In this study, we employed an integrated approach, combining histological analyses encompassing both conventional histological sections and transmission electron microscopy, along with transcriptomics to study the structural changes and molecular processes of gill resorption during metamorphosis. Our results showed gill resorption in M. fisspesiss is intricately linked with a cascade of physiological changes, including the loss of respiratory functionality, cell death, and a restructuring of metabolic processes during the metamorphic climax. These findings underscored ta synchronized orchestration of events pivotal to the successful progression of metamorphosis in frogs.
The gill structural changes and loss of respiratory function
In pro-metamorphic stages, frog tadpoles rely on their gills as the primary respiratory organ. These structures undergo significant degeneration, nearly disappearing entirely [14] or leaving only a small remnant [27] in metamorphic climax. In this study, we observed extensive structural differentiation in the M. fissipes gills from stages S37 to 39. This was characterized by highly branched gill filaments densely covered with capillaries. The projections of gill filaments featured a surface layer of pseudostratified columnar epithelial cells, significantly augmenting the contact area with gases (Fig. 1B). Ultrastructural studies of gill epithelial cells during this stage revealed the presence of microvilli, substantial substance secretion, and numerous mitochondria in the cytoplasm. These structural characteristics indicate a robust respiratory function in the gills during these stages. At stage 41, we observed the initiation of gill filament shrinkage, reduced spacing between filaments, and contraction of gill epithelial cells (Fig. 1C). Remarkably, despite these changes, the overall gill structure remained intact, suggesting effective gas exchange in water during this stage. As metamorphosis progressed, gill filaments continued to shrink, and blood vessel density decreased, particularly in metamorphic climax. Gill epithelial cells assumed irregular shapes, accompanied by the condensation and reduction in size of cell nuclei. Additionally, a decrease in mitochondrial presence and the appearance of cytoplasmic cavities were noted (Fig. 1C). Consistent with these observations, the gills in R. catesbeiana at TK stage XXII (metamorphic climax) showed decreased weight and reduced vascularization [14]. Microvascular casting in Xenopus laevis gills further revealed that the branching of gill filament row veins reached its maximum at stage 58, gradually decreasing in number and frequency until stage 62 (metamorphic climax) [15]. This suggests an accelerated regression of the blood-air barrier in gills and the subsequent loss of respiratory function during metamorphic climax.
Transcriptomic analysis of the gills unveils dynamic gene expression patterns, notably highlighting the significant upregulation of hemoglobin and mucin genes during pro-metamorphosis, contrasting with their lowest expression levels observed in metamorphic climax (Fig. 3A, B). Hemoglobin, a protein predominantly found in red blood cells, plays a pivotal role in the transport of oxygen and carbon dioxide [28, 29]. The heightened expression of hemoglobin genes during pro-metamorphosis implies an augmented capacity for gas exchange, contributing to the respiratory efficiency of the gills during this developmental stage. Mucins, characterized by their high molecular weight and extensive glycosylation, are synthesized by epithelial tissues in most animals. Serving as integral components of numerous gel-like secretions, mucins fulfill diverse functions, including lubrication, acting as a protective barrier against pathogens and toxic substances, and maintaining the hydration layer of the epithelium. Moreover, their role as a permeable gel layer facilitates the exchange of gases and nutrients between the upper epithelium and the underlying layers [30, 31]. The substantial upregulation of mucin genes in tadpole gills during pro-metamorphosis underscores their active participation in respiratory processes, further supporting the notion of a robust respiratory function. Conversely, the observed decline in the expression levels of hemoglobin and mucin genes during metamorphic climax suggests a diminishing respiratory capacity of the gills. This reduction aligns with the structural changes in gill filaments.
Collectively, these findings suggest a progressive loss of respiratory function in the gills as metamorphosis advances, contributing to a deeper understanding of amphibian adaptation to terrestrial life.
The molecular mechanisms underlying cell death in gill resorption
TH is the key factor to initiate cell death pathways during amphibian metamorphosis [7]. It is evidenced that TH exerts its effects via TH receptor (TR) and there is a pair of TR subtypes encoded by separate genes, TRα and TRβ. Our results showed that as metamorphosis proceeded, the transcriptional level of TRβ in M. fissipes gills increased and peaked in metamorphic climax (Fig. 4A). It has been shown that the treatment of pre-metamorphic tadpoles with the TRβ-specific agonist, GC-1, induces the gill resorption and tail shortening in vivo [32]. These findings suggest that apoptosis of larval cells in the gills is mediated predominantly by TRβ. In addition, we observed an upregulation in the transcription of BCL-2-related genes (BCL-2-like protein 10 and BCL-2-like protein 11) in S44 (Fig. 4B). BCL-2 protein family plays a crucial role in determining cellular apoptosis, which is vital for organ development, tissue homeostasis, and immune function [33, 34]. The upregulation of transcription levels of BCL-2-related genes suggests the activation of the apoptosis process in metamorphic climax.
