Cathepsin Family Members Research Articles

Expanding the Library of Covalent Cysteine Cathepsin Probes Featuring Sulfoxonium Ylide Electrophiles.

Covalent activity-based probes are invaluable tools to monitor protease activity in vitro and in vivo. We recently discovered that dimethyl sulfoxonium ylides (SYs) bind selectively to cysteine cathepsin proteases in a mechanism-dependent manner. Herein, we present the synthetic routes and characterization of an expanded library of SY probes with a greater diversity in recognition sequences. The probes exhibit a range of potency and selectivity for the cathepsin family members. We also investigated the impact of fluorophore positioning on probes bearing P1 lysine. When sulfonated cyanine 5 was attached via the lysine side chain, the resulting probe was selective for cathepsin S. When attached to the α-amine, with the side chain amine either free or Boc-protected, the probes reacted with both cathepsin S and X. Bulk in the P1 position is thus well tolerated by cathepsin S but not cathepsin X. We examined the impact of Cy5 sulfonation on probe properties, demonstrating that unsulfonated probes exhibit greater cellular uptake, which affects their relative selectivity. Finally, we demonstrated that SY probes exhibit minimal labeling of cathepsin S in freshly prepared lysates, but this increases during the prolonged incubation of lysates. This work extends our understanding of SY probes and informs future probe development.

Reduced expression of cathepsin F predicts poor prognosis in patients with clear cell renal cell carcinoma

Abnormalities in the extracellular matrix (ECM) play important roles in the regulation and progression of clear cell renal cell carcinoma (ccRCC). The cysteine cathepsin is one of the major proteases involved in ECM remodeling and has been shown to be aberrantly expressed in multiple cancer types. However, the clinical significance and biological function of distinct cysteine cathepsins in ccRCC remain poorly understood. In this study, several bioinformatics databases, including UALCAN, TIMER, GEPIA and the Human Protein Atlas datasets, were used to analyze the expression and prognostic value of different cysteine cathepsin family members in ccRCC. We found that the expression level of CTSF was downregulated in tumor tissues and closely related to the poor survival of ccRCC patients. Further in vitro experiments suggested that CTSF overexpression suppressed the proliferation and migration of ccRCC cells. Moreover, the expression of CTSF was shown to be associated with several immune-infiltrating cells and immunomodulators in ccRCC. These results indicated that CTSF might be a promising diagnostic and prognostic marker in ccRCC.

Therapeutic potential of targeting cathepsin S in pulmonary fibrosis.

Cathepsin S (CTSS), a lysosomal protease, belongs to a family of cysteine cathepsin proteases that promote degradation of damaged proteins in the endolysosomal pathway. Aberrant CTSS expression and regulation are associated with the pathogenesis of several diseases, including lung diseases. CTSS overexpression causes a variety of pathological processes, including pulmonary fibrosis, with increased CTSS secretion and accelerated extracellular matrix remodeling. Compared to many other cysteine cathepsin family members, CTSS has unique features that it presents limited tissue expression and retains its enzymatic activity at a neutral pH, suggesting its decisive involvement in disease microenvironments. In this review, we investigated the role of CTSS in lung disease, exploring recent studies that have indicated that CTSS mediates fibrosis in unique ways, along with its structure, substrates, and distinct regulation. We also outlined examples of CTSS inhibitors in clinical and preclinical development and proposed CTSS as a potential therapeutic target for pulmonary fibrosis.

Genomic and transcriptomic identification of the cathepsin superfamily in the Mediterranean mussel Mytilus galloprovincialis

Cathepsins are lysosomal enzymes that participate in important physiological processes, such as development, tissue remodelling, senescence and innate and adaptive immunity. The description of these proteins in molluscs is fragmented and incomplete. In the present work, we identified most of the cathepsin family members in the bivalve Mytilus galloprovincialis by screening published genomic and transcriptomic information. In this specie, the cathepsin family is composed of 41 proteins showing a high diversification of cathepsins D, L and F, not previously observed in other taxonomic groups. Specific set of cathepsins are constitutively expressed in the different mussel tissues. Transcriptomic analyses suggested coordinated activity of the different cathepsins and their sequential activation during larval development. Cathepsins also play an important role in the immune response of bivalves, and different immune pathways seem to be activated in response to Vibrio splendidus infection.

