The HH-GLI2-CKS1B network regulates the proliferation-to-maturation transition of cardiomyocytes.

Cardiomyocyte (CM) proliferation and maturation are highly linked processes, however, the extent to which these processes are controlled by a single signaling axis is unclear. Here, we show the previously undescribed role of Hedgehog (HH)-GLI2-CKS1B cascade in regulation of the toggle between CM proliferation and maturation. Here we show downregulation of GLI-signaling in adult human CM, adult murine CM, and in late-stage hiPSC-CM leading to their maturation. In early-stage hiPSC-CM, inhibition of HH- or GLI-proteins enhanced CM maturation with increased maturation indices, increased calcium handling, and transcriptome. Mechanistically, we identified CKS1B, as a new effector of GLI2 in CMs. GLI2 binds the CKS1B promoter to regulate its expression. CKS1B overexpression in late-stage hiPSC-CMs led to increased proliferation with loss of maturation in CMs. Next, analysis of datasets of patients with heart disease showed a significant enrichment of GLI2-signaling in patients with ischemic heart failure (HF) or dilated-cardiomyopathy (DCM) disease, indicating operational GLI2-signaling in the stressed heart. Thus, the Hh-GLI2-CKS1B axis regulates the proliferation-maturation transition and provides targets to enhance cardiac tissue engineering and regenerative therapies.

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.

Cis-regulatory control of mammalian Trps1 gene expression.

TRPS1 serves as the causative gene for tricho-rhino phalangeal syndrome, known for its craniofacial and skeletal abnormalities. The Trps1 gene encodes a protein that represses Wnt signaling through strong interactions with Wnt signaling inhibitors. The identification of genomic cis-acting regulatory sequences governing Trps1 expression is crucial for understanding its role in embryogenesis. Nevertheless, to date, no investigations have been conducted concerning these aspects of Trps1. To identify deeply conserved noncoding elements (CNEs) within the Trps1 locus, we employed a comparative genomics approach, utilizing slowly evolving fish such as coelacanth and spotted gar. These analyses resulted in the identification of eight CNEs in the intronic region of the Trps1 gene. Functional characterization of these CNEs in zebrafish revealed their regulatory potential in various tissues, including pectoral fins, heart, and pharyngeal arches. RNA in-situ hybridization experiments revealed concordance between the reporter expression pattern induced by the identified set of CNEs and the spatial expression pattern of the trps1 gene in zebrafish. Comparative in vivo data from zebrafish and mice for CNE7/hs919 revealed conserved functions of these enhancers. Each of these eight CNEs was further investigated in cell line-based reporter assays, revealing their repressive potential. Taken together, in vivo and in vitro assays suggest a context-dependent dual functionality for the identified set of Trps1-associated CNE enhancers. This functionally characterized set of CNE-enhancers will contribute to a more comprehensive understanding of the developmental roles of Trps1 and can aid in the identification of noncoding DNA variants associated with human diseases.

Sall4 regulates posterior trunk mesoderm development by promoting mesodermal gene expression and repressing neural genes in the mesoderm.

The trunk axial skeleton develops from paraxial mesoderm cells. Our recent study demonstrated that conditional knockout of the stem cell factor Sall4 in mice by TCre caused tail truncation and a disorganized axial skeleton posterior to the lumbar level. Based on this phenotype, we hypothesized that, in addition to the previously reported role of Sall4 in neuromesodermal progenitors, Sall4 is involved in the development of the paraxial mesoderm tissue. Analysis of gene expression and SALL4 binding suggests that Sall4 directly or indirectly regulates genes involved in presomitic mesoderm differentiation, somite formation and somite differentiation. Furthermore, ATAC-seq in TCre; Sall4 mutant posterior trunk mesoderm shows that Sall4 knockout reduces chromatin accessibility. We found that Sall4-dependent open chromatin status drives activation and repression of WNT signaling activators and repressors, respectively, to promote WNT signaling. Moreover, footprinting analysis of ATAC-seq data suggests that Sall4-dependent chromatin accessibility facilitates CTCF binding, which contributes to the repression of neural genes within the mesoderm. This study unveils multiple mechanisms by which Sall4 regulates paraxial mesoderm development by directing activation of mesodermal genes and repression of neural genes.

Sall4 restricts glycolytic metabolism in limb buds through transcriptional regulation of glycolytic enzyme genes.

