located within a few hundred base pairs of the transcriptional start site. Promoter activity is modulated by sequence-specific transcription factors that physically interact either with the protein complexes that make up the core transcriptional machinery or with the promoter sequence itself.

In eukaryotes, the activity of a promoter can be modified by transcription factors binding to DNA sequences (frequently termed cis-regulatory modules or enhancers) that are located from hundreds to hundreds of thousands of base pairs away from the promoter. These regulatory modules can either increase or decrease the rate of transcription for a target gene, depending on the cellular state and the activities of the bound transcription factors. There are several mechanisms by which transcription factors bound to regulatory modules exert their effects. .irst, many transcription factors interact directly with the core transcriptional machinery by recruiting the latters protein complexes to the promoter. Second, transcription factors may bend or twist the DNA, altering the way in which other transcription factors interact with the DNA. .inally, transcription factors can alter local chromatin structure by modifying histones (typically through methylation, acetylation, and substitution of histone subunits) to permit or restrict access to the DNA. Modifications of chromosome structure also occur at much larger scales. Most eukaryotes exhibit distinct chromosomal regions that are usually either transcriptionally active (euchromatin) or inactive (heterochromatin). In animals, heterochromatin is typically found near centromeres and other regions of low sequence complexity.

Less clear are the mechanisms by which the regulation provided by a cis-regulatory module is restricted to specific target genes. Several examples of insulators -sequences that prevent neighboring modules from affecting transcription -have been identified (reviewed in [2]). Insulators seem to function not by deactivating cis-regulatory modules but by preventing their influence from being propagated along the chromosome. It is not known how common insulators are in the Drosophila (or any other) genome. Some insulator-binding proteins localize to a few hundred chromosomal positions, and these positions coincide with genomic sequences that are not heavily compacted by chromatin structure (the interbands of polytene chromosomes) [3]. There is substantial evidence that, although gene expression can be tightly controlled, neighboring genes or chromatin regions are important for the expression of individual genes. .or example, otherwise identical transgenes inserted into different chromosomal sites show varying levels of expression [4].

Two recent observations lend credence to the idea that genomes may be divided into domains important for controlling the expression of groups of adjacent genes. .irst, there is evidence from budding yeast that some genes are found in pairs or triplets of adjacent genes that display similar expression patterns [5]. Second, about 50 much larger regions of the human genome show a strong clustering of highly expressed genes [6], which is caused by clustering of genes that are expressed in nearly all tissues [7]. We have examined the fraction of genes in the Drosophila genome that are subject to regulation that reflects large domains, using data from high-density oligonucleotide microarrays that reflect over 80 experimental conditions, and have found more than 20% of the genes clustered into co-regulated groups of 10-30 genes.

Results

Many neighboring genes show similar expression patterns

We collected relative gene-expression profiles covering 88 distinct experimental conditions from 267 Affymetrix GeneChip Drosophila Genome Arrays (see Materials and methods section). When the genes in this dataset were organized according to their positions along the chromosome, we observed numerous groups of physically adjacent genes that shared strikingly similar expression profiles. We sought to measure the magnitude of this effect by identifying all groups of physically adjacent genes that showed pair-wise correlations between their expression profiles that were higher than expected by chance.

Visual inspection of the entire dataset using TreeView software [8] revealed that groups of adjacent genes with similar expression patterns appeared frequently in our real dataset but rarely in a randomized dataset. The size of these groups varied, but appeared to average about 10 genes. In order to systematically identify groups of adjacent, similarly expressed genes, we calculated the average pair-wise Pearson correlation of gene expression for genes in a sliding ten-gene window across the genome. The Pearson correlation is a commonly used metric for determining the similarity between two gene expression profiles [8], and the average pair-wise correlation is the average of the Pearson correlations of all 45 possible pairs of genes within the tengene set. We estimated the probability of the average correlation scores by randomly sampling one million times from the dataset and calculating the average pair-wise correlation for windows of ten genes. We also created a random dataset of the same size, by randomly shuffling the associations from genes to expression profiles, and used this to illustrate the significance of our results. Our analyses show that groups of physically adjacent genes with similar expression are common; nearly 1,100 such groups are significant at a p value of 10 -2 (Table 1). In more conservative analyses (requiring an uncorrected p value of 10 -4 ), where we expect to observe only one group by chance, in fact we observed 124 groups (Table 1).

To ensure that ten-gene windows were appropriate, we repeated the analysis using windows of various sizes. As the window size increases from two to eight genes, the net number of genes in groups (that is, the genes in groups in the ordered dataset minus genes in groups from the random dataset) increases linearly. At a window size of about ten genes, the net number of genes begins to plateau (.igure 1). This suggests that most groups include about ten genes, so we used a window size of ten for the remainder of our analysis. There are no qualitative differences in the nature of groups identified by larger window sizes.

