40†years of the homeobox: either it is wrong or it is quite interesting.

Many genes that regulate development share a 180 bp DNA sequence, called the homeobox, encoding a 60 amino acid DNA-binding domain ( McGinnis et al., 1984c; Scott and Weiner, 1984). Because the homeobox is long enough to hybridize to related, but different, genes, it has been a powerful tool for discovering developmental regulators. This year is the 40th anniversary of the first homeobox report. Here, I describe work carried out at Indiana University that led to the discovery of the homeobox. The accompanying Perspective from McGinnis and Levine describes the independent discovery made at the Biozentrum in Basel ( McGinnis and Levine, 2024). At the time, the competition was lively but, as we all met each other - and realized that no one cares more about your work than competitors - we fortunately became friends and have enjoyed many years of following and respecting each other's work.

Common Regulatory Targets of NFIA, NFIX and NFIB during Postnatal Cerebellar Development.

Transcriptional regulation plays a central role in controlling neural stem and progenitor cell proliferation and differentiation during neurogenesis. For instance, transcription factors from the nuclear factor I (NFI) family have been shown to co-ordinate neural stem and progenitor cell differentiation within multiple regions of the embryonic nervous system, including the neocortex, hippocampus, spinal cord and cerebellum. Knockout of individual Nfi genes culminates in similar phenotypes, suggestive of common target genes for these transcription factors. However, whether or not the NFI family regulates common suites of genes remains poorly defined. Here, we use granule neuron precursors (GNPs) of the postnatal murine cerebellum as a model system to analyse regulatory targets of three members of the NFI family: NFIA, NFIB and NFIX. By integrating transcriptomic profiling (RNA-seq) of Nfia- and Nfix-deficient GNPs with epigenomic profiling (ChIP-seq against NFIA, NFIB and NFIX, and DNase I hypersensitivity assays), we reveal that these transcription factors share a large set of potential transcriptional targets, suggestive of complementary roles for these NFI family members in promoting neural development.

Neuropilins are positive regulators of Hedgehog signal transduction.

The Hedgehog (Hh) pathway is essential for vertebrate embryogenesis, and excessive Hh target gene activation can cause cancer in humans. Here we show that Neuropilin 1 (Nrp1) and Nrp2, transmembrane proteins with roles in axon guidance and vascular endothelial growth factor (VEGF) signaling, are important positive regulators of Hh signal transduction. Nrps are expressed at times and locations of active Hh signal transduction during mouse development. Using cell lines lacking key Hh pathway components, we show that Nrps mediate Hh transduction between activated Smoothened (Smo) protein and the negative regulator Suppressor of Fused (SuFu). Nrp1 transcription is induced by Hh signaling, and Nrp1 overexpression increases maximal Hh target gene activation, indicating the existence of a positive feedback circuit. The regulation of Hh signal transduction by Nrps is conserved between mammals and bony fish, as we show that morpholinos targeting the Nrp zebrafish ortholog nrp1a produce a specific and highly penetrant Hh pathway loss-of-function phenotype. These findings enhance our knowledge of Hh pathway regulation and provide evidence for a conserved nexus between Nrps and this important developmental signaling system.

Single-molecule imaging of Hedgehog pathway protein Smoothened in primary cilia reveals binding events regulated by Patched1.

Accumulation of the signaling protein Smoothened (Smo) in the membrane of primary cilia is an essential step in Hedgehog (Hh) signal transduction, yet the molecular mechanisms of Smo movement and localization are poorly understood. Using ultrasensitive single-molecule tracking with high spatial/temporal precision (30 nm/10 ms), we discovered that binding events disrupt the primarily diffusive movement of Smo in cilia at an array of sites near the base. The affinity of Smo for these binding sites was modulated by the Hh pathway activation state. Activation, by either a ligand or genetic loss of the negatively acting Hh receptor Patched-1 (Ptch), reduced the affinity and frequency of Smo binding at the base. Our findings quantify activation-dependent changes in Smo dynamics in cilia and highlight a previously unknown step in Hh pathway activation.