Furthermore, the tumor necrosis factor (TNF) pathway plays a significant role in inducing programmed cell death and tumor necrosis factor receptor superfamily are proved to be the major death receptors involved in the extrinsic pathway of tail apoptosis in X. laevis tadpoles [35, 36]. Based on transcriptomic analysis, we identified four major categories of genes associated with the TNF signaling pathway: TNF receptor superfamily members (TNFRSF5, TNFRSF6B, TNFRSF16, TNFRSF18, and TNFRSF19), TNF alpha-induced proteins (TNFAIP2, TNFAIP3, and TNFAIP8), and TNF receptor-associated factors (TRAF1 and TRAF5) (Fig. 4C). Among these, TNFSF5/C40LG binding to TNFRSF5 can activate extracellular regulated protein kinases in macrophages and B cells, inducing immunoglobulin secretion [37]. TNFRSF16 can mediate neuronal cell death [38]. TNFRSF18 is involved in the interaction between activated T lymphocytes and endothelial cells, regulating T-cell receptor-mediated cell death and activating the NF-kappa-B pathway through the TRAF2/NIK pathway [39]. TNFRSF19, by mediating the activation of JNK and NF-kappa-B pathways, further promotes caspase-independent cell death [40]. Both TRAF1 and TRAF5 participate in regulating the activation of NF-kappa-B and JNK pathways, playing a role in the process of regulating cell apoptosis [41, 42]. The upregulation of these genes suggests the activation of the TNF signaling pathway during the metamorphic climax. TNFAIP3 can interact with TRAF1/TRAF2 and inhibit the activation of the NF-kappa-B pathway [43, 44]. Meanwhile, the transcription of TNFAIP3 and TNFAIP8 in the gills of M. fissepess tadpoles is downregulated in S44. Considering that TNFAIP8 can inhibit the activity of caspase-8, thereby negatively regulating TNF-mediated apoptosis [45], these results further support the activation of the TNF signaling pathway and apoptosis during metamorphic climax.
Matrix metalloproteinases (MMPs) are a type of collagenase and belong to the metzincins superfamily. They act as extracellular matrix-degrading enzymes, responsible for breaking down various protein components of the extracellular matrix during tissue apoptosis [46]. The upregulation of matrix metalloproteinases during the metamorphic climax similarly suggests their involvement in the absorption processes in the gills during this stage (Fig. 4D).
In summary, we illuminated the intricate processes involved in gill resorption that manifests through two distinct mechanisms analogous to the phenomenon of tail regression, denoted as "suicide" and "murder" [11, 12] (Fig. 4E). In the former, apoptosis is triggered directly by thyroid hormone action on gill tissues. Within this context, our study delineates two primary apoptotic pathways: the extrinsic pathway, initiated by membrane death receptors such as TNF receptor superfamily, and the intrinsic pathway, instigated within mitochondria by members of the BCL-2 family. This dual-pathway model underscores the complexity of the molecular cascades governing cellular apoptosis in the context of gill regression. Furthermore, we also delineated a distinct facet of gill resorption characterized as "murder," wherein cell death ensues through the degradation of the extracellular matrix and the consequential loss of cellular anchorage. These findings enhanced our understanding of the regulatory dynamics of gill resorption, offering insights into the intricate interplay of molecular pathways and cellular events that govern this biological process.
The metabolic switches during gill resorption
The gills undergo metabolic changes during both the execution of respiratory functions and the process of significant resorption, involving alterations in both substance and energy metabolism. Based on the results of transcriptomic analysis, we explored the transcriptional patterns of metabolism-related genes in the gills at different developmental stages. Ribosomal protein genes, including RPS27, PPS34, RPL2, RPL15, and RPL38, crucial constituents of ribosomes, exhibited transcriptional upregulation during the pre-metamorphic stages (Stages 37 to 39) but underwent downregulation at the metamorphic climax (Stage 43) (Fig. 5C). This upregulation suggests an augmentation in cellular protein synthesis capability, indicative of concurrent gill tissue growth alongside tadpole development during the pro-metamorphic stages. Respiration is a process entailing energy production and storage. Core genes integral to the mitochondrial respiratory chain, including ATP synthase subunit (MT-ATP6), NADH-ubiquinone oxidoreductase chains (MT-ND1 and MT-ND2), and Cytochrome c oxidase subunits (MT-CO1 and MT-CO3), maintained elevated expression levels in the gills. This sustained upregulation possibly is consistent with increased respiratory demands in tadpoles during the pro-metamorphic stages (Fig. 5C). In contrast to energy metabolism, most genes related to substrate metabolism, maintained relatively low transcription levels during the pro-metamorphic stages and experienced substantial upregulation during the metamorphic climax. For instance, G6PD catalyzed the rate-limiting step of the oxidative pentose-phosphate pathway, offering an alternative route for carbohydrate dissimilation beyond glycolysis [47]. Glucose-6-phosphate exchanger (SLC37A2 and SLC37A4) may transport cytoplasmic glucose-6-phosphate into the lumen of the endoplasmic reticulum [48], while facilitated glucose transporter members of the solute carrier family 2 (SLC2A1 and SLC2A6) exhibited the ability to transport a wide range of aldoses, including both pentoses and hexoses [49]. The transcriptional upregulation of these genes pointed to enhanced carbohydrate transport and metabolism. Additionally, genes involved in lipid transport and metabolism, including fatty acid-binding proteins (FABP1 and FABP7), SCD5, thioesterase B, and Lipocalin [50, 51], as well as those associated with amino acid transport and metabolism, such as Aminopeptidase (ANPEP), Carboxypeptidase (CPE and CPZ), Sodium-coupled monocarboxylate transporter (SLC5A8), and Y + L amino acid transporter (SLC7A6) [52–55], exhibited similar expression patterns (Fig. 5C). Enhanced substrate transport and metabolism aligns with the rapid apoptosis observed in the gills during the metamorphic climax, wherein many macromolecules undergo hydrolysis, producing corresponding substrate molecules. The enhanced metabolism and transport of substrates facilitate the translocation of substrate molecules to other rapidly growing and metabolically active organs during tissue apoptosis (Fig. 5D). Therefore, during the metamorphic climax, the gills, serving as an energy-supplying organ, recycle substrate molecules through the resorption process, providing metabolic fuel for the frog's metamorphic development.