Ptch2 Deficiency Triggers Lipoma Formation and Adipogenic Transcriptome Reprogramming in Nile Tilapia (Oreochromis niloticus)

1. Introduction

Adipose tissue is a dynamic endocrine organ primarily composed of adipocytes, serving as the central hub for systemic energy homeostasis through lipid storage and mobilization [1,2]. The differentiation and development of adipocytes, a process termed adipogenesis, are tightly regulated by complex transcriptional cascades [3,4]. Dysregulation of this process is implicated not only in prevalent metabolic diseases like obesity and type 2 diabetes but also in localized pathologies such as lipoma formation [5,6]. Lipoma is a benign mesenchymal tumor characterized by the uncontrolled growth of adipose tissue [7,8], and its pathogenesis is not yet clear.

Hedgehog (Hh) signaling pathway, a highly conserved pathway crucial for cell fate determination, tissue patterning, and stem cell regulation, is a potential regulator in adipose biology and tumorigenesis [9,10,11,12]. In canonical Hh signaling, the binding of ligands to the Patched (Ptch) receptor relieves its inhibition on Smoothened (Smo), ultimately activating Gli transcription factors [13,14]. In adipose biology, canonical Hh signaling acts as a potent inhibitor of white adipocyte differentiation [15]. In mice, the canonical Hh signaling, which is transduced via primary cilia, inhibits adipogenesis, whereas omega-3 fatty acids, which are sensed via the free fatty acid receptor 4 (FFAR4) in the cilium, promote adipogenesis [16,17]. In humans, Gli2-mediated Hh signaling is enriched in adipose progenitor cells and in dedifferentiated liposarcoma, where it promotes malignant progression by driving stromal formation and suppressing immune infiltration [18].

Patched2 (Ptch2) is a pivotal transmembrane receptor in the Hh pathway, functioning as the primary negative regulator of the pathway [13]. Ptch2 plays a crucial tumor-suppressive role in the development and progression of various cancers. In basal cell carcinoma, the absence of Ptch2 leads to abnormal activation of the Hh signaling pathway, which promotes tumor growth [19,20]. In breast cancer, truncating mutations in Ptch2 disrupt its transmembrane structure, activating the MAPK, PI3K/mTOR pathways and driving tumor cell proliferation [21,22]. These studies indicate that the normal function of Ptch2 is vital for inhibiting tumor development, and its mutations or deletions can activate oncogenic signaling pathways, thereby promoting tumorigenesis. However, the role of Ptch2 in lipomas remains elusive.

Nile tilapia (Oreochromis niloticus) is a valuable vertebrate model for genetic and physiological studies due to its well-annotated genome, suitability for CRISPR/Cas9-mediated genome editing [23,24]. In this study, we systematically investigated the effects of Ptch2 deficiency on adipogenesis and lipid metabolism. Our results showed that Ptch2 deficiency leads to the formation of coelomic lipomas in Nile tilapia. Further histological and transcriptomic analyses demonstrated that Ptch2 acts as a key inhibitor of the adipogenesis program in vivo. These findings highlight a novel and critical role for Ptch2 in lipid metabolic homeostasis and tumor suppression.

2. Materials and Methods

2.1. Animals

Nile tilapias (Oreochromis niloticus) were reared in circulating aerated fresh water tank under natural light at 26 ± 0.5 °C. The water quality parameters were monitored daily (pH: 7.2 ± 0.5; dissolved oxygen: 6.5–7.0 mg/L). Fish were fed three times a day with commercial feed (Shengsuo, Yantai, China). All progenies were obtained by crossing XY male with XX female. Larvae and juveniles were raised under the same conditions as the parental fish. All animal experiments were carried out in accordance with the regulations of the Guide for Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Southwest University (NO. IACUC-20181015-12).

