Parallels between oncogene-driven cardiac hyperplasia and heart regeneration in zebrafish.

The human heart is poorly regenerative and cardiac tumors are extremely rare. Whether the adult zebrafish myocardium is responsive to oncogene overexpression and how this condition affects its intrinsic regenerative capacity remains unknown. Here, we have established a strategy of inducible and reversible expression of HRASG12V in zebrafish cardiomyocytes. This approach stimulated a hyperplastic cardiac enlargement within 16 days. The phenotype was suppressed by rapamycin-mediated inhibition of TOR signaling. As TOR signaling is also required for heart restoration after cryoinjury, we compared transcriptomes of hyperplastic and regenerating ventricles. Both conditions were associated with upregulation of cardiomyocyte dedifferentiation and proliferation factors, as well as with similar microenvironmental responses, such as deposition of nonfibrillar Collagen XII and recruitment of immune cells. Among the differentially expressed genes, many proteasome and cell-cycle regulators were upregulated only in oncogene-expressing hearts. Preconditioning of the heart with short-term oncogene expression accelerated cardiac regeneration after cryoinjury, revealing a beneficial synergism between both programs. Identification of the molecular bases underlying the interplay between detrimental hyperplasia and advantageous regeneration provides new insights into cardiac plasticity in adult zebrafish.

Platyfish bypass the constraint of the caudal fin ventral identity in teleosts.

BACKGROUND: The caudal fin of teleosts is characterized by dorsoventral symmetry. Despite this external morphology, the principal rays of this appendage connect to bones below the notochord, indicating the ventral (hypochordal) identity of this organ. RESULTS: Here, we report that this typical architecture of the caudal fin is not fully conserved in the platyfish (Xiphophorus maculatus) and the guppy (Poecilia reticulata), representatives of the Poeciliidae family. We show that in these species, 3-4 principal rays connect to bones above the notochord, suggesting an epichordal contribution. Consistently, as examined in platyfish, dorsal identity genes zic1/4 were highly expressed in these rays, providing molecular evidence of their epichordal origin. Developmental analysis revealed that the earliest rays above the notochord emerge at the 10-ray stage of fin morphogenesis. In contrast to zebrafish and medaka, platyfish and guppies display a mirrored shape of dorsal and ventral processes of the caudal endoskeleton. Our study suggests that an ancestral bauplan expanded in poeciliids by advancing its symmetrical pattern. CONCLUSION: The platyfish evolved a fin architecture with the epichordal origin of its upper principal rays and a high level of symmetry in the caudal endoskeleton. This innovative architecture highlights the adaptation of the teleost skeleton.

Hydrodynamic stress and phenotypic plasticity of the zebrafish regenerating fin.

Understanding how extrinsic factors modulate genetically encoded information to produce a specific phenotype is of prime scientific interest. In particular, the feedback mechanism between abiotic forces and locomotory organs during morphogenesis to achieve efficient movement is a highly relevant example of such modulation. The study of this developmental process can provide unique insights on the transduction of cues at the interface between physics and biology. Here, we take advantage of the natural ability of adult zebrafish to regenerate their amputated fins to assess its morphogenic plasticity upon external modulations. Using a variety of surgical and chemical treatments, we could induce phenotypic responses to the structure of the fin. Through the ablation of specific rays in regenerating caudal fins, we generated artificially narrowed appendages in which the fin cleft depth and the positioning of rays bifurcations were perturbed compared with normal regenerates. To dissect the role of mechanotransduction in this process, we investigated the patterns of hydrodynamic forces acting on the surface of a zebrafish fin during regeneration by using particle tracking velocimetry on a range of biomimetic hydrofoils. This experimental approach enabled us to quantitatively compare hydrodynamic stress distributions over flapping fins of varying sizes and shapes. As a result, viscous shear stress acting on the distal margin of regenerating fins and the resulting internal tension are proposed as suitable signals for guiding the regulation of ray growth dynamics and branching pattern. Our findings suggest that mechanical forces are involved in the fine-tuning of the locomotory organ during fin morphogenesis.

Dynamics of actinotrichia regeneration in the adult zebrafish fin.

