Cachd1 interacts with Wnt receptors and regulates neuronal asymmetry in the zebrafish brain.

Neurons on the left and right sides of the nervous system often show asymmetric properties, but how such differences arise is poorly understood. Genetic screening in zebrafish revealed that loss of function of the transmembrane protein Cachd1 resulted in right-sided habenula neurons adopting left-sided identity. Cachd1 is expressed in neuronal progenitors, functions downstream of asymmetric environmental signals, and influences timing of the normally asymmetric patterns of neurogenesis. Biochemical and structural analyses demonstrated that Cachd1 can bind simultaneously to Lrp6 and Frizzled family Wnt co-receptors. Consistent with this, lrp6 mutant zebrafish lose asymmetry in the habenulae, and epistasis experiments support a role for Cachd1 in modulating Wnt pathway activity in the brain. These studies identify Cachd1 as a conserved Wnt receptor-interacting protein that regulates lateralized neuronal identity in the zebrafish brain.

Loss of the crumbs cell polarity complex disrupts epigenetic transcriptional control and cell cycle progression in the developing retina.

The crumbs cell polarity complex plays a crucial role in apical-basal epithelial polarity, cellular adhesion, and morphogenesis. Homozygous variants in human CRB1 result in autosomal recessive Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP), with no established genotype-phenotype correlation. The associated protein complexes have key functions in developmental pathways; however, the underlying disease mechanism remains unclear. Using the oko meduzym289/m289 (crb2a-/- ) zebrafish, we performed integrative transcriptomic (RNA-seq data) and methylomic [reduced representation bisulphite sequencing (RRBS)] analysis of whole retina to identify dysregulated genes and pathways. Delayed retinal cell specification was identified in both the crb2a-/- zebrafish and CRB1 patient-derived retinal organoids, highlighting the dysfunction of cell cycle modulation and epigenetic transcriptional control. Differential DNA methylation analysis revealed novel hypermethylated pathways involving biological adhesion, Hippo, and transforming growth factor ß (TGFß) signalling. By integrating gene expression with DNA methylation using functional epigenetic modules (FEM), we identified six key modules involving cell cycle control and disturbance of TGFß, bone morphogenetic protein (BMP), Hippo, and SMAD protein signal transduction pathways, revealing significant interactome hotspots relevant to crb2a function and confirming the epigenetic control of gene regulation in early retinal development, which points to a novel mechanism underlying CRB1-retinopathies. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Collaborative Research and Development of a Novel, Patient-Centered Digital Platform (MyEyeSite) for Rare Inherited Retinal Disease Data: Acceptability and Feasibility Study.

BACKGROUND: Inherited retinal diseases (IRDs) are a leading cause of blindness in children and working age adults in the United Kingdom and other countries, with an appreciable socioeconomic impact. However, by definition, IRD data are individually rare, and as a result, this patient group has been underserved by research. Researchers need larger amounts of these rare data to make progress in this field, for example, through the development of gene therapies. The challenge has been how to find and make these data available to researchers in the most productive way. MyEyeSite is a research collaboration aiming to design and develop a digital platform (the MyEyeSite platform) for people with rare IRDs that will enable patients, doctors, and researchers to aggregate and share specialist eye health data. A crucial component of this platform is the MyEyeSite patient application, which will provide the means for patients with IRD to interact with the system and, in particular, to collate, manage, and share their personal specialist IRD data both for research and their own health care. OBJECTIVE: This study aims to test the acceptability and feasibility of the MyEyeSite platform in the target IRD population through a collaborative patient-centered study. METHODS: Qualitative data were generated through focus groups and workshops, and quantitative data were obtained through a survey of patients with IRD. Participants were recruited through clinics at Moorfields Eye Hospital National Health Service (NHS) Foundation Trust and the National Institute for Health Research (NIHR) Moorfields Biomedical Research Centre through their patient and public involvement databases. RESULTS: Our IRD focus group sample (n=50) highlighted the following themes: frustration with the current system regarding data sharing within the United Kingdom's NHS; positive expectations of the potential benefits of the MyEyeSite patient application, resulting from increased access to this specialized data; and concerns regarding data security, including potentially unethical use of the data outside the NHS. Of the surveyed 80 participants, 68 (85%) were motivated to have a more active role in their eye care and share their data for research purposes using a secure technology, such as a web application or mobile app. CONCLUSIONS: This study demonstrates that patients with IRD are highly motivated to be actively involved in managing their own data for research and their own eye care. It demonstrates the feasibility of involving patients with IRD in the detailed design of the MyEyeSite platform exemplar, with input from the patient with IRD workshops playing a key role in determining both the functionality and accessibility of the designs and prototypes. The development of a user-centered technological solution to the problem of rare health data has the potential to benefit not only the patient with IRD community but also others with rare diseases.