Abstract 328: Selective fluorogenic probe for rapid detection of cathepsin L activity

Abstract Cathepsin L (CTL) is a cysteine protease that demonstrates upregulated activity and altered trafficking in various cancers. Its upregulation in many cancers correlates positively with tumor grade while its excessive secretion makes elevated serum levels a possible biomarker for aggressive cancer types. The overlapping substrate specificity of cathepsin family members makes selective detection of activity from a single cathepsin difficult, and CTL activity is particularly difficult to parse from its close homologue CTV and the ubiquitous CTB. This complicates clinical sample assays for CTL activity, which generally rely on simple fluorescent probes lacking specificity for CTL over other cathepsins. We have developed a fluorogenic probe, CTLAP, that is rapidly activated by CTL and displays good selectivity over CTB and CTV, the closest competing analytes for CTL. The novel chemical structure of CTLAP facilitates markedly higher signal generation and improved selectivity for CTL when compared to Z-FR-AMC, the commercial probe commonly used to detect CTL activity in mixed samples. Optimum selectivity for CTL is achieved within 10 min of incubation, suggesting that CTLAP is amenable for rapid detection of CTL, even in the presence of competing cathepsins. Citation Format: Kelton A. Schleyer, Ben Fetrow, Peter Fatland, Jun Liu, Maya Chaaban, Biwu Ma, Lina Cui. Selective fluorogenic probe for rapid detection of cathepsin L activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 328.

Making Connections: p53 and the Cathepsin Proteases as Co-Regulators of Cancer and Apoptosis.

Simple SummaryThis article describes an emerging area of significant interest in cancer and cell death and the relationships shared by these through the p53 and cathepsin proteins. While it has been demonstrated that the p53 protein can directly induce the leakage of cathepsin proteases from the lysosome, directly triggering cell death, little is known about what factors set the threshold at which the lysosome can become permeabilized. It appears that the expression levels of cathepsin proteases may be central to this process, with some of them being transcriptionally regulated by p53. The consequences of such a mechanism have serious implications for lysosomal-mediated apoptosis and have significant input into the design of therapeutics and their strategic use. In this review, we highlight the importance of extending such findings to other cathepsin family members and the need to assess the roles of p53 isoforms and mutants in furthering this mechanism.While viewed as the “guardian of the genome”, the importance of the tumor suppressor p53 protein has increasingly gained ever more recognition in modulating additional modes of action related to cell death. Slowly but surely, its importance has evolved from a mutated genetic locus heavily implicated in a wide array of cancer types to modulating lysosomal-mediated cell death either directly or indirectly through the transcriptional regulation of the key signal transduction pathway intermediates involved in this. As an important step in determining the fate of cells in response to cytotoxicity or during stress response, lysosomal-mediated cell death has also become strongly interwoven with the key components that give the lysosome functionality in the form of the cathepsin proteases. While a number of articles have been published highlighting the independent input of p53 or cathepsins to cellular homeostasis and disease progression, one key area that warrants further focus is the regulatory relationship that p53 and its isoforms share with such proteases in regulating lysosomal-mediated cell death. Herein, we review recent developments that have shaped this relationship and highlight key areas that need further exploration to aid novel therapeutic design and intervention strategies.

Integrative p53, micro-RNA and Cathepsin Protease Co-Regulatory Expression Networks in Cancer.

Simple SummaryThis article describes an emerging area of significant interest in cancer and cell death and the relationships shared by these through the transcriptional regulation of cathepsin protease genes by micro-RNAs that are connected to p53 activation. While it has been demonstrated that the p53 protein can directly regulate some cathepsin genes and the expression of their upstream regulatory micro-RNAs, very little is known about what input the p53 isoform proteins may have in regulating this relationship. Herein, we draw attention to this important regulatory aspect in the context of describing mechanisms that are being established for the micro-RNA regulation of cathepsin protease genes and their collective use in diagnostic or prognostic assays.As the direct regulatory role of p53 and some of its isoform proteins are becoming established in modulating gene expression in cancer research, another aspect of this mode of gene regulation that has captured significant interest over the years is the mechanistic interplay between p53 and micro-RNA transcriptional regulation. The input of this into modulating gene expression for some of the cathepsin family members has been viewed as carrying noticeable importance based on their biological effects during normal cellular homeostasis and cancer progression. While this area is still in its infancy in relation to general cathepsin gene regulation, we review the current p53-regulated micro-RNAs that are generating significant interest through their regulation of cathepsin proteases, thereby strengthening the link between activated p53 forms and cathepsin gene regulation. Additionally, we extend our understanding of this developing relationship to how such micro-RNAs are being utilized as diagnostic or prognostic tools and highlight their future uses in conjunction with cathepsin gene expression as potential biomarkers within a clinical setting.

