Organ Size Determination Research Articles

Transcriptional and phytohormone regulatory network involved in LITTLELEAF-mediated organ size development in cucumber (Cucumis sativus)

Organ size, especially fruit size, is an economically important trait in many horticulture crops like cucumber (Cucumis sativus L.) which affects both fruit yield and quality. Organ size determination is the outcome of complex interplay of many factors such as transcription regulators, phytohormone and the environments. The cucumber littleleaf (ll) mutation exhibits small organ size, which encodes a defective WD40 repeat domain-containing protein, but the regulatory mechanisms of LL-dependent organ size control are largely unknown. To test the functions of the ll gene in different genetic backgrounds and explore its regulatory network, we developed near isogenic lines (NIL) of ll gene by introgressing the recessive allele into a Chinese Long inbred line 9930 with marker assisted selection (MAS). Both foreground and background selections were practiced which allowed development of a NIL (HAULL) at BC2F2 which were homologous at the ll locus and captured >96% genetic background of the recurrent parent. Comparative morphological analysis between the NILs indicated that the mutant allele for reduced organ size was independent of the genetic background. Transcriptome profiling using RNA-Seq in the unpollinated ovaries of the two NILs identified differentially expressed genes which were highly enriched with genes known to regulate organ size, as well as transcription factors, and genes in various phytohormone biosynthesis/signaling pathways. RNA-Seq and phytohormone assays found that auxin and cytokinin played more important roles in LL-regulated fruit growth. These results will be helpful for further explore the molecular mechanism of organ size in cucumber.

Bioelectricity in Developmental Patterning and Size Control: Evidence and Genetically Encoded Tools in the Zebrafish Model.

Developmental patterning is essential for regulating cellular events such as axial patterning, segmentation, tissue formation, and organ size determination during embryogenesis. Understanding the patterning mechanisms remains a central challenge and fundamental interest in developmental biology. Ion-channel-regulated bioelectric signals have emerged as a player of the patterning mechanism, which may interact with morphogens. Evidence from multiple model organisms reveals the roles of bioelectricity in embryonic development, regeneration, and cancers. The Zebrafish model is the second most used vertebrate model, next to the mouse model. The zebrafish model has great potential for elucidating the functions of bioelectricity due to many advantages such as external development, transparent early embryogenesis, and tractable genetics. Here, we review genetic evidence from zebrafish mutants with fin-size and pigment changes related to ion channels and bioelectricity. In addition, we review the cell membrane voltage reporting and chemogenetic tools that have already been used or have great potential to be implemented in zebrafish models. Finally, new perspectives and opportunities for bioelectricity research with zebrafish are discussed.

Abstract 1474: Hippo-mediated control of the mutant TERT promoter via MYC and GABP

Abstract Most cancers achieve replicative immortality through reactivation of telomerase reverse transcriptase (TERT), the rate limiting subunit in telomerase activity. Tumors from more than 50 distinct cancer types reactivate TERT expression via mutations in the TERT promoter. Understanding how the mutations lead to activation and immortality is a central question in cancer research. Oncogenic signaling pathways including EGFR/AMPK, MAPK, and C-MYC/ASL, have been linked to telomere maintenance via their regulation of the mutant TERT promoter. Two mutually exclusive point mutations in the TERT promoter generate a de novo E26 Transformation Specific (ETS) binding motif that recruits the GA-Binding Protein (GABP) multimeric complex to reactivate TERT expression (Bell et al, Science 2015). Recruitment of GABP to the mutant TERT promoter facilitates the recruitment of C-MYC to the promoter (Shi et al, Mol. Cell 2022), which aids in transcriptional activation. The Hippo pathway plays a key role in development, organ size determination, and homeostasis, and has been found dysregulated in numerous cancer types. GABP is a member of the Hippo pathway, with GABP activating the promoter of the Hippo transcriptional effector Yes-associated protein (YAP1) and the Hippo pathway core kinases stimulating GABP nuclear exit (Wu et al., Cell Reports 2013). We have found that YAP and its paralogue transcriptional coactivator with PDZ-binding motif (TAZ) are linked to mutant TERT promoter activation in glioblastoma via transcriptional activation of both GABPA and MYC. These observations suggest the Hippo pathway supports tumor immortality in glioblastoma. While immortality has proven to be an elusive target, further investigations of the signaling networks that converge on the activation of the mutant TERT promoter may reveal therapeutic targets that block tumor growth and have secondary benefit by their impact on tumor immortality. Citation Format: Nicholas O. Stevers, Andrew Mancini, Joseph F. Costello. Hippo-mediated control of the mutant TERT promoter via MYC and GABP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1474.