Identification of 4 novel human ocular coloboma genes ANK3, BMPR1B, PDGFRA, and CDH4 through evolutionary conserved vertebrate gene analysis.

PURPOSE: Ocular coloboma arises from genetic or environmental perturbations that inhibit optic fissure (OF) fusion during early eye development. Despite high genetic heterogeneity, 70% to 85% of patients remain molecularly undiagnosed. In this study, we have identified new potential causative genes using cross-species comparative meta-analysis. METHODS: Evolutionarily conserved differentially expressed genes were identified through in silico analysis, with in situ hybridization, gene knockdown, and rescue performed to confirm spatiotemporal gene expression and phenotype. Interrogation of the 100,000 Genomes Project for putative pathogenic variants was performed. RESULTS: Nine conserved differentially expressed genes between zebrafish and mouse were identified. Expression of zebrafish ank3a, bmpr1ba/b, cdh4, and pdgfaa was localized to the OF, periocular mesenchyme cells, or ciliary marginal zone, regions traversed by the OF. Knockdown of ank3, bmpr1b, and pdgfaa revealed a coloboma and/or microphthalmia phenotype. Novel pathogenic variants in ANK3, BMPR1B, PDGFRA, and CDH4 were identified in 8 unrelated coloboma families. We showed BMPR1B rescued the knockdown phenotype but variant messenger RNAs failed, providing evidence of pathogenicity. CONCLUSION: We show the utility of cross-species meta-analysis to identify several novel coloboma disease-causing genes. There is a potential to increase the diagnostic yield for new and unsolved patients while adding to our understanding of the genetic basis of OF morphogenesis.

Smartphone screen testing, a novel pre-diagnostic method to identify SARS-CoV-2 infectious individuals.

The COVID-19 pandemic will likely take years to control globally, and constant epidemic surveillance will be required to limit the spread of SARS-CoV-2, especially considering the emergence of new variants that could hamper the effect of vaccination efforts. We developed a simple and robust - Phone Screen Testing (PoST) - method to detect SARS-CoV-2-positive individuals by RT-PCR testing of smartphone screen swab samples. We show that 81.3-100% of individuals with high-viral-load SARS-CoV-2 nasopharyngeal-positive samples also test positive for PoST, suggesting this method is effective in identifying COVID-19 contagious individuals. Furthermore, we successfully identified polymorphisms associated with SARS-CoV-2 Alpha, Beta, and Gamma variants, in SARS-CoV-2-positive PoST samples. Overall, we report that PoST is a new non-invasive, cost-effective, and easy-to-implement smartphone-based smart alternative for SARS-CoV-2 testing, which could help to contain COVID-19 outbreaks and identification of variants of concern in the years to come.

Expanding the phenotypic spectrum consequent upon de novo WDR37 missense variants.

Structural eye disorders are increasingly recognised as having a genetic basis, although current genetic testing is limited in its success. De novo missense variants in WDR37 are a recently described cause of a multisystemic syndromic disorder featuring ocular coloboma. This study characterises the phenotypic spectrum of this disorder and reports 2 de novo heterozygous variants (p.Thr115Ile, p.Ser119Tyr) in three unrelated Caucasian individuals. All had a clinical phenotype consisting of bilateral iris and retinal coloboma, developmental delay and additional, variable multisystem features. The variants fall within a highly conserved region upstream of the WD-repeat domains, within an apparent mutation cluster. Consistent with the literature, intellectual disability, structural eye disorders, epilepsy, congenital heart disease, genitorenal anomalies and dysmorphic facial features were observed. In addition, a broader developmental profile is reported with a more specific musculoskeletal phenotype described in association with the novel variant (p.Thr115Ile). We further expand the phenotypic spectrum of WDR37-related disorders to include those with milder developmental delay and strengthen the association of ocular coloboma and musculoskeletal features. We promote the inclusion of WDR37 on gene panels for intellectual disability, epilepsy and structural eye disorders.