2.2. Cloning and sequence analysis

The putative coding sequence containing the complete open reading frame (ORF) of Nile tilapia ptch2 was obtained through a search in GenBank (Gene ID: 100692939). The sequence was amplified via RT-PCR using primers specific to ptch2 (Table S1), followed by sequencing and bioinformatic analyses. The ClustalX 1.83 program and GeneDoc 2.6 software were utilized to perform multiple amino acid sequence alignment and identity analysis. Additionally, MEGA 7.0 software was employed to construct neighbor-joining phylogenetic trees, while the Ensembl Genome Browser (http://www.ensembl.org) was used for gene syntenic analyses.

2.3. Hematoxylin and Eosin (H&E) Staining

The ptch2^-/- fish were generated through CRISPR/Cas9-mediated knockout and hybridization, as described in our previous study [25]. Wild-type (WT) and ptch2^-/- fish at 90 days after hatching (dah) were fixed in Bouin’s solution for 24 h at room temperature with agitation. The fixed samples were then processed as follows: serial dehydration in 60%, 70%, 80%, 90%, and 95% ethanol for 1 h each, followed by three 1-h immersions in 100% ethanol; sequential clearance in a xylene and ethanol mixture (1:1) for 30 min, followed by two 10-min immersions in xylene; and infiltration in paraffin for 2 h. The samples were sectioned at a thickness of 5 µm using a Leica Microsystems microtome (Wetzlar, Germany) and subsequently stained with H&E. Images were captured using an Olympus BX53 light microscope (Tokyo, Japan).

2.4. RNA-seq

Adipose tissues were harvested separately from WT and ptch2^-/- fish at 90 dah, with three biological replicates performed for each group. Total RNA was extracted from each group using RNAiso Plus (Takara). Libraries were sequenced on an Illumina platform (GENEBOOK Biotechnology), and clean reads were aligned to the reference genome using HISAT2. Gene expression levels, quantified as fragments per kilobase per million mapped reads (FPKM), were determined using featureCounts. Differentially expressed genes (DEGs) were analyzed with DESeq2 and identified based on the following criteria: P value < 0.05 and |log2(fold change) | ≥ 1.5. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway enrichment analyses were conducted on the DEGs using an online analysis platform (https://www.genescloud.cn/login).

Total RNA from the aforementioned samples was reverse-transcribed into complementary DNA (cDNA) using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara). Quantitative real-time PCR (qRT-PCR) was performed on an ABI-7500 system (Applied Biosystems) following the SYBR^® Premix Ex TaqTM II (Takara Bio Inc., Japan) protocol. Relative mRNA levels were normalized to β-actin and calculated using the formula R = 2^−ΔΔCt (primers listed in Table S1).

2.5. Serum Biochemistry

Blood was collected from the tail vein of WT and ptch2^-/- fish at 90 dah, and serum was separated by centrifugation. Levels of glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), lipase, triglycerides (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL) and low-density lipoprotein cholesterol (LDL) were measured using the biochemical analyzer SMT-120VP (Seamaty, China).

2.6. Data Analysis

Statistical analyses were conducted using the GraphPad Prism 8 software package (GraphPad Software, La Jolla, CA, USA). Data are presented as mean ± SD derived from at least three independent experiments; each performed in triplicate. Statistical significance was assessed using a two-tailed Student’s t-test. The levels of statistical significance are indicated as follows: **P < 0.01; *P < 0.05; NS, not significant.

3. Results

3.1. Sequence Analyses

The ORF of Nile tilapia ptch2 was successfully cloned and validated. The resulting protein, composed of 1491 amino acids, exhibits a high sequence identity with vertebrate Ptch2 homologs, exceeding 65% (Table 1), and is phylogenetically clustered with these homologs, distinct from the Ptch1 clade and the Drosophila Ptch outgroup (Figure 1A). A comparative analysis of neighboring genes across species reveals that the chromosome 18, which contains ptch2 in Nile tilapia, maintains good synteny with zebrafish chromosome 2 and human chromosome 1, both of which also harbor Ptch2 (Figure 1B). Structural analysis indicates that Nile tilapia Ptch2 possesses characteristic domains, including a N-terminal cytoplasmic domain (N^cyto) and 12 transmembrane domains, akin to its homolog Ptch1, although notable divergence is observed in the C-terminal cytoplasmic domains (C^cyto) (Figure 1C). These findings suggest that Nile tilapia Ptch2 is orthologous to mammalian Ptch2, while exhibiting divergence from Ptch1.