CHIQUITA1 maintains the temporal transition between proliferation and differentiation in Arabidopsis thaliana.

Body size varies widely among species, populations and individuals, depending on the environment. Transitioning between proliferation and differentiation is a crucial determinant of final organ size, but how the timing of this transition is established and maintained remains unknown. Using cell proliferation markers and genetic analysis, we show that CHIQUITA1 (CHIQ1) is required to maintain the timing of the transition from proliferation to differentiation in Arabidopsis thaliana. Combining kinematic and cell lineage-tracking studies, we found that the number of actively dividing cells in chiquita1-1 plants decreases prematurely compared with wild-type plants, suggesting CHIQ1 maintains the proliferative capacity in dividing cells and ensures that cells divide a specific number of times. CHIQ1 belongs to a plant-specific gene family of unknown molecular function and genetically interacts with three close members of its family to control the timing of proliferation exit. Our work reveals the interdependency between cellular and organ-level processes underlying final organ size determination.

The Hippo Pathway Effectors YAP and TAZ Regulate LH Release by Pituitary Gonadotrope Cells in Mice.

The Hippo transcriptional coactivators YAP and TAZ exert critical roles in morphogenesis, organ size determination and tumorigenesis in many tissues. Although Hippo kinase cascade activity was recently reported in the anterior pituitary gland in mice, the role of the Hippo effectors in regulating gonadotropin production remains unknown. The objective of this study was therefore to characterize the roles of YAP and TAZ in gonadotropin synthesis and secretion. Using a conditional gene targeting approach (cKO), we found that gonadotrope-specific inactivation of Yap and Taz resulted in increased circulating levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in adult male mice, along with increased testosterone levels and testis weight. Female cKO mice had increased circulating LH (but not FSH) levels, which were associated with a hyperfertility phenotype characterized by higher ovulation rates and larger litter sizes. Unexpectedly, the loss of YAP/TAZ did not appear to affect the expression of gonadotropin subunit genes, yet both basal and GnRH-induced LH secretion were increased in cultured pituitary cells from cKO mice. Likewise, pharmacologic inhibition of YAP binding to the TEAD family of transcription factors increased both basal and GnRH-induced LH secretion in LβT2 gonadotrope-like cells in vitro without affecting Lhb expression. Conversely, mRNA levels of ChgA and SgII, which encode key secretory granule cargo proteins, were decreased following pharmacologic inhibition of YAP/TAZ, suggesting a mechanism whereby YAP/TAZ regulate the LH secretion machinery in gonadotrope cells. Together, these findings represent the first evidence that Hippo signaling may play a role in regulating pituitary LH secretion.

The Leptinotarsa forkhead transcription factor O exerts a key function during larval-pupal-adult transition

Forkhead box O (FoxO) protein, a major downstream transcription factor of insulin/insulin-like growth factor signaling/target of rapamycin pathway (IIS/TOR), is involved in the regulation of larval growth and the determination of organ size. FoxO also interacts with 20-hydroxyecdysone (20E) and juvenile hormone (JH) signal transduction pathways, and hence is critical for larval development in holometabolans. However, whether FoxO plays a critical role during larval metamorphosis needs to be further determined in Leptinotarsa decemlineata. We found that 20E stimulated the expression of LdFoxO. RNA interference (RNAi)-aided knockdown of LdFoxO at the third-instar stage repressed 20E signaling and reduced larval weight. Although the resultant larvae survived through the third-fourth instar ecdysis, around 70% of the LdFoxO depleted moribund beetles developmentally arrested at prepupae stage. These LdFoxO depleted beetles were completely wrapped in the larval exuviae, gradually darkened and finally died. Moreover, approximately 12% of the LdFoxO RNAi beetles died as pharate adults. Ingestion of either 20E or JH by the LdFoxO depletion beetles excessively rescued the corresponding hormonal signals, but could not alleviate larval performance and restore defective phenotypes. Therefore, FoxO plays an important role in regulation of larval-pupal-adult transformation in L. decemlineata, in addition to mediation of IIS/TOR pathway and stimulation of ecdysteroidogenesis.

