<b><i>Supporting data for</i></b> "Phenotypic Analysis of <i>Tspyl1</i> Knockout Mice"

<p dir="ltr"><i>TSPYL1</i>, encoding Testis-specific Y-encoded-like protein 1, is mutated in Sudden Infant Death with Dysgenesis of the Testes syndrome (SIDDT). However, its biological roles remain poorly understood. Our previous study identified TSPYL1 as a modulator of TGFβ signalling. Mechanistically, TSPYL2 protein is stabilized upon TSPYL1 depletion. TSPYL2 mediates TGFβ responses which are dependent on TP53 in specific human cell lines. Building on this, the present study investigated the physiological consequences of <i>Tspyl1</i> knockout (KO) in mice and explored the involvement of TGFβ signalling. The functional relationships of TSPYL1 with TSPYL2 as well as TP53 were tested genetically in mice. All <i>Tspyl1</i> KO mice on a pure C57BL/6N background died before weaning. Phenotypic analysis revealed multiple defects, including growth retardation, hypoglycaemia, hypothermia, lymphopenia, and lung defects as characterized by impaired alveolarization and reduced proliferation of alveolar epithelial cells. The metabolic dysregulation, immune abnormalities and lung defects have not been reported in SIDDT cases, highlighting potential species-specific roles of TSPYL1.<i> </i><i>Tspyl1</i> KO mice displayed dysregulated TGFβ signalling with stage-specific alterations. During embryogenesis (embryonic day 14.5 to embryonic day 16.5), <i>Tgfbr1</i> and <i>Smad7</i> transcripts were elevated in <i>Tspyl1</i> KO lungs, suggesting upregulation of TGFβ signalling. However, postnatally, these transcripts were suppressed, while phospho-Smad3 levels remained unchanged. Intriguingly, <i>Tspyl1</i> KO embryonic fibroblasts showed heightened TGFβ responsiveness, as shown by increased TGFBR1 expression and elevated Smad3 phosphorylation upon TGFβ stimulation. The data suggested that TSPYL1 negatively regulated TGFβ responsiveness through lowering TGFBR1 expression during embryonic development. An interesting finding was the upregulation of TSPYL2 protein, not linked to an increased mRNA, in <i>Tspyl1</i> KO mice across multiple organs (lung, liver, kidney). This upregulation of TSPYL2 was observed on both pure C57BL/6N and mixed 129SvEvXC57BL/6N genetic backgrounds, as well as in <i>Tspyl1</i>-deficient NIH-3T3 cells and mouse embryonic fibroblasts. Genetic rescue experiments demonstrated that double knockout of <i>Tspyl1</i> and <i>Tspyl2</i> in mice restored viability and fertility, underscoring the critical role of TSPYL2 in mediating <i>Tspyl1</i> KO phenotype. In contrast, <i>Tspyl1</i> and <i>Trp53</i> double KO mice failed to rescue mortality or TSPYL2 upregulation, ruling out p53-dependent mechanisms. This study establishes TSPYL1 as a multifunctional regulator of neonatal survival, with TSPYL2 as a critical mediator of lethality in <i>Tspyl1</i><i> </i>knockout mice. However, the disconnection between perturbed TGFβ signalling and phenotypic outcome points to unresolved mechanistic complexities. Further work is needed to elucidate the molecular basis of TSPYL2 protein stabilization and its broader implications in developmental disorders.</p>