The output of Hedgehog signaling is controlled by the dynamic association between Suppressor of Fused and the Gli proteins.

The transcriptional program orchestrated by Hedgehog signaling depends on the Gli family of transcription factors. Gli proteins can be converted to either transcriptional activators or truncated transcriptional repressors. We show that the interaction between Gli3 and Suppressor of Fused (Sufu) regulates the formation of either repressor or activator forms of Gli3. In the absence of signaling, Sufu restrains Gli3 in the cytoplasm, promoting its processing into a repressor. Initiation of signaling triggers the dissociation of Sufu from Gli3. This event prevents formation of the repressor and instead allows Gli3 to enter the nucleus, where it is converted into a labile, differentially phosphorylated transcriptional activator. This key dissociation event depends on Kif3a, a kinesin motor required for the function of primary cilia. We propose that the Sufu-Gli3 interaction is a major control point in the Hedgehog pathway, a pathway that plays important roles in both development and cancer.

Multiple Surface Regions on the Niemann-Pick C2 Protein Facilitate Intracellular Cholesterol Transport.

The cholesterol storage disorder Niemann-Pick type C (NPC) disease is caused by defects in either of two late endosomal/lysosomal proteins, NPC1 and NPC2. NPC2 is a 16-kDa soluble protein that binds cholesterol in a 1:1 stoichiometry and can transfer cholesterol between membranes by a mechanism that involves protein-membrane interactions. To examine the structural basis of NPC2 function in cholesterol trafficking, a series of point mutations were generated across the surface of the protein. Several NPC2 mutants exhibited deficient sterol transport properties in a set of fluorescence-based assays. Notably, these mutants were also unable to promote egress of accumulated intracellular cholesterol from npc2(-/-) fibroblasts. The mutations mapped to several regions on the protein surface, suggesting that NPC2 can bind to more than one membrane simultaneously. Indeed, we have previously demonstrated that WT NPC2 promotes vesicle-vesicle interactions. These interactions were abrogated, however, by mutations causing defective sterol transfer properties. Molecular modeling shows that NPC2 is highly plastic, with several intense positively charged regions across the surface that could interact favorably with negatively charged membrane phospholipids. The point mutations generated in this study caused changes in NPC2 surface charge distribution with minimal conformational changes. The plasticity, coupled with membrane flexibility, probably allows for multiple cholesterol transfer routes. Thus, we hypothesize that, in part, NPC2 rapidly traffics cholesterol between closely appositioned membranes within the multilamellar interior of late endosomal/lysosomal proteins, ultimately effecting cholesterol egress from this compartment.

Engineered knottin peptide enables noninvasive optical imaging of intracranial medulloblastoma.

Central nervous system tumors carry grave clinical prognoses due to limited effectiveness of surgical resection, radiation, and chemotherapy. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. We demonstrate that mouse cerebellar medulloblastoma (MB) can be targeted and illuminated with a fluorescent, engineered cystine knot (knottin) peptide that binds with high affinity to αvß3, αvß5, and α5ß1 integrin receptors. This integrin-binding knottin peptide, denoted EETI 2.5F, was evaluated as a molecular imaging probe in both orthotopic and genetic models of MB. Following tail vein injection, fluorescence arising from dye-conjugated EETI 2.5F was localized to the tumor compared with the normal surrounding brain tissue, as measured by optical imaging. The imaging signal intensity correlated with tumor volume. Due to its unique ability to bind to α5ß1 integrin, EETI 2.5F showed superior in vivo and ex vivo brain tumor imaging contrast compared with other engineered integrin-binding knottin peptides and with c(RGDfK), a well-studied integrin-binding peptidomimetic. Next, EETI 2.5F was fused to an antibody fragment crystallizable (Fc) domain (EETI 2.5F-Fc) to determine if a larger integrin-binding protein could also target intracranial brain tumors. EETI 2.5F-Fc, conjugated to a fluorescent dye, illuminated MB following i.v. injection and was able to distribute throughout the tumor parenchyma. In contrast, brain tumor imaging signals were not detected in mice injected with EETI 2.5F proteins containing a scrambled integrin-binding sequence, demonstrating the importance of target specificity. These results highlight the potential of using EETI 2.5F and EETI 2.5-Fc as targeted molecular probes for brain tumor imaging.