Determination of organ size: a need to focus on growth rate, not size.

The regulation of growth and the determination of organ-size in animals is an area of research that has received much attention during the past two and a half decades. Classic regeneration and cell-competition studies performed during the last century suggested that for size to be determined, organ-size is sensed and this sense of size feeds back into the growth control mechanism such that growth stops at the "correct" size. Recent work using Drosophila imaginal discs as a system has provided a particularly detailed cellular and molecular understanding of growth. Yet, a clear mechanistic basis for size-sensing has not emerged. I re-examine these studies from a different perspective and ask whether there is scope for alternate modes of size control in which size does not need to be sensed.

Abstract 4472: The hippo pathway effector TAZ regulates ferroptosis in renal cell carcinoma

Abstract Ferroptosis is a newly appreciated lipid peroxidation-dependent regulated cell death with relevance for many human diseases, which may be a tumor suppression mechanism and may have antitumor potential. Elucidation of the mechanism is therefore important. Here, we reported that ferroptosis sensitivity is significantly influenced by cell density. The effectors of the Hippo pathway, YAP/TAZ, are the molecular sensors of cell density that regulate cell proliferation, survival, and organ size determination. Therefore, we determined the potential role of YAP/TAZ in regulating ferroptosis sensitivities. RCC cell lines and RCC PDX tumor cells mainly express TAZ that undergoes density-dependent nuclear translocation. Importantly, the removal of TAZ confers ferroptosis resistance, while the expression of constructively active TAZ sensitizes cells to ferroptosis. Mechanistically, we found that TAZ knockdown reduced ferroptosis through the repression of its target gene, EMP1. EMP1 affects ferroptosis through the regulation of NOX4 and lipid peroxidation. Collectively, we have shown that the ferroptosis can be regulated by non-genetic factors, such as cell density, through the Hippo pathway and suggest the therapeutic potential of ferroptosis-inducing agent, such as erastin, for TAZ-activated tumors. Citation Format: Wen-Hsuan Yang, Jen-Tsan Chi. The hippo pathway effector TAZ regulates ferroptosis in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4472.

Hippo Signaling in the Liver - A Long and Ever-Expanding Story.

The first description of Hippo signaling in mammals a little more than 10 years ago showed a striking phenotype in the liver, linking the role of this signaling pathway to organ size control and carcinogenesis. Even though Hippo signaling has been extensively studied in the liver and other organs over the recent years, many open questions remain in our understanding of its role in hepatic physiology and disease. The functions of Hippo signaling extend well beyond cancer and organ size determination: components of upstream Hippo signaling and the downstream effectors YAP and TAZ are involved in a multitude of cell and non-cell autonomous functions including cell proliferation, survival, development, differentiation, metabolism, and cross-talk with the immune system. Moreover, regulation and biological functions of Hippo signaling are often organ or even cell type specific – making its role even more complex. Here, we give a concise overview of the role of Hippo signaling in the liver with a focus on cell-type specific functions. We outline open questions and future research directions that will help to improve our understanding of this important pathway in liver disease.

Revising the PLAC8 gene family: from a central role in differentiation, proliferation, and apoptosis in mammals to a multifunctional role in plants.

PLAC8 is a cysteine-rich protein described as a central mediator of tumor evolution in mammals; as such, it represents a promising candidate for diagnostic and therapeutic targeting. The human PLAC8 gene is also involved in contact hypersensitivity response and presents a role in psoriatic skin. In plants, PLAC8 motif-containing proteins are involved in the determination of organ size and growth, response to infection, Ca2+ influx, Cd resistance, and zinc detoxification. In general, PLAC8 motif-containing proteins present the conserved CCXXXXCPC or CLXXXXCPC region. However, there is no devised nomenclature for the PLAC8 motif-containing proteins. Here, through the analysis of 445 sequences, we show that PLAC8 motif-containing proteins constitute a unique gene family, and we propose a unified nomenclature. This is the first report indicating the existence of different groups of PLAC8 proteins, which we have called types I, II, and III. Type I genes are found in mammals, fungi, plants, and algae, and types II and III are exclusive to plants. Our study describes for the first time PLAC8 type III proteins. Whether these sequences maintain their known functional role or possess distinct functions of types I and II genes remains unclear.