Recent studies illustrate the importance of regulation of cellular metabolism, especially glycolysis and pathways branching from glycolysis, during vertebrate embryo development. For example, glycolysis generates cellular energy ATP. Glucose carbons are also directed to the pentose phosphate pathway, which is needed to sustain anabolic processes in the rapidly growing embryos. However, our understanding of the exact status of glycolytic metabolism as well as genes that regulate glycolytic metabolism are still incomplete. Sall4 is a zinc finger transcription factor that is highly expressed in undifferentiated cells in developing mouse embryos, such as blastocysts and the post-implantation epiblast. TCre; Sall4 conditional knockout mouse embryos exhibit various defects in the posterior part of the body, including hindlimbs. Using transcriptomics approaches, we found that many genes encoding glycolytic enzymes are upregulated in the posterior trunk, including the hindlimb-forming region, of Sall4 conditional knockout mouse embryos. In situ hybridization and qRT-PCR also confirmed upregulation of expression of several glycolytic genes in hindlimb buds. A fraction of those genes are bound by SALL4 at the promoters, gene bodies or distantly-located regions, suggesting that Sall4 directly regulates expression of several glycolytic enzyme genes in hindlimb buds. To further gain insight into the metabolic status associated with the observed changes at the transcriptional level, we performed a comprehensive analysis of metabolite levels in limb buds in wild type and Sall4 conditional knockout embryos by high-resolution mass spectrometry. We found that the levels of metabolic intermediates of glycolysis are lower, but glycolytic end-products pyruvate and lactate did not exhibit differences in Sall4 conditional knockout hindlimb buds. The increased expression of glycolytic genes would have caused accelerated glycolytic flow, resulting in low levels of intermediates. This condition may have prevented intermediates from being re-directed to other pathways, such as the pentose phosphate pathway. Indeed, the change in glycolytic metabolite levels is associated with reduced levels of ATP and metabolites of the pentose phosphate pathway. To further test whether glycolysis regulates limb patterning downstream of Sall4, we conditionally inactivated Hk2, which encodes a rate-limiting enzyme gene in glycolysis and is regulated by Sall4. The TCre; Hk2 conditional knockout hindlimb exhibited a short femur, and a lack of tibia and anterior digits in hindlimbs, which are defects similarly found in the TCre; Sall4 conditional knockout. The similarity of skeletal defects in Sall4 mutants and Hk2 mutants suggests that regulation of glycolysis plays a role in hindlimb patterning. These data suggest that Sall4 restricts glycolysis in limb buds and contributes to patterning and regulation of glucose carbon flow during development of limb buds.

Structural studies of SALL family protein zinc finger cluster domains in complex with DNA reveal preferential binding to an AATA tetranucleotide motif.

The Spalt-like 4 transcription factor (SALL4) plays an essential role in controlling the pluripotent property of embryonic stem cells via binding to AT-rich regions of genomic DNA, but structural details on this binding interaction have not been fully characterized. Here, we present crystal structures of the zinc finger cluster 4 (ZFC4) domain of SALL4 (SALL4ZFC4) bound with different dsDNAs containing a conserved AT-rich motif. In the structures, two zinc fingers of SALL4ZFC4 recognize an AATA tetranucleotide. We also solved the DNA-bound structures of SALL3ZFC4 and SALL4ZFC1. These structures illuminate a common preference for the AATA tetranucleotide shared by ZFC4 of SALL1, SALL3, and SALL4. Furthermore, our cell biology experiments demonstrate that the DNA-binding activity is essential for SALL4 function as DNA-binding defective mutants of mouse Sall4 failed to repress aberrant gene expression in Sall4-/- mESCs. Thus, these analyses provide new insights into the mechanisms of action underlying SALL family proteins in controlling cell fate via preferential targeting to AT-rich sites within genomic DNA during cell differentiation.

Etv2 regulates enhancer chromatin status to initiate Shh expression in the limb bud.

