Organogenesis : A New Theme to Study Genetic Expression in Adult Liver Regeneration After Partial Hepatectomy

Researchers in different disciplines studied liver’s genetic expression of organogenesis in embryogenesis; however, organogenesis has not been studied as an independent and complementary process during adult liver regeneration. This paper reviewed studies and extracted information related to organogenesis in adult liver regeneration because of organogenesis’ significant role in cancer and tissue regeneration. The paper discussed the accumulated and current status of knowledge of organogenesis after partial hepatectomy, it also dived into the gaps associated with this knowledge and compare it to cellular proliferation process of hepatocytes in liver regeneration. A new theme proposed to signify the role of organogenesis as a separate and parallel process to cellular proliferation in liver regeneration. In addition; the paper reviewed evidence-based contributions that explored the organogenesis and described the associated changes with the targeted factors or genes.


I. Introduction
The liver has a unique ability to regenerate and to retain a specific three-dimensional shape after partial hepatectomy; researchers investigated the liver's abilities because of the multiple factors involved and the complexities of the processes.The focus of this review is organogenesis, and because it is part of the regeneration process, and it overlaps and intermingles with cellular proliferation, both processes were reviewed.While organogenesis defined as the formation of organs 1,2 , but this definition formulated in the context of embryogenesis 3 and these definitions do not specify which process in the regeneration referred to (three-dimensional constitution, cellular proliferation or regeneration as a whole).We propose a definition to define the term organogenesis as following: [Organogenesis is the process in which the cells constitute the three-dimensional shape, and this in turn may contribute to the function, of an organ in the normal spatiotemporal parameters of that organ].With this definition, we generalize the process to both embryonic organogenesis and adult organogenesis and specialize and differentiate the term organogenesis apart from cellular proliferation and liver regeneration.By utilizing this definition, we look for how and why cells move and organize to form the destined three-dimensional shape.Most previous studies focused on the proliferation and differentiation of liver cells (for citations, please refer to Table 1, 2, and 3).In addition, this paper will discuss a theme in which the organogenesis process would be recognized as a separate yet parallel and complementary process in all phases of liver regeneration to help identify factors associated with each process.PROX1 and LYVE1 correlate with regeneration process and can be used as biomarkers to identify formation of new liver sinusoidal endothelial cells.).There is gap in current knowledge because few studies identified causality of specific factors in the processes, especially morphogens.Furthermore, some factors associated with triggering and termination of organogenesis are still unknown (see Table 3), and most importantly, there is a lack of controlled studies to identify factors that control how the liver achieves its final three-dimensional shape.Some studies experimented with models of threedimensional cultures to study a variety of effects (Li et  process need to be investigated (Weiss et al., 2016 10 ).The paper reviewed and critiqued the early measures of regeneration activities, the hormonal role, the factors role, the genes involved, the threedimensional modeling of adult liver organogenesis, and the stages and phases of adult liver organogenesis.Then, the paper discussed and proposed a new theme to consider adult liver organogenesis as a separate and parallel process to cell proliferation in liver regeneration.The information was tabulated for readability.

II. Early Methodologies and Measurements of Regeneration Activities
Emergence of new discoveries related to phosphorylation and mRNA directed the focus of researchers toward the application of these concepts to explore their association with liver regeneration.Studies found that high phosphorylation activities are associated with cell proliferation; however, these studies did not state whether or not an association with organogenesis exists (see Table 1).Table 1 reveals important studies in regeneration and their lack of indication to organogenesis in regeneration process.An exception to these studies is Shu et al.'s (2009) 11 contribution of an important observation associated with STAT3 phosphorylation (see orange highlight in Table 1): their team observed that defective regeneration correlates with steatosis, this observation extended to the group with adiponectin-deficiency, but this study did not describe the pattern in which the defective liver regenerates.

III. Hormones
Hormonal effects were studied to explore whether hormones have an enhancing or inhibitory effect on the regeneration process, but studies were not specific as to whether or not hormones have any effect on organogenesis (Table 2).Proliferation could give rise to mass, but organogenesis is the process that determines how the liver forms its three-dimensional figure, lobar shapes, and chirality.

IV. Factors Associated with Regeneration Process Pace
While many studies investigated different factors involved in liver regeneration, only a few researched factors involved in organogenesis (Table 3).lack of vHNF1 leads to formation of a defective hepatic bud and an abnormal gut regionalization.
Meng (2017) 12 found that PROX1 and LYVE1 correlate with liver regeneration and can be used as biomarkers to identify formation of new liver sinusoidal endothelial cells.However, Meng (2017) 12 did not indicate how the endothelial cells constitute sinusoidal final shape.The abovementioned studies found factors correlated with organogenesis (see orange highlight in Table 3).In addition, these studies demonstrate the importance of organogenesis in liver regeneration.The rest of the studies in Table 3 showed association of factors with regeneration process; however, they did not indicate the association with organogenesis.