Tissue-Specific Requirement for the GINS Complex During Zebrafish Development.

Efficient and accurate DNA replication is particularly critical in stem and progenitor cells for successful proliferation and survival. The replisome, an amalgam of protein complexes, is responsible for binding potential origins of replication, unwinding the double helix, and then synthesizing complimentary strands of DNA. According to current models, the initial steps of DNA unwinding and opening are facilitated by the CMG complex, which is composed of a GINS heterotetramer that connects Cdc45 with the mini-chromosome maintenance (Mcm) helicase. In this work, we provide evidence that in the absence of GINS function DNA replication is cell autonomously impaired, and we also show that gins1 and gins2 mutants exhibit elevated levels of apoptosis restricted to actively proliferating regions of the central nervous system (CNS). Intriguingly, our results also suggest that the rapid cell cycles during early embryonic development in zebrafish may not require the function of the canonical GINS complex as neither zygotic Gins1 nor Gins2 isoforms seem to be present during these stages.

Developmentally regulated Tcf7l2 splice variants mediate transcriptional repressor functions during eye formation.

Tcf7l2 mediates Wnt/ß-Catenin signalling during development and is implicated in cancer and type-2 diabetes. The mechanisms by which Tcf7l2 and Wnt/ß-Catenin signalling elicit such a diversity of biological outcomes are poorly understood. Here, we study the function of zebrafish tcf7l2alternative splice variants and show that only variants that include exon five or an analogous human tcf7l2 variant can effectively provide compensatory repressor function to restore eye formation in embryos lacking tcf7l1a/tcf7l1b function. Knockdown of exon five specific tcf7l2 variants in tcf7l1a mutants also compromises eye formation, and these variants can effectively repress Wnt pathway activity in reporter assays using Wnt target gene promoters. We show that the repressive activities of exon5-coded variants are likely explained by their interaction with Tle co-repressors. Furthermore, phosphorylated residues in Tcf7l2 coded exon5 facilitate repressor activity. Our studies suggest that developmentally regulated splicing of tcf7l2 can influence the transcriptional output of the Wnt pathway.

De Novo Missense Variants in FBXW11 Cause Diverse Developmental Phenotypes Including Brain, Eye, and Digit Anomalies.

The identification of genetic variants implicated in human developmental disorders has been revolutionized by second-generation sequencing combined with international pooling of cases. Here, we describe seven individuals who have diverse yet overlapping developmental anomalies, and who all have de novo missense FBXW11 variants identified by whole exome or whole genome sequencing and not reported in the gnomAD database. Their phenotypes include striking neurodevelopmental, digital, jaw, and eye anomalies, and in one individual, features resembling Noonan syndrome, a condition caused by dysregulated RAS signaling. FBXW11 encodes an F-box protein, part of the Skp1-cullin-F-box (SCF) ubiquitin ligase complex, involved in ubiquitination and proteasomal degradation and thus fundamental to many protein regulatory processes. FBXW11 targets include ß-catenin and GLI transcription factors, key mediators of Wnt and Hh signaling, respectively, critical to digital, neurological, and eye development. Structural analyses indicate affected residues cluster at the surface of the loops of the substrate-binding domain of FBXW11, and the variants are predicted to destabilize the protein and/or its interactions. In situ hybridization studies on human and zebrafish embryonic tissues demonstrate FBXW11 is expressed in the developing eye, brain, mandibular processes, and limb buds or pectoral fins. Knockdown of the zebrafish FBXW11 orthologs fbxw11a and fbxw11b resulted in embryos with smaller, misshapen, and underdeveloped eyes and abnormal jaw and pectoral fin development. Our findings support the role of FBXW11 in multiple developmental processes, including those involving the brain, eye, digits, and jaw.

Transcriptome profiling of zebrafish optic fissure fusion.