Sonic hedgehog (Shh) is essential for limb development, and the mechanisms that govern the propagation and maintenance of its expression has been well studied; however, the mechanisms that govern the initiation of Shh expression are incomplete. Here we report that ETV2 initiates Shh expression by changing the chromatin status of the developmental limb enhancer, ZRS. Etv2 expression precedes Shh in limb buds, and Etv2 inactivation prevents the opening of limb chromatin, including the ZRS, resulting in an absence of Shh expression. Etv2 overexpression in limb buds causes nucleosomal displacement at the ZRS, ectopic Shh expression, and polydactyly. Areas of nucleosome displacement coincide with ETS binding site clusters. ETV2 also functions as a transcriptional activator of ZRS and is antagonized by ETV4/5 repressors. Known human polydactyl mutations introduce novel ETV2 binding sites in the ZRS, suggesting that ETV2 dosage regulates ZRS activation. These studies identify ETV2 as a pioneer transcription factor (TF) regulating the onset of Shh expression, having both a chromatin regulatory role and a transcriptional activation role.

Aberrant Nuclear Translocation of E2F1 and Its Association in Cushing's Disease.

Nonsurgical medical treatments are often performed for Cushing's disease due to high recurrence rates. However, current medical treatment that targets corticotroph adenomas are limited. To develop a treatment that specifically targets corticotrophs in Cushing's disease, it is necessary to identify corticotroph lineage-specific proteins, which are involved in the Cushing's tumor phenotype. We have previously reported that the expression of E2F transcription factor 1 (E2F1), one of the cell cycle regulatory proteins, was increased in corticotrophs in Cushing's disease model mice and was involved in the regulation of POMC gene expression. Phosphorylation of Ser337 of E2F1 (pS337-E2F1) facilitates its binding to the POMC promoter, which was suggested to contribute to elevated POMC expression in corticotrophs. Here, we report that E2F1 expression is specific to the corticotroph lineage in normal human pituitaries and that the E2F1 protein is localized in the cytosol in normal corticotrophs. We show that pS337-E2F1 is localized in the nucleus specifically in Cushing's tumors, while it is localized in the perinuclear cytoplasm in the normal pituitary. This observation demonstrates that pS337 is a marker for Cushing's tumors and suggests that phosphorylation of E2F1 may be a target for developing a novel pharmacological treatment for tumorigenesis and hormone dysregulation of Cushing's disease.

A Case of Hypophysitis Associated With SARS-CoV-2 Vaccination.

Background/Objective: Although SARS-CoV-2 vaccines have been developed with multiple novel technologies and rapidly disseminated worldwide, the full profile of adverse effects has not been known. Recently, there are sporadic but increasing reports of endocrinopathy in relation to SARS-CoV-2 vaccination. Here we report a rare case of hypophysitis with acute onset of diabetes insipidus, immediately after SARS-CoV-2 vaccination. Case Report: A 48-year-old female patient had been in her usual state of health until she received the first SARS-CoV-2 vaccine. Two days after vaccination, she started to have flu-like symptoms, including severe headache and myalgia as well as persistent headache, polydipsia, and polyuria. She was diagnosed with diabetes insipidus, and magnetic resonance imaging revealed thickening of the pituitary stalk. Three months after vaccination, her symptoms had somewhat improved, but she still had pituitary stalk thickening on magnetic resonance imaging. Discussion: Given the timing of the occurrence of diabetes insipidus, we believe that the patient's hypophysitis may be associated with SARS-CoV-2 vaccination. We also found 19 cases of endocrinopathy after SARS-CoV-2 vaccination by literature search. The reported endocrine organs were the thyroid, pituitary, and adrenals. Twelve cases of diabetes were also reported. Among 3 pituitary cases, diabetes insipidus was reported only in our case. Conclusion: We report a rare case of SARS-CoV-2 vaccine-triggered hypophysitis, which led to diabetes insipidus. SARS-CoV-2 vaccine-related endocrinopathy seems, indeed, possible. Endocrinopathy is associated with infrequent complications; however, it may be underestimated in the post-SARS-CoV-2-vaccinated population. Further studies are warranted to better understand SARS-CoV-2 vaccine-related endocrinopathy.

Normal embryonic development and neonatal digit regeneration in mice overexpressing a stem cell factor, Sall4.