Many of the ten-gene groups that have high average pairwise correlations of gene expression represent physically overlapping stretches of genes (that is, genes n through n + 9 make up one group and genes n + 1 through n + 10 form another). .or all further analyses, therefore, we collapsed all groups that bordered one another into a single group. This substantially reduced the number of groups, showing that the effect on expression extends well beyond ten genes ( Table 2). Nearly 1,100 ten-gene groups are significant at p < 10 -2 , but these collapse into only 211 groups with an average group size of greater than 15 genes. As the p values decrease the average group size also decreases, but even at p < 10 -4 there are, on average, 12 genes in each group (553 genes in 46 groups; see Table 2).

The 44 groups (681 genes in total) that map to the left arm of chromosome two and have a p value of less than 10 -2 are shown, using a ratiogram [8] aligned to the chromosome arm, in .igure 2. The distribution of groups along the chromosomes appears random and there is little bias for genes in a group to be on the same strand. The length of genomic sequence occupied by similarly expressed gene groups is highly variable. The average group size is nearly 125 kilobase pairs (kbp) in length, with a standard deviation of about 90 kbp, while the smallest group is 22 kbp and the largest is over 450 kbp. As might be expected, there is a relationship between the number of genes in a group and the length of genomic DNA covered by each group (Pearson correlation 0.59).

Gene groups are not explained by gene function or homology

Many genes that are related by function share similar expression patterns, and it is plausible that the same is true for homologous genes, particularly those that arose from recent duplications. In Drosophila there are 2,207 genes for which there is a homolog within the genome and the two homologs are separated by less than 10 genes. To determine whether homologs account for our observations, we repeated our analysis on a dataset from which homologs that are physically near one another were removed. This dataset is just under 12,000 genes, and although there is a significant decrease in the numbers of Table 1 The number of ten-gene groups of adjacent, similarly expressed genes that are found in ordered and randomized datasets, or are expected to be found in a randomized dataset The 'Expected' column gives an approximate number.

Figure 1

The number of genes identified as being in groups when different window sizes are used. In order to identify groups of adjacent, similarly expressed genes, the average pair-wise correlation of gene expression was calculated for genes in a sliding window across the genome, and this process was repeated for windows of different sizes. The net number of genes (that is, the number of genes in groups in the ordered dataset minus the number of genes in groups from the random dataset) is plotted against window size.  genes found to be in groups in this dataset (Table 3), 176 groups remain, containing about 2,500 genes.

We considered an extreme model to account for our observations -that evolutionary selection has organized gene groups according to the biological processes the genes are involved in, so that their expression can be coordinately regulated. We sought to test this model using the Gene Ontology (GO) database [9,10] as a source of annotations of biological processes. We first used the hypergeometric distribution to calculate the probability of observing each GO term as enriched in each group, on the basis of the number of genes in the group, the number of genes in that group that are annotated with that GO term, and the number of genes in the genome that are annotated with that GO term. We then selected all GO process terms associated with a group at p < 0.05 where at least two genes had the selected GO term. Of the 211 groups identified in our full dataset and the 176 groups from the homologs-removed dataset, 43 and 11 GO terms, respectively, have associations to groups that meet the above criteria. These numbers are modestly higher than would be expected by placing a random selection of genes into groups, where we would expect 7 ± 2 from the full dataset and 4 ± 2 from the homologs-removed dataset. The observed enrichment is clearly dependent on homologs, however, given the nearly four-fold decrease in observed associations when homologs are excluded from the analysis. Thus, with the present level of functional annotation, the vast majority of gene groups we observe are not composed of genes with similar biological processes, and the extreme model is not supported.