Incomplete fusion of the optic fissure leads to ocular coloboma, a congenital eye defect that affects up to 7.5 per 10,000 births and accounts for up to 10 percent of childhood blindness. The molecular and cellular mechanisms that facilitate optic fissure fusion remain elusive. We have profiled global gene expression during optic fissure morphogenesis by transcriptome analysis of tissue dissected from the margins of the zebrafish optic fissure and the opposing dorsal retina before (32 hours post fertilisation, hpf), during (48 hpf) and after (56 hpf) optic fissure fusion. Differential expression analysis between optic fissure and dorsal retinal tissue resulted in the detection of several known and novel developmental genes. The expression of selected genes was validated by qRT-PCR analysis and localisation investigated using in situ hybridisation. We discuss significantly overrepresented functional ontology categories in the context of optic fissure morphogenesis and highlight interesting transcripts from hierarchical clustering for subsequent analysis. We have identified netrin1a (ntn1a) as highly differentially expressed across optic fissure fusion, with a resultant ocular coloboma phenotype following morpholino antisense translation-blocking knockdown and downstream disruption of atoh7 expression. To support the identification of candidate genes in human studies, we have generated an online open-access resource for fast and simple quantitative querying of the gene expression data. Our study represents the first comprehensive analysis of the zebrafish optic fissure transcriptome and provides a valuable resource to facilitate our understanding of the complex aetiology of ocular coloboma.

Compensatory growth renders Tcf7l1a dispensable for eye formation despite its requirement in eye field specification.

The vertebrate eye originates from the eye field, a domain of cells specified by a small number of transcription factors. In this study, we show that Tcf7l1a is one such transcription factor that acts cell-autonomously to specify the eye field in zebrafish. Despite the much-reduced eye field in tcf7l1a mutants, these fish develop normal eyes revealing a striking ability of the eye to recover from a severe early phenotype. This robustness is not mediated through genetic compensation at neural plate stage; instead, the smaller optic vesicle of tcf7l1a mutants shows delayed neurogenesis and continues to grow until it achieves approximately normal size. Although the developing eye is robust to the lack of Tcf7l1a function, it is sensitised to the effects of additional mutations. In support of this, a forward genetic screen identified mutations in hesx1, cct5 and gdf6a, which give synthetically enhanced eye specification or growth phenotypes when in combination with the tcf7l1a mutation.

Antagonism between Gdf6a and retinoic acid pathways controls timing of retinal neurogenesis and growth of the eye in zebrafish.

Maintaining neurogenesis in growing tissues requires a tight balance between progenitor cell proliferation and differentiation. In the zebrafish retina, neuronal differentiation proceeds in two stages with embryonic retinal progenitor cells (RPCs) of the central retina accounting for the first rounds of differentiation, and stem cells from the ciliary marginal zone (CMZ) being responsible for late neurogenesis and growth of the eye. In this study, we analyse two mutants with small eyes that display defects during both early and late phases of retinal neurogenesis. These mutants carry lesions in gdf6a, a gene encoding a BMP family member previously implicated in dorsoventral patterning of the eye. We show that gdf6a mutant eyes exhibit expanded retinoic acid (RA) signalling and demonstrate that exogenous activation of this pathway in wild-type eyes inhibits retinal growth, generating small eyes with a reduced CMZ and fewer proliferating progenitors, similar to gdf6a mutants. We provide evidence that RA regulates the timing of RPC differentiation by promoting cell cycle exit. Furthermore, reducing RA signalling in gdf6a mutants re-establishes appropriate timing of embryonic retinal neurogenesis and restores putative stem and progenitor cell populations in the CMZ. Together, our results support a model in which dorsally expressed gdf6a limits RA pathway activity to control the transition from proliferation to differentiation in the growing eye.

Transcription factor 7-like 1 is involved in hypothalamo-pituitary axis development in mice and humans.

Aberrant embryonic development of the hypothalamus and/or pituitary gland in humans results in congenital hypopituitarism (CH). Transcription factor 7-like 1 (TCF7L1), an important regulator of the WNT/ß-catenin signaling pathway, is expressed in the developing forebrain and pituitary gland, but its role during hypothalamo-pituitary (HP) axis formation or involvement in human CH remains elusive. Using a conditional genetic approach in the mouse, we first demonstrate that TCF7L1 is required in the prospective hypothalamus to maintain normal expression of the hypothalamic signals involved in the induction and subsequent expansion of Rathke's pouch progenitors. Next, we reveal that the function of TCF7L1 during HP axis development depends exclusively on the repressing activity of TCF7L1 and does not require its interaction with ß-catenin. Finally, we report the identification of two independent missense variants in human TCF7L1, p.R92P and p.R400Q, in a cohort of patients with forebrain and/or pituitary defects. We demonstrate that these variants exhibit reduced repressing activity in vitro and in vivo relative to wild-type TCF7L1. Together, our data provide support for a conserved molecular function of TCF7L1 as a transcriptional repressor during HP axis development in mammals and identify variants in this transcription factor that are likely to contribute to the etiology of CH.