Sall4 encodes a transcription factor and is known to participate in the pluripotency network of embryonic stem cells. Sall4 expression is known to be high in early stage post-implantation mouse embryos. During early post-gastrulation stages, Sall4 is highly expressed in the tail bud and distal limb buds, where progenitor cells are maintained in an undifferentiated status. The expression of Sall4 is rapidly downregulated during embryonic development. We previously demonstrated that Sall4 is required for limb and posterior axial skeleton development by conditional deletion of Sall4 in the T (Brachyury) lineage. To gain insight into Sall4 functions in embryonic development and postnatal digit regeneration, we genetically overexpressed Sall4 in the mesodermal lineage by the TCre transgene and a novel knockin allele of Rosa26-loxP-stop-loxP-Sall4. In significant contrast to severe defects by Sall4 loss of function reported in previous studies, overexpression of Sall4 resulted in normal morphology and pattern in embryos and neonates. The length of limb long bones showed subtle reduction in Sall4-overexpression mice. It is known that the digit tip of neonatal mice has level-specific regenerative ability after experimental amputation. We observed Sall4 expression in the digit tip by using a sensitive Sall4-LacZ knock-in reporter expression. Sall4 overexpression did not alter the regenerative ability of the terminal phalange that normally regenerates after amputation. Moreover, Sall4 overexpression did not confer regenerative ability to the second phalange that normally does not regenerate after amputation. These genetic experiments show that overexpression of Sall4 does not alter the development of the appendicular and axial skeleton, or neonatal digit regeneration. The results suggest that Sall4 acts as a permissive factor rather than playing an instructive role.

The Roles of Dominance of the Nitric Oxide Fractions Nitrate and Nitrite in the Epilepsy-Prone EL Mouse Brain.

BACKGROUND: Oxidative stress is thought to be closely related to epileptogenesis. We have previously reported that nitric oxide (NO) levels are higher in epilepsy-prone EL mice between the ages of 3 and 8 weeks than in control mice. However, NO is divided into two fractions, nitrite (NO2) and nitrate (NO3), which appear to play different roles in epileptogenesis. METHODS: NO2 and NO3 levels were measured, in EL mice and the control mice, in the parietal cortex, which is thought to be the primary epileptogenetic center in EL mice, and measured in the hippocampus, which is thought to be the secondary center. RESULTS: NO3 levels in the hippocampus and parietal cortex of the immature EL mice (3 to 8 weeks of age) were significantly higher than those in the control mice; NO2 levels were significantly higher in the EL mice throughout the study period. The NO3 levels were significantly higher than the NO2 levels in the immature EL mice, but after the onset of ictogenesis at 10 weeks of age, the relative levels of the two fractions reversed. CONCLUSION: The reversal of the NO fraction distribution at the onset of seizures that we observed may be related to the developmental process of seizure susceptibility in the neural network of EL mice.

Two Distinctive POMC Promoters Modify Gene Expression in Cushing Disease.

CONTEXT: Mechanisms underlying pituitary corticotroph adenoma adrenocorticotropin (ACTH) production are poorly understood, yet circulating ACTH levels closely correlate with adenoma phenotype and clinical outcomes. OBJECTIVE: We characterized the 5' ends of proopiomelanocortin (POMC) gene transcripts, which encode the precursor polypeptide for ACTH, in order to investigate additional regulatory mechanisms of POMC gene transcription and ACTH production. METHODS: We examined 11 normal human pituitary tissues, 32 ACTH-secreting tumors, as well as 6 silent corticotroph adenomas (SCAs) that immunostain for but do not secrete ACTH. RESULTS: We identified a novel regulatory region located near the intron 2/exon 3 junction in the human POMC gene, which functions as a second promoter and an enhancer. In vitro experiments demonstrated that CREB binds the second promoter and regulates its transcriptional activity. The second promoter is highly methylated in SCAs, partially demethylated in normal pituitary tissue, and highly demethylated in pituitary and ectopic ACTH-secreting tumors. In contrast, the first promoter is demethylated in all POMC-expressing cells and is highly demethylated only in pituitary ACTH-secreting tumors harboring the ubiquitin-specific protease 8 (USP8) mutation. Demethylation patterns of the second promoter correlate with clinical phenotypes of Cushing disease. CONCLUSION: We identified a second POMC promoter regulated by methylation status in ACTH-secreting pituitary tumors. Our findings open new avenues for elucidating subcellular regulation of the hypothalamic-pituitary-adrenal axis and suggest the second POMC promoter may be a target for therapeutic intervention to suppress excess ACTH production.

Acute elevation of interleukin 6 and matrix metalloproteinase 9 during the onset of pituitary apoplexy in Cushing's disease.