Similarly expressed gene groups can be identified from smaller datasets

Our dataset is derived from RNA samples taken from embryos or adults (primarily males). The groups in our dataset show a pattern of gene expression that mirrors this
CT15882 CG4947 CG4947 CT15884 CG5001 CG5001 CT16455 CG5139 CG5139 CT16096 CG5011 CG5011 CT33975 CG14342 CG14342 CT33976 CG14343 CG14343 CT16503 CG5156 CG5156 CT16527 CG5397 CG5397 CT17158 CG5423 CG5423 CT33977 CG14344 CG14344 CT33978 CG14345 CG14345 CT17230 CG5430 a5 CT17252 CG5440 CG5440 CT17290 CG5450 Cdlc2 CT17558 CG5556 CG5556 CT17554 CG5561 CG5561 CT17492 CG5564 CG5564 CT17332 CG5565 CG5565 CT33980 CG16933 NLaz CT33979 CG14346 CG14346 CT17328 CG5574 CG5574 (a) (b) (c) CT35452 CG15402 CG15402 CT38165 CG3151 Rbp9 CT10673 CG3181 Ts CT10659 CG3178 Rrp1 CT10601 CG3157 Tub23 CT27262 CG9641 CG9641 CT10615 CG3165 CG3165 CT27264 CG9643 CG9643 CT12153 CG3733 Chd1 CT42330 CG18642 Bem46 CT12137 CG3736 okr CT11970 CG3558 CG3558 CT35453 CG17265 CG17265 CT38181 CG17224 CG17224 CT35454 CG17264 CG17264 CT38179 CG17223 CG17223 CT38167 CG3542 CG3542 CT12091 CG3605 CG3605
bifurcation: most genes are expressed at higher levels in either adults or embryos. We wished to determine whether our observations of groups reflect this division, so we divided our dataset in two, creating one dataset of embryo experiments and one of adult experiments. It should be noted that four of the adult experiments contained RNA from males and from females, which contain a substantial number of oocytes, whereas the rest of the dataset was only from males. We calculated the average pair-wise correlations for all groups of genes in each of the two new datasets; Table 4 summarizes the number of genes in groups for the embryo and adult datasets (both randomized and ordered). The gene numbers are remarkably similar to those found for the entire dataset, as are the numbers of groups (see the Additional data files with this article online).

We wished to know if there was a correlation between the gene groups identified in the adult, embryo, and combined datasets. To do this we tabulated all genes identified in each dataset at each of three p values (10 -2 , 10 -3 and 10 -4 ) and calculated the Pearson correlation between each pair of datasets at each p value ( Table 5). The average correlation between either the embryo or the adult dataset and the combined dataset is about 0.35, while the average correlation between the adult and embryo datasets is lower (about 0.23). The number of genes involved makes little difference, because the correlations are similar at each p value, despite the vastly different numbers of genes identified at different p values. In all, 890 genes are present in a group defined by one of the three datasets at p < 10 -4 . After correcting for genes expected to be found in groups by chance, about 2,250 genes are identified in one of the three datasets at a p value of 10 -3 and about 4,000 genes are identified at 10 -2 .

Correlations with known chromosome structures

We attempted to determine whether the locations of similarly expressed gene groups correlate with known chromosome structures. Polytene chromosomes show a distinct, reproducible pattern of extended and compacted regions. The compacted regions contain the vast majority of the DNA, although the amount of DNA in each band can vary by more than one order of magnitude. The mean DNA content of each band is approximately 25 kbp [11,12] as compared with approximately 125 kbp for each group of co-expressed genes. We calculated the number of bands that overlap (or are contained in) each group and compared this with the number of bands that overlap (or are contained in) a randomly placed group matched for size. There was very little difference in the average number of bands overlapping each co-expressed group or each randomly placed group (5.9 versus 6.6).

It has been proposed that Drosophila chromosomes are attached to a nuclear scaffold at precise locations [13], but there is very limited mapping data on the position of these attachments. Mirkovitch et al. [13] mapped four attachment sites in a 320 kbp region near the rosy gene on chromosome 3R, dividing the region into a number of discrete domains of average size 50 kbp, each containing many genes. We wished to determine whether the groups we identified might correspond to distinct regions between attachment sites, as several of our groups fall in the region Journal of Biology 2002, 1:5 Table 3 The number of groups of genes, and total numbers of genes in groups, from a dataset containing no physically close homologs    [13]. We attempted to align these regions but there are no clear overlaps; the sizes and positions of the domains identified between attachment sites did not correspond to the groups we found.

Discussion

We have found that over 20% of the genes in the Drosophila genome appear to fall into groups of 10-30 genes such that the genes within each group are expressed similarly across a wide range of experimental conditions. Our data do not reveal the mechanism(s) responsible for the observed similarities in expression of adjacent genes but we believe the findings are most consistent with regulation at the level of chromatin structure, for the following reasons. .irst, the regions showing similarities in expression are quite large, containing on average 15 genes, with each gene presumably having its own core promoter. Second, it is frequently the case that one or two genes in a group display a high level of differential expression (see .igure 2c). If the chromatin in a region of the chromosome that contained many genes was opened so that a single target gene could be expressed, it might increase the accessibility of the promoters and enhancers of other genes to the transcriptional machinery, leading to modest parallel increases in their expression. Such an effect could account for the observations we have made.