Immune recovery in patients with mantle cell lymphoma receiving long-term ibrutinib and venetoclax combination therapy

AbstractCombination venetoclax plus ibrutinib for the treatment of mantle cell lymphoma (MCL) has demonstrated efficacy in the relapsed or refractory setting; however, the long-term impact on patient immunology is unknown. In this study, changes in immune subsets of MCL patients treated with combination venetoclax and ibrutinib were assessed over a 4-year period. Multiparameter flow cytometry of peripheral blood mononuclear cells showed that ≥12 months of treatment resulted in alterations in the proportions of multiple immune subsets, most notably CD4+ and CD8+ effector and central memory T cells and natural killer cells, and normalization of T-cell cytokine production in response to T-cell receptor stimulation. Gene expression analysis identified upregulation of multiple myeloid genes (including S100 and cathepsin family members) and inflammatory pathways over 12 months. Four patients with deep responses stopped study drugs, resulting in restoration of normal immune subsets for all study parameters except myeloid gene/pathway expression, suggesting long-term combination venetoclax and ibrutinib irreversibly affects this population. Our findings demonstrate that long-term combination therapy is associated with immune recovery in MCL, which may allow responses to subsequent immunotherapies and suggests that this targeted therapy results in beneficial impacts on immunological recovery. This trial was registered at www.clinicaltrials.gov as #NCT02471391.

Expression profile of cathepsins indicates the potential of cathepsins B and D as prognostic factors in breast cancer patients.

Breast cancer is one of the most prevalent types of cancer in women and contributes to 32% of all female cancer cases. Cathepsins, a family of proteins, are known to have a critical role in human cancers. However, previous studies on the systematic analysis of the role of cathepsin family members in breast cancer are limited. The aim of the present study was to identify biological markers to predict prognosis and treatment response of breast cancer patients, as well as to elucidate novel therapeutic targets. The present study analyzed the expression of six members of cathepsin family, including cathepsins B, G, D, K, L and V in 188 breast cancer tissue specimens using immunohistochemistry. The data showed that all members of the tested cathepsin families featured cytoplasmic staining. Notably, expression of cathepsin L was associated with advanced tumor stages, while cathepsins B and K expression levels were associated with positive estrogen receptor expression; in addition, cathepsin K expression was also demonstrated to be associated with progesterone receptor expression. Cathepsins V and D expression levels were found to be associated with breast cancer metastasis, while the expression levels of cathepsins B and D were associated with poor disease-free survival in breast cancer patients. In addition, univariate analysis demonstrated that breast cancer metastasis to the bone and the expression of cathepsin B protein were associated with poor disease-free survival. In conclusion, the results of the present study indicated that the altered expression of cathepsins, in particular cathepsins B and D, contributed to the progression of breast cancer and poor disease-free survival in breast cancer patients.

Deficiency for the cysteine protease cathepsin L impairs Myc-induced tumorigenesis in a mouse model of pancreatic neuroendocrine cancer.

Motivated by the recent implication of cysteine protease cathepsin L as a potential target for anti-cancer drug development, we used a conditional MycERTAM;Bcl-xL model of pancreatic neuroendocrine tumorigenesis (PNET) to assess the role of cathepsin L in Myc-induced tumor progression. By employing a cysteine cathepsin activity probe in vivo and in vitro, we first established that cathepsin activity increases during the initial stages of MycERTAM;Bcl-xL tumor development. Among the cathepsin family members investigated, only cathepsin L was predominately produced by beta-tumor cells in neoplastic pancreata and, consistent with this, cathepsin L mRNA expression was rapidly upregulated following Myc activation in the beta cell compartment. By contrast, cathepsins B, S and C were highly enriched in tumor-infiltrating leukocytes. Genetic deletion of cathepsin L had no discernible effect on the initiation of neoplastic growth or concordant angiogenesis. However, the tumors that developed in the cathepsin L-deficient background were markedly reduced in size relative to their typical wild-type counterparts, indicative of a role for cathepsin L in enabling expansive tumor growth. Thus, genetic blockade of cathepsin L activity is inferred to retard Myc-driven tumor growth, encouraging the potential utility of pharmacological inhibitors of cysteine cathepsins in treating late stage tumors.

