A third of patients with the pediatric cerebellar tumor Medulloblastoma (MB) have mutations that activate Sonic hedgehog (SHH) signaling (SHH-MB subgroup). The contribution of secondary mutations to tumor severity, however is not clear. PTEN mutations are enriched in the SHH-1 subtype that has the lowest survival rate. Widespread heterozygous loss of Pten in two SHH-MB mouse models increases penetrance and excellerates onset of differentiated tumors. We delineated cellular and transcriptional changes that accelerate tumor growth and cause differentiation using a sporadic SHH-MB mouse model expressing oncogenic SmoM2 in rare cerebellar granule cell precursors (GCPs) and scRNA-seq analysis. Homozygous but not heterozygous sporadic loss of Pten resulted in rapid acceleration of tumor growth and end stage disease by 40 days, compared to ∼25% survival in control SmoM2 mice at 100 days. Heterozygous PTEN mutations therefore should negatively impact disease outcome primarily with germline mutations. Loss of Pten in normal or SmoM2-expressing GCPs increased proliferation and enhanced progenitor state initially but by 12 days Pten mutant SmoM2 tumors were highly differentiated due to increased survival of non-proliferating GCPs. Furthermore, macrophage infiltration and cytotoxicity appeared reduced in differentiated regions of tumors lacking Pten, indicating cell nonautonomous changes could also contribute to accelerated tumor growth.

Decreased brain levels of coenzyme Q10 (CoQ10), an endogenously synthesized lipophilic antioxidant1,2, underpin encephalopathy in primary CoQ10 deficiencies3,4 and are associated with common neurodegenerative diseases and the ageing process5,6. CoQ10 supplementation does not increase CoQ10 pools in the brain or in other tissues. The recent discovery of the mammalian CoQ10 headgroup synthesis pathway, in which 4-hydroxyphenylpyruvate dioxygenase-like protein (HPDL) makes 4-hydroxymandelate (4-HMA) to synthesize the CoQ10 headgroup precursor 4-hydroxybenzoate (4-HB)7, offers an opportunity to pharmacologically restore CoQ10 synthesis and mechanistically treat CoQ10 deficiencies. To test whether 4-HMA or 4-HB supplementation promotes CoQ10 headgroup synthesis in vivo, here we administered 4-HMA and 4-HB to Hpdl-/- mice, which model an ultra-rare, lethal mitochondrial encephalopathy in humans. Both 4-HMA and 4-HB were incorporated into CoQ9 and CoQ10 in the brains of Hpdl-/- mice. Oral treatment of Hpdl-/- pups with 4-HMA or 4-HB enabled 90-100% of Hpdl-/- mice to live to adulthood. Furthermore, 4-HB treatment stabilized and improved the neurological symptoms of a patient with progressive spasticity due to biallelic HPDL variants. Our work shows that 4-HMA and 4-HB can modify the course of mitochondrial encephalopathy driven by HPDL variants and demonstrates that CoQ10 headgroup intermediates can restore CoQ10 synthesis in vivo.

Although specific transcription factors (TFs) are known to regulate cell fate decisions, the degree to which they can stimulate formation of specific cell organelles is less clear. We used a multiomics comparison of the transcriptomes of ciliated and unciliated embryonic cells to identify TFs up-regulated in ciliated cells. We also used conditional genetics in mouse embryos and stem cells and found that the TFs SP5 and SP8 regulate cilia formation and gene expression. In embryos lacking Sp5 and Sp8, primary and motile cilia were shorter than normal and reduced in number across cell types, contributing to situs inversus and hydrocephalus. Moreover, expression of SP8 was sufficient to induce primary cilia in unciliated cells. This work will facilitate the study of cilia assembly using stem cell models and promote further understanding of human ciliopathies.

While specific transcription factors are known to regulate cell fate decisions, the degree to which they can stimulate formation of specific cell organelles is less clear. We used a multi-omics comparison of the transcriptomes of ciliated and non-ciliated embryonic cells to identify transcription factors upregulated in ciliated cells, and conditional genetics in mouse embryos and stem cells to demonstrate that SP5/8 regulate cilia formation and gene expression. In Sp5/8 mutant embryos primary and motile cilia are shorter than normal and reduced in number across cell types, contributing to situs inversus and hydrocephalus. Moreover, expression of SP8 is sufficient to induce primary cilia in unciliated cells. This work opens new avenues for studying cilia assembly using stem cell models and offers new insights into human ciliopathies.