Discussions of transcriptional regulation often emphasize the belief that the process is tightly controlled and essentially error-free. We believe that the degree of precision, at least at a quantitative level, may be less than is generally assumed. .or example, only a few genes show an obvious phenotype when heterozygous, and heterozygosity generally results in a two-fold reduction in expression level [14]. Moreover, there are numerous examples in the literature of genes that, when misexpressed either temporally or spatially, do not generate a phenotype. Although it is difficult to prove that individuals carrying such traits are as fit as their normal relatives, it is likely that the precise regulation of many genes is allowed to vary considerably. If we presume that the groups we have observed arise because of selection on the regulation of a small subset of genes in each group, then the vast majority of genes are in effect being carried along for a ride. The regulation of transcription may be precise when it is needed and sloppy when it is not important.

If coordinated gene expression is unimportant, there should be no selection that drives the groups of co-regulated genes we observed to be evolutionarily conserved. It will be possible to test this when the D. pseudoobscura sequence is completed. If the groups of genes we identify here are found to be more syntenic in the D. melanogaster and D. pseudoobscura genomes than expected, that would support the idea that the observed coordinated expression is advantageous.

Although we have assayed a relatively large number of biological samples, we cannot infer the profiles of unique cellular states. As further experiments are carried out it may be that our observation of similarly regulated groups will grow to include all genes -that is, the entire euchromatic genome may be structured in such domains.

Materials and methods

Data collection

We collected a dataset composed of 88 experimental conditions hybridized to a total of 267 GeneChip Drosophila Genome Arrays (Affymetrix, Santa Clara, CA, USA) [15]. This dataset came from six independent investigations that will be described in detail elsewhere (A. Supplemental data including software used in this study and the underlying expression dataset is available at our website [17] and from the ArrayExpress database [18] with the accession id E-RUBN-1.

Data processing

Genes are represented on the GeneChip Drosophila Genome Array by one or more transcripts, which in turn are represented by a probe set. Each probe set has 14 pairs of perfect match (PM) and mismatch (MM) oligonucleotides. Data were collected at the level of the transcript, but for ease in the text, the data are referred to by gene. Intensity data for each feature on the array were calculated from the images generated by the GeneChip scanner, using the GeneChip Microarray Suite. These intensity data were loaded into a MySQL database where information on each of the features was also stored. The difference between the PM and MM oligonucleotides (probe pair) was calculated, and the mean PM-MM intensity for each array was set to a constant value by linearly scaling array values. The mean intensity of individual probe pairs was calculated across all arrays, and the log 2 ratio of each value to this mean was stored. Next, all log ratios for each probe pair set (transcript) were averaged, creating one measurement for each transcript on each array. The final dataset was generated by averaging data for each transcript on replicate arrays and subtracting the average log ratio of each gene in the dataset.

Definition of homologs

BLAST scores based on predicted protein sequence were obtained from Gadfly (Release 2) [19]. We used these scores to define a homolog pair as those gene pairs for which BLAST E values are less than 10 -7 .

Identification of adjacent similarly regulated genes

We calculated the average pair-wise correlation of geneexpression profiles for all genes that were within n genes (an n-gene window) of one another using the Pearson correlation. Significance (p values) was estimated by sampling random sets of n genes 1 million times to determine the likelihood distribution for the dataset. We also calculated the average pair-wise correlation for a random dataset in which the associations between genes and expression profiles were shuffled. We have calculated the number of genes in groups at each of the three p values, namely 10 -2 , 10 -3 , and 10 -4 , for window sizes ranging from 2 to 25 genes.

Next, we set out to show that homologs did not account for the increase in the number of gene groups with higher than expected average correlations. We searched for cases in which homologs (as defined above) were near each other in the genome by scanning the set of genes for each chromosome from one end to another. If a gene showed homology to another gene that appeared less than 10 genes ahead, it was removed from the dataset, although no break in gene order was created. .or example, in a set of 11 genes where the third and fourth were homologs, gene 3 would be removed, and a ten-gene group would consist of genes 1, 2 and 4 through 11. In total, 1,369 genes were removed from the dataset. This homologs removed dataset was subjected to the average pair-wise algorithm, as was a randomized version of it.

We also constructed two non-overlapping subsets of the total data matrix. All hybridizations were divided into either the embryo or adult dataset on the basis of the source of the RNA used in that hybridization. In total, 35 experiments remained in the embryo dataset and 53 experiments remained in the adult dataset. The random pairwise correlation algorithm was applied independently to each of these datasets as well as to randomized versions of each dataset.