Wing tips: The wing disc as a platform for studying Hedgehog signaling.

Hedgehog (Hh) signal transduction is necessary for the development of most mammalian tissues and can go awry and cause birth defects or cancer. Hh signaling was initially described in Drosophila, and much of what we know today about mammalian Hh signaling was directly guided by discoveries in the fly. Indeed, Hh signaling is a wonderful example of the use of non-vertebrate model organisms to make basic discoveries that lead to new disease treatment. The first pharmaceutical to treat hyperactive Hh signaling in Basal Cell Carcinoma was released in 2012, approximately 30 years after the isolation of Hh mutants in Drosophila. The study of Hh signaling has been greatly facilitated by the imaginal wing disc, a tissue with terrific experimental advantages. Studies using the wing disc have led to an understanding of Hh ligand processing, packaging into particles for transmission, secretion, reception, signal transduction, target gene activation, and tissue patterning. Here we describe the imaginal wing disc, how Hh patterns this tissue, and provide methods to use wing discs to study Hh signaling in Drosophila. The tools and approaches we highlight form the cornerstone of research efforts in many laboratories that use Drosophila to study Hh signaling, and are essential for ongoing discoveries.

Neuronal and epithelial cell rescue resolves chronic systemic inflammation in the lipid storage disorder Niemann-Pick C.

Chronic systemic inflammation is thought to be a major contributor to metabolic and neurodegenerative diseases. Since inflammatory components are shared among different disorders, targeting inflammation is an attractive option for mitigating disease. To test the significance of inflammation in the lipid storage disorder (LSD) Niemann-Pick C (NPC), we deleted the macrophage inflammatory gene Mip1a/Ccl3 from NPC diseased mice. Deletion of Ccl3 had been reported to delay neuronal loss in Sandhoff LSD mice by inhibiting macrophage infiltration. For NPC mice, in contrast, deleting Ccl3 did not retard neurodegeneration and worsened the clinical outcome. Depletion of visceral tissue macrophages also did not alter central nervous system (CNS) pathology and instead increased liver injury, suggesting a limited macrophage infiltration response into the CNS and a beneficial role of macrophage activity in visceral tissue. Prevention of neuron loss or liver injury, even at late stages in the disease, was achieved through specific rescue of NPC disease in neurons or in liver epithelial cells, respectively. Local epithelial cell correction was also sufficient to reduce the macrophage-associated pathology in lung tissue. These results demonstrate that elevated inflammation and macrophage activity does not necessarily contribute to neurodegeneration and tissue injury, and LSD defects in immune cells may not preclude an appropriate inflammatory response. We conclude that inflammation remains secondary to neuronal and epithelial cell dysfunction and does not irreversibly contribute to the pathogenic cascade in NPC disease. Without further exploration of possible beneficial roles of inflammatory mediators, targeting inflammation may not be therapeutically effective at ameliorating disease severity.

Mutations in prickle orthologs cause seizures in flies, mice, and humans.

Epilepsy is heritable, yet few causative gene mutations have been identified, and thus far no human epilepsy gene mutations have been found to produce seizures in invertebrates. Here we show that mutations in prickle genes are associated with seizures in humans, mice, and flies. We identified human epilepsy patients with heterozygous mutations in either PRICKLE1 or PRICKLE2. In overexpression assays in zebrafish, prickle mutations resulted in aberrant prickle function. A seizure phenotype was present in the Prickle1-null mutant mouse, two Prickle1 point mutant (missense and nonsense) mice, and a Prickle2-null mutant mouse. Drosophila with prickle mutations displayed seizures that were responsive to anti-epileptic medication, and homozygous mutant embryos showed neuronal defects. These results suggest that prickle mutations have caused seizures throughout evolution.