The skeleton of adult zebrafish fins comprises lepidotrichia, which are dermal bones of the rays, and actinotrichia, which are non-mineralized spicules at the distal margin of the appendage. Little is known about the regenerative dynamics of the actinotrichia-specific structural proteins called Actinodins. Here, we used immunofluorescence analysis to determine the contribution of two paralogous Actinodin proteins, And1/2, in regenerating fins. Both proteins were detected in the secretory organelles in the mesenchymal cells of the blastema, but only And1 was detected in the epithelial cells of the wound epithelium. The analysis of whole mount fins throughout the entire regenerative process and longitudinal sections revealed that And1-positive fibers are complementary to the lepidotrichia. The analysis of another longfin fish, a gain-of-function mutation in the potassium channel kcnk5b, revealed that the long-fin phenotype is associated with an extended size of actinotrichia during homeostasis and regeneration. Finally, we investigated the role of several signaling pathways in actinotrichia formation and maintenance. This revealed that the pulse-inhibition of either TGFß/Activin-ßA or FGF are sufficient to impair deposition of Actinodin during regeneration. Thus, the dynamic turnover of Actinodin during fin regeneration is regulated by multiple factors, including the osteoblasts, growth rate in a potassium channel mutant, and instructive signaling networks between the epithelium and the blastema of the regenerating fin.

Zebrafish fin regeneration involves transient serotonin synthesis.

The zebrafish is a vertebrate organism capable of regenerating many of its organs. Notably, it can undergo epimorphic regeneration of its fins after amputation. This process occurs through the formation of a wound epithelium and the dedifferentiation of mesenchymal and bone-forming cells, which form a proliferative blastema. Here, we report that the entry into the regenerative process involves the local synthesis of serotonin (5-hydroxytryptamine, 5-HT) in the injury-associated tissue. One day after wounding, intracellular accumulation of serotonin was induced in the stump below the amputation plane. During blastema formation, serotonin was detected in the mesenchyme at the vicinity of the amputation plane and in the apical wound epithelium. During the advanced outgrowth phase, this monoamine was no longer present in the blastema, suggesting a temporal involvement of serotonin in the postinjury area. We show the expression of two serotonin synthesizing enzymes, tryptophan hydroxylase 1a and 1b in the blastema, suggesting the local production of this monoamine. Neither depletion of serotonin by chemical inhibition of tryptophan hydroxylase, nor ectopic administration of this monoamine affected fin regeneration, indicating it does not play a role during this process. Finally, we found that the presence of serotonin during regeneration depends on fibroblast growth factor and retinoic acid signaling. Overall, our study demonstrates that the initiation of fin regeneration is associated with a transient synthesis of serotonin in the regrowing tissue.

In vivo quantification of mechanical properties of caudal fins in adult zebrafish.

The caudal fins of adult zebrafish are supported by multiple bony rays that are laterally interconnected by soft interray tissue. Little is known about the fin's mechanical properties that influence bending in response to hydrodynamic forces during swimming. Here, we developed an experimental setup to measure the elastic properties of caudal fins in vivo by applying micro-Newton forces to obtain bending stiffness and a tensional modulus. We detected overall bending moments of 1.5×10-9-4×10-9 N m2 along the proximal-distal axis of the appendage showing a non-monotonous pattern that was not due to the geometry of the fin itself. Surgical disruption of the interray tissues along the proximal-distal axis revealed no significant changes to the overall bending stiffness, which we confirmed by determining a tensional modulus of the interray tissue. Thus, the biophysical values suggest that the flexibility of the fin during its hydrodynamic performance predominantly relies on the mechanical properties of the rays.

Regeneration versus scarring in vertebrate appendages and heart.

Injuries to complex human organs, such as the limbs and the heart, result in pathological conditions, for which we often lack adequate treatments. While modern regenerative approaches are based on the transplantation of stem cell-derived cells, natural regeneration in lower vertebrates, such as zebrafish and newts, relies predominantly on the intrinsic plasticity of mature tissues. This property involves local activation of the remaining material at the site of injury to promote cell division, cell migration and complete reproduction of the missing structure. It remains an unresolved question why adult mammals are not equally competent to reactivate morphogenetic programmes. Although organ regeneration depends strongly on the proliferative properties of cells in the injured tissue, it is apparent that various organismic factors, such as innervation, vascularization, hormones, metabolism and the immune system, can affect this process. Here, we focus on a correlation between the regenerative capacity and cellular specialization in the context of functional demands, as illustrated by appendages and heart in diverse vertebrates. Elucidation of the differences between homologous regenerative and non-regenerative tissues from various animal models is essential for understanding the applicability of lessons learned from the study of regenerative biology to clinical strategies for the treatment of injured human organs.

A dual epimorphic and compensatory mode of heart regeneration in zebrafish.