Cathepsin S: therapeutic, diagnostic, and prognostic potential.

Cathepsin S is a member of the cysteine cathepsin protease family. It is a lysosomal protease which can promote degradation of damaged or unwanted proteins in the endo-lysosomal pathway. Additionally, it has more specific roles such as MHC class II antigen presentation, where it is important in the degradation of the invariant chain. Unsurprisingly, mis-regulation has implicated cathepsin S in a variety of pathological processes including arthritis, cancer, and cardiovascular disease, where it becomes secreted and can act on extracellular substrates. In comparison to many other cysteine cathepsin family members, cathepsin S has uniquely restricted tissue expression and is more stable at a neutral pH, which supports its involvement and importance in localised disease microenvironments. In this review, we examine the known involvement of cathepsin S in disease, particularly with respect to recent work indicating its role in mediating pain, diabetes, and cystic fibrosis. We provide an overview of current literature with regards cathepsin S as a therapeutic target, as well as its role and potential as a predictive diagnostic and/or prognostic marker in these diseases.

Clinical significance of detection of cathepsin X and cystatin C in the sera of patients with lung cancer

背景与目的组织蛋白酶X（Cathepsin X, Cat X）是最近发现的一种组织蛋白酶（Cathepsins, Cats）家族成员。近年来研究表明Cat X与多种恶性肿瘤发生、发展有关。本研究旨在探讨肺癌患者血清Cat X及cystatin C的表达与临床特征及预后的关系。方法采用ELISA法定量检测84例肺癌患者及36例健康对照者血清Cat X及cystatin C表达。结果肺癌患者血清Cat X和cystatin C水平明显高于健康人（P < 0.01）；Cat X水平与肺癌病理类型之间有相关的趋势（P=0.076）。血清cystatin C水平与肺癌TNM分期正相关（P=0.01），cystatin C/Cat X与淋巴结转移之间有相关趋势（P=0.058）。Cat X表达水平与肺癌患者总生存期（overall survival, OS）相关，高水平Cat X肺癌患者OS更短。Cox单因素回归示Cat X高表达以及TNM分期是影响肺癌预后独立因素，Cox多因素回归显示，仅TNM分期是患者预后的独立危险因素。结论肺癌患者中血清Cat X和cystatin C水平升高，检测肺癌患者Cat X和cystatin C血清水平对于指导临床肺癌诊断、评估预后有重要意义。

Manila clam, Ruditapes philippinarum Cathepsin D: Molecular analysis and immune response against brown ring disease causing Vibrio tapetis challenge

Cathepsins are lysosomal/cysteine proteases belong to papain family (C1 family) that is involved in intracellular protein degradation, antigen processing, hormone maturation, and immune responses. In this study, member of cathepsin family was identified from Manila clam (Mc-Cathepsin D) and investigated the immune response against brown ring disease (BRD) causing Vibrio tapetis challenge. The identified Mc-Cathepsin D gene encodes characteristic features typical for the cathepsin family including eukaryotic and viral aspartyl protease signature domain and two highly conserved active sites (<TEX>$^{84}VVFDTGSSNLWV^{95}$</TEX> and <TEX>$^{270}IADTGTSLLAG^{281}$</TEX>). Moreover, MC-Cathepsin D shows higher identity values (-50-70%) and conserved amino acids with known cathepsin D members. Transcriptional results (by quantitative real-time RT-PCR) showed that Mc-Cathepsin D was expressed at higher levels in gills and hemocytes than mantle, adductor muscle, foot, and siphon. After the V. tapetis challenge under laboratory conditions, Mc-Cathepsin D mRNA was up-regulated in gills and hemocytes. Present study indicates that Mc-Cathepsin D is constitutively expressed in different tissues and potentially inducible when infecting BRD by V. tapetis. It is further suggesting that Mc-Cathepsin D may be involved in multiple role including immune response reactions against BRD.