Patching the gaps in Hedgehog signalling.

The Hedgehog (Hh) pathway plays central roles in animal development and stem-cell function. Defects in Hh signalling lead to birth defects and cancer in humans. The first and often genetically damaged step in this pathway is the interaction between two membrane proteins - Patched (Ptc), encoded by a tumour suppressor gene, and Smoothened (Smo), encoded by a proto-oncogene. Recent work linking Hh signalling to sterol metabolites and protein-trafficking events at the primary cilium promises to shed light on the biochemical basis of how Patched inhibits Smoothened, and to provide new avenues for cancer treatment.

Full p53 transcriptional activation potential is dispensable for tumor suppression in diverse lineages.

Over half of all human cancers, of a wide variety of types, sustain mutations in the p53 tumor suppressor gene. Although p53 limits tumorigenesis through the induction of apoptosis or cell cycle arrest, its molecular mechanism of action in tumor suppression has been elusive. The best-characterized p53 activity in vitro is as a transcriptional activator, but the identification of numerous additional p53 biochemical activities in vitro has made it unclear which mechanism accounts for tumor suppression. Here, we assess the importance of transcriptional activation for p53 tumor suppression function in vivo in several tissues, using a knock-in mouse strain expressing a p53 mutant compromised for transcriptional activation, p53(25,26). p53(25,26) is severely impaired for the transactivation of numerous classical p53 target genes, including p21, Noxa, and Puma, but it retains the ability to activate a small subset of p53 target genes, including Bax. Surprisingly, p53(25,26) can nonetheless suppress tumor growth in cancers derived from the epithelial, mesenchymal, central nervous system, and lymphoid lineages. Therefore, full transactivation of most p53 target genes is dispensable for p53 tumor suppressor function in a range of tissue types. In contrast, a transcriptional activation mutant that is completely defective for transactivation, p53(25,26,53,54), fails to suppress tumor development. These findings demonstrate that transcriptional activation is indeed broadly critical for p53 tumor suppressor function, although this requirement reflects the limited transcriptional activity observed with p53(25,26) rather than robust transactivation of a full complement of p53 target genes.

Twelve tips for teaching with ultrasound in the undergraduate curriculum.

With ever increasing use of imaging as a diagnostic tool in medicine, medical schools are being urged to further integrate imaging into their curricula. Ultrasound is one such way of doing so-enabling students to bridge the gap between form and function, medical school and clinical practice. As a non-invasive imaging technique, with low risk when compared to other methods of imaging, ultrasound is ideal for integration into basic science and clinical teaching. The twelve tips given in this article offer advice on the practicalities of running a successful ultrasound imaging session in an appropriate environment, promoting safety and curriculum integration.

Kif3a is necessary for initiation and maintenance of medulloblastoma.

Medulloblastoma (MB) cells arise from granule neuron precursors (GNPs) that have lost growth control. During normal development, GNPs divide in response to Sonic hedgehog (SHH), a ligand that binds to the patched (PTCH) receptor on GNPs. If one copy of the Ptch gene is lost, as in human Gorlin's syndrome and in Ptch(+/-) mice, MBs may form. Proper transduction of the SHH signal critically depends on primary cilia. Loss of primary cilia results in improper signal reception and failure to properly activate SHH target genes. KIF3a, part of a kinesin motor, is required for formation of primary cilia. Here, we use tamoxifen-induced ablation of Kif3a in GNPs of postnatal Ptch(+/-) mouse cerebella to show that KIF3a is necessary for MB formation. To investigate the importance of primary cilia in established tumors, we deleted Kif3a from cultured cells and from tumor cell grafts. The loss of Kif3a from established tumors led to their growth arrest and regression. MBs behave as if they are addicted to the presence of primary cilia. These results underscore the potential utility of agents that disrupt cilia for the treatment of Hh pathway-related MBs.