Tcf7l2 is required for left-right asymmetric differentiation of habenular neurons.

BACKGROUND: Although left-right asymmetries are common features of nervous systems, their developmental bases are largely unknown. In the zebrafish epithalamus, dorsal habenular neurons adopt medial (dHbm) and lateral (dHbl) subnuclear character at very different frequencies on the left and right sides. The left-sided parapineal promotes the elaboration of dHbl character in the left habenula, albeit by an unknown mechanism. Likewise, the genetic pathways acting within habenular neurons to control their asymmetric differentiated character are unknown. RESULTS: In a forward genetic screen for mutations that result in loss of habenular asymmetry, we identified two mutant alleles of tcf7l2, a gene that encodes a transcriptional regulator of Wnt signaling. In tcf7l2 mutants, most neurons on both sides differentiate with dHbl identity. Consequently, the habenulae develop symmetrically, with both sides adopting a pronounced leftward character. Tcf7l2 acts cell automously in nascent equipotential neurons, and on the right side, it promotes dHbm and suppresses dHbl differentiation. On the left, the parapineal prevents this Tcf7l2-dependent process, thereby promoting dHbl differentiation. CONCLUSIONS: Tcf7l2 is essential for lateralized fate selection by habenular neurons that can differentiate along two alternative pathways, thereby leading to major neural circuit asymmetries.

In vivo Wnt signaling tracing through a transgenic biosensor fish reveals novel activity domains.

The creation of molecular tools able to unravel in vivo spatiotemporal activation of specific cell signaling events during cell migration, differentiation and morphogenesis is of great relevance to developmental cell biology. Here, we describe the generation, validation and applications of two transgenic reporter lines for Wnt/ß-catenin signaling, named TCFsiam, and show that they are reliable and sensitive Wnt biosensors for in vivo studies. We demonstrate that these lines sensitively detect Wnt/ß-catenin pathway activity in several cellular contexts, from sensory organs to cardiac valve patterning. We provide evidence that Wnt/ß-catenin activity is involved in the formation and maintenance of the zebrafish CNS blood vessel network, on which sox10 neural crest-derived cells migrate and proliferate. We finally show that these transgenic lines allow for screening of Wnt signaling modifying compounds, tissue regeneration assessment as well as evaluation of potential Wnt/ß-catenin genetic modulators.

HESX1- and TCF3-mediated repression of Wnt/ß-catenin targets is required for normal development of the anterior forebrain.

The Wnt/ß-catenin pathway plays an essential role during regionalisation of the vertebrate neural plate and its inhibition in the most anterior neural ectoderm is required for normal forebrain development. Hesx1 is a conserved vertebrate-specific transcription factor that is required for forebrain development in Xenopus, mice and humans. Mouse embryos deficient for Hesx1 exhibit a variable degree of forebrain defects, but the molecular mechanisms underlying these defects are not fully understood. Here, we show that injection of a hesx1 morpholino into a 'sensitised' zygotic headless (tcf3) mutant background leads to severe forebrain and eye defects, suggesting an interaction between Hesx1 and the Wnt pathway during zebrafish forebrain development. Consistent with a requirement for Wnt signalling repression, we highlight a synergistic gene dosage-dependent interaction between Hesx1 and Tcf3, a transcriptional repressor of Wnt target genes, to maintain anterior forebrain identity during mouse embryogenesis. In addition, we reveal that Tcf3 is essential within the neural ectoderm to maintain anterior character and that its interaction with Hesx1 ensures the repression of Wnt targets in the developing forebrain. By employing a conditional loss-of-function approach in mouse, we demonstrate that deletion of ß-catenin, and concomitant reduction of Wnt signalling in the developing anterior forebrain of Hesx1-deficient embryos, leads to a significant rescue of the forebrain defects. Finally, transcriptional profiling of anterior forebrain precursors from mouse embryos expressing eGFP from the Hesx1 locus provides molecular evidence supporting a novel function of Hesx1 in mediating repression of Wnt/ß-catenin target activation in the developing forebrain.