Zebrafish heart regeneration relies on the capacity of cardiomyocytes to proliferate upon injury. To understand the principles of this process after cryoinjury-induced myocardial infarction, we established a spatio-temporal map of mitotic cardiomyocytes and their differentiation dynamics. Immunodetection of phosphohistone H3 and embryonic ventricular heavy chain myosin highlighted two distinct regenerative processes during the early phase of regeneration. The injury-abutting zone comprises a population of cardiac cells that reactivates the expression of embryo-specific sarcomeric proteins and it displays a 10-fold higher mitotic activity in comparison to the injury-remote zone. The undifferentiated cardiomyocytes resemble a blastema-like structure between the original and wound tissues. They integrate with the fibrotic tissue through the fibronectin-tenascin C extracellular matrix, and with the mature cardiomyocytes through upregulation of the tight junction marker, connexin 43. During the advanced regenerative phase, the population of undifferentiated cardiomyocytes disperses within the regenerating myocardium and it is not detected after the termination of regeneration. Although the blastema represents a transient landmark of the regenerating ventricle, the remaining mature myocardium also displays an enhanced mitotic index when compared to uninjured hearts. This suggests an unexpected contribution of a global proliferative activity to restore the impaired cardiac function. Based on these findings, we propose a new model of zebrafish heart regeneration that involves a combination of blastema-dependent epimorphosis and a compensatory organ-wide response.

Bone morphogenetic protein signaling promotes morphogenesis of blood vessels, wound epidermis, and actinotrichia during fin regeneration in zebrafish.

Zebrafish fin regeneration involves initial formation of the wound epidermis and the blastema, followed by tissue morphogenesis. The mechanisms coordinating differentiation of distinct tissues of the regenerate are poorly understood. Here, we applied pharmacologic and transgenic approaches to address the role of bone morphogenetic protein (BMP) signaling during fin restoration. To map the BMP transcriptional activity, we analyzed the expression of the evolutionarily conserved direct phospho-Smad1 target gene, id1, and its homologs id2a and id3. This analysis revealed the BMP activity in the distal blastema, wound epidermis, osteoblasts, and blood vessels of the regenerate. Blocking the BMP function with a selective chemical inhibitor of BMP type I receptors, DMH1, suppressed id1 and id3 expression and arrested regeneration after blastema formation. We identified several previously uncharacterized functions of BMP during fin regeneration. Specifically, BMP signaling is required for remodeling of plexus into structured blood vessels in the rapidly growing regenerate. It organizes the wound epithelium by triggering wnt5b expression and promoting Collagen XIV-A deposition into the basement membrane. BMP represents the first known signaling that induces actinotrichia formation in the regenerate. Our data reveal a multifaceted role of BMP for coordinated morphogenesis of distinct tissues during regeneration of a complex vertebrate appendage.

The regenerative capacity of the zebrafish heart is dependent on TGFß signaling.

Mammals respond to a myocardial infarction by irreversible scar formation. By contrast, zebrafish are able to resolve the scar and to regenerate functional cardiac muscle. It is not known how opposing cellular responses of fibrosis and new myocardium formation are spatially and temporally coordinated during heart regeneration in zebrafish. Here, we report that the balance between the reparative and regenerative processes is achieved through Smad3-dependent TGFß signaling. The type I receptor alk5b (tgfbr1b) is expressed in both fibrotic and cardiac cells of the injured heart. TGFß ligands are locally induced following cryoinjury and activate the signaling pathway both in the infarct area and in cardiomyocytes in the vicinity of the trauma zone. Inhibition of the relevant type I receptors with the specific chemical inhibitor SB431542 qualitatively altered the infarct tissue and completely abolished heart regeneration. We show that transient scar formation is an essential step to maintain robustness of the damaged ventricular wall prior to cardiomyocyte replacement. Taking advantage of the reversible action of the inhibitor, we dissected the multifunctional role of TGFß signaling into three crucial processes: collagen-rich scar deposition, Tenascin C-associated tissue remodeling at the infarct-myocardium interface, and cardiomyocyte proliferation. Thus, TGFß signaling orchestrates the beneficial interplay between scar-based repair and cardiomyocyte-based regeneration to achieve complete heart regeneration.

Specific NuRD components are required for fin regeneration in zebrafish.