The neonatal mouse cerebellum shows remarkable regenerative potential upon injury at birth, wherein a subset of Nestin-expressing progenitors (NEPs) undergoes adaptive reprogramming to replenish granule cell progenitors that die. Here, we investigate how the microenvironment of the injured cerebellum changes upon injury and contributes to the regenerative potential of normally gliogenic-NEPs and their adaptive reprogramming. Single-cell transcriptomic and bulk chromatin accessibility analyses of the NEPs from injured neonatal cerebella compared to controls show a temporary increase in cellular processes involved in responding to reactive oxygen species (ROS), a known damage-associated molecular pattern. Analysis of ROS levels in cerebellar tissue confirms a transient increase 1 day after injury at postnatal day 1, overlapping with the peak cell death in the cerebellum. In a transgenic mouse line that ubiquitously overexpresses human mitochondrial catalase (mCAT), ROS is reduced 1 day after injury to the granule cell progenitors, and we demonstrate that several steps in the regenerative process of NEPs are curtailed, leading to reduced cerebellar growth. We also provide preliminary evidence that microglia are involved in one step of adaptive reprogramming by regulating NEP replenishment of the granule cell precursors. Collectively, our results highlight that changes in the tissue microenvironment regulate multiple steps in adaptive reprogramming of NEPs upon death of cerebellar granule cell progenitors at birth, highlighting the instructive roles of microenvironmental signals during regeneration of the neonatal brain.

The capacity of the brain to compensate for insults during development depends on the type of cell loss, whereas the consequences of genetic mutations in the same neurons are difficult to predict. We reveal powerful compensation from outside the mouse cerebellum when the excitatory cerebellar output neurons are ablated embryonically and demonstrate that the main requirement for these neurons is for motor coordination and not basic learning and social behaviors. In contrast, loss of the homeobox transcription factors Engrailed1/2 (EN1/2) in the cerebellar excitatory lineage leads to additional deficits in adult learning and spatial working memory, despite half of the excitatory output neurons being intact. Diffusion MRI indicates increased thalamo-cortico-striatal connectivity in En1/2 mutants, showing that the remaining excitatory neurons lacking En1/2 exert adverse effects on extracerebellar circuits regulating motor learning and select non-motor behaviors. Thus, an absence of cerebellar output neurons is less disruptive than having cerebellar genetic mutations.

Genomic rearrangements are a hallmark of most childhood tumors, including medulloblastoma, one of the most common brain tumors in children, but their causes remain largely unknown. Here, we show that PiggyBac transposable element derived 5 (Pgbd5) promotes tumor development in multiple developmentally accurate mouse models of Sonic Hedgehog (SHH) medulloblastoma. Most Pgbd5-deficient mice do not develop tumors, while maintaining normal cerebellar development. Ectopic activation of SHH signaling is sufficient to enforce cerebellar granule cell progenitor-like cell states, which exhibit Pgbd5-dependent expression of distinct DNA repair and neurodevelopmental factors. Mouse medulloblastomas expressing Pgbd5 have increased numbers of somatic structural DNA rearrangements, some of which carry PGBD5-specific sequences at their breakpoints. Similar sequence breakpoints recurrently affect somatic DNA rearrangements of known tumor suppressors and oncogenes in medulloblastomas in 329 children. This identifies PGBD5 as a medulloblastoma mutator and provides a genetic mechanism for the generation of oncogenic DNA rearrangements in childhood cancer.

More than 40 years ago, studies of the Drosophila engrailed and Hox genes led to major discoveries that shaped the history of developmental biology. We learned that these genes define the state of determination of cells that populate particular spatially defined regions: the identity of segmental domains by Hox genes, and the identity of posterior developmental compartments by engrailed. Hence, the boundaries that delimit spatial domains depend on engrailed. Here, we review the engrailed field, which now includes orthologs in Drosophila and mouse, as well as many other animals. We focus on fly and mouse and highlight additional functions that span early stages of embryogenesis and neural development.