Lef1-dependent Wnt/ß-catenin signalling drives the proliferative engine that maintains tissue homeostasis during lateral line development.

During tissue morphogenesis and differentiation, cells must self-renew while contemporaneously generating daughters that contribute to the growing tissue. How tissues achieve this precise balance between proliferation and differentiation is, in most instances, poorly understood. This is in part due to the difficulties in dissociating the mechanisms that underlie tissue patterning from those that regulate proliferation. In the migrating posterior lateral line primordium (PLLP), proliferation is predominantly localised to the leading zone. As cells emerge from this zone, they periodically organise into rosettes that subsequently dissociate from the primordium and differentiate as neuromasts. Despite this reiterative loss of cells, the primordium maintains its size through regenerative cell proliferation until it reaches the tail. In this study, we identify a null mutation in the Wnt-pathway transcription factor Lef1 and show that its activity is required to maintain proliferation in the progenitor pool of cells that sustains the PLLP as it undergoes migration, morphogenesis and differentiation. In absence of Lef1, the leading zone becomes depleted of cells during its migration leading to the collapse of the primordium into a couple of terminal neuromasts. We show that this behaviour resembles the process by which the PLLP normally ends its migration, suggesting that suppression of Wnt signalling is required for termination of neuromast production in the tail. Our data support a model in which Lef1 sustains proliferation of leading zone progenitors, maintaining the primordium size and defining neuromast deposition rate.

A useful approach to identify novel small-molecule inhibitors of Wnt-dependent transcription.

The Wnt signaling pathway is frequently deregulated in cancer due to mutations in genes encoding APC, beta-catenin, and axin. To identify small-molecule inhibitors of Wnt signaling as potential therapeutics, a diverse chemical library was screened using a transcription factor reporter cell line in which the activity of the pathway was induced at the level of Disheveled protein. A series of deconvolution studies was used to focus on three compound series that selectively killed cancer cell lines with constitutive Wnt signaling. Activities of the compounds included the ability to induce degradation of beta-catenin that had been stabilized by a glycogen synthase kinase-3 (GSK-3) inhibitor. This screen illustrates a practical approach to identify small-molecule inhibitors of Wnt signaling that can seed the development of agents suitable to treat patients with Wnt-dependent tumors.

Early stages of zebrafish eye formation require the coordinated activity of Wnt11, Fz5, and the Wnt/beta-catenin pathway.

During regional patterning of the anterior neural plate, a medially positioned domain of cells is specified to adopt retinal identity. These eye field cells remain coherent as they undergo morphogenetic events distinct from other prospective forebrain domains. We show that two branches of the Wnt signaling pathway coordinate cell fate determination with cell behavior during eye field formation. Wnt/beta-catenin signaling antagonizes eye specification through the activity of Wnt8b and Fz8a. In contrast, Wnt11 and Fz5 promote eye field development, at least in part, through local antagonism of Wnt/beta-catenin signaling. Additionally, Wnt11 regulates the behavior of eye field cells, promoting their cohesion. Together, these results allow us to postulate a model in which Wnt11 and Fz5 signaling promotes early eye development through the coordinated antagonism of signals that suppress retinal identity and promotion of coherence of eye field cells.

Expression and splice variant analysis of the zebrafish tcf4 transcription factor.

Wnt signalling has been implicated in antero-posterior patterning of the vertebrate embryonic body axis and in a number of other developmental processes. One of the downstream effectors of Wnt signalling is the beta-catenin protein which complexes with members of the Lef/tcf transcription factor family. In the zebrafish, specification of the head has been shown to be dependent on the Tcf3 protein which acts as a repressor of the posteriorizing activity of Wnt (Nature 407 (2000) 913). Here, we report the cloning and expression pattern of the zebrafish tcf4 gene. In embryos, we find that the tcf4 gene is highly regulated at the level of RNA splicing such that the variant proteins that are produced contain or lack domains proposed to be essential in repression or activation of transcription. Expression of tcf4 mRNA is first detected in a graded fashion in the anterior brain and subsequently becomes restricted to the dorsal diencephalon and anterior midbrain. There is also transient expression in the anterior rhombomeres of the hindbrain and in the developing gut.