The plant LIM proteins: unlocking the hidden attractions.

The plant LIMs comprise two sub-families with one (DA1/DAR) and two (2LIM) LIM domains. This review comprehensively discussed the structure and potential role of this protein family in diverse area of plant biology. The description of first eukaryote lineage-specific plant LIM domain (LIN11, ISL1, and MEC3) proteins was observed in Helianthus long back. The successive study of LIM proteins in diverse plants has shown its vital relation to development, metabolism and defence. This nascent gene family has been worked out for their role in actin dynamics, organ size determination and transcription regulation. On grounds of protein architecture, two sub-families have been delineated as DA1/DAR (one LIM domain) and 2LIMs (two LIM domains). The genomic and expression study guides to the identification of diverse sub-categories. The significance of 2LIMs in regulation of actin dynamics leading to pollen growth and development has prospects to understand the plant reproductive behaviour. Interestingly, new facet of these LIMs as a transcriptional regulator in biological pathway/biosynthesis was also reported. Recently, the cumulative contribution of these features was also recognized for obtaining good quality fibre, thus giving translational outlook to this family. The DA1/DAR proteins are orchestrated with additional domains and provide a key role in regulation of organ size and tolerance to biotic and abiotic stress. This review will focus the journey of plant LIMs till date and will cover details of its structure, type, classification and functional relevance. This will provide insight to identify the potential of this gene family in the improvement of desired crop features.

Drosophila eye size is determined by Innexin 2-dependent Decapentaplegic signalling

Organogenesis relies on specific genetic and molecular programmes, which orchestrate growth and cellular differentiation over developmental time. This is particularly important during Drosophila eye development in which cell–cell inductive events and long-range signalling have to be integrated to regulate proper cell proliferation, differentiation and morphogenesis. How these processes are coordinated is still not very well understood. Here we identify the gap junction protein Innexin2 (Inx2) as an important regulator of eye development. Depleting inx2 during eye development reduces eye size whereas elevating inx2 levels increases eye size. Loss- and gain-of-function experiments demonstrate that inx2 is required functionally in larval eye disc cells where it localises apico-laterally. inx2 regulates disc cell proliferation as well as morphogenetic furrow movement and as a result the amount of differentiated photoreceptors. inx2 interacts genetically with the Dpp pathway and we find that proper activation of the Dpp pathway transducer Mad at the furrow and expression of Dpp receptors Thickveins and Punt in the anterior disc compartment require inx2. We further show that inx2 is required for the transcriptional activation of dpp and punt in the eye disc. Our results highlight the crucial role of gap junction proteins in regulating morphogen-dependent organ size determination.

A miR-130a-YAP positive feedback loop promotes organ size and tumorigenesis.

Organ size determination is one of the most intriguing unsolved mysteries in biology. Aberrant activation of the major effector and transcription co-activator YAP in the Hippo pathway causes drastic organ enlargement in development and underlies tumorigenesis in many human cancers. However, how robust YAP activation is achieved during organ size control remains elusive. Here we report that the YAP signaling is sustained through a novel microRNA-dependent positive feedback loop. miR-130a, which is directly induced by YAP, could effectively repress VGLL4, an inhibitor of YAP activity, thereby amplifying the YAP signals. Inhibition of miR-130a reversed liver size enlargement induced by Hippo pathway inactivation and blocked YAP-induced tumorigenesis. Furthermore, the Drosophila Hippo pathway target bantam functionally mimics miR-130a by repressing the VGLL4 homolog SdBP/Tgi. These findings reveal an evolutionarily conserved positive feedback mechanism underlying robustness of the Hippo pathway in size control and tumorigenesis.

YAP immunoreactivity is directly related to pilomatrixoma size and proliferation rate.