Significantly enriched GO terms among gene groups

GO terms for all genes were obtained from the GO database [10]. Using the hypergeometric distribution, the probability of observing each GO term with each group was calculated. Briefly, the probability p that a GO term is significantly enriched among a specified set of genes can be calculated with the following formula:
A i G -A n -1 p = 1 -͚ k -1 i = 0 G n
where k is the number of genes in the group, G is the total number of genes, n is the number of genes in the group with a given annotation and A is the total number of genes with a given annotation. Because many sets of GO terms (> 1,000) were tested on many groups of genes (> 200), there is a problem of multiple testing. All GO terms significantly associated with a group of similarly expressed genes at a p value of less than 5 x 10 -4 were recorded.

Correlation of groups with known chromosomal structures

We determined the number of polytene bands present in each group of similarly expressed genes. The coordinates of each group were determined by using the transcription start sites (from Gad.ly Release 2) [19] of the genes at each end of a group. We then determined how many bands overlapped each group based on the positions reported [11,12]. We also calculated the number of bands that overlap randomly placed groups (with the same sizes as the real groups).

Additional data files

The following are provided as supplemental materials; a tab-delimited text file of the underlying expression data; the perl scripts used to process the data; and a text file used to generate .igure 2. All expression data are reported as log base 2 and are mean centered (the mean expression value for each gene in all experiments is zero). The first column of each expression data file is the CT identifier of each transcript. The second column is a description field, which includes the CT identifier, CG identifier, gene name, and brief Gene Ontology annotations. The remainder of the columns contain expression data, classified by the column header (either adult or embryo). The data used to generate .igure 2 can be loaded into the TreeView software [8] to visualize individual groups (null data rows indicate boundaries between groups). The software and underlying expression dataset are also available at our website [17] and from the ArrayExpress database [18] with the accession ID E-RUBN-1.

Journal of Biology 2002 , 1: 5 Figure 2
200252Similarly expressed adjacent genes on the left arm of Drosophila chromosome 2 (2L). (a) Ratiograms show the relative expression of all gene groups on 2L that are significant at p < 10 -2 . In each ratiogram, columns represent individual experimental conditions and rows represent individual genes. For each square on the resulting grid, red denotes relative expression higher than the average for a gene in an experiment, green denotes lower relative expression and black indicates that the expression is equal to the average. The black bar represents the chromosome, and the ticks along its left side mark 1 megabase (Mb) distances. The black shapes link the positions of groups on 2L to the expanded views of certain groups that are shown in (b,c). (b) An expanded view of about 5 Mb. (c) The genes in two groups are shown in detail. The CT (computed transcript identifier), CG (computed gene identifier), and gene name are shown for each of the genes in these two groups. Each of the two expanded sections represents one group.

Table 2
2The number of groups of genes, and total numbers of genes in groups, that are identified at various levels of significance (p values)Groups
Genes

Significance
Ordered
Randomized
Ordered
Randomized
(p value)
dataset
dataset
dataset
dataset

10 -4
46
0
553
0

10 -3
93
5
1,219
51

10 -2
211
53
3,228

Table 4
4The number of genes within groups identified in either 'adult' or 'embryo' experimentsEmbryo
Adult

Significance
Ordered
Randomized
Ordered
Randomized
(p value)
dataset
dataset
dataset
dataset

10 -4
285
0
371
0

10 -3
1,159
52
1,139
114

10 -2
3,108
686
3,144
938

Table 5
5The correlation between sets of genes identified in the adult, embryo and combined datasetsSignificance
Combined:
Combined:
Adult:
(p value)
adult
embryo
embryo

10 -4
0.33
0.41
0.24

10 -3
0.34
0.34
0.23

10 -2
0.38
0.28
0.22

AcknowledgementsWe thank Adina Bailey, Michael Brodsky, Amy Tang, and Pavel Tomancak for sharing data prior to publication. P.T.S. was a recipient of an NSF Biocomputing postdoctoral fellowship. G.M.R. is an investigator of the Howard Hughes Medical Institute.
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Efficacy of ImageJ in the assessment of apoptosis