BACKGROUND: Epimorphic regeneration of a missing appendage in fish and urodele amphibians involves the creation of a blastema, a heterogeneous pool of progenitor cells underneath the wound epidermis. Current evidence indicates that the blastema arises by dedifferentiation of stump tissues in the vicinity of the amputation. In response to tissue loss, silenced developmental programs are reactivated to form a near-perfect copy of the missing body part. However, the importance of chromatin regulation during epimorphic regeneration remains poorly understood. RESULTS: We found that specific components of the Nucleosome Remodeling and Deacetylase complex (NuRD) are required for fin regeneration in zebrafish. Transcripts of the chromatin remodeler chd4a/Mi-2, the histone deacetylase hdac1/HDAC1/2, the retinoblastoma-binding protein rbb4/RBBP4/7, and the metastasis-associated antigen mta2/MTA were specifically co-induced in the blastema during adult and embryonic fin regeneration, and these transcripts displayed a similar spatial and temporal expression patterns. In addition, chemical inhibition of Hdac1 and morpholino-mediated knockdown of chd4a, mta2, and rbb4 impaired regenerative outgrowth, resulting in reduction in blastema cell proliferation and in differentiation defects. CONCLUSION: Altogether, our data suggest that specialized NuRD components are induced in the blastema during fin regeneration and are involved in blastema cell proliferation and redifferentiation of osteoblast precursor cells. These results provide in vivo evidence for the involvement of key epigenetic factors in the cellular reprogramming processes occurring during epimorphic regeneration in zebrafish.

Skeletal muscle regeneration after extensive cryoinjury of caudal myomeres in adult zebrafish.

Skeletal muscles can regenerate after minor injuries, but severe structural damage often leads to fibrosis in mammals. Whether adult zebrafish possess the capacity to reproduce profoundly destroyed musculature remains unknown. Here, a new cryoinjury model revealed that several myomeres efficiently regenerated within one month after wounding the zebrafish caudal peduncle. Wound clearance involved accumulation of the selective autophagy receptor p62, an immune response and Collagen XII deposition. New muscle formation was associated with proliferation of Pax7 expressing muscle stem cells, which gave rise to MyoD1 positive myogenic precursors, followed by myofiber differentiation. Monitoring of slow and fast muscles revealed their coordinated replacement in the superficial and profound compartments of the myomere. However, the final boundary between the muscular components was imperfectly recapitulated, allowing myofibers of different identities to intermingle. The replacement of connective with sarcomeric tissues required TOR signaling, as rapamycin treatment impaired new muscle formation, leading to persistent fibrosis. The model of zebrafish myomere restoration may provide new medical perspectives for treatment of traumatic injuries.

Autologous Blood Transfusion During Elective Surgery in a Candidate for Living Donor Transplantation: A Case Report.

BACKGROUND: Blood transfusions are risk factors for alloimmunization and unfavorable outcomes in solid organ transplant recipients. PURPOSE: We propose the adoption of autologous blood transfusion (ABT) in transplant candidates and recipients referred to elective surgery. METHODS: We present a case of a 45-year-old man with chronic kidney disease stage 5 due to polycystic kidney disease, who was qualified for a native kidney nephrectomy (NKN) before kidney transplantation. Before the scheduled surgery, the patient was referred to a blood donation center for blood collection. RESULTS: During 2 consecutive visits, autologous blood was collected uneventfully, and this allowed for the preparation of 2 units of red blood cell concentrates and a unit of plasma. Pre- and post-donation hemoglobin values were 11.9 and 10.4 g/dL, respectively. The NKN procedure was complicated by intra-abdominal bleeding from an accessory aberrant artery of the kidney. Hemoglobin dropped to 6.8 g/dL and was treated with ABT, followed by artery embolization. This allowed for an increase of hemoglobin to 8.3 mg/dL and avoidance of allotransfusion. Six weeks after NKN, the patient underwent successful kidney transplantation from a living donor. Panel reactive antibodies before transplantation were 0%, and graft function has been excellent during 20 months of observation. CONCLUSION: An autologous blood collection is a feasible option for patients with chronic kidney disease. ABT should be considered the procedure of choice when qualifying potential waiting list candidates and solid organ recipients for elective surgeries.

IGF signaling between blastema and wound epidermis is required for fin regeneration.