Organ size regulation is a highly coordinated process involving complex mechanisms. Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif, also known as WWTR1 (TAZ) have recently been linked to organ size determination and cell proliferation. Pilomatrixoma (PM) is a benign tumor of the adnexal appendages with a certain degree of differentiation toward the matrix of the hair follicle. PM presents as a dermal nodule that usually ranges from 0.5 to 2 cm, rarely exceeding 3 cm. We recently observed a case of unusual "giant" (6.5 cm) PM. Our hypothesis was that YAP and TAZ could be related to PM growth. We analyzed YAP and TAZ immunohistochemical expression in the giant and in ten usual size PMs in relation with tumor size and proliferation rate. YAP nuclear expression was remarkably higher in the giant PM in comparison with usual size PMs and statistically correlated, in a direct manner, with size and proliferation rate of PMs. Contrariwise, TAZ nuclear expression seemed stochastic. Our findings suggest that YAP could play a role in PM growth.

Physalis floridana Cell Number Regulator1 encodes a cell membrane-anchored modulator of cell cycle and negatively controls fruit size.

Physalis species show a significant variation in berry size; however, the underlying molecular basis is unknown. In this work, we showed that cell division difference in the ovaries might contribute to the ultimate berry size variation within Physalis species, and that mRNA abundance of Physalis floridana Cell Number Regulator1 (PfCNR1), the putative orthologue of the tomato fruit weight 2.2 (FW2.2), was negatively correlated with cell division in the ovaries. Moreover, heterochronic expression variation of the PfCNR1 genes in the ovaries concomitantly correlated with berry weight variation within Physalis species. In transgenic Physalis, multiple organ sizes could be negatively controlled by altering PfCNR1 levels, and cell division instead of cell expansion was primarily affected. PfCNR1 was shown to be anchored in the plasma membrane and to interact with PfAG2 (an AGAMOUS-like protein determining ovary identity). The expression of PfCYCD2;1, a putative orthologue of the mitosis-specific gene CyclinD2;1 in the cell cycle was negatively correlated with the PfCNR1 mRNA levels. PfAG2 was found to selectively bind to the CArG-box in the PfCYCD2;1 promoter and to repress PfCYCD2;1 expression, thus suggesting a PfAG2-mediated pathway for PfCNR1 to regulate cell division. The interaction of PfCNR1 with PfAG2 enhanced the repression of PfCYCD2;1 expression. The nuclear import of PfAG2 was essential in the proposed pathway. Our data provide new insights into the developmental pathways of a cell membrane-anchored protein that modulates cell division and governs organ size determination. This study also sheds light on the link between organ identity and organ growth in plants.

Cell competition in vertebrate organ size regulation.

The study of animal organ size determination has provided evidence of the existence of organ-intrinsic mechanisms that 'sense' and adjust organ growth. Cell competition, a form of cell interaction that equalizes cell population growth, has been proposed to play a role in organ size regulation. Cell competition involves a cell-context dependent response triggered by perceived differences in cell growth and/or proliferation rates, resulting in apoptosis in growth-disadvantaged cells and compensatory expansion of the more 'fit' cells. The mechanisms that allow cells to compare growth are not yet understood, but a number of genes and pathways have been implicated in cell competition. These include Myc, the members of the Hippo, JAK/STAT and WNT signaling pathways, and the Dlg/Lgl/Scrib and the Crb/Std/PatJ membrane protein complexes. Cell competition was initially characterized in the Drosophila imaginal disc, but several recent studies have shown that cell competition occurs in mouse embryonic stem cells and in the embryonic epiblast, where it plays a role in the regulation of early embryo size. In addition, competition-like behavior has been described in the adult mouse liver and the hematopoietic stem cell compartment. These data indicate that cell competition plays a more universal role in organ size regulation. In addition, as some authors have suggested that similar types of competitive behavior may operate in during tumorigenesis, there may be additional practical reasons for understanding this fundamental process of intercellular communication.

An extended steepness model for leg-size determination based on Dachsous/Fat trans-dimer system

What determines organ size has been a long-standing biological question. Lawrence et al. (2008) proposed the steepness hypothesis suggesting that the protocadherin Dachsous/Fat (Ds/Ft) system may provide some measure of dimension to the cells in relation to the gradient. In this paper we extended the model as a means of interpreting experimental results in cricket leg regeneration. We assumed that (1) Ds/Ft trans-heterodimers or trans-homodimers are redistributed during cell division, and (2) growth would cease when a differential of the dimer across each cell decreases to a certain threshold. We applied our model to simulate the results obtained by leg regeneration experiments in a cricket model. The results were qualitatively consistent with the experimental data obtained for cricket legs by RNA interference methodology. Using our extended steepness model, we provided a molecular-based explanation for leg size determination even in intercalary regeneration and for organ size determination.