V. Genetics
Insufficient studies identified genes involved in organogenesis (including signaling factors or inhibitors associated with gene activities).Arai et al. ( 2003) 13 conducted extensive gene expression analysis during liver regeneration of rats and found 496 out of 2,304 genes showed a measurable expression with up-and down-regulation, including 317 genes showed at least 2-fold change.
Nevertheless, Arai et al. ( 2003) 13 did not indicate how these genes are associated with the organogenesis of the liver after partial hepatectomy.Youn et al. ( 2003) 14 reported high expression of abdB hox genes during liver regeneration of murine rats; however, hoxa1 and b had the same level pre-and post-organogenesis process, indicating both hoxa1 and b are not involved in this organogenesis process.Group IX and X-including hoxad10 and 10-of hox genes are regenerationspecific.Matz-Soja (2016) 15 reviewed signaling pathways of morphogenesis in adult liver mathematical model (e.g., Hedgehog and Wnt/β-Catenin pathways).These signaling pathways were lagging yet had similarities with those that occur in embryogenesis.Matz-Soja (2016) 15  B3, A4, and A5] that were expressed before and after hepatectomy.The focus of the majority of previous studies was liver regeneration, but the study of Homeobox containing 1 demonstrated the importance of organogenesis factors in halting tumors; in addition, some of hox genes are associated with regeneration; however, their role in organogenesis needs to be determined.Furthermore, there is lack of studies that compare genes and their expression of organogenesis in adults versus embryos.

VI. Three-Dimensional Modeling of Adult Liver Organogenesis
Multiple studies were conducted to investigate three-dimensional cultures of the liver.A study found that hepatocytes' size increases dramatically 18 hours after hepatectomy; it was followed by a noticeable increment in volume which happens 24 hours after partial hepatectomy (Vizzotto et al.,1989)  22 .Hepatocyte stellate cells play a mediator role in communication between hepatocytes and endothelial cells in terms of endothelial cells morphogenesis (Kasuya et al., 2011) 23 .
Parsons-Wingerter & Saltzman (1993) 24 reported that aggregate cultures retained two folds of functional parameter compared to singlet cultures; in addition, functional recovery starts when growth is completed.However, the abovementioned studies did not indicate how the volume cells orient, regionalize, or take specific shape.Three-dimensional organotypic liver culture was used to understand liver sinusoidal endothelial cells' (LSECs) and Kupfer cells' (KCs) behavior in three- however, specific mechanisms and pathways of organogenesis in this study is still unraveled and needs to be determined.

VII. Three or Six Phases and Two Processes
Most of previous research conducted on liver regeneration concentrated on the tissue's competency to proliferate and the proliferation process.Three distinct and sequential stages were A networks triad was suggested to explain the mechanism of liver regeneration: cytokine, growth factor, and metabolic (Fausto et al., 2006)  27 .There was no indication on how are these networks are correlated with organogenesis.Furchtgott et al. ( 2009) 28 proposed a model of liver regeneration based on interaction of multiple factors, the model divided regeneration process into three phases: 1) quiescent; 2) primed; and 3) replicating.This model lacks a termination phase as a separate phase, and the model did not discuss organogenesis in liver regeneration.
There was a difference in the staging timelines and phase counts between liver regeneration models in the previously mentioned studies mainly because the phases were established according to the timing of the samples' retrieval, and these models did not reflect the actual timeline of  5 .These studies demonstrated that some areas and regions in the liver are controlled by different factors during organogenesis; which makes the process more unique.
The paper proposes a theme (Figure 1) in which organogenesis process would be identified as independent, yet parallel and complementary process to proliferation in all phases of liver regeneration, this theme will help identify factors associated with each process.In this theme, organogenesis starts at prime stage, and continue to direct the expansion of three-dimensional growth and ends the process in termination stage.Factors that affect organogenesis are different from those associated with proliferation because of specificity of these factors to three-dimensional spatial control other than random mass expansion (mass can be achieved by proliferation alone).