In mammals, the loss of a limb is irreversible. By contrast, urodele amphibians and teleost fish are capable of nearly perfect regeneration of lost appendages. This ability depends on direct interaction between the wound epithelium and mesenchymal progenitor cells of the blastema. It has been known for decades that contact between the wound epithelium and the underlying blastema is essential for successful regeneration. However, the underlying mechanisms are poorly understood. Here, we show that upon amputation the blastema induces expression of the ligand Igf2b, which then activates IGF signaling specifically in cells of the adjacent apical epithelium. Inhibition of IGF signaling by either morpholino antisense technology, or by specific chemical inhibitors of Igf1 receptor function NVP-AEW541 and NVP-ADW742, impairs fin regeneration. At the cellular level, this block in regeneration is reflected by a lack of the distinctive basal epithelium, increased apoptosis in the wound epidermis and reduced proliferation of blastema cells. Furthermore, induction of the blastemal and wound epidermal markers cannot be supported in the absence of IGF signaling. These data provide evidence that Igf2b expressed in the blastema promotes the properties of the adjacent wound epidermis, which subsequently are necessary for blastema function. Thus, IGF signaling upregulated upon fin amputation represents a signal from the blastema to the wound epithelium, a crucial step in appendage regeneration.

Regeneration of the dermal skeleton and wound epidermis formation depend on BMP signaling in the caudal fin of platyfish.

Fin regeneration has been extensively studied in zebrafish, a genetic model organism. Little is known about regulators of this process in distant fish taxa, such as the Poeciliidae family, represented by the platyfish. Here, we used this species to investigate the plasticity of ray branching morphogenesis following either straight amputation or excision of ray triplets. This approach revealed that ray branching can be conditionally shifted to a more distal position, suggesting non-autonomous regulation of bone patterning. To gain molecular insights into regeneration of fin-specific dermal skeleton elements, actinotrichia and lepidotrichia, we localized expression of the actinodin genes and bmp2 in the regenerative outgrowth. Blocking of the BMP type-I receptor suppressed phospho-Smad1/5 immunoreactivity, and impaired fin regeneration after blastema formation. The resulting phenotype was characterized by the absence of bone and actinotrichia restoration. In addition, the wound epidermis displayed extensive thickening. This malformation was associated with expanded Tp63 expression from the basal epithelium towards more superficial layers, suggesting abnormal tissue differentiation. Our data add to the increasing evidence for the integrative role of BMP signaling in epidermal and skeletal tissue formation during fin regeneration. This expands our understanding of common mechanisms guiding appendage restoration in diverse clades of teleosts.

A Cryoinjury Model for Studying Skeletal Muscle Regeneration of the Caudal Peduncle in Adult Zebrafish.

Skeletal muscle undergoes renewal and restoration after minor injury through the activation of satellite-like stem cells. Severe injuries of the musculature often lead to fibrosis in humans. In comparison to mammals, zebrafish possess a higher innate capacity for organ regeneration, providing a powerful model for studying tissue restoration after extensive damage to the organ. Here, a cryoinjury model is described to induce profound damage to four myomeres of the caudal peduncle in adult zebrafish. A custom-made cryoprobe was designed to fit the body shape and reproducibly injure the lateral musculature from the skin to the midline. Importantly, the body integrity remained intact, and the fish continued their swimming activity. Changes to the skeletal muscle were assessed by histological staining and fluorescence staining of sarcomeric proteins on tissue sections. This method will open up new avenues of research aiming to understand how the degeneration of the skeletal muscle induces reparative responses and, thus, the reactivation of the myogenic program in adult zebrafish.

The regeneration-responsive element careg monitors activation of Müller glia after MNU-induced damage of photoreceptors in the zebrafish retina.

In contrast to mammals, zebrafish can regenerate their damaged photoreceptors. This capacity depends on the intrinsic plasticity of Müller glia (MG). Here, we identified that the transgenic reporter careg, a marker of regenerating fin and heart, also participates in retina restoration in zebrafish. After methylnitrosourea (MNU) treatment, the retina became deteriorated and contained damaged cell types including rods, UV-sensitive cones and the outer plexiform layer. This phenotype was associated with the induction of careg expression in a subset of MG until the reconstruction of the photoreceptor synaptic layer. Single-cell RNA sequencing (scRNAseq) analysis of regenerating retinas revealed a population of immature rods, defined by high expression of rhodopsin and the ciliogenesis gene meig1, but low expression of phototransduction genes. Furthermore, cones displayed deregulation of metabolic and visual perception genes in response to retina injury. Comparison between careg:EGFP expressing and non-expressing MG demonstrated that these two subpopulations are characterized by distinct molecular signatures, suggesting their heterogenous responsiveness to the regenerative program. Dynamics of ribosomal protein S6 phosphorylation showed that TOR signaling became progressively switched from MG to progenitors. Inhibition of TOR with rapamycin reduced the cell cycle activity, but neither affected careg:EGFP expression in MG, nor prevented restoration of the retina structure. This indicates that MG reprogramming, and progenitor cell proliferation might be regulated by distinct mechanisms. In conclusion, the careg reporter detects activated MG, and provides a common marker of regeneration-competent cells in diverse zebrafish organs, including the retina.

