A Nonsynonymous Substitution of Lhx3 Leads to Changes in Body Size in Dogs and Mice.

Lhx3 is a LIM-homeodomain transcription factor that affects body size in mammals by regulating the secretion of pituitary hormones. Akita, Shiba Inu, and Mame Shiba Inu dogs are Japanese native dog breeds that have different body sizes. To determine whether Lhx3 plays a role in the differing body sizes of these three dog breeds, we sequenced the Lhx3 gene in the three breeds, which led to the identification of an SNP in codon 280 (S280N) associated with body size. The allele frequency at this SNP differed significantly between the large Akita and the two kinds of smaller Shiba dogs. To validate the function of this SNP on body size, we introduced this change into the Lhx3 gene of mice. Homozygous mutant mice (S279N+/+) were found to have significantly increased body lengths and weights compared to heterozygous mutant (S279N+/-) and wild-type (S279N-/-) mice several weeks after weaning. These results demonstrate that a nonsynonymous substitution in Lhx3 plays an important role in regulating body size in mammals.

Forced LMX1A expression induces dorsal neural fates and disrupts patterning of human embryonic stem cells into ventral midbrain dopaminergic neurons.

The differentiation of human pluripotent stem cells into ventral mesencephalic dopaminergic (DA) fate is relevant for the treatment of Parkinson's disease. Shortcuts to obtaining DA cells through direct reprogramming often include forced expression of the transcription factor LMX1A. Although reprogramming with LMX1A can generate tyrosine hydroxylase (TH)-positive cells, their regional identity remains elusive. Using an in vitro model of early human neural tube patterning, we report that forced LMX1A expression induced a ventral-to-dorsal fate shift along the entire neuroaxis with the emergence of roof plate fates despite the presence of ventralizing molecules. The LMX1A-expressing progenitors gave rise to grafts containing roof plate-derived choroid plexus cysts as well as ectopically induced TH-positive neurons of a forebrain identity. Early activation of LMX1A prior to floor plate specification was necessary for the dorsalizing effect. Our work suggests using caution in employing LMX1A for the induction of DA fate, as this factor may generate roof plate rather than midbrain fates.

Structural domain in the Titin N2B-us region binds to FHL2 in a force-activation dependent manner.

Titin N2B unique sequence (N2B-us) is a 572 amino acid sequence that acts as an elastic spring to regulate muscle passive elasticity. It is thought to lack stable tertiary structures and is a force-bearing region that is regulated by mechanical stretching. In this study, the conformation of N2B-us and its interaction with four-and-a-half LIM domain protein 2 (FHL2) are investigated using AlphaFold2 predictions and single-molecule experimental validation. Surprisingly, a stable alpha/beta structural domain is predicted and confirmed in N2B-us that can be mechanically unfolded at forces of a few piconewtons. Additionally, more than twenty FHL2 LIM domain binding sites are predicted to spread throughout N2B-us. Single-molecule manipulation experiments reveals the force-dependent binding of FHL2 to the N2B-us structural domain. These findings provide insights into the mechano-sensing functions of N2B-us and its interactions with FHL2.

Lhx2 promotes axon regeneration of adult retinal ganglion cells and rescues neurodegeneration in mouse models of glaucoma.

The axons of retinal ganglion cells (RGCs) form the optic nerve, transmitting visual information from the eye to the brain. Damage or loss of RGCs and their axons is the leading cause of visual functional defects in traumatic injury and degenerative diseases such as glaucoma. However, there are no effective clinical treatments for nerve damage in these neurodegenerative diseases. Here, we report that LIM homeodomain transcription factor Lhx2 promotes RGC survival and axon regeneration in multiple animal models mimicking glaucoma disease. Furthermore, following N-methyl-D-aspartate (NMDA)-induced excitotoxicity damage of RGCs, Lhx2 mitigates the loss of visual signal transduction. Mechanistic analysis revealed that overexpression of Lhx2 supports axon regeneration by systematically regulating the transcription of regeneration-related genes and inhibiting transcription of Semaphorin 3C (Sema3C). Collectively, our studies identify a critical role of Lhx2 in promoting RGC survival and axon regeneration, providing a promising neural repair strategy for glaucomatous neurodegeneration.