VIII. Discussion
Organogenesis is a distinct process that encompasses multiple factors that need to be studied during proliferation.Signaling pathway, cell-cell interactions, extracellular matrix and multiple factors are associated and required to achieve the organogenesis process.This paper discussed a theme to recognize organogenesis in adult's liver as separate, yet parallel and complementary process to proliferation in order to achieve liver regeneration.The theme will help explain organogenesis's factors rather than proliferation alone in regeneration.And (c) Time recording is important to identify factors in each phase; however, causality of factors in their phase is more important.Further studies are warranted to determine each factor and its relationship to its pathway.
not stated Tumor Necrosis Factor-alpha (TNF-α) and Inteleukin-6 (IL-6), Transforming Growth Factor Beta (TGF-β) and related TGFβ family members (Tao et al., 2017) 53 TNF-α and IL-6 act as complete mitogen, (TGF-β) and related TGF-β family members associated with termination of liver regeneration not stated Studies investigated adult liver organogenesis, and factors associated with it were scarce (Lokmane et al., 2008 4 ; Wang et al., 2015 5 ).Signaling and genes expression that control threedimensional modeling of liver are important for many reasons, including the identification of termination factors that may help in controlling both proliferation and organogenesis in tumor tissue, and the identification of signaling factors that may help in initiating organogenesis of regenerative tissue for organ donation.Researchers also proposed phases to describe these processes (Xu et al., 2005 6 ; Zimmermann, 2002 7 described during liver regeneration: (1) initiation stage involves replicating competency;(2)   proliferation stage includes the expansion of cells population; and (3) termination stage: here the cellular growth is halted to terminate regeneration at a set point; however, organogenesis is still vague process(Zimmermann, 2002) 7 .In their review,Pahlavan et al. (2006) 25 used staging to discuss factors associated with regeneration; the team also stated the process is ambiguous, but they predicted the presence of inhibitors to control the size of regeneration.A three-stages model was diagramed with factors and pathways associated with these factors in the regeneration (e.g., TNFalpha and IL6 activate TNFR, STAT3, NF-kB and AP1 in priming [initiation] stage) (Pagano et al., 2012) 26 .Xu et al. (2005) 6 recognized six temporal phases based on gene expression: 1) immediate early; 2) early; 3) intermediate; 4) early-late; 5) late; and 6) terminal.
Based on retrieved studies, four important observations were concluded after review stratification: (a) Organogenesis is an overlooked process in adult liver regeneration.(b) The factors associated with organogenesis lack causality relationship and mechanisms.(c) Multiple factors are probably associated with organogenesis; most are unknown.(d) Structural architecture and heterogeneity of cells and tissues (including neovascularization and sinusoidal endothelial Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 16 January 2019 Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 16 January 2019 doi:10.20944/preprints201809.0351.v3integration) to achieve final liver function are controlled by organogenesis.Further studies are warranted to determine each factor and its relationship to its pathway.Three conclusions were reached: (a) Phases of liver regeneration fall under two parallel processes: organogenesis and cell proliferation.(b) Current regeneration staging classification depends on timing of tissue retrieval from experimental groups for testing and does not reflect actual system biology of liver regeneration.

Table 1 .
Summary of Phosphorylation Processes in Liver Regeneration and Organogenesis

Table 2 .
Hormonal Effect on Liver Regeneration and Organogenesis

Table 3 .
Factors Associated with Liver Regeneration and Organogenesis

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5ang et al. (2015)5observed that the absence of FGF signaling associated with halting of only anterior murine liver bud; FGF signaling was specific for anterior hepatic bud and it has no effect on the posterior portion.The posterior 4epatic bud might be controlled by another signaling pathway.Lokmane et al. (2008)4investigated hepatocyte nuclear factor (vHnf1) gene and its effect on regeneration process and concluded that a Preprints (www.

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19 the parenchyma.β-Catenin is important in the expression of enzymes that metabolize drugs and in localizing liver reactions to xenobiotic inducers(Godoy et al., 2013)18.Aging is associated with a decrease in liver ability to regenerate and an increase in transcription of inflammatory and immune response; this aging effect on liver regeneration remains ambiguous, but it might be contributed to a depressed level of extracellular factors and hormones(Pibiri et al., 2015)19.
18uld affect organogenesis.Godoy et al. (2013)18reviewed morphogens-particularly Wnts (p.1327) and their team identified canonical Wnts' signaling pathway as a major determinant in Preprints (www.preprints.org)| NOT PEER-REVIEWED | and a tumor suppressor to hepatocyte growth.Mizuta et al. (1996) 21 used competitive reversetranscriptase polymerase chain reaction to clone and sequence homebox genes at different phases after partial hepatectomy of rat liver, high levels of caudal related gene (RCdx-3) at one hour, while low levels of hox gene (RHoxB5) was noticed; however, other identified hox genes [RHoxA1, B2,

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40ents.Another factor was the technology that was used in analyzing the data and the kind of data were analyzed.However, these staging models will aid in the classification and assigning of different factors to their temporal window in future studies.Sugimoto et al., 2006)29, BMP-7 included in table3due to lack of consensus on the description of its role -whether or not it is a hormone-.Adult hepatocytes play a major role in organogenesis (Utoh et al., 2016)30.Lokmane et al. (2008)4reported that the lack of vHNF1 leads to formation of a defective hepatic bud and an abnormal gut regionalization.Organogenesis requires Fibroblast Growth Factor (FGF) signaling for anterior but not posterior specification of the murine liver bud(Wang et al., 2015)