Neuropilin-2 contributes to tumorigenicity in a mouse model of Hedgehog pathway medulloblastoma.

The Hedgehog (Hh) signaling pathway has been implicated in the most common childhood brain tumor, medulloblastoma (MB). Given the toxicity of post-surgical treatments for MB, continued need exists for new, targeted therapies. Based upon our finding that Neuropilin (Nrp) transmembrane proteins are required for Hh signal transduction, we investigated the role of Nrp in MB cells. Cultured cells derived from a mouse Ptch (+/-) ;LacZ MB (Med1-MB), effectively modeled the Hh pathway-related subcategory of human MBs in vitro. Med1-MB cells maintained constitutively active Hh target gene transcription, and consistently formed tumors within one month after injection into mouse cerebella. The proliferation rate of Med1-MBs in culture was dependent upon Nrp2, while reducing Nrp1 function had little effect. Knockdown of Nrp2 prior to cell implantation significantly increased mouse survival, compared to transfection with a non-targeting siRNA. Knocking down Nrp2 specifically in MB cells avoided any direct effect on tumor vascularization. Nrp2 should be further investigated as a potential target for adjuvant therapy in patients with MB.

Hedgehog pathway-regulated gene networks in cerebellum development and tumorigenesis.

Many genes initially identified for their roles in cell fate determination or signaling during development can have a significant impact on tumorigenesis. In the developing cerebellum, Sonic hedgehog (Shh) stimulates the proliferation of granule neuron precursor cells (GNPs) by activating the Gli transcription factors. Inappropriate activation of Shh target genes results in unrestrained cell division and eventually medulloblastoma, the most common pediatric brain malignancy. We find dramatic differences in the gene networks that are directly driven by the Gli1 transcription factor in GNPs and medulloblastoma. Gli1 binding location analysis revealed hundreds of genomic loci bound by Gli1 in normal and cancer cells. Only one third of the genes bound by Gli1 in GNPs were also bound in tumor cells. Correlation with gene expression levels indicated that 116 genes were preferentially transcribed in tumors, whereas 132 genes were target genes in both GNPs and medulloblastoma. Quantitative PCR and in situ hybridization for some putative target genes support their direct regulation by Gli. The results indicate that transformation of normal GNPs into deadly tumor cells is accompanied by a distinct set of Gli-regulated genes and may provide candidates for targeted therapies.

Anatomically defined neuron-based rescue of neurodegenerative Niemann-Pick type C disorder.

Niemann-Pick type C disease is a fatal lysosomal storage disorder caused by loss of NPC1 function. The disorder severely affects multiple body systems, particularly the nervous system. To test whether rescue of NPC1 activity in neurons, astrocytes, or other cell types can correct the neurological defects, a Tet-inducible Npc1-YFP transgene was introduced into Npc1(-/-) mice for the cell type-specific rescue of NPC1 loss. NPC1-YFP produced in neurons prevented neuron degeneration, slowed reactive glial activity, and ameliorated the disease. NPC1-YFP produced in astrocytes or in cells of visceral tissue did not. These results suggest that loss of NPC1 activity from neurons is the primary cause of the neuropathology and that rescue of NPC1 function in neurons is sufficient to mitigate the disease. The ability of neurons to survive and function in a cell-autonomous fashion allowed the use of this newly engineered rescue system to further define the brain regions or neuron populations required to ameliorate a neurological symptom. NPC1-YFP produced specifically in cerebellar Purkinje neurons reduced ataxia, increased weight, and prolonged life, but it did not prevent the eventual decline and premature death of Npc1(-/-) mice. Significant increase in lifespan correlated with sustained reduction of inflammation in the thalamus. Neuron rescue of other forebrain areas provided little benefit. Future work targeting increasingly discrete neuronal networks should reveal which CNS areas are critical for survival. This work may have broad implications for understanding the anatomical and cellular basis of neurological signs and symptoms of other neurodegenerative and lysosomal disorders.