Iman M Helmy
Adel M
Abdel Azim
Efficacy of ImageJ in the assessment of apoptosis
R E S E A R C H Open AccessApoptosisHep2 cellImage analysisNAF (nuclear area factor)
Objective: To verify the efficacy of ImageJ 1.43 n in determining the extent of apoptosis which is a complex and multistep process. Study Design: Cisplatin in different concentrations was used to induce apoptosis in cultured Hep2 cells. Cell viability assay and nuclear image analysis of stained Hep2 cells were used to discriminate apoptotic cells and cells suspected to be undergoing apoptosis from control cells based on parameters such as nuclear area, circularity, perimeter and nuclear area factor (NAF), in association with visual morphology. Results: Image analysis revealed a progressive and highly significant decrease in nuclear area factor detected in apoptotic cells and in cells suspected of undergoing apoptosis compared to the control cells (P-values < 0.01). Some of the other studied parameters showed also the same trend. This decrease was assumed to indicate DNA loss. Image analysis results correlated positively and significantly with the results obtained by cell viability assay (R = 0.958, P-value = 0.042). NAF was the most reliable parameter in assessment of apoptosis. Conclusion: Nuclear area factor can be calculated using powerful free and open-source software. Consequently, a quantitative measure of apoptosis can be obtained that is linked to morphological changes. ImageJ 1.43 n may therefore provide a useful tool for the assessment and discrimination of apoptotic cells. Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5929043086367338

Introduction

Apoptosis is an active process of self-destruction, whereby cells undergo physiological cell death. It occurs during development and regulation of tissue homeostasis or as a result of changes in environmental stimuli [1].

Induction of apoptosis is due to the absence of specific interactions between transmembrane receptors of the integrin family and extracellular matrix proteins [2]. The triggering of apoptosis requires the activation of specific enzymes called caspases [3], differently associated as inactive form with signal transduction complexes or with cellular organelles [1].

The rate of cell apoptosis is a significant parameter in many experiments involving cell cultures. Cell death kinetics can be measured by counting the number of cells and/ or area occupied on each culture dish by analyzing images taken at different moments of their evolution, apoptosis leads to condensation and fragmentation of cell bodies, producing regions populated with unstructured smaller objects [4].

Examining cells by microscopy has long been a primary method for studying cellular function. When cells are stained appropriately, visual analysis can reveal biological mechanisms [5].

Cells in advanced stages of apoptosis exhibit morphological changes characterized by nuclear and cytoplasmic condensation and cell fragmentation into membrane bound apoptotic bodies. These morphological changes can be observed by light microscopy and are correlated initially with large and subsequently, very small chromosomal DNA fragments [6]. Indeed, other morphological features of apoptosis occur in the absence of detectable DNA fragmentation or a decrease in DNA content [7].

Still, for most applications, image cytometry (automated cell image analysis) is strongly preferable to analysis by eye. In fact, in some cases image cytometry is absolutely required to extract the full spectrum of information present in biological images for many reasons such as: it yields many informative measures of cells, consistent quantitative measures for every image, measures each rather than producing a score for the entire image and finally is much less labor-intensive and higher-throughput [5].

Currently there are many systems available to an anatomic pathologist for the purpose of image analysis in nuclear morphometry. Many of these systems, however, require expensive software and hardware attachments for image acquisition, analysis and storage. Consequently, a cost-effective alternative for image analysis would be a welcome tool for pathologists and researchers alike. In this connection, "ImageJ" is a freely available java-based public-domain image processing and analysis program developed at the National Institutes of Health (NIH). At present, ImageJ's Macintosh platform counterpart, "NIH Image" is widely used in biologic research. A number of nuclear morphometric descriptions can be evaluated on stained sections after downloading specific plug-ins from the ImageJ website. The macros and plug-ins are available as source files and download to the ImageJ folder [8].

Image analysis of stained nuclei should be capable of revealing changes in chromatin organization and in DNA amounts during apoptosis. Such analysis should allow the detection of the early stages of apoptosis by associating slight changes in nuclear morphology with a decrease in DNA content [9].

There is a number of biochemical and image based essays for apoptosis that vary widely in complexity, specificity and cost [10]. It has previously shown that the nuclear area factor (NAF) can be an early indicator of cell morphological changes occurring during apoptosis [11]. Furthermore, calculation of NAF is relatively straightforward, and may be accomplished with a number of image analysis programs.

As long as the cell nucleus remains somewhat intact and the DNA does not become completely fragmented, calculation of NAF should remain a valid marker of apoptotic changes, even though the greatest extent of these changes may occur early in the apoptosis process [10] A systematic undercount of nuclei by image analysis method may produce variation, due in part to the inability to accurately determine a nuclear count when nuclei touch one another. Hence, the image analysis method required thin sections to minimize this problem [12].

In a previous study, there was a disparagement for the public image analysis software program ImageJ 1.37 v, the author proclaimed that while ImageJ was free available, it did not include all of the preanalysis filters and features which are often essential for separating cells before calculating NAF or other measurements especially in cases where cells may aggregate or clump in vitro either in normal conditions or after apoptotic stimuli, and tools available in Image Proplus for separating and splitting objects, are useful for calculating NAF [10].