Visual acuity and contrast sensitivity of adult zebrafish.

BACKGROUND: The aim of this study was to evaluate the visual acuity of adult zebrafish by assessing the optokinetic reflex. Using a modified commercially available optomotor device (OptoMotry®), virtual three-dimensional gratings of variable spatial frequency or contrast were presented to adult zebrafish. In a first experiment, visual acuity was evaluated by changing the spatial frequency at different angular velocities. Thereafter, contrast sensitivity was evaluated by changing the contrast level at different spatial frequencies. RESULTS: At the different tested angular velocities (10, 15, 20, 25, and 30 d/s) and a contrast of 100%, visual acuity values ranged from 0.56 to 0.58 c/d. Contrast sensitivity measured at different spatial frequencies (0.011, 0.025, 0.5, 0.1, 0.2, 0.3, 0.4, 0.5 and 0.55 c/d) with an angular velocity of 10 d/s and 25 d/s revealed an inverted U-shaped contrast sensitivity curve. The highest mean contrast sensitivity (±SD) values of 20.49 ± 4.13 and 25.24 ± 8.89 were found for a spatial frequency of 0.05 c/d (angular velocity 10 d/s) and 0.1 c/d (angular velocity 25 d/s), respectively. CONCLUSIONS: Visual acuity and contrast sensitivity measurements in adult zebrafish with the OptoMotry® device are feasible and reveal a remarkably higher VA compared to larval zebrafish and mice.

The zebrafish heart regenerates after cryoinjury-induced myocardial infarction.

BACKGROUND: In humans, myocardial infarction is characterized by irreversible loss of heart tissue, which becomes replaced with a fibrous scar. By contrast, teleost fish and urodele amphibians are capable of heart regeneration after a partial amputation. However, due to the lack of a suitable infarct model, it is not known how these animals respond to myocardial infarction. RESULTS: Here, we have established a heart infarct model in zebrafish using cryoinjury. In contrast to the common method of partial resection, cryoinjury results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts. As in mammals, the initial stages of the injury response include thrombosis, accumulation of fibroblasts and collagen deposition. However, at later stages, cardiac cells can enter the cell cycle and invade the infarct area in zebrafish. In the subsequent two months, fibrotic scar tissue is progressively eliminated by cell apoptosis and becomes replaced with a new myocardium, resulting in scarless regeneration. We show that tissue remodeling at the myocardial-infarct border zone is associated with accumulation of Vimentin-positive fibroblasts and with expression of an extracellular matrix protein Tenascin-C. Electrocardiogram analysis demonstrated that the reconstitution of the cardiac muscle leads to the restoration of the heart function. CONCLUSIONS: We developed a new cryoinjury model to induce myocardial infarction in zebrafish. Although the initial stages following cryoinjury resemble typical healing in mammals, the zebrafish heart is capable of structural and functional regeneration. Understanding the key healing processes after myocardial infarction in zebrafish may result in identification of the barriers to efficient cardiac regeneration in mammals.

Epithelial tube morphogenesis during Drosophila tracheal development requires Piopio, a luminal ZP protein.

The formation of branched epithelial networks is fundamental to the development of many organs, such as the lung, the kidney or the vasculature. Little is known about the mechanisms that control cell rearrangements during tubulogenesis and regulate the size of individual tubes. Recent studies indicate that whereas the basal surface of tube cells interacts with the surrounding tissues and helps to shape the ramification pattern of tubular organs, the apical surface has an important role in the regulation of tube diameter and tube growth. Here we report that two proteins, Piopio (Pio) and Dumpy (Dp), containing a zona pellucida (ZP) domain are essential for the generation of the interconnected tracheal network in Drosophila melanogaster. Pio is secreted apically, accumulates in the tracheal lumen and possibly interacts with Dp through the ZP domains. In the absence of Pio and Dp, multicellular tubes do not rearrange through cell elongation and cell intercalation to form narrow tubes with autocellular junctions; instead they are transformed into multicellular cysts, which leads to a severe disruption of the branched pattern. We propose that an extracellular matrix containing Pio and Dp provides a structural network in the luminal space, around which cell rearrangements can take place in an ordered fashion without losing interconnections. Our results suggest that a similar structural role might be attributed to other ZP-domain proteins in the formation of different branched organs.