The cholesterol trafficking protein NPC1 is required for Drosophila spermatogenesis.

Niemann-Pick C (NPC) disease is a lethal neurodegenerative disorder affecting cellular sterol trafficking. Besides neurodegeneration, NPC patients also exhibit other pleiotropic conditions, indicating that NPC protein is required for other physiological processes. Previous studies indicated that a sterol shortage that in turn leads to a shortage of steroid hormones (for example, ecdysone in Drosophila) is likely to be the cause of NPC disease pathology. We have shown that mutations in Drosophila npc1, one of the two NPC disease-related genes, leads to larval lethal and male infertility. Here, we reported that npc1 mutants are defective in spermatogenesis and in particular in the membrane-remodeling individualization process. Interestingly, we found that ecdysone, the steroid hormone responsible for the larval lethal phenotype in npc1 mutants, is not required for individualization. However, supplying 7-dehydrocholesterol can partially rescue the male infertility of npc1 mutants, suggesting that a sterol shortage is responsible for the spermatogenesis defects. In addition, the individualization defects of npc1 mutants were enhanced at high temperature, suggesting that the sterol shortage may lead to temperature-sensitive defects in the membrane-remodeling process. Together, our study reveals a sterol-dependent, ecdysone-independent mechanism of NPC1 function in Drosophila spermatogenesis.

Complement is dispensable for neurodegeneration in Niemann-Pick disease type C.

BACKGROUND: The immune system has been implicated in neurodegeneration during development and disease. In various studies, the absence of complement (that is, C1q deficiency) impeded the elimination of apoptotic neurons, allowing survival. In the genetic lysosomal storage disease Niemann-Pick C (NPC), caused by loss of NPC1 function, the expression of complement system components, C1q especially, is elevated in degenerating brain regions of Npc1-/- mice. Here we test whether complement is mediating neurodegeneration in NPC disease. FINDINGS: In normal mature mice, C1q mRNA was found in neurons, particularly cerebellar Purkinje neurons (PNs). In Npc1-/- mice, C1q mRNA was additionally found in activated microglia, which accumulate during disease progression and PN loss. Interestingly, C1q was not enriched on or near degenerating neurons. Instead, C1q was concentrated in other brain regions, where it partially co-localized with a potential C1q inhibitor, chondroitin sulfate proteoglycan (CSPG). Genetic deletion of C1q, or of the downstream complement pathway component C3, did not significantly alter patterned neuron loss or disease progression. Deletion of other immune response factors, a Toll-like receptor, a matrix metalloprotease, or the apoptosis facilitator BIM, also failed to alter neuron loss. CONCLUSION: We conclude that complement is not involved in the death and clearance of neurons in NPC disease. This study supports a view of neuroinflammation as a secondary response with non-causal relationship to neuron injury in the disease. This disease model may prove useful for understanding the conditions in which complement and immunity do contribute to neurodegeneration in other disorders.

Hedgehog signal transduction by Smoothened: pharmacologic evidence for a 2-step activation process.

The Hedgehog (Hh) signaling pathway controls growth, cell fate decisions, and morphogenesis during development. Damage to Hh transduction machinery can lead to birth defects and cancer. The transmembrane protein Smoothened (Smo) relays the Hh signal and is an important drug target in cancer. Smo enrichment in primary cilia is thought to drive activation of target genes. Using small-molecule agonists and antagonists to dissect Smo function, we find that Smo enrichment in cilia is not sufficient for signaling and a distinct second step is required for full activation. This 2-step mechanism--localization followed by activation--has direct implications for the design and use of anticancer therapeutics targeted against Smo.