The neurons of the three cerebellar nuclei (CN) are the primary output neurons of the cerebellum. The excitatory neurons (e) of the medial (m) CN (eCNm) were recently divided into molecularly defined subdomains in the adult; however, how they are established during development is not known. We define molecular subdomains of the mouse embryonic eCNm using single-cell RNA-sequencing and spatial expression analysis, showing that they evolve during embryogenesis to prefigure the adult. Furthermore, eCNm are transcriptionally divergent from cells in the other nuclei by embryonic day 14.5. We previously showed that loss of the homeobox genes En1 and En2 leads to loss of approximately half of the embryonic eCNm. We demonstrate that mutation of En1/2 in the embryonic eCNm results in death of specific posterior eCNm molecular subdomains and downregulation of TBR2 (EOMES) in an anterior embryonic subdomain, as well as reduced synaptic gene expression. We further reveal a similar function for EN1/2 in mediating TBR2 expression, neuron differentiation and survival in the other excitatory neurons (granule and unipolar brush cells). Thus, our work defines embryonic eCNm molecular diversity and reveals conserved roles for EN1/2 in the cerebellar excitatory neuron lineage.

The major cause of treatment failure and mortality among medulloblastoma patients is metastasis intracranially or along the spinal cord. The molecular mechanisms driving tumor metastasis in Sonic hedgehog-driven medulloblastoma (SHH-MB) patients, however, remain largely unknown. In this study we define a tumor suppressive role of KMT2D (MLL2), a gene frequently mutated in the most metastatic beta-subtype. Strikingly, genetic mouse models of SHH-MB demonstrate that heterozygous loss of Kmt2d in conjunction with activation of the SHH pathway causes highly penetrant disease with decreased survival, increased hindbrain invasion and spinal cord metastasis. Loss of Kmt2d attenuates neural differentiation and shifts the transcriptional/chromatin landscape of primary and metastatic tumors toward a decrease in differentiation genes and tumor suppressors and an increase in genes/pathways implicated in advanced stage cancer and metastasis (TGF beta, Notch, Atoh1, Sox2, and Myc). Thus, secondary heterozygous KMT2D mutations likely have prognostic value for identifying SHH-MB patients prone to develop metastasis.

Magnetic Resonance Imaging (MRI) resolution continues to improve, making it important to understand the cellular basis for different MRI contrast mechanisms. Manganese-enhanced MRI (MEMRI) produces layer-specific contrast throughout the brain enabling in vivo visualization of cellular cytoarchitecture, particularly in the cerebellum. Due to the unique geometry of the cerebellum, especially near the midline, 2D MEMRI images can be acquired from a relatively thick slice by averaging through areas of uniform morphology and cytoarchitecture to produce very high-resolution visualization of sagittal planes. In such images, MEMRI hyperintensity is uniform in thickness throughout the anterior-posterior axis of sagittal sections and is centrally located in the cerebellar cortex. These signal features suggested that the Purkinje cell layer, which houses the cell bodies of the Purkinje cells and the Bergmann glia, is the source of hyperintensity. Despite this circumstantial evidence, the cellular source of MRI contrast has been difficult to define. In this study, we quantified the effects of selective ablation of Purkinje cells or Bergmann glia on cerebellar MEMRI signal to determine whether signal could be assigned to one cell type. We found that the Purkinje cells, not the Bergmann glia, are the primary of source of the enhancement in the Purkinje cell layer. This cell-ablation strategy should be useful for determining the cell specificity of other MRI contrast mechanisms.

Modeling has proposed that the amount of neural tissue folding is set by the level of differential-expansion between tissue layers and that the wavelength is set by the thickness of the outer layer. Here we used inbred mouse strains with distinct amounts of cerebellar folding to investigate these predictions. We identified a critical period where the folding amount diverges between the strains. In this period, regional changes in the level of differential-expansion between the external granule layer (EGL) and underlying core correlate with the folding amount in each strain. Additionally, the thickness of the EGL is regionally adjusted during the critical period alongside corresponding changes in wavelength. While the number of SHH-expressing Purkinje cells predicts the folding amount, the proliferation rate in the EGL is the same between the strains. However, regional changes in the cell division angle within the EGL predicts both the tangential-expansion and thickness of the EGL. Cell division angle is likely a tunable mechanism whereby both the level of differential-expansion and thickness of the EGL are regionally tuned to set the amount and wavelength of folding.