PURPOSE: Pituitary apoplexy is a rare endocrine emergency. The purpose of this study is to characterize physiological changes involved in pituitary apoplexy, especially during the acute phase. METHODS: A Cushing's disease patient experienced corticotroph releasing hormone (CRH)-induced pituitary apoplexy during inferior petrosal sinus sampling (IPSS). The IPSS blood samples from the Cushing's disease patient were retrospectively analyzed for cytokine markers. For comparison, we also analyzed cytokine markers in blood samples from two pituitary ACTH-secreting microadenoma patients and one patient with an ectopic ACTH-secreting tumor. RESULTS: Acute elevation of interleukin 6 (IL-6) and matrix metalloproteinase 9 (MMP9) was observed in the IPSS blood sample on the apoplectic hemorrhagic site of the tumor. In contrast, such a change was not observed in the blood samples from the contralateral side of the apoplexy patient and in other IPSS samples from two non-apoplexy Cushing's disease patient and a patient with ectopic Cushing's syndrome. CONCLUSION: IL-6 and MMP9 may be involved in the acute process of pituitary apoplexy in Cushing's disease.

Multimodal Non-Surgical Treatments of Aggressive Pituitary Tumors.

Up to 35% of aggressive pituitary tumors recur and significantly affect mortality and quality of life. Management can be challenging and often requires multimodal treatment. Current treatment options, including surgery, conventional medical therapies such as dopamine agonists, somatostatin receptor agonists and radiotherapy, often fail to inhibit pituitary tumor growth. Recently, anti-tumor effects of chemotherapeutic drugs such as Temozolomide, Capecitabine, and Everolimus, as well as peptide receptor radionuclide therapy on aggressive pituitary tumors have been increasingly investigated and yield mixed, although sometimes promising, outcomes. The purpose of this review is to provide thorough information on non-surgical medical therapies and their efficacies and used protocols for aggressive pituitary adenomas from pre-clinical level to clinical use.

The FGF-AKT pathway is necessary for cardiomyocyte survival for heart regeneration in zebrafish.

Zebrafish have a remarkable ability to regenerate the myocardium after injury by proliferation of pre-existing cardiomyocytes. Fibroblast growth factor (FGF) signaling is known to play a critical role in zebrafish heart regeneration through promotion of neovascularization of the regenerating myocardium. Here, we define an additional function of FGF signaling in the zebrafish myocardium after injury. We find that FGF signaling is active in a small fraction of cardiomyocytes before injury, and that the number of FGF signaling-positive cardiomyocytes increases after amputation-induced injury. We show that ERK phosphorylation is prominent in endothelial cells, but not in cardiomyocytes. In contrast, basal levels of phospho-AKT positive cardiomyocytes are detected before injury, and the ratio of phosphorylated AKT-positive cardiomyocytes increases after injury, indicating a role of AKT signaling in cardiomyocytes following injury. Inhibition of FGF signaling reduced the number of phosphorylated AKT-positive cardiomyocytes and increased cardiomyocyte death without injury. Heart injury did not induce cardiomyocyte death; however, heart injury in combination with inhibition of FGF signaling caused significant increase in cardiomyocyte death. Pharmacological inhibition of AKT signaling after heart injury also caused increased cardiomyocyte death. Our data support the idea that FGF-AKT signaling-dependent cardiomyocyte survival is necessary for subsequent heart regeneration.

A nontoxic fungal natural product modulates fin regeneration in zebrafish larvae upstream of FGF-WNT developmental signaling.

BACKGROUND: The regeneration of larvae zebrafish fin emerged as a new model of regeneration in the last decade. In contrast to genetic tools to study fin regeneration, chemical probes to modulate and interrogate regeneration processes are not well developed. RESULTS: We set up a zebrafish larvae fin regeneration assay system and tested activities of natural product compounds and extracts, prepared from various microbes. Colomitide C, a recently isolated product from a fungus obtained from Antarctica, inhibited larvae fin regeneration. Using fluorescent reporter transgenic lines, we show that colomitide C inhibited fibroblast growth factor (FGF) signaling and WNT/ß-catenin signaling, which were activated after larvae fin amputation. By using the endothelial cell reporter line and immunofluorescence, we showed that colomitide C did not affect migration of the blood vessel and nerve into the injured larvae fin. Colomitide C did not show any cytotoxic activities when tested against FGF receptor-amplified human cancer cell lines. CONCLUSION: Colomitide C, a natural product, modulated larvae fin regeneration likely acting upstream of FGF and WNT signaling. Colomitide C may serve as a template for developing new chemical probes to study regeneration and other biological processes.