Proximal tubular FHL2, a novel downstream target of hypoxia inducible factor 1, is a protector against ischemic acute kidney injury.

Four-and-a-half LIM domains protein 2 (FHL2) is an adaptor protein that may interact with hypoxia inducible factor 1α (HIF-1α) or ß-catenin, two pivotal protective signaling in acute kidney injury (AKI). However, little is known about the regulation and function of FHL2 during AKI. We found that FHL2 was induced in renal tubular cells in patients with acute tubular necrosis and mice model of ischemia-reperfusion injury (IRI). In cultured renal proximal tubular cells (PTCs), hypoxia induced FHL2 expression and promoted the binding of HIF-1 to FHL2 promoter. Compared with control littermates, mice with PTC-specific deletion of FHL2 gene displayed worse renal function, more severe morphologic lesion, more tubular cell death and less cell proliferation, accompanying by downregulation of AQP1 and Na, K-ATPase after IRI. Consistently, loss of FHL2 in PTCs restricted activation of HIF-1 and ß-catenin signaling simultaneously, leading to attenuation of glycolysis, upregulation of apoptosis-related proteins and downregulation of proliferation-related proteins during IRI. In vitro, knockdown of FHL2 suppressed hypoxia-induced activation of HIF-1α and ß-catenin signaling pathways. Overexpression of FHL2 induced physical interactions between FHL2 and HIF-1α, ß-catenin, GSK-3ß or p300, and the combination of these interactions favored the stabilization and nuclear translocation of HIF-1α and ß-catenin, enhancing their mediated gene transcription. Collectively, these findings identify FHL2 as a direct downstream target gene of HIF-1 signaling and demonstrate that FHL2 could play a critical role in protecting against ischemic AKI by promoting the activation of HIF-1 and ß-catenin signaling through the interactions with its multiple protein partners.

miR-652-3p Suppressed the Protective Effects of Isoflurane Against Myocardial Injury in Hypoxia/Reoxygenation by Targeting ISL1.

This research is concentrated on investigating the role and mechanism of miR-652-3p in the protective effects of isoflurane (ISO) against myocardial ischemia-reperfusion (I/R) injury. H9c2 cells underwent pretreatment with varying concentrations of ISO, and subsequently, a hypoxia/reoxygenation (H/R) model was constructed. The levels of miR-652-3p, ISL LIM homeobox 1 (ISL1), and inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) were evaluated through reverse transcription polymerase chain reaction (RT-qPCR). Enzyme-linked immunosorbent assay was employed to investigate concentrations of myocardial injury markers, such as creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI). Cell counting kit-8 was used to evaluate cell viability, while flow cytometry was utilized to measure apoptosis. Additionally, a dual luciferase reporter assay was conducted to validate the targeting relationship between ISL1 and miR-652-3p. Herein, we confirmed that the level of miR-652-3p was gradually increased with prolonged hypoxia; nevertheless, this increase was suppressed by ISO pretreatment (P < 0.05). Additionally, ISO pretreatment prevented the decrease in cell viability, increase in apoptosis, and overproduction of IL-6, TNF-α, CK-MB, and cTnI induced by H/R (P < 0.05). However, the inhibitory effects of ISO were counteracted by the increased levels of miR-652-3p (P < 0.05). ISL1 is a potential target of miR-652-3p. H/R induction suppressed ISL1 levels compared to the control, but ISO treatment increased its expression (P < 0.05). Overexpression of ISL1 inhibited the elimination of the protective effect of ISO on myocardial damage induced by the elevation of miR-652-3p (P < 0.05). The findings of this research confirm that miR-652-3p attenuated the protective effect of ISO on cardiomyocytes in myocardial ischemia by targeting ISL1.

LIM homeobox 8 reduced apoptosis and promoted periodontal tissue regeneration function of dental pulp stem cells.