In concept, calculation of NAF could as easily be used with nuclear dye such as hematoxylin or Giemsa, which we used here. A benefit of these blue dyes is that they allow for simultaneous staining of the same cells with independent markers for apoptosis such as FITC-based TUNEL [10].

NAF was calculated as the product of area (in pixel 2 ) roundness. For calculating NAF from ImageJ data, since roundness was not an available function, as an alternative circularity was used.

The existence of a variety of apoptosis inducers and of variable expression according to the cell type considered has led to a reconsideration of the morphological criteria for the detection of apoptosis. Even in studies using in situ labeling of DNA strand breaks as a marker, there is a heavy reliance on morphological features for the identification of apoptotic nuclei. This study was conducted to proof the efficacy of ImageJ 1.43 n as open source software in assessment of apoptosis which is a complex and multistep process.

Material and methods

A. Cell line

-Squamous cell carcinoma cell line Hep2 used in this research work was kindly supplied from Cell Culture Department-VACSERA, Egypt. Hep2 cells were grown in Minimum essential medium modified with Hank's salts (MEM-H) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine and sodium bicarbonate. The Hep2 cells were treated with Cisplatin (10 μgm, 5 μgm and 2.5 μgm) for 24 hours. Some Hep2 cells remain untreated and used as control.

B. Determination of Cytotoxic Effect of Cisplatin on HEp2

Cell Line 1. Cellular viability assay -The treated dead cells detected were discarded by plate washing using phosphate buffer saline PH 7.2 ± 0.2, secondly the cells were fixed with 10% formalin for 1 hour then fixative was discarded. Plates were washed as previous, and then stained with 0.05% crystal violet solution in 20% ethanol for 10 minute. The extracellular dye was removed by thoroughly rinsing of the cell monolayer using tap water. The remaining cell-attached dye was dissolved in 0.1% acetic acid solution in 50% ethanol. The optical density was read at 540 nm using ELISA reader. Cell viability percentage was evaluated as the percentage of the mean value of optical density of treated wells divided by the mean value of optical density of negative cell control.

Morphometric analysis

-Treated Hep-2 cells were separately stained with heamatoxylin and eosin, and Giemsa stains to detect the histomorphological changes in the Hep-2 cells caused by the cytotoxic drug. For Giemsa staining, the slides were fixed with methyl alcohol for 3-5 minutes, immersed in diluted Giemsa stain for 10-20 minutes and then washed with buffered water (PH 6.8). After that, slides were dried, mounted with Canada balsam and cover slips were placed.

The surface area, perimeter (the length of the outside boundary of the selection) and circularity of the nuclei were automatically measured and then NAF was calculated by the product of nuclear area and circularity (steps of measurements are shown in Figures 1a, b, c and 1d).

In ImageJ 1.43 n, the formula for circularity is 4π (area/perimeter 2 ). A value of 1.0 indicates a perfect circle, which can be associated with apoptosis. Note that the circularity field was first added to ImageJ ver1.35e. As the circularity value approaches 0.0, it indicates an increasingly elongated polygon. The user notes for Ima-geJ software states that measuring circularity may lead to invalid values for very small particles. These aberrant readings can be eliminated by excluding particles that are below a specific minimum area [10].

Statistical analysis

Statistical analysis was performed using (SPSS 17). ANOVA test was achieved to test the differences in the morphometric measurements in the control cells and cells undergoing apoptosis induced by the effect of Cisplatin. Then, it was followed by Tukey post-hoc test. Correlation between cell viability test and NAF was achieved. Statistica7 software was used to generate the graphs of statistical data.

Results

Three phenotypes were detected in the culture differing in their chromatin packing states and intranuclear distribution, the phenotype (I) was characterized by a few small chromatin granules in a homogeneously distributed, delicate chromatin background (Figure 2a). Another phenotype (II), designated as suspected apoptosis, exhibited larger areas of condensed chromatin and a reduced nuclear area compare to type I nuclei (Figure 2b). In the third phenotype (III) the nuclei clumps of condensed chromatin distributed at the nuclear periphery. Entire III nuclei frequently appeared condensed into clusters of round corpuscles (Figure 2c). The apoptotic nuclei chosen for measurements showed no or minimum signs of nuclear envelope disruption. Figure 3 shows control cells for comparison.

A negative correlation between the concentration of Cisplatin and cell viability percentage (as drug concentration decrease the viability percentage increase) from concentration 10 μgm to 2.5 μgm, then with any increase in concentration there was no increase in the number of apoptotic cells.

For calculations using ImageJ, both the average area and circularity of Hep2 treated cells decreased compared to control cells, resulting in dramatic reduction of NAF from 7,023 in control cells to 1,497 in Hep2 cells treated with Cisplatin (table 1). Since, the NAF is the product of area and circularity, a decrease in NAF was observed using ImageJ analysis (Figure 4).