3.2. ptch2 Deficiency Leads to Visceral and Perirenal Lipomatosis

Homozygous ptch2 mutants were viable at early stages but exhibited a striking internal phenotype by 90 dah. External morphology showed no significant difference from WT siblings. However, upon opening the body cavity, WT fish exhibited an intact peritoneum (Figure 2A), while ptch2^-/- mutants displayed prominent, encapsulated fatty masses (lipomas) attached to the inner body wall, with some causing visible rupture of the peritoneal lining (Figure 2B, B’). Furthermore, the kidneys of mutant fish were abnormally encapsulated by adipose tissue, unlike the clean surface of WT kidneys (Figure 2C, D). These results demonstrate that Ptch2 deficiency induces ectopic lipoma formation in the coelom and perirenal region.

Histological examination confirmed the macroscopic observations. H&E staining of peritoneal tissue from WT fish showed a normal, thin mesothelial layer (Figure 3A). In contrast, ptch2^-/- mutants exhibited a massive accumulation of adipocytes forming a discrete lipoma above the peritoneal layer (Figure 3B). The glomeruli, anterior renal tubules, and mesorenal tubules in the kidneys of WT fish developed normally (Figure 3C, E). Although kidney histology appeared normal in ptch2^-/- mutants (Figure 3F), the organ surface was covered by adipocyte clusters (Figure 3D).

Analysis of adipose tissue depots revealed a significant alteration in adipocyte morphology. Contrary to hypertrophy, adipocytes in ptch2^-/- fish were significantly smaller in area compared to those in WT (Figure 3G, H). Quantification confirmed a significant decrease in adipocyte area in mutants (Figure 3I). Notably, some mutant adipocytes contained increased nuclei within the lipid vacuole (Figure 3H), a feature occasionally associated with active adipogenesis or remodeling.

3.3. Transcriptomic Reprogramming in ptch2^-/- Adipose Tissue

To investigate the molecular basis of the phenotype, we performed RNA-seq on abdominal adipose tissue. Hierarchical clustering clearly separated WT and mutant samples (Figure 4A). We identified 2675 DEGs, with 1621 upregulated and 1054 downregulated in ptch2^-/- adipose tissue (Figure 4B). GO term enrichment analysis of upregulated DEGs revealed strong association with biological processes such as “complement activation” and “cell cycle” (Figure 4C). Consistently, KEGG pathway analysis showed significant enrichment for “PPAR signaling pathway”, “cell cycle”, “DNA replication”, “fatty acid biosynthesis”, “glycerolipid metabolism” and “biosynthesis of unsaturated fatty acids”. (Figure 4D). These data indicate a global transcriptional shift towards enhanced lipid synthesis and adipogenesis in the absence of ptch2.

Focusing on the core lipogenic pathway, RNA-seq data showed a significant increase in the expression of key regulators and enzymes, including novo fatty acid synthesis (acaca, fasn, acsl3), fatty acid desaturation (scd, fadsd6, elovl6), and triglyceride synthesis (dgat2, lpl, fabp4a, slc27a4, acsbg2) (Figure 5A). This upregulation was independently validated by qRT-PCR, which confirmed a highly significant increase in the mRNA levels of these genes in ptch2^-/- adipose tissue compared to WT (Figure 5B).

3.4. Systemic Metabolic Changes in ptch2 Mutants

Serum biochemical analysis showed that ptch2^-/- mutants had elevated blood glucose levels (Figure 6A) and increased activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), along with reduced alkaline phosphatase (ALP) activity (Figure 6B). No significant changes were observed in total protein, albumin, lipase, or lipid profiles (triglycerides, total cholesterol, HDL, LDL) (Figure 6B–D).

4. Discussion

This study reveals that Ptch2 deficiency in Nile tilapia results in a distinct pathological syndrome characterized by visceral lipomatosis and systemic metabolic dysregulation. The formation of coelomic and perirenal lipomas, alongside reduced post-juvenile survival (Figure S1), underscores a critical role for Ptch2 in maintaining adipose tissue homeostasis.

The mutant phenotype is not merely one of adipose hypertrophy but of true neoplastic transformation. Histological analysis confirmed that the ectopic fatty masses exhibited key diagnostic features of lipoma, including densely packed, small adipocytes and the presence of multinucleated cells (Figure 3G-I). Critically, H&E staining indicated that the internal architecture of the kidney remained histologically intact in mutants (Figure 3F), yet the organ was extensively displaced and encased by the lipomatous growth (Figure 2D, 3D). This distinction suggests that mortality likely stems not from a primary developmental defect or intrinsic organ failure, but from a progressive pathological process in which the mechanical compression and displacement of vital organs by uncontrolled adipose overgrowth ultimately compromises their function.

To define the molecular drivers of this transformation, we performed transcriptional profiling of mutant adipose tissue. The profound shift in gene expression, with over 2,600 differentially expressed genes, points to a comprehensive adipogenic reprogramming. KEGG pathway analysis pinpointed the specific metabolic routes activated, showing significant enrichment in “Fatty acid biosynthesis,” “Glycerolipid metabolism,” and “Biosynthesis of unsaturated fatty acids” (Figure 4D). Crucially, the PPAR signaling pathway—a master transcriptional regulator of adipogenesis—was among the most significantly enriched. This finding is mechanistically coherent, as it aligns with the concerted upregulation of a suite of direct PPAR target genes and core lipogenic enzymes, including fasn and acaca (for de novo synthesis), scd and fadsd6 (for desaturation), and dgat2 (for triglyceride synthesis) (Figure 5). We therefore propose that the loss of Ptch2 relieves a constitutive inhibition on the adipogenic transcriptional program, leading to sustained activation of the PPAR pathway. This, in turn, initiates a feed-forward loop driving uncontrolled lipid synthesis, which ultimately manifests as lipoma formation. However, the precise molecular mechanism linking Ptch2 to PPAR pathway repression remains a key question for future study.

When considering the broader genetic context, the severe embryonic lethality of ptch1 mutants in tilapia has prevented a direct comparison of the two Hh receptors in postnatal adipose biology. Our work establishes Ptch2 as a non-redundant, essential suppressor of pathological adipogenesis in vivo. Whether Ptch1 plays a complementary role, or whether functional compensation occurs, remains an open and interesting avenue for future investigation.

Finally, the systemic metabolic alterations, notably elevated blood glucose and liver transaminases (Figure 6), demonstrate that the local disruption of adipose homeostasis has whole-organism consequences. Hyperglycemia suggests the development of insulin resistance, which is a common sequela of dysfunctional adipose tissue [26]. Concurrently, elevated AST and ALT levels signal secondary hepatic stress [27], likely resulting from altered systemic lipid flux or inflammatory signals originating from the pathological fat depots. These findings integrate the local tumor phenotype into a framework of systemic metabolic disease, enhancing the translational relevance of this model.

5. Conclusions

In summary, our results delineate a novel pathway in which Ptch2 acts as a guardian of adipose tissue integrity. Its deficiency initiates a transcriptional cascade through the activation of the PPAR pathway, resulting in lipomatous transformation and subsequent metabolic dysfunction. This study not only identifies Ptch2 as a critical regulator of fat biology but also establishes a robust aquatic model for dissecting the mechanisms of adipogenesis and systemic fat metabolic comorbidities.