Stem cell-mediated tissue regeneration is a promising strategy for repairing tissue defects and functional reconstruction in periodontitis, a common disease that leads to the loss of alveolar bone and teeth. However, stem cell apoptosis, widely observed during tissue regeneration, impairs its efficiency. Therefore, the regulation of stem cell apoptosis is critical for improving regeneration efficiency. The LIM homeobox 8 gene LHX8, belongs to the LIM homeobox family, which was involved in tooth morphogenesis. Here, we found that LHX8 was significantly expressed in dental pulp. LHX8 knockdown significantly increased dental pulp mesenchymal stem cells (DPSCs) apoptosis, as confirmed by RT-PCR, western blotting, flow cytometry, and transmission electron microscopy. Additionally, LHX8 overexpression inhibited apoptosis and enhanced the osteo/odontogenic differentiation potential of hDPSCs in vitro. Furthermore, LHX8-overexpression could enhance the periodontal tissue regeneration efficiency of hDPSCs in mice with periodontitis. In conclusion, the present study indicates that LHX8 inhibits stem cell apoptosis and promotes functional tissue formation in stem cell-based tissue regeneration engineering, suggesting a new therapeutic target to increase the efficacy of periodontal tissue regeneration.

A global gene regulatory program and its region-specific regulator partition neurons into commissural and ipsilateral projection types.

Understanding the genetic programs that drive neuronal diversification into classes and subclasses is key to understand nervous system development. All neurons can be classified into two types: commissural and ipsilateral, based on whether their axons cross the midline or not. However, the gene regulatory program underlying this binary division is poorly understood. We identified a pair of basic helix-loop-helix transcription factors, Nhlh1 and Nhlh2, as a global transcriptional mechanism that controls the laterality of all floor plate-crossing commissural axons in mice. Mechanistically, Nhlh1/2 play an essential role in the expression of Robo3, the key guidance molecule for commissural axon projections. This genetic program appears to be evolutionarily conserved in chick. We further discovered that Isl1, primarily expressed in ipsilateral neurons within neural tubes, negatively regulates the Robo3 induction by Nhlh1/2. Our findings elucidate a gene regulatory strategy where a conserved global mechanism intersects with neuron class-specific regulators to control the partitioning of neurons based on axon laterality.

Four and a half LIM domains 2 (FHL2) attenuates tumorigenesis of gastrointestinal stromal tumors (GISTs) by negatively regulating KIT signaling.

Gastrointestinal stromal tumors (GISTs) are predominately induced by KIT mutants. In this study, we found that four and a half LIM domains 2 (FHL2) was highly expressed in GISTs and KIT signaling dramatically increased FHL2 transcription while FHL2 inhibited KIT transcription. In addition, our results showed that FHL2 associated with KIT and increased the ubiquitination of both wild-type KIT and primary KIT mutants in GISTs, leading to decreased expression and activation of KIT although primary KIT mutants were less inhibited by FHL2 than wild-type KIT. In the animal experiments, loss of FHL2 expression in mice carrying germline KIT/V558A mutation which can develop GISTs resulted in increased tumor growth, but increased sensitivity of GISTs to imatinib treatment which is used as the first-line targeted therapy of GISTs, suggesting that FHL2 plays a role in the response of GISTs to KIT inhibitor. Unlike wild-type KIT and primary KIT mutants, we further found that FHL2 didn't alter the expression and activation of drug-resistant secondary KIT mutants. Taken together, our results indicated that FHL2 acts as the negative feedback of KIT signaling in GISTs while primary KIT mutants are less sensitive and secondary KIT mutants are resistant to the inhibition of FHL2.

ERN1 knockdown modifies the hypoxic regulation of homeobox gene expression in U87MG glioblastoma cells.