There was a significant decrease in the nuclear area factor detected in the apoptotic cells and cells undergoing apoptosis, induced by different concentrations of Cisplatin, compared to the control cells (P value < 0.01), this decrease denoting DNA loss. There was also a significant positive correlation between the results of cell viability assay and morphometric assessment of apoptosis by ImageJ ( Figure 5) (R = 0.958, P-value = 0.042).

Discussion

Apoptosis proceeds through typical alterations of nuclear morphology [13] characterized by a progressive condensation of chromatin that induces nuclei to shrink into little single balls or cluster of grapes [14]. Thus a nuclear stain such as heamatoxylin can well be chosen to evidence alterations in nuclear morphology [1].

The first apoptotic stage is compatible with an initial step of caspase-3 activation in the cytoplasm, characterized by normal nuclear morphology faintly stained by heamatoxylin. The second stage, characterized by cytoplasm and nuclear over-staining, may represent the step of increasing caspase-3 activation in the cytoplasm and its presence into the nucleus. The third stage identified fits in well with the typical picture of the late apoptotic phase, characterized by nuclear envelope fragmentation [15].

ImageJ software is freely available on the web, and does not allow particle analysis of color images.    Therefore, images were converted to 8-bit grayscale, and then thresholded. From these thresholded images, Ima-geJ then outlines and numbers each object. ImageJ also has the function of "cleaning edges", thus cells on the edge of the image and cut by the image border can be excluded from the final analysis. The old version of Ima-geJ did not have a "split" function used to separate closely touching cells. Thus cells that cannot be separated by thresholding remain as larger groups, and also need to be excluded from the final analysis, but this drawback was enhanced in the recent version. The new version of ImageJ has the watershed function that can split the closely touching cells. Thus, cells that cannot be separated by thresholding can be split by the watershed function. The larger number of available functions for cell counting added to the new version of ImageJ results in more objects that can be included in the count.

In the old version of ImageJ, while objects can be theoretically separated using thresholding, it was a destructive process, which makes all objects smaller when thresholding is increased, in the new version there is the option of automatic thresholding using sample of the desired color, and consequently thresholding is more accurate.

For calculations using ImageJ, both the average area and circularity of Hep2 cells decreased compared to control cells, resulting in dramatic reduction of NAF from 7,023 in control cells to 1,497 in Hep2 cells treated with Cisplatin. Since, the NAF is the product of area and circularity, a decrease in NAF was observed using ImageJ analysis.

The technique reported here may have utility for measuring cell morphological changes such as those occurring in cancer. For example, the nuclear morphometry measures of mean nuclear area, mean nuclear elongation factor, and mean nuclear regularity factor were used as potential predictors for clinical outcomes in localized renal carcinoma [16]. However, in a consensus report of prognostic factors in prostate cancer, it was deemed that factors such as nuclear circularity and nuclear area required further studies before routine use could be recommended [17].

In short, NIH Image/ImageJ delivers a lot despite being a freeware. Knowledge of its potential uses will go a long way in establishing objective, reproducible, costeffective and timesaving methods of automated imagebased IHC evaluation and morphometry for histopathologists.

Figure 1
1(a) Giemsa stained section of Hep2-treated cells showing sampling of the color to be thresholded. (b) Thresholding of the color and watershed of 8-bit image. (c) Analyzing particles after excluding edges. (d) Drawing outlines and automatic numbering of the desired objects.

Figure 2
2H&E stained section of Hep2 cells showing the 3 phenotypes detected in culture with different chromatin packing.

Figure 3
3H&E stained section showing control cells in culture.

Figure 4
4Error bars showing the effect of different concentrations on the nuclear area factor.

Table 1
1Descriptive Statistics for Nuclear Area Factor at Different Concentrations*N
Mean
Std. Deviation
Std. Error

control
27
7,023 a
1,740
335

2.5
400
3,583 b
3,696
185

5
345
2,598 c
3,912
211
10
292
1,497 d
2,622
153

* Means with different superscripts are statistically different at the level of a
0.05

Authors' contributions I H participated in the design of the study, coordination, sequence alignment and drafted the manuscript. A A performed the statistical analysis and reviewing the manuscript. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.
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Cite this article as: Helmy and Abdel Azim: Efficacy of ImageJ in the assessment of apoptosis. Diagnostic Pathology. 15Cite this article as: Helmy and Abdel Azim: Efficacy of ImageJ in the assessment of apoptosis. Diagnostic Pathology 2012 7:15.