Cathepsin proteases promote angiogenic sprouting and laser-induced choroidal neovascularisation in mice

Cysteine cathepsins are a family of proteases involved in intracellular protein turnover and extracellular matrix degradation. Cathepsin B (Ctsb) and cathepsin Z (Ctsz) promote tumorigenesis and Ctsb is a known modulator of tumor angiogenesis. We therefore investigated the angiomodulatory function of these cathepsins in vitro as well as in a mouse model of laser-induced choroidal neovascularization (laser-CNV). Ctsb−/−, Ctsz−/−, Ctsb/Ctsz double-knockout (Ctsb/z DKO), and wild type (WT) mice underwent argon laser treatment to induce choroidal neovascularization (CNV). The neovascularized area was quantified individually for each lesion at 14 days after laser coagulation. In vitro the effects of cathepsin inhibitors on angiogenesis were analysed by endothelial cell (EC) spheroid sprouting and EC invadosome assays. Retinas from cathepsin KO mice did not show gross morphological abnormalities. In the laser CNV model, however, Ctsb/z DKO mice displayed a significantly reduced neovascularized area compared to WT (0.027 mm2 vs. 0.052 mm2; p = 0.012), while single knockouts did not differ significantly from WT. In line, VEGF-induced EC spheroid sprouting and invadosome formation were not significantly altered by a specific cathepsin B inhibitor alone, but significantly suppressed when more than one cathepsin was inhibited. Our results demonstrate that laser-CNV formation is significantly reduced in Ctsb/z DKO mice. In line, EC sprouting and invadosome formation are blunted when more than one cathepsin is inhibited in vitro. These results reveal an angiomodulatory potential of cathepsins with partial functional redundancies between different cathepsin family members.

Cathepsins mediate tumor metastasis

Cathepsins are highly expressed in various human cancers, associated with tumor metastasis. It is superfamily, concluding A, B, C, D, E, F, G, H, L, K, O, S, V, and W family members. As a group of lysosomal proteinases or endopeptidases, each member has a different function, playing different roles in distinct tumorigenic processes such as proliferation, angiogenesis, metastasis, and invasion. Cathepsins belong to a diverse number of enzyme subtypes, including cysteine proteases, serine proteases and aspartic proteases. The contribution of cathepsins to invasion in human cancers is well documented, although the precise mechanisms by which cathepsins exert their effects are still not clear. In the present review, the role of cathepsin family members in cancer is discussed.

Determination of cathepsin V activity and intracellular trafficking by N-glycosylation

Cathepsin V (L2), a lysosomal cysteine protease, is a member of cathepsin family, relating to cancer invasion and metastasis. Cathepsin V contains two predicted N-glycosylation sites, but it has not been reported whether cathepsin V is glycosylated or not. In this study, we clarified the role of N-glycosylation of cathepsin V for its functions. We demonstrated that cathepsin V is N-glycosylated at both Asn221 and Asn292 using mass spectrometry and site-directed mutagenesis. N-glycosylation of cathepsin V was important for transportation to lysosome, secretion, and activity in HT1080 cells. These data demonstrated that functions of cathepsin V are controlled by N-glycosylation.

Cathepsins S, L, and K and Their Pathophysiological Relevance in Obesity

Members of the cysteine protease cathepsin family play an important part in human pathophysiology. Among those, Cathepsin K has a major role in enzymatic bone degradation. Cathepsin K was recently found to be expressed in adipose tissue and increased in obesity. This led to the hypothesis that Cathepsin K and potentially other forms of cathepsin might contribute to obesity and obesity-linked complications. In this context, we identified Cathepsin S gene as one of the most deregulated gene in the adipose tissue of obese subjects, showing a strong correlation with body mass index, while Cathepsin L was also found in high abundance in the adipose tissue of diet-induced and genetically obese mice. Cathepsin K or Cathepsin L deletion in genetically modified mice reduces adiposity and ameliorates glucose homeostasis. The pharmacological inhibition of these cathepsins attenuates body weight gain both in diet-induced and in genetically obese mice and impairs adipose differentiation in cell culture experiments. These data in mice and humans enlighten the unexpected implication of cathepsin family members to promote fat accretion and contribute to the deterioration of glycemic parameters in obesity. Thus, inhibition of specific cathepsins might prove an efficient adjunct to diet and exercise in human obesity, notwithstanding it remains to decipher the cellular and molecular mechanisms implicated.