OBJECTIVE.: Homeobox genes play an important role in health and disease including oncogenesis. The present investigation aimed to study ERN1-dependent hypoxic regulation of the expression of genes encoding homeobox proteins MEIS (zinc finger E-box binding homeobox 2) and LIM homeobox 1 family, SPAG4 (sperm associated antigen 4) and NKX3-1 (NK3 homeobox 1) in U87MG glioblastoma cells in response to inhibition of ERN1 (endoplasmic reticulum to nucleus signaling 1) for evaluation of their possible significance in the control of glioblastoma growth. METHODS.: The expression level of homeobox genes was studied in control (transfected by vector) and ERN1 knockdown U87MG glioblastoma cells under hypoxia induced by dimethyloxalylglycine (0.5 mM for 4 h) by quantitative polymerase chain reaction and normalized to ACTB. RESULTS.: It was found that hypoxia down-regulated the expression level of LHX2, LHX6, MEIS2, and NKX3-1 genes but up-regulated the expression level of MEIS1, LHX1, MEIS3, and SPAG4 genes in control glioblastoma cells. At the same time, ERN1 knockdown of glioblastoma cells significantly modified the sensitivity of all studied genes to a hypoxic condition. Thus, ERN1 knockdown of glioblastoma cells removed the effect of hypoxia on the expression of MEIS1 and LHX1 genes, but increased the sensitivity of MEIS2, LHX2, and LHX6 genes to hypoxia. However, the expression of MEIS3, NKX3-1, and SPAG4 genes had decreased sensitivity to hypoxia in ERN1 knockdown glioblastoma cells. Moreover, more pronounced changes under the conditions of ERN1 inhibition were detected for the pro-oncogenic gene SPAG4. CONCLUSION.: The results of the present study demonstrate that hypoxia affected the expression of homeobox genes MEIS1, MEIS2, MEIS3, LHX1, LHX2, LHX6, SPAG4, and NKX3-1 in U87MG glioblastoma cells in gene-specific manner and that the sensitivity of all studied genes to hypoxia condition is mediated by ERN1, the major pathway of the endoplasmic reticulum stress signaling, and possibly contributed to the control of glioblastoma growth. A fundamentally new results of this work is the establishment of the fact regarding the dependence of hypoxic regulation of SPAG4 gene expression on ER stress, in particular ERN1, which is associated with suppression of cell proliferation and tumor growth.

Titin's cardiac-specific N2B element is critical to mechanotransduction during volume overload of the heart.

The heart has the ability to detect and respond to changes in mechanical load through a process called mechanotransduction. In this study, we focused on investigating the role of the cardiac-specific N2B element within the spring region of titin, which has been proposed to function as a mechanosensor. To assess its significance, we conducted experiments using N2B knockout (KO) mice and wildtype (WT) mice, subjecting them to three different conditions: 1) cardiac pressure overload induced by transverse aortic constriction (TAC), 2) volume overload caused by aortocaval fistula (ACF), and 3) exercise-induced hypertrophy through swimming. Under conditions of pressure overload (TAC), both genotypes exhibited similar hypertrophic responses. In contrast, WT mice displayed robust left ventricular hypertrophy after one week of volume overload (ACF), while the KO mice failed to undergo hypertrophy and experienced a high mortality rate. Similarly, swim exercise-induced hypertrophy was significantly reduced in the KO mice. RNA-Seq analysis revealed an abnormal ß-adrenergic response to volume overload in the KO mice, as well as a diminished response to isoproterenol-induced hypertrophy. Because it is known that the N2B element interacts with the four-and-a-half LIM domains 1 and 2 (FHL1 and FHL2) proteins, both of which have been associated with mechanotransduction, we evaluated these proteins. Interestingly, while volume-overload resulted in FHL1 protein expression levels that were comparable between KO and WT mice, FHL2 protein levels were reduced by over 90% in the KO mice compared to WT. This suggests that in response to volume overload, FHL2 might act as a signaling mediator between the N2B element and downstream signaling pathways. Overall, our study highlights the importance of the N2B element in mechanosensing during volume overload, both in physiological and pathological settings.

Higher-order thalamocortical circuits are specified by embryonic cortical progenitor types in the mouse brain.