The cerebellum has a simple cytoarchitecture consisting of a folded cortex with three cell layers that surrounds a nuclear structure housing the output neurons. The excitatory neurons are generated from a unique progenitor zone, the rhombic lip, whereas the inhibitory neurons and astrocytes are generated from the ventricular zone. The growth phase of the cerebellum is driven by lineage-restricted progenitor populations derived from each zone. Research during the past decade has uncovered the importance of cell-to-cell communication between the lineages through largely unknown signaling mechanisms for regulating the scaling of cell numbers and cell plasticity during mouse development and following injury in the neonatal (P0-P14) cerebellum. This Review focuses on how the interplay between cell types is key to morphogenesis, production of robust neural circuits and replenishment of cells after injury, and ends with a discussion of the implications of the greater complexity of the human cerebellar progenitor zones for development and disease.

Targeted protein degradation methods offer a unique avenue to assess a protein's function in a variety of model systems. Recently, these approaches have been applied to mammalian cell culture models, enabling unprecedented temporal control of protein function. However, the efficacy of these systems at the tissue and organismal levels in vivo is not well established. Here, we tested the functionality of the degradation tag (dTAG) degron system in mammalian development. We generated a homozygous knock-in mouse with a FKBP12F36V tag fused to negative elongation factor b (Nelfb) locus, a ubiquitously expressed regulator of transcription. In our validation of targeted endogenous protein degradation across mammalian development and adulthood, we demonstrate that irrespective of the route of administration the dTAG system is non-toxic, rapid, and efficient in embryos from the zygote-to-mid-gestation stages. Additionally, acute depletion of NELFB revealed a specific role in zygote-to-2-cell development and zygotic genome activation (ZGA).

The neocortex consists of a vast number of diverse neurons that form distinct layers and intricate circuits at the single-cell resolution to support complex brain functions(1). Diverse cell-surface molecules are thought to be key for defining neuronal identity, and they mediate interneuronal interactions for structural and functional organization2-6. However, the precise mechanisms that control the fine neuronal organization of the neocortex remain largely unclear. Here, by integrating in-depth single-cell RNA-sequencing analysis, progenitor lineage labelling and mosaic functional analysis, we report that the diverse yet patterned expression of clustered protocadherins (cPCDHs)-the largest subgroup of the cadherin superfamily of cell-adhesion molecules7-regulates the precise spatial arrangement and synaptic connectivity of excitatory neurons in the mouse neocortex. The expression of cPcdh genes in individual neocortical excitatory neurons is diverse yet exhibits distinct composition patterns linked to their developmental origin and spatial positioning. A reduction in functional cPCDH expression causes a lateral clustering of clonally related excitatory neurons originating from the same neural progenitor and a significant increase in synaptic connectivity. By contrast, overexpression of a single cPCDH isoform leads to a lateral dispersion of clonally related excitatory neurons and a considerable decrease in synaptic connectivity. These results suggest that patterned cPCDH expression biases fine spatial and functional organization of individual neocortical excitatory neurons in the mammalian brain.

Evidence for a cerebellar role during cardiopulmonary challenges has long been established, but studies of cerebellar involvement in eupneic breathing have been inconclusive. Here we investigated temporal aspects of eupneic respiration in the Atoh1-En1/2 mouse model of cerebellar neuropathology. Atoh1-En1/2 conditional knockout mice have conditional loss of the developmental patterning genes Engrailed1 and 2 in excitatory cerebellar nuclear neurons, which leads to loss of a subset of medial and intermediate excitatory cerebellar nuclear neurons. A sample of three Atoh1-derived extracerebellar nuclei showed no cell loss in the conditional knockout compared to control mice. We measured eupneic respiration in mutant animals and control littermates using whole-body unrestrained plethysmography and compared the average respiratory rate, coefficient of variation, and the CV2, a measure of intrinsic rhythmicity. Linear regression analyses revealed that Atoh1-En1/2 conditional knockouts have decreased overall variability (p = 0.021; b = -0.045) and increased intrinsic rhythmicity compared to their control littermates (p < 0.001; b = -0.037), but we found no effect of genotype on average respiratory rate (p = 0.064). Analysis also revealed modestly decreased respiratory rates (p = 0.025; b = -0.82), increased coefficient of variation (p = 0.0036; b = 0.060), and increased CV2 in female animals, independent of genotype (p = 0.024; b = 0.026). These results suggest a cerebellar involvement in eupneic breathing by controlling rhythmicity. We argue that the cerebellar involvement in controlling the CV2 of respiration is indicative of an involvement of coordinating respiration with other orofacial rhythms, such as swallowing.

Genomic rearrangements are a hallmark of most solid tumors, including medulloblastoma, one of the most common brain tumors in children. Childhood cancers involve dysregulated cell development, but their mutational causes remain largely unknown. One of the most common forms of medulloblastoma is caused by ectopic activation of Sonic Hedgehog (SHH) signaling in cerebellar granule cell progenitors, associated with genetic deletions, amplifications, and other oncogenic chromosomal rearrangements. Here, we show that PiggyBac Transposable Element Derived 5 (Pgbd5) promotes tumor development in multiple developmentally-accurate mouse models of SHH medulloblastoma. Most mice with Pgbd5 deficiency do not develop tumors, while Pgbd5 -deficient mice maintain largely normal cerebellar development. Mouse medulloblastomas expressing Pgbd5 exhibit significantly increased numbers of somatic structural DNA rearrangements, with PGBD5-specific transposon sequences at their breakpoints. Similar sequence breakpoints recurrently affect somatic DNA rearrangements of known tumor suppressors and oncogenes in medulloblastomas in 329 children. Therefore, this study identifies PGBD5 as a primary medulloblastoma mutator and provides a genetic mechanism responsible for the generation of somatic oncogenic DNA rearrangements in childhood cancer. One-Sentence Summary Induction of somatic oncogenic mutations by the DNA transposase PGBD5 in cerebellar progenitor cells promotes medulloblastoma development.

Support for basic science has been eclipsed by initiatives aimed at specific medical problems. The latest example is the dismantling of the Skirball Institute at NYU School of Medicine. Here, we reflect on the achievements and mission underlying the Skirball to gain insight into the dividends of maintaining a basic science vision within the academic enterprises.

“Genetics doesn’t exist in isolation, but without genetics, you don’t know where your reality is,” said Kathryn Anderson after she was awarded the 2016 Edwin G. Conklin medal by the Society for Developmental Biology (Maartens, 2016Maartens A. An interview with Kathryn Anderson.Development. 2016; 143: 4095-4096Crossref Scopus (0) Google Scholar). Kathryn was a fearless geneticist who made seminal contributions to Drosophila embryogenesis, immunology, and mammalian development. Kathryn was the founding chair of the Developmental Biology Program at the Sloan Kettering Institute, which has become renowned for its groundbreaking research. In addition to her scientific contributions, Kathryn was an outstanding advisor to those who worked in her lab, to her colleagues at Sloan Kettering, and for the broader scientific community. Kathryn’s early work investigating the dorsal-ventral body plan in Drosophila led to the discovery of key developmental genes, including Toll. She elegantly dissected the Toll signaling cascade and its roles in both Drosophila embryo dorsal-ventral patterning and adult immune response against invading pathogens. These discoveries laid the foundation for studies of innate immunity in mammals, where a family of Toll-like receptors (TLRs) was found to act as sensors of microbial pathogens and transduce the signal through analogous pathway components as those identified in flies. Having made landmark discoveries in these two important fields, Kathryn shocked many people when she made the leap to investigating mouse development. Her decision to apply forward genetic screens in mice was both ambitious and risky. As her trainees, we were fortunate to witness this important transition. Kathryn led the discovery of many new genes critical to key aspects of mouse development, including the dorsal-ventral patterning of the developing nervous system, and the morphogenetic events that shape the mammalian body plan. Highlighting the importance of forward genetic approaches in mammals, her screens identified new components in the vertebrate Hedgehog pathway that are not conserved in flies, including those that establish the requirement for primary cilia in vertebrate Hedgehog signal transduction. Although Kathryn’s research has had broad impact on biology, it has always focused on her curiosity around the earliest developmental events driving the elaboration of the body plan of animals. Born in La Jolla, CA in 1952, Kathryn Virginia Anderson became interested in science in eighth grade. She would later credit her budding interest in developmental biology (in an interview with Ushma Neill) to the captivating photographs of human embryos and fetuses taken by Lennart Nilsson, published in Life Magazine’s 1965 cover story “Drama of Life Before Birth.” This interest was further nurtured by her supportive parents and an excellent high school biology teacher, Michael Lorch. Kathryn was introduced to developmental biology as the first graduate student of Judith Lengyel at the University of California, Los Angeles (UCLA) in 1977. For her graduate work, Kathryn pioneered the use of modern molecular approaches to measure the rates of RNA synthesis in relation to DNA replication in early fly embryos. For her postdoctoral research, Kathryn joined Christiane Nüsslein-Volhard’s newly established group in the Friedrich Miescher Laboratory of the Max Planck Society in Tübingen, Germany in 1981. Starting from a large-scale genetic screen for maternal effect mutants with Nüsslein-Volhard and continuing the work in her own lab at the University of California, Berkeley, Kathryn embarked on an awesome journey of discovery, identifying an array of genes required for the establishment of the dorsal-ventral body axis in fly embryos, using genetics, biochemistry, and embryology. Her findings were foundational for defining the Toll pathway cascade and understanding how the body plan of Drosophila embryos is laid out. An inflection point in Kathryn’s career took place in the early ‘90s after she took a sabbatical with Rosa Beddington at the Medical Research Council’s National Institute for Medical Research in Mill Hill. Her initial plans were to investigate the roles of Toll in mice, although it turned out that the Toll pathway is not required for patterning early mouse embryos. The experience at Mill Hill piqued Kathryn’s interest in the control of early mouse embryo polarity. At that point, most mammalian genetics revolved around performing targeted mutagenesis (knockout) of previously identified genes, especially homologs of those identified from genetic screens in flies. However, Kathryn’s experience with the Toll pathway highlighted the need for a new approach that could uncover mammalian-specific developmental mechanisms. She reasoned that a forward genetic approach focused on recessive mutations would allow for the discovery of essential mammalian developmental genes without relying on prior assumptions about their function. There were obvious hurdles of conducting systematic screens in mice to examine recessive embryonic phenotypes. In particular, even after the discovery of interesting mutant phenotypes, the search for the mutations was arduous due to the lack of a sequenced mouse genome. Therefore, many people at the time, including those serving as reviewers for funding agencies, thought it was unrealistic to use mutagenesis screens to study mouse embryogenesis. Upon returning to Berkeley, Kathryn and a graduate student, Andrew Kasarskis, convinced themselves that genetic redundancy in mice should not be an insurmountable barrier to mouse forward genetics and that ENU mutagenesis was efficient enough to conduct a pilot screen (on a shoestring budget) to identify recessive mutations that disrupt mouse development (Kasarskis et al., 1998Kasarskis A. Manova K. Anderson K.V. A phenotype-based screen for embryonic lethal mutations in the mouse.Proc. Natl. Acad. Sci. USA. 1998; 95: 7485-7490Crossref PubMed Scopus (157) Google Scholar). The success of the pilot screen motivated Kathryn to expand the mutagenesis efforts after she moved to the Sloan Kettering Institute in New York in 1996. As the first generation of “mouse” trainees, we made fast progress characterizing the mutant phenotypes, but discovering the causal mutations remained painfully slow at the beginning, which fed the skepticism in the scientific community. Some warned us that joining such a risky endeavor could ruin our careers. Others would express well-intentioned concerns at our poster presentations at scientific conferences. However, Kathryn was always able to see the bigger picture. While not obvious to many, Kathryn recognized that the mouse genome sequencing project would soon accelerate the discovery of genes responsible for the mutant phenotype. Indeed, we vividly remember the weeks after the release of the first human genome draft, when our work moved from the bench to the computer, and we soon discovered the mutated genes in the mutants we were characterizing. The audacity of Kathryn’s decision, when she first conceived the idea of using forward genetics to study mouse embryogenesis, was based on her confidence in the genetic approach and her foresight that different pieces of the puzzle—the mutant phenotypes, the mapping, and the sequence information—would come together. Beyond taking the risky screening approach, the ultimate success of the screens was driven by Kathryn’s intuition to select the most revealing mutants to study and her open-minded approaches to understanding the biology behind the morphological abnormalities observed. Mutations that disrupted gastrulation, axis formation, and mesoderm migration were some of Kathryn’s favorites. Surprisingly, the study of these mutations did not reveal many signaling molecules or transcription factors, but rather discovered genes involved in cytoskeletal reorganization, cell migration, and epithelial remodeling. Collectively, these findings emphasized the dynamic nature of mouse development and the impact of morphogenetic processes in early cell fate decisions in mammals. Another striking difference between mouse and fly development was uncovered through the studies of Hedgehog pathway mutants. The screens identified not only components that were conserved between flies and mammals, but also many genes unique to mammalian Hedgehog signaling. This was surprising because the Hedgehog pathway was initially discovered in the fly, and numerous screens in flies had identified the core Hedgehog pathway components. Many of these mammalian-specific Hedgehog signaling components were involved in function of the primary cilium, a microtubule-based organelle present on most mammalian cells (Huangfu and Anderson, 2005Huangfu D. Anderson K.V. Cilia and Hedgehog responsiveness in the mouse.Proc. Natl. Acad. Sci. USA. 2005; 102: 11325-11330Crossref PubMed Scopus (631) Google Scholar; Huangfu et al., 2003Huangfu D. Liu A. Rakeman A.S. Murcia N.S. Niswander L. Anderson K.V. Hedgehog signalling in the mouse requires intraflagellar transport proteins.Nature. 2003; 426: 83-87Crossref PubMed Scopus (1042) Google Scholar), and it soon became apparent that primary cilia play a central role in Hedgehog signal transduction in mammals. This discovery helped to bring cilia into the spotlight and underscored their clinical significance, as we now appreciate that defective ciliary assembly and function underlie a long list of inherited disorders, collectively known as ciliopathies. This landmark discovery prompted Kathryn’s lab and the broader scientific community to investigate the roles of cilia as signal-processing organelles. Kathryn was an inspirational and supportive mentor who led by example. She always conducted her own experiments in the laboratory alongside us, providing ample room for impromptu conversations. Some of the most valuable advice we remember receiving from Kathryn originated from these informal discussions, which sometimes ended with us posting “bets” on our whiteboard to predict experimental outcomes. Many of the bets involved the prediction of her beloved double mutant phenotypes. Kathryn always understood that biology could be unpredictable, so making predictions was not about being right, but rather about being able to rationalize one’s predictions with the underlying biology. Kathryn made us see why open questions, paradoxes, controversies, and models (both elegant and flawed) were inherently interesting and worthy of devoting one’s life to study. Kathryn never used many words, but the ones she had were sharp and insightful. In our discussions, she would jump in to correct errors in logic or provide some key facts, but she would also be patient and encouraging when we stumbled upon an original hypothesis, and she made space for us to own our projects and ideas. A testament to Kathryn’s successful mentorship is that many of the graduate students and postdoctoral fellows trained in her lab have gone on to establish their own labs at academic institutions or have become leaders in industry and government. Her generosity extended beyond her own lab. She was a visionary leader of her department, which has become a model for gender equality. Similar to her approach inside the lab, she gave junior faculty members in her department space to grow, while also making sure they had the support to succeed. Kathryn will be remembered as a fearless, visionary geneticist. Her phenotype-first approach to developmental biology embodied the spirit of Viktor Hamburger’s famous words, “Our real teacher has been and still is the embryo, who is, incidentally, the only teacher who is always right.” For many of us, Kathryn was also a generous and supportive mentor, with a contagious love for science. She recognized the unique strength in each of us and encouraged all of us to achieve our full potential. Her legacy of fearlessness in scientific pursuit will continue to be an inspiration for her trainees and the numerous scientists influenced by her work. We thank Tamara Caspary, Anna-Katerina Hadjantonakis, Andrew Kasarskis, Andrew Rakeman, and Louisa Wu for helpful comments.