IRX3/5 regulate mitotic chromatid segregation and limb bud shape.

Pattern formation is influenced by transcriptional regulation as well as by morphogenetic mechanisms that shape organ primordia, although factors that link these processes remain under-appreciated. Here we show that, apart from their established transcriptional roles in pattern formation, IRX3/5 help to shape the limb bud primordium by promoting the separation and intercalation of dividing mesodermal cells. Surprisingly, IRX3/5 are required for appropriate cell cycle progression and chromatid segregation during mitosis, possibly in a nontranscriptional manner. IRX3/5 associate with, promote the abundance of, and share overlapping functions with co-regulators of cell division such as the cohesin subunits SMC1, SMC3, NIPBL and CUX1. The findings imply that IRX3/5 coordinate early limb bud morphogenesis with skeletal pattern formation.

A Rare Case of Recurrent Pituitary Collision Tumors.

Pituitary collision tumors are sporadically reported and rare. We present a case of pituitary collision tumors with nonfunctioning pituitary adenoma (NFPA) and craniopharyngioma. In order to look for any common activated pathway, we examined WNT/ß-CATENIN signaling activation, known to be involved in tumorigenesis in both craniopharyngioma and NFPA. We found nuclear accumulation of ß-CATENIN protein and expression of LEF1 protein, markers of active ß-CATENIN signaling in the craniopharyngioma but not in the pituitary adenomas. In our case, the NFPA is invasive macroadenoma, which is a frequently identified type of pituitary adenoma in collision tumor cases. Recurrence of this tumor was first observed after 8 years of follow-up. Based on this case, we suggest that pituitary collision tumors require long-term follow-up.

Development of the Proximal-Anterior Skeletal Elements in the Mouse Hindlimb Is Regulated by a Transcriptional and Signaling Network Controlled by Sall4.

The vertebrate limb serves as an experimental paradigm to study mechanisms that regulate development of the stereotypical skeletal elements. In this study, we simultaneously inactivated Sall4 using Hoxb6Cre and Plzf in mouse embryos, and found that their combined function regulates development of the proximal-anterior skeletal elements in hindlimbs. The Sall4; Plzf double knockout exhibits severe defects in the femur, tibia, and anterior digits, distinct defects compared to other allelic series of Sall4; Plzf We found that Sall4 regulates Plzf expression prior to hindlimb outgrowth. Further expression analysis indicated that Hox10 genes and GLI3 are severely downregulated in the Sall4; Plzf double knockout hindlimb bud. In contrast, PLZF expression is reduced but detectable in Sall4; Gli3 double knockout limb buds, and SALL4 is expressed in the Plzf; Gli3 double knockout limb buds. These results indicate that Plzf, Gli3, and Hox10 genes downstream of Sall4, regulate femur and tibia development. In the autopod, we show that Sall4 negatively regulates Hedgehog signaling, which allows for development of the most anterior digit. Collectively, our study illustrates genetic systems that regulate development of the proximal-anterior skeletal elements in hindlimbs.

Tuba8 Drives Differentiation of Cortical Radial Glia into Apical Intermediate Progenitors by Tuning Modifications of Tubulin C Termini.

Most adult neurons and glia originate from radial glial progenitors (RGs), a type of stem cell typically extending from the apical to the basal side of the developing cortex. Precise regulation of the choice between RG self-renewal and differentiation is critical for normal development, but the mechanisms underlying this transition remain elusive. We show that the non-canonical tubulin Tuba8, transiently expressed in cortical progenitors, drives differentiation of RGs into apical intermediate progenitors, a more restricted progenitor type lacking attachment to the basal lamina. This effect depends on the unique C-terminal sequence of Tuba8 that antagonizes tubulin tyrosination and Δ2 cleavage, two post-translational modifications (PTMs) essential for RG fiber maintenance and the switch between direct and indirect neurogenesis and ultimately distinct neuronal lineage outcomes. Our work uncovers an instructive role of a developmentally regulated tubulin isotype in progenitor differentiation and provides new insights into biological functions of the cellular tubulin PTM "code."