The sensory cortex receives synaptic inputs from both first-order and higher-order thalamic nuclei. First-order inputs relay simple stimulus properties from the periphery, whereas higher-order inputs relay more complex response properties, provide contextual feedback, and modulate plasticity. Here, we reveal that a cortical neuron's higher-order input is determined by the type of progenitor from which it is derived during embryonic development. Within layer 4 (L4) of the mouse primary somatosensory cortex, neurons derived from intermediate progenitors receive stronger higher-order thalamic input and exhibit greater higher-order sensory responses. These effects result from differences in dendritic morphology and levels of the transcription factor Lhx2, which are specified by the L4 neuron's progenitor type. When this mechanism is disrupted, cortical circuits exhibit altered higher-order responses and sensory-evoked plasticity. Therefore, by following distinct trajectories, progenitor types generate diversity in thalamocortical circuitry and may provide a general mechanism for differentially routing information through the cortex.

Spatial enhancer activation influences inhibitory neuron identity during mouse embryonic development.

The mammalian telencephalon contains distinct GABAergic projection neuron and interneuron types, originating in the germinal zone of the embryonic basal ganglia. How genetic information in the germinal zone determines cell types is unclear. Here we use a combination of in vivo CRISPR perturbation, lineage tracing and ChIP-sequencing analyses and show that the transcription factor MEIS2 favors the development of projection neurons by binding enhancer regions in projection-neuron-specific genes during mouse embryonic development. MEIS2 requires the presence of the homeodomain transcription factor DLX5 to direct its functional activity toward the appropriate binding sites. In interneuron precursors, the transcription factor LHX6 represses the MEIS2-DLX5-dependent activation of projection-neuron-specific enhancers. Mutations of Meis2 result in decreased activation of regulatory enhancers, affecting GABAergic differentiation. We propose a differential binding model where the binding of transcription factors at cis-regulatory elements determines differential gene expression programs regulating cell fate specification in the mouse ganglionic eminence.

Knockdown of Lhx6 during embryonic development results in neurophysiological alterations and behavioral deficits analogous to schizophrenia in adult rats.

A decreased expression of specific interneuron subtypes, containing either the calcium binding protein parvalbumin (PV) or the neurotransmitter somatostatin (SST), are observed in the cortex and hippocampus of both patients with schizophrenia and rodent models used to study the disorder. Moreover, preclinical studies suggest that this loss of inhibitory function is a key pathological mechanism underlying the symptoms of schizophrenia. Interestingly, decreased expression of Lhx6, a key transcriptional regulator specific to the development and migration of PV and SST interneurons, is seen in human postmortem studies and following multiple developmental disruptions used to model schizophrenia preclinically. These results suggest that disruptions in interneuron development in utero may contribute to the pathology of the disorder. To recapitulate decreased Lhx6 expression during development, we used in utero electroporation to introduce an Lhx6 shRNA plasmid and knockdown Lhx6 expression in the brains of rats on gestational day 17. We then examined schizophrenia-like neurophysiological and behavioral alterations in the offspring once they reached adulthood. In utero Lhx6 knockdown resulted in increased ventral tegmental area (VTA) dopamine neuron population activity and a sex-specific increase in locomotor response to a psychotomimetic, consistent with positive symptomology of schizophrenia. However, Lhx6 knockdown had no effect on social interaction or spatial working memory, suggesting behaviors associated with negative and cognitive symptom domains were unaffected. These results suggest that knockdown of Lhx6 during development results in neurophysiological and behavioral alterations consistent with the positive symptom domain of schizophrenia in adult rats.

Excitatory Spinal Lhx9-Derived Interneurons Modulate Locomotor Frequency in Mice.

Locomotion allows us to move and interact with our surroundings. Spinal networks that control locomotion produce rhythm and left-right and flexor-extensor coordination. Several glutamatergic populations, Shox2 non-V2a, Hb9-derived interneurons, and, recently, spinocerebellar neurons have been proposed to be involved in the mouse rhythm generating networks. These cells make up only a smaller fraction of the excitatory cells in the ventral spinal cord. Here, we set out to identify additional populations of excitatory spinal neurons that may be involved in rhythm generation or other functions in the locomotor network. We use RNA sequencing from glutamatergic, non-glutamatergic, and Shox2 cells in the neonatal mice from both sexes followed by differential gene expression analyses. These analyses identified transcription factors that are highly expressed by glutamatergic spinal neurons and differentially expressed between Shox2 neurons and glutamatergic neurons. From this latter category, we identified the Lhx9-derived neurons as having a restricted spinal expression pattern with no Shox2 neuron overlap. They are purely glutamatergic and ipsilaterally projecting. Ablation of the glutamatergic transmission or acute inactivation of the neuronal activity of Lhx9-derived neurons leads to a decrease in the frequency of locomotor-like activity without change in coordination pattern. Optogenetic activation of Lhx9-derived neurons promotes locomotor-like activity and modulates the frequency of the locomotor activity. Calcium activities of Lhx9-derived neurons show strong left-right out-of-phase rhythmicity during locomotor-like activity. Our study identifies a distinct population of spinal excitatory neurons that regulates the frequency of locomotor output with a suggested role in rhythm-generation in the mouse alongside other spinal populations.