Nucleic acid recognition by Toll-like receptors is coupled to stepwise processing by cathepsins and asparagine endopeptidase

Toll-like receptor (TLR) 9 requires proteolytic processing in the endolysosome to initiate signaling in response to DNA. However, recent studies conflict as to which proteases are required for receptor cleavage. We show that TLR9 proteolysis is a multistep process. The first step removes the majority of the ectodomain and can be performed by asparagine endopeptidase (AEP) or cathepsin family members. This initial cleavage event is followed by a trimming event that is solely cathepsin mediated and required for optimal receptor signaling. This dual requirement for AEP and cathepsins is observed in all cell types that we have analyzed, including mouse macrophages and dendritic cells. In addition, we show that TLR7 and TLR3 are processed in an analogous manner. These results define the core proteolytic steps required for TLR9 function and suggest that receptor proteolysis may represent a general regulatory strategy for all TLRs involved in nucleic acid recognition.

Molecular characterization and expression analysis of Cathepsin B and L cysteine proteases from rock bream ( Oplegnathus fasciatus)

Cathepsins are lysosomal cysteine proteases of the papain family that play an important role in intracellular protein degradation and turn over within the lysosomal system. In the present study, full-length sequences of cathepsin B (RbCathepsin B) and L (RbCathepsin L) were identified after transcriptome sequencing of rock bream Oplegnathus fasciatus mixed tissue cDNA. Cathepsin B was composed of 330 amino acid residues with 36 kDa predicted molecular mass. RbCathepsin L contained 336 amino acid residues encoding for a 38 kDa predicted molecular mass protein. The sequencing analysis results showed that both cathepsin B and L contain the characteristic papain family cysteine protease signature and active sites for the eukaryotic thiol proteases of cysteine, asparagine and histidine. In addition, RbCathepsin L contained EF hand Ca 2+ binding and cathepsin propeptide inhibitor domains. The rock bream cathepsin B and L showed the highest amino acid identity of 90 and 95% to Lutjanus argentimaculatus cathepsin B and Lates calcarifer cathepsin L, respectively. By phylogenetic analysis, cathepsin B and L exhibited a high degree of evolutionary relationship to respective cathepsin family members of the papain superfamily. Quantitative real-time RT-PCR analysis results confirmed that the expression of cathepsin B and L genes was constitutive in all examined tissues isolated from un-induced rock bream. Moreover, activation of RbCathepsin B and L mRNA was observed in both lipopolysaccharide (LPS) and Edwardsiella tarda challenged liver and blood cells, indicating a role of immune response in rock bream.

Transient reduction of placental angiogenesis in PAI-1-deficient mice

Murine placentation is associated with the invasion of maternal endometrium by trophoblasts and an extensive maternal and fetal angiogenesis. Plasminogen activator inhibitor-1 (PAI-1) is transiently produced by spongiotrophoblasts and trophoblast giant cells at 10.5-11.5 days postcoitum (dpc). Knowing the key contribution of PAI-1 in the regulation of angiogenesis, we have now analyzed the consequence of PAI-1 deficiency on murine placentation. Morphological and quantitative computer-assisted image analysis revealed abnormal placental morphology in PAI-1-/- mice at 10.5 and 12.5 dpc. At 10.5 dpc, the genetic ablation of PAI-1 resulted in a transient reduction of both maternal and fetal vascularizations in the placenta and increased trophoblast cell density. This was associated with a poorer development of the labyrinth and an extension of the decidua. A larger spongiotrophoblast layer appeared at 12.5 dpc in PAI-1-deficient mice. Placental morphology was normalized at 14.5 dpc. Microarray analyses performed on laser capture microdissected labyrinths revealed that 46 genes were differentially expressed between the two genotypes at 10.5 dpc. However, only 11 genes were still differently modulated at 14.5 dpc, when normalization of placental morphology had taken place. This transcriptomic profiling highlighted a dysregulation in the expression of placenta-related cathepsin family members. Altogether our data provide evidence for a transient impaired placental morphology in PAI-1-deficient mice that is then normalized, leading to normal embryonic development.