Dual function of Yap in the regulation of lens progenitor cells and cellular polarity

Hippo-Yap signaling has been implicated in organ size determination via its regulation of cell proliferation, growth and apoptosis (Pan, 2007). The vertebrate lens comprises only two major cell types, lens progenitors and differentiated fiber cells, thereby providing a relatively simple system for studying size-controlling mechanisms. In order to investigate the role of Hippo-Yap signaling in lens size regulation, we conditionally ablated Yap in the developing mouse lens. Lens progenitor-specific deletion of Yap led to near obliteration of the lens primarily due to hypocellularity in the lens epithelium (LE) and accompanying lens fiber (LF) defects. A significantly reduced LE progenitor pool resulted mainly from failed self-renewal and increased apoptosis. Additionally, Yap-deficient lens progenitor cells precociously exited the cell cycle and expressed the LF marker, β-Crystallin. The mutant progenitor cells also exhibited multiple cellular and subcellular alterations including cell and nuclear shape change, organellar polarity disruption, and disorganized apical polarity complex and junction proteins such as Crumbs, Pals1, Par3 and ZO-1. Yap-deficient LF cells failed to anchor to the overlying LE layer, impairing their normal elongation and packaging. Furthermore, our localization study results suggest that, in the developing LE, Yap participates in the cell context-dependent transition from the proliferative to differentiation-competent state by integrating cell density information. Taken together, our results shed new light on Yap's indispensable and novel organizing role in mammalian organ size control by coordinating multiple events including cell proliferation, differentiation, and polarity.

NEDD4 E3 ligase inhibits the activity of the Hippo pathway by targeting LATS1 for degradation

Proper regulation of cell proliferation, cell apoptosis, and cell death are vital for the development and survival of living organisms. Failure or dysfunction of any of these processes can have devastating effects, including cancer. The Hippo pathway, first discovered in Drosophila, has been found to be a major growth-regulatory signaling pathway that controls these crucial processes and has been implicated in cell-progress regulation and organ size determination. Abnormal regulation of this pathway has been found in several cancer types. However, the mechanisms that regulate the pathway and its core members yet have to be elucidated. One of the main core components of this pathway is LATS1, a serine/threonine kinase. Therefore, understanding how LATS1 activity is regulated is expected to shed light on new mechanisms that regulate the Hippo pathway. In the current work, we identified several potential LATS1 regulators and proved that NEDD4 E3 ubiquitin ligase controls LATS1 stability. We demonstrate that NEDD4 directly interacts with LATS1, leading to ubiquitination and decreased levels of LATS1 and, thus, increased YAP localization in the nucleus, which subsequently increases the transcriptional activity of YAP. As such, we show that NEDD4 acts as an additional regulator of the Hippo pathway on the protein level via interactions between WW domain-containing and PPxY motif-containing proteins. These findings might be applied in the development of new therapeutic approaches through the activation of LATS1.

Rho signalling restriction by the RhoGAP Stard13 integrates growth and morphogenesis in the pancreas

The development of functional organ architecture relies on coordinated morphogenesis and growth. In the developing pancreas, the branching epithelium is organised in discrete domains, delineating one specific domain of progenitor cells at the tip of the branches. The molecular mechanisms underlying the coordinated action of branching and proliferation in organ formation are largely unknown. Here, we identify the RhoGAP protein Stard13 as an essential regulator of pancreas tissue architecture in the mammalian embryo. Conditional ablation of Stard13 expression in the pancreas disrupts epithelial morphogenesis and tip-domain organisation, resulting in hampered proliferation of tip progenitors and subsequent organ hypoplasia. Stard13 acts by regulating Rho signalling spatially and temporally during pancreas development. Our findings provide new insights into the mechanisms that shape pancreatic epithelium to create a mature organ and establish a functional link between Rho-mediated control of epithelial remodelling and organ size determination, involving reciprocal interaction of actin-MAL/SRF and MAPK signalling pathways.