Case report: A novel R246L mutation in the LMX1B homeodomain causes isolated nephropathy in a large Chinese family.

BACKGROUND: Genetic factors contribute to chronic kidney disease (CKD) and end-stage renal disease (ESRD). Advances in genetic testing have enabled the identification of hereditary kidney diseases, including those caused by LMX1B mutations. LMX1B mutations can lead to nail-patella syndrome (NPS) or nail-patella-like renal disease (NPLRD) with only renal manifestations. CASE PRESENTATION: The proband was a 13-year-old female who was diagnosed with nephrotic syndrome at the age of 6. Then she began intermittent hormone and drug therapy. When she was 13 years old, she was admitted to our hospital due to sudden chest tightness, which progressed to end-stage kidney disease (ESRD), requiring kidney replacement therapy. Whole-Exome Sequencing (WES) results suggest the presence of LMX1B gene mutation, c.737G > T, p.Arg246Leu. Tracing her family history, we found that her father, grandmother, uncle and 2 cousins all had hematuria, or proteinuria. In addition to the grandmother, a total of 9 members of the family performed WES. The members with kidney involved all carry the mutated gene. Healthy members did not have the mutated gene. It is characterized by co-segregation of genotype and phenotype. We followed the family for 9 year, the father developed ESRD at the age of 50 and started hemodialysis treatment. The rest patients had normal renal function. No extra-renal manifestations associated with NPS were found in any member of the family. CONCLUSIONS: This study has successfully identified missense mutation, c.737G > T (p.Arg246Leu) in the homeodomain, which appears to be responsible for isolated nephropathy in the studied family. The arginine to leucine change at codon 246 likely disrupts the DNA-binding homeodomain of LMX1B. Previous research has documented 2 types of mutations at codon R246, namely R246Q and R246P, which are known to cause NPLRD. The newly discovered mutation, R246L, is likely to be another novel mutation associated with NPLRD, thus expanding the range of mutations at the crucial renal-critical codon 246 that contribute to the development of NPLRD. Furthermore, our findings suggest that any missense mutation occurring at the 246th amino acid position within the homeodomain of the LMX1B gene has the potential to lead to NPLRD.

Generation of a ISL1 homozygous knockout stem cell line (WAe009-A-1G) by episomal vector-based CRISPR/Cas9 system.

The ISL LIM homeobox 1 (ISL1) gene belongs to the LIM/homeodomain transcription factor family and plays a pivotal role in conveying multipotent and proliferative properties of cardiac precursor cells. Mutations in ISL1 are linked to congenital heart disease. To further explore ISL1's role in the human heart, we have created a homozygous ISL1 knockout (ISL1-KO) human embryonic stem cell line using the CRISPR/Cas9 system. Notably, this ISL1-KO cell line retains normal morphology, pluripotency, and karyotype. This resource serves as a valuable tool for investigating ISL1's function in cardiomyocyte differentiation.

Single-Cell Transcriptome Analysis of Small Cell Neuroendocrine Carcinoma of the Endometrium Reveals ISL1 as a Potential Biomarker for Diagnosis and Treatment.

BACKGROUND: As a dedifferentiated tumor, small cell endometrial neuroendocrine tumors (NETs) are rare and frequently diagnosed at an advanced stage with a poor prognosis. Current treatment recommendations are often extrapolated from histologically similar tumors in other sites or based on retrospective studies. The exploration for diagnostic and therapeutic markers in small cell NETs is of great significance. METHODS: In this study, we conducted single-cell RNA sequencing on a specimen obtained from a patient diagnosed with small cell endometrial neuroendocrine carcinoma (SCNEC) based on pathology. We revealed the cell map and intratumoral heterogeneity of the cancer cells through data analysis. Further, we validated the function of ISL LIM Homeobox 1 (ISL1) in vitro in an established neuroendocrine cell line. Finally, we examined the association between ISL1 and tumor staging in small cell lung cancer (SCLC) patient samples. RESULTS: We observed the significant upregulation of ISL1 expression in tumor cells that showed high expression of the neuroepithelial markers. Additionally, in vitro cell function experiments demonstrated that the high ISL1 expression group exhibited markedly higher cell proliferation and migration abilities compared to the low expression group. Finally, we showed that the expression level of ISL1 was correlated with SCLC stages. CONCLUSIONS: ISL1 protein in NETs shows promise as a potential biomarker for diagnosis and treatment.

Sall genes regulate hindlimb initiation in mouse embryos.

Vertebrate limbs start to develop as paired protrusions from the lateral plate mesoderm at specific locations of the body with forelimb buds developing anteriorly and hindlimb buds posteriorly. During the initiation process, limb progenitor cells maintain active proliferation to form protrusions and start to express Fgf10, which triggers molecular processes for outgrowth and patterning. Although both processes occur in both types of limbs, forelimbs (Tbx5), and hindlimbs (Isl1) utilize distinct transcriptional systems to trigger their development. Here, we report that Sall1 and Sall4, zinc finger transcription factor genes, regulate hindlimb initiation in mouse embryos. Compared to the 100% frequency loss of hindlimb buds in TCre; Isl1 conditional knockouts, Hoxb6Cre; Isl1 conditional knockout causes a hypomorphic phenotype with only approximately 5% of mutants lacking the hindlimb. Our previous study of SALL4 ChIP-seq showed SALL4 enrichment in an Isl1 enhancer, suggesting that SALL4 acts upstream of Isl1. Removing 1 allele of Sall4 from the hypomorphic Hoxb6Cre; Isl1 mutant background caused loss of hindlimbs, but removing both alleles caused an even higher frequency of loss of hindlimbs, suggesting a genetic interaction between Sall4 and Isl1. Furthermore, TCre-mediated conditional double knockouts of Sall1 and Sall4 displayed a loss of expression of hindlimb progenitor markers (Isl1, Pitx1, Tbx4) and failed to develop hindlimbs, demonstrating functional redundancy between Sall1 and Sall4. Our data provides genetic evidence that Sall1 and Sall4 act as master regulators of hindlimb initiation.

Epigenetic-based age prediction in blood samples: Model development.

Aging is a complex process influenced by genetic, epigenetic, and environmental factors that lead to tissue deterioration and frailty. Epigenetic mechanisms, such as DNA methylation, play a significant role in gene expression regulation and aging. This study presents a new age estimation model developed for the Turkish population using blood samples. Eight CpG sites in loci TOM1L1, ELOVL2, ASPA, FHL2, C1orf132, CCDC102B, cg07082267, and RASSF5 were selected based on their correlation with age. Methylation patterns of these sites were analyzed in blood samples from 100 volunteers, grouped into age categories (20-35, 36-55, and ≥56). Sensitivity analysis indicated a reliable performance with DNA inputs ≥1 ng. Statistical modeling, utilizing Multiple Linear Regression, underscores the reliability of the primary 6-CpG model, excluding cg07082267 and TOM1L1. This model demonstrates strong correlations with chronological age (r = 0.941) and explains 88% of the age variance with low error rates (MAE = 4.07, RMSE = 5.73 years). Validation procedures, including a training-test split and fivefold cross-validation, consistently confirm the model's accuracy and consistency. The study indicates minimal variation in error scores across age cohorts and no significant gender differences. The developed model showed strong predictive accuracy, with the ability to estimate age within certain prediction intervals. This study contributes to the age prediction by using DNA methylation patterns, which can have disparate applications, including